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14.current Applications of Pharmaceutical Biotechnology

This document provides information about Volume 171 of the book series Advances in Biochemical Engineering/Biotechnology. It lists the editorial board members and describes the aims and scope of reviewing trends in modern biotechnology and biochemical engineering. The preface discusses current clinical relevance and manufacturing challenges of biopharmaceuticals such as monoclonal antibodies, cytokines, growth factors, hormones, blood products, therapeutic enzymes, vaccines, and advanced therapy medicinal products involving cells, tissues, and genes. The book aims to provide up-to-date information on various applications of pharmaceutical biotechnology for healthcare professionals and researchers.

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0% found this document useful (0 votes)
553 views520 pages

14.current Applications of Pharmaceutical Biotechnology

This document provides information about Volume 171 of the book series Advances in Biochemical Engineering/Biotechnology. It lists the editorial board members and describes the aims and scope of reviewing trends in modern biotechnology and biochemical engineering. The preface discusses current clinical relevance and manufacturing challenges of biopharmaceuticals such as monoclonal antibodies, cytokines, growth factors, hormones, blood products, therapeutic enzymes, vaccines, and advanced therapy medicinal products involving cells, tissues, and genes. The book aims to provide up-to-date information on various applications of pharmaceutical biotechnology for healthcare professionals and researchers.

Uploaded by

ltbnhu
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Volume 171

Advances in Biochemical
Engineering/Biotechnology

Series Editor
T. Scheper
Hannover, Germany

Editorial Board
S. Belkin
Jerusalem, Israel

T. Bley
Dresden, Germany

J. Bohlmann
Vancouver, Canada

M. B. Gu
Seoul, Korea (Republic of)

W.-S. Hu
Minneapolis, USA

B. Mattiasson
Lund, Sweden

H. Seitz
Potsdam, Germany

R. Ulber
Kaiserslautern, Germany

A.-P. Zeng
Hamburg, Germany

J.-J. Zhong
Shanghai, China

W. Zhou
Shanghai, China

Aims and Scope


This book series reviews current trends in modern biotechnology and
biochemical engineering. Its aim is to cover all aspects of these
interdisciplinary disciplines, where knowledge, methods and expertise are
required from chemistry, biochemistry, microbiology, molecular biology,
chemical engineering and computer science.
Volumes are organized topically and provide a comprehensive
discussion of developments in the field over the past 3–5 years. The series
also discusses new discoveries and applications. Special volumes are
dedicated to selected topics which focus on new biotechnological products
and new processes for their synthesis and purification.
In general, volumes are edited by well-known guest editors. The series
editor and publisher will, however, always be pleased to receive suggestions
and supplementary information. Manuscripts are accepted in English.
In references, Advances in Biochemical Engineering/Biotechnology is
abbreviated asAdv. Biochem. Engin./Biotechnol. and cited as a journal.
More information about this series at http://www.springer.com/series/10
Editors
Ana Catarina Silva, João Nuno Moreira, José Manuel Sousa Lobo and
Hugo Almeida

Current Applications of Pharmaceutical


Biotechnology
Editors
Ana Catarina Silva
UCIBIO, REQUIMTE, MEDTECH, Laboratory of Pharmaceutical
Technology, Department of Drug Sciences, Faculty of Pharmacy, University
of Porto, Porto, Portugal
FP-ENAS (UFP Energy, Environment and Health Research Unit),
CEBIMED (Biomedical Research Centre), Faculty of Health Sciences,
Fernando Pessoa University, Porto, Portugal

João Nuno Moreira


Center for Neurosciences and Cell Biology (CNC) and Faculty of Pharmacy
(FFUC), University of Coimbra, Coimbra, Portugal

José Manuel Sousa Lobo


UCIBIO, REQUIMTE, MEDTECH, Laboratory of Pharmaceutical
Technology, Department of Drug Sciences, Faculty of Pharmacy, University
of Porto, Porto, Portugal

Hugo Almeida
UCIBIO, REQUIMTE, MEDTECH, Laboratory of Pharmaceutical
Technology, Department of Drug Sciences, Faculty of Pharmacy, University
of Porto, Porto, Portugal

ISSN 0724-6145 e-ISSN 1616-8542


Advances in Biochemical Engineering/Biotechnology
ISBN 978-3-030-40463-5 e-ISBN 978-3-030-40464-2
https://doi.org/10.1007/978-3-030-40464-2

© Springer Nature Switzerland AG 2020

This work is subject to copyright. All rights are reserved by the Publisher,
whether the whole or part of the material is concerned, specifically the
rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and
transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or
hereafter developed.

The use of general descriptive names, registered names, trademarks, service


marks, etc. in this publication does not imply, even in the absence of a
specific statement, that such names are exempt from the relevant protective
laws and regulations and therefore free for general use.

The publisher, the authors, and the editors are safe to assume that the advice
and information in this book are believed to be true and accurate at the date
of publication. Neither the publisher nor the authors or the editors give a
warranty, expressed or implied, with respect to the material contained
herein or for any errors or omissions that may have been made. The
publisher remains neutral with regard to jurisdictional claims in published
maps and institutional affiliations.

This Springer imprint is published by the registered company Springer


Nature Switzerland AG.
The registered company address is: Gewerbestrasse 11, 6330 Cham,
Switzerland
Preface
Biopharmaceuticals have been showing high therapeutic potential,
especially for medical conditions associated with unmet medical needs.
Examples of these conditions include cancer and autoimmune, metabolic,
dermatological, neurological, and genetic diseases. Thereby, current clinical
relevance of biopharmaceuticals is unquestionable. Since the 1980s, several
biological medicines have been approved and, due to patents expiration,
many biosimilars have been marketed.
Manufacture and formulation of biopharmaceuticals comprising
therapeutic proteins generated by DNA recombination is challenging.
Depending on the protein’s molecular weight and structure complexity,
industrial manufacturing processes have employed different host cells.
Furthermore, biopharmaceuticals formulation is fundamental to achieve an
effective medicine, being necessary to stabilize the protein conformation,
enabling its efficacy while avoiding immunogenicity and other safety
problems. Commercial biopharmaceuticals are typically for parenteral
administration, although several efforts have been made to find alternative
administration routes and/or new delivery systems enabling improved
pharmacokinetic properties and better patient compliance.
Therapeutic applications of biopharmaceuticals are highly diverse and
include active substances such as monoclonal antibodies, cytokines, growth
factors, hormones, blood products, therapeutic enzymes, as well as
vaccines, cellular therapies and products of gene therapy. In respect of
biopharmaceuticals (including biosimilars and orphans), the European
Medicines Agency (EMA) and the Food and Drug Administration (FDA)
have approved so far 89 cytokines, 78 hormones (insulin, glucagon, growth
hormone, gonadotropins, and thyroid-stimulating hormone and parathyroid
hormone), 49 blood products (blood factors, anticoagulants, and
thrombolytic agents), and 22 therapeutic enzymes. Current research has
been also focused on identifying new clinical applications for existing
therapeutic proteins.
A number of different advances in the development, manufacturing, and
characterization of vaccines have enabled the prevention of infections and
promoted human health worldwide. Approved recombinant vaccines
include Shingrix, Ebola (Ad5-EBOV), meningococcal subgroup B, human
papilloma virus (HPV), dengue, enterovirus 71 (EV71), and hepatitis B
(HBV), while vaccines against rVSV-ZEBOV Ebola, respiratory syncytial
virus (RSV), and malaria are under clinical evaluation.
In addition to biopharmaceuticals and vaccines, advanced therapy
medicinal products based on the use of cells, tissues, or genes offer new
therapeutic possibilities. For example, cell-based therapies have been used
in regenerative medicine, disease modelling, and drug screening studies.
This has also constituted an opportunity to engineer new manufacturing
concepts as 3D bioprinting technology, which have succeeded in generating
living tissues and organs from a combination of cells, biomaterial bioinks,
and growth factors. Examples of bioprinted tissues include nerve, skin,
heart, bone, cartilage, skeletal muscle and soft tissues such as liver, cornea,
intestine, and bladder.
In the last years, gene therapy medicines, such as DNA-based therapies,
antisense oligonucleotides, small interfering RNA, and T-cell-based
therapies, have shown promising results to such a point that they have
started to change the treatment paradigm, as in cancer. Furthermore, the use
of gene-editing approaches, such as the CRISPR (clustered regularly
interspaced short palindromic repeats), has started to be assessed at the
clinical level. Besides cancer, the clinical usefulness of gene therapy is also
being assessed in monogenic infections, inflammatory, cardiovascular, and
neurodegenerative diseases.
Within the scope of the different therapeutic tools herein addressed,
pharmacogenomics assumes a relevant role on their performance, in terms
of both efficacy and safety. In fact, the use of a medicine adapted to the
genetic variants of each patient, reducing adverse effects and improving
treatment outcomes, supports pharmacogenomics as a relevant component
of personalized medicine.
According to the wide range of pharmaceutical biotechnology
applications, this book aims at providing the most relevant up-to-date
information for healthcare professionals, students, and researchers.
We would like to thank all the authors of this book for contributing with
high quality chapters. We are also very grateful to all the reviewers, who
had the kindness of critically evaluating the chapters. In addition, we would
like to thank to the Series Editor, Professor Thomas Scheper and to the
Associate Editors, Dr. Sofia Costa and Dr. Tanja Weyandt, for giving us the
opportunity of organizing this volume, and to the publishing team (Ms.
Alamelu Damodharan and Ms. S. Dhivya Geno) for their kind help.
Ana Catarina Silva
João Nuno Moreira
José Manuel Sousa Lobo
Hugo Almeida
Porto, Portugal, Coimbra, Portugal, Porto, Portugal, Porto, Portugal
Contents
Industrial Challenges of Recombinant Proteins
Scott R. Rudge and Michael R. Ladisch
Insights on the Formulation of Recombinant Proteins
Rita Ribeiro, Teresa Raquel Abreu, Ana Catarina Silva, João Gonçalves
and João Nuno Moreira
Therapeutic Antibody Engineering and Selection Strategies
Joana Ministro, Ana Margarida Manuel and Joao Goncalves
Cytokines and Growth Factors
A. C. Silva and J. M. Sousa Lobo
Hormones, Blood Products, and Therapeutic Enzymes
Ana Catarina Silva, Cládia Pina Costa, Hugo Almeida,
João Nuno Moreira and José Manuel Sousa Lobo
Advances in Vaccines
Helen H. Mao and Shoubai Chao
Human Pluripotent Stem Cells: Applications and Challenges for
Regenerative Medicine and Disease Modeling
Cláudia C. Miranda, Tiago G. Fernandes, M. Margarida Diogo and
Joaquim M. S. Cabral
Addressing the Manufacturing Challenges of Cell-Based Therapies
Miguel de Almeida Fuzeta, André Dargen de Matos Branco,
Ana Fernandes-Platzgummer, Cláudia Lobato da Silva and
Joaquim M. S. Cabral
Bioprinting Technologies in Tissue Engineering
Bengi Yilmaz, Aydin Tahmasebifar and Erkan Türker Baran
Gene Therapy
Ana del Pozo-Rodríguez, Alicia Rodríguez-Gascón, Julen Rodríguez-
Castejón, Mónica Vicente-Pascual, Itziar Gómez-Aguado,
Luigi S. Battaglia and María Ángeles Solinís
The Impact of Pharmacogenomics in Personalized Medicine
Dev Bukhsh Singh
Index
© Springer Nature Switzerland AG 2019
A. C. Silva et al. (eds.), Current Applications of Pharmaceutical Biotechnology, Advances in
Biochemical Engineering/Biotechnology 171
https://doi.org/10.1007/10_2019_120

Industrial Challenges of Recombinant


Proteins
Scott R. Rudge1 and Michael R. Ladisch2
(1) RMC Pharmaceutical Solutions, Inc., Longmont, CO, USA
(2) Purdue University Laboratory of Renewable Resource Engineering,
West Lafayette, IN, USA

Scott R. Rudge (Corresponding author)


Email: [email protected]

Michael R. Ladisch (Corresponding author)


Email: [email protected]

Abstract
The use of recombinant DNA methods to produce large quantities of
protein for therapeutic uses has revolutionized medicine. Industrial
challenges for manufacture of biotherapeutic proteins are related to the
characteristics of these proteins and the increasing quantities required to
address needs of patients, worldwide. A brief overview of therapies in
which proteins are employed helps to frame some of the challenges facing
their industrial production. The number of molecules and their applications
have significantly expanded over the last 15–20 years, together with the
quantities used to address specific indications. Challenges addressed
include achieving cell density, protein expression, separations of cells and
protein, protein purification, and segmentation of protein production into
smaller quantities with the evolution of personalized medicine and products
designed for increasingly small patient populations. This chapter highlights
some of the current challenges.
Graphical Abstract

Keywords Biologics – Bioprocessing – Cell culture – Cell separation –


Chromatograhpy – Expression systems – Fermentation – Filtration – mAbs
– Recombinant proteins – Separations

1 Introduction
Since the early commercialization of recombinant human insulin,
recombinant human growth hormone, and recombinant bovine growth
hormone, the field has expanded to include over 174 US-approved
therapeutic proteins, including 98 antibodies (including biosimilars) [1, 2].
While insulin was among the first recombinant products, its lower
molecular weight (~6 kD), long history of use, and molecular
characterization positioned it for scaling up [3]. Subsequent early products
such as erythropoietin and tissue plasminogen activator, with their higher
molecular weights and complex structures, required production through
mammalian cell culture and the many attendant scaling and regulatory
factors that needed to be addressed. Along the way, the development and
laboratory culture of hybridoma cells enabled the production of monoclonal
antibodies, proteins with molecular weights in the 120–150 kD range, and
for which initial applications were limited to research tools. It required
about 20 years of further molecular engineering as biopharmaceuticals to
develop this class of proteins. Further advances have been made in yeast,
specifically Pichia pastoris (reclassified as Komagataella pastoris), to
enable the production of large and small proteins including antibodies and
proinsulins. Gene therapy has now emerged with potential to not only treat
diseases but perhaps provide cures. With the emergence of gene therapy has
come the need for vectors consisting of nanoparticulate viral capsids, i.e.,
structured protein assemblies, for delivery of nucleic acid payloads.
Manufacturing approaches have employed similar unit operations
throughout the development of biotherapeutic products since their
introduction about 40 years ago. What has changed are cells and culture
methods and the separations media utilized during the manufacturing
processes.

2 Classes of Products (Agonists and Antagonists)


Therapeutic recombinant proteins generally fall within two classes, those
meant for replacement therapy (agonists) and those meant for antagonist
therapy.

2.1 Agonists
Replacement therapies are those in which the intended patient has lost the
ability to produce a protein, and that lack of ability results in disease. Type I
diabetes is an excellent example of such a disease, in that the patient has
lost the ability to produce insulin due to an autoimmune destruction of
pancreatic islet cells. In such patients exogenous insulin is required for the
patient’s body to properly regulate glucose in the blood stream. These
patients were treated with insulin purified from porcine pancreas before the
introduction of recombinant protein production [2]. The injection of insulin
at intervals related to meal times and times of fasting (overnight, for
example) allows these patients to regain some control, albeit imperfect, of
their blood glucose levels. In this case, the ability to inject a recombinant
form of human insulin replaces the patient’s ability to produce their own
insulin. In general, low doses of proteins in this category are required, as
the protein being replaced physiologically has a specific function. These
proteins are also called “agonists,” as their specific function is to make
something happen. Other examples of agonists are erythropoietin,
granulocyte-colony stimulating factor, growth hormone, and factor VIII.
New agonist therapies are not common, as most diseases requiring a
replacement protein are well-known and fatal and are/were being treated
with a purified protein from an animal, plasma fractionation, or cadavers.
Additionally, these diseases are usually in small patient numbers, as a rare
genetic defect may be involved or an autoimmunity. The search for new
agonists continues, as these treatments are almost certain to be clinically
successful with few side effects. Progress in understanding the function of
the brain and the neurological system may be the last frontier for agonist
therapies.

2.2 Antagonists
The other class of recombinant proteins is called antagonists. These proteins
are used to block some physiological action. Antagonists bind to receptors
or other proteins, or other molecules in the blood stream, and prevent them
from having their normal physiological function. An excellent example of
an antagonist therapy is the chemotherapeutic antibody Avastin or
antihuman vascular endothelial growth factor (VEGF) immunoglobulin G1.
VEGF is a protein that stimulates the growth of new blood vessels, which
are produced by tumors growing in the body, which require a source of
nutrition from the blood stream. Avastin blocks the action of VEGF, thereby
depriving tumors of the source of nutrition they need to grow [4].
Antagonist therapies typically require higher doses than agonists, as the
antagonist molecule has to battle stoichiometry and biology to bind and
neutralize all the target molecules in the patient. As the target molecules are
neutralized by the antagonist, the body tends to make more as the biological
feedback loop indicates that the desired effect is not being achieved.
Additionally, the complete elimination of a physiological mechanism is
usually not desirable. For example, patients taking Avastin will have
difficulty growing new healthy tissue as well as tumor mass.

2.3 Humanized Antibodies


The development of humanized antibodies to treat human disease has been
a second major milestone in the history of recombinant protein therapies.
Early attempts to use antibodies to seek out and bind targets in humans
were set back by immune responses to the nonhuman epitopes in the
antibody structure. Methods for “humanizing” antibodies, including
critically in the “complement defining region” or CDR, have made the
construction of antibodies specific for distinct biological targets possible
[5].
Over the last 20 years, antibody antagonists have largely displaced
recombinant agonists as the top selling protein pharmaceuticals, as shown
in Table 1 (compare [6] to [7]) with the pipeline of therapeutic mAbs now at
560 in various stages of clinical trials [8, 37].
Table 1 Top selling biopharmaceuticals from 1997 and 2011

1997 [6] 2011 [7]


1 Erythropoietin (agonist) Anti-TNF monoclonal antibody (antagonist)
2 Granulocyte-colony stimulating factor Anti-TNF receptor monoclonal antibody
(agonist) (antagonist)
3 Insulin (agonist) Anti-VEGF monoclonal antibody (antagonist)
4 α-Interferon (agonist) Anti-CD20 monoclonal antibody (antagonist)
5 Hepatitis B vaccine (agonist) Anti-HER2 receptor monoclonal antibody
(antagonist)
6 Glucocerebrosidase (agonist) Insulin (agonist)
7 Tissue plasminogen activator (agonist) PEGylated granulocyte-colony stimulating factor
(agonist)
8 Growth hormone (agonist) Erythropoietin (agonist)
9 GPIIb/IIIa antibody (antagonist) Anti-VEGF monoclonal antibody fragment
(antagonist)
10 Interferon β-1a (agonist) Interferon β-1a

Today, the top ten selling recombinant protein pharmaceuticals are


mostly monoclonal antibodies. Antibody products accounted for about
$114.6 billion in sales in 2018, while all biopharmaceuticals were about
$237 billion. Total pharmaceutical sales were $1,200 billion for reference.
Interestingly, all of these products received their marketing approval within
the last 15 years, which demonstrates the rapid growth of the product
category and may indicate the large potential still remaining.
However, as with most technologies, the targets for antibodies and other
antagonists are becoming harder to identify, and the targets that are being
pursued are in higher concentrations, requiring higher doses of antagonists
[9]. Doses as high as one or more grams per day are becoming more
common. High doses can be problematic because (1) they require greater
purity, (2) they are harder to administer to the patient, and (3) they require
lower cost of goods. The third constraint is easily understood, the treatment
of disease is an economic proposition, and the market, or society, will only
bare so much cost. The cost of treating versus not treating must be carefully
balanced, and in cases where other therapies exist, a price is already set.
The market cares little about the mass of protein required to produce the
desired effect, and the cost of goods for an active antibody can vary
between $100 and $1,000/g, excluding testing, dosage form manufacturing,
and other expenses [10].
Higher doses also require higher purity. This is best illustrated with the
impurity endotoxin, which is produced in very large quantities when
Escherichia coli is used to produce the recombinant protein. Endotoxin is
part of the cell wall of gram-negative bacteria and causes an immune
response and inflammation when present in the blood stream of humans.
This activity of endotoxin, called pyrogenicity, is measured in endotoxin
units (EU) by a few standardized biological assays. The total amount of
endotoxin that a patient can be exposed to per 1 h administration is limited
by medical standards to 5 EU/kg (the kg refers to the weight of the patient).
A 50 kg patient may be exposed to a total of 250 EU. If the dose of the
protein is 10 mg, the protein must be purified to at most 25 EU/mg, but if
the protein dose is 2 g/day, then the most endotoxin the product can contain
is 0.125 EU/mg.

2.4 Biosimilars
With a regulatory pathway now open for biosimilar products, there will be a
focus on cost of goods for biologics like never before. When patent
exclusivity expires, the cost of making and purifying the protein will
become a more significant part of the selling price. The cost for making a
purified therapeutic protein ranges from about $100 to $1,000/g, with cell
culture-derived proteins requiring higher expenditures. Therefore, reduction
of cost of goods at higher purity requirements will be a major challenge for
biopharmaceutical manufacturing in the immediate future.

2.5 Pharmacoeconomics
Finally, the political climate is one in which justification of high
reimbursement for pharmaceuticals is increasingly difficult.
Pharmacoeconomics may not be sufficient to justify reimbursement in all
cases. Therefore, due to high doses, biologics intended for treating diseases
where other treatments exist, pressure from biosimilars, and price restraints,
the cost of manufacturing a therapeutic protein is under pressure. This
chapter will review some current efforts to decrease the cost of
manufacturing.

3 Upstream Manufacturing
Upstream manufacturing includes fermentation, cell culture, or another
means of expressing the therapeutic protein at high titer and reasonable
purity. Upstream manufacturing typically also includes the removal of the
production cells or biomass. This may be done with a centrifuge, a filter, by
flocculation and settling, or another unit operation.

3.1 Expression Systems


The expression of the protein is the first and primary step in producing the
product in its desired form. Expression systems consist of a host cell and
the foreign DNA that encodes the product. The most commonly used host
cells are E. coli and Chinese hamster ovary (CHO) cells. E. coli is used
when the protein requires little posttranslational modifications, while CHO
cells are used when posttranslational modifications are required for the
structure or function of the protein. Antibodies require fairly extensive
posttranslational modifications. For example, immunoglobulin Gs (the most
common therapeutic antibody and the simplest) require posttranslational
modifications in the form of the assembly of two heavy chains with two
light chains to form a hetero-tetramer, and glycosylation of an asparagine
located on the heavy chain constant region, so typically antibodies are made
in CHO. Insulin only requires enzymatic activation, which can be done in
manufacturing with the correct protease, so typically, insulin is made in E.
coli.
Protein expression is complicated, and there are several steps that have
to be optimized to get the maximum expression of the desired protein.
There are several considerations:
The codons in the gene for the desired protein must be optimized for the
host cell.
The gene must possess flanking regions, such as a poly-adenosine tail
and a ribosome binding site.
The gene must be under the control of a promoter that will cause its
expression, either constitutively or in response to an inducer or
derepressor.
If it is desired to export the product to another compartment of the cell, or
to secrete the product, a leader sequence must be attached to direct the
protein to that cellular compartment.
The gene must have the proper reading frame to ensure the appropriate
sequence is transcribed and translated.
There are numerous expression systems available, both proprietary and
in the public domain. The expression of every recombinant protein is
different and unpredictable, but the expression of antibodies tends to be
fairly consistent from antibody to antibody. A titer of 3 g/L can be expected
for an antibody expressed from nonproprietary expression constructs in
CHO cells [11]. Titers as high as 15–25 g/L have been reported in vendor
marketing data; however the corresponding product quality is unknown.

3.2 Promoters
A common promoter for protein expression in CHO cells is the
cytomegalovirus (CMV) promoter [12]. This promoter is constitutive,
meaning it is always activated and does not require induction. The titer is
also dependent on the cell density – the more cells, the more product. A
typical cell density for CHO cells is 20 × 106 cells/mL. However, high cell
density cultures with up to 100 × 106 cells/mL are now being developed
which can increase the titer a commensurate fivefold. These cell densities
are a relatively new development, and their impact on downstream
processing is still being determined. Cell culture medium costs about
$100/L on average, so the media alone can contribute $10–$33/g to the cost
of the product, depending on the titer. Media used to achieve high cell
density is more expensive, but not five times more expensive, so the
investment in high cell density is justified.
Product expression in E. coli is typically greater than 5 g/L and can be
as high as 15 g/L. At very high expression levels in E. coli, the product is
usually found in inclusion bodies, small precipitate particles that have to be
solubilized and renatured to make them usable. A common expression
system for E. coli is the T7 promoter, which is induced with iso-propyl thio
glycerol (IPTG) [13]. IPTG derepresses the T7 promoter, allowing
transcription of the gene and subsequent translation. An alternate promoter
system is the xyz switch where amino acid content in the media is used to
induce insulin expression [3]. In most bacterial systems, the expression of
the foreign protein is toxic to the cells, and so inducible rather than
constitutive promoters are used. Inducer is added to the fermentation after
high cell density is reached, at which point the cells do not grow
significantly. The final wet weight of solids in the fermentation is typically
12–16% (120–160 g/L) of which the expressed protein is about 10% (12–
16 g/L as previously stated). On a dry weight basis, the proportion of
expressed protein is closer to 20–25% by weight. E. coli media is much
cheaper to prepare, usually $5–$10/L. If the expression level is reasonably
high, the cost of the media will only be $1–$2/g.
Efforts to increase protein expression are ongoing and remain important.
When the protein expression is increased, the purification is easier.
However, additional impurities are typically generated with highly
expressed proteins as the protein synthesis machinery is overtaken by the
higher level of expression. One common impurity is the mis-incorporation
of norleucine for methionine residues [14]. Covalent protein aggregates are
also prevalent in highly expressed proteins. Some attention to the likely
downstream yield purity must be paid when designing high expression
systems.

3.3 High Cell Density Reactors


Another way to increase product throughput is to grow cells to very high
cell density. In mammalian cell culture, this means densities on the order of
100 × 106 cells/mL, and for bacterial and yeast fermentations, this means
ODs greater than 100 and approaching 300 or 400, and wet cell weights
around 50%. These higher cell densities are made possible by new media
formulations that are rich in key nutrients, as well as advances in oxygen
transfer into and heat removal from the reactor vessel.
Increased cell density gives productivity gains linearly in proportion to
the increase of numbers of cells. CHO cultures are routinely run at
20 × 106 cells/mL, so the ability to achieve 100 × 106 cells/mL offers an
opportunity to increase the productive time in the bioreactor given that
protein expression and cell growth occur concurrently, unlike E. coli where
expression follows accumulation of cells and subsequent induction. Since
mammalian cell culture expression is typically constitutive, the culture is
producing product throughout, again in proportion to cell density. The area
under the cell density vs. time curve gives the basis for the product
expression. As shown in Fig. 1, 43% of the productivity of a 25-day culture
can be accounted for in the 6 days the culture achieves >80 × 106 cells/mL.

Fig. 1 Typical cell density vs. time curve and protein production, assuming a doubling time of 24 h
and specific productivity of 1 pg/cell/day

Bacterial expression systems are typically induced, and when they are
induced, the growth of the cells slows or stops, as metabolism is taken over
by the protein synthesis machinery of the cell. The fermentation may only
last some hours after induction of the expression system; 10–20 h is typical.
In this case, the specific productivity is unchanged as the productivity gains
are directly proportional to the cell density at induction.
Both high cell density techniques are limited in the further advantage
that they can bring to protein production. At 50% wet weight, another
doubling in bacterial fermentation productivity by this method is not
possible. Extending the length of time such high cell densities can be
maintained with cells in maximum productivity will be the most practical
way to increase production in the future.
High cell density cell culture poses challenges in cell separation. These
will be discussed in a subsequent section.
4 Single Use Manufacturing
Many new products are designed for increasingly small patient populations.
These products are known as orphan products, and they are highly
reimbursed. Gene and cell therapies are also beginning to emerge in this
space. Efficient expression systems have made it possible to produce a
worldwide supply of these drugs in a few batches per year. Since it is not
practical to dedicate an entire clean facility to produce a few batches of a
product per year, multi-product facilities have become more common, and
contract manufacturing organizations are being tasked for production on a
more frequent basis.

4.1 Single-Use Bioreactors


Keeping different products segregated from one another is a critical
requirement of multi-product facilities, which means extensive and detailed
cleaning of the equipment during product change-over. Another solution is
to make inexpensive, disposable equipment. Disposable bioreactors have
been in use at commercial scale for about 10 years for mammalian cell
culture. Disposable systems increase the flexibility of multi-product
manufacturing facilities by removing the requirement for post-use cleaning
of the bioreactor. These bioreactors are typically made of plastic and come
pre-sterilized by gamma irradiation. While larger bioreactors have been
made, the most common large single-use bioreactor has a 2,000 L working
volume, a version of which is available from at least three different
manufacturers. These are accompanied by other single-use unit operations,
such as filtration pods, chromatography systems, and vessels. A fully
“disposable factory” may still be cost prohibitive, but it could become less
expensive in the future.
While flexibility is promoted as a feature of disposable systems,
particularly bioreactors, such systems are not as flexible as might be
imagined [15]. The bioreactors require a superstructure made of steel to
support them. The single-use bioreactors are very heavy and require a crane
to lift them into the support structure and then to lift them out again.
Shipping is expensive and can result in pinholes and creases in the plastic,
which lead to bioreactor rupture or sterility failure. And while some early
economic analysis made the case that single-use plastic was more
economical and environmentally friendly than cleaning fixed stainless-steel
reactors, these analyses [16] ignored shipping, gamma irradiation, the clean
room required to fabricate the single-use reactors, and the effect of the
plastic load on the biosphere.
Single-use systems are not readily available for large-scale microbial
fermentation. As bacteria grow many times faster than mammalian cells,
their oxygen transfer and heat transfer requirements are much larger. Plastic
bioreactors are not able to hold significant pressure which facilitates oxygen
transfer, and the magnetic drive that is used to agitate plastic bioreactors
cannot deliver the torque required for mixing and heat and oxygen transfer.

5 Perfusion Reactors and Continuous Processing


One method for increasing bioreactor productivity is to increase the time
the fermentation or bioreaction remains at high cell density and maximum
productivity. One obvious way to do this is to continuously supply fresh
nutrient medium while removing spent medium and product (if secreted)
while retaining cells. There are two challenges in this endeavor, one is to
continuously provide sterile medium, and the other is to remove spent
medium while retaining cells and maintaining sterility.
The nutrients required for mammalian cell culture are complex and not
stable to autoclaving. Therefore, the introduction of fresh medium is
typically via filtration. Mammalian cultures are also susceptible to viruses,
and so viral reduction devices such as “high-temperature-short-time” and
UVC irradiators are being designed to continuously introduce sterile, viral-
free media into the bioreactor [17]. This challenge appears to have been
substantially met.
Sterile cell retention is a different challenge. The bioreactor contains
cells, along with dead cells, cell fragments, lipids, and other debris. The cell
separation device must operate efficiently in this environment. Several
methods that are currently in use are:
Cross-flow filtration
Centrifugation and sedimentation
Acoustic separation
It is important to separate the cells without lysing them. When lysed,
cells release proteases that can degrade the product and additional
impurities that must be subsequently removed by downstream purification
processes. Ideally, the retention device would retain only cells and allow
removal of debris and unproductive solids. Otherwise, the bioreaction will
be choked off by accumulation of the unproductive solids.

5.1 Cell Retention by Cross-Flow Filtration


The alternating tangential flow filtration device may be the most widely
used device for perfusion reactors today. It works by drawing culture fluid
across a micro-filter in tangential flow fashion. The culture fluid
accumulates in an expanding bulb downstream of the filter. Cells are
retained, and spent culture fluid with secreted product crosses into the
filtrate. Then the cycle is reversed, the bulb is collapsed, and the retained
cells are returned to the bioreactor. Since bioreactors typically operate under
slight positive pressure, the driving force for filtration is vacuum on the
filtrate side of the membrane. An example is shown in Fig. 2 [18].
Fig. 2 An alternating tangential flow filtration device [18]

The alternating tangential flow filter allows high throughput and


essentially total retention of cells. Perfusion flow rates of two
volumes/reactor volume/day are readily achievable. The technology is easy
to scale up as the principals of tangential flow filtration are well-known.
The stream is already filtered and so already clarified, so no further
filtration is needed. When a filter clogs, which will happen ultimately, it
must be replaced without stopping operations. This can be done with tube
welding technology, or steam in place valves, although it is inconvenient
and requires the purchase of a second device.
The alternating tangential flow filter retains all solids, including
nonproductive solids, which can accumulate and choke the cell culture if
not removed. Additionally, as the perfusion rate increases, the alternating
flow filter must operate faster, increasing shear on the cells and creating
additional debris. This is typically addressed by adding a cell syphon stream
where cells, debris, and medium are all removed. This is an imperfect
solution, however, and eventually the cell debris will overtake the cell
culture process.

5.2 Cell Retention by Centrifugation or Sedimentation


Centrifugation is also used as a cell retention mechanism. These centrifuge
systems use a modest centrifugal force (approximately 100–300× g) to
sediment cells and other solids and to enable withdrawal of a clear fluid
stream. Care must be taken not to damage the cells and not to pack them too
tightly so that they can freely flow back to the bioreactor. The moving parts
of a centrifuge also make maintaining sterility difficult. Because of these
challenges, sedimentation has also been used as a cell retention mechanism.
Inclined settling uses an inclined plane to settle and return live cells to a
bioreactor, while allowing product-containing medium to be removed from
the overflow. This method also allows dead cells and debris to be removed
with the product stream. A recent improvement has been made to this
system by allowing the flow over stacked cones, much like in a disk-stack
centrifuge but without the added centrifugal force [19, 20]. An example is
shown in Fig. 3.
Fig. 3 Compact settler diagram [20]. Media containing cells are fed through port 142. Product and
nonproductive solids may be withdrawn through port 140, and live cells can be returned to the
bioreactor through port 139

The downside to inclined settlers is the slow settling, which results in


fairly large volumes required to achieve the appropriate residence time. The
large volume/long residence time results in the settling device required
being nearly as large as the bioreactor itself, meaning cells spend significant
time in an environment where pH and dissolved oxygen are not being
actively controlled. Attaining high flow rates has also been difficult in
inclined settling systems, meaning high rates of turnover of bioreactor
contents are not achievable. However, as a solid/liquid separating device
with no moving parts that can be made of inexpensive, light weight
disposable materials, compact settlers have some advantages that cannot be
ignored.

5.3 Cell Retention by Acoustic Separation


A final method for cell retention is the application of an ultrasound field
[21]. The ultrasound is able to retain particles of a certain size against a
small flow field. This method is also difficult to scale up, since the
dimensions increase to provide a small enough linear velocity for the flow
field, the device becomes large relative to the bioreactor, and the acoustic
field heats the fluid throughout, while heat can only be removed from the
periphery of the device. This method for cell retention is rarely used outside
very small-scale lab experiments.

5.4 Perfusion Reaction Control


Continuous processing is being explored as a method for faster and less
expensive manufacturing of biologics such as therapeutic proteins and
antibodies [1]. The advantage of continuous processing is a relatively high
productivity at a smaller overall volume. Continuous processing may
improve overall quality by reducing hold times between batch steps and
increase flexibility by allowing for longer production times at higher
volumetric productivities [22].
Continuous processing requires that a steady state be achieved. In
typical steady-state reactions, reactants are fed to a reactor to produce
product at a constant rate, which can then be removed and purified. In order
to maintain the steady state, feedback control is used to account for small
fluctuations in conditions that can cause variability in the rate of
production, such as the concentration of reactants, temperature and pressure
of feed streams, and fouling of surfaces, for example. The state of
development of feedback control for bioreactors (and other bioprocessing
steps) is in its infancy, and much more work is needed in order to achieve
true continuous processing in biopharmaceuticals. To further develop
feedback control, more information will be needed on the effects of media,
temperature, metabolite concentrations, debris and unproductive solids,
oxygen transfer, pH and osmolarity on product quality, and production over
time. This information is likely to be complex and process specific and may
need to be studied on the genetic level, where different genes in a cell are
upregulated and downregulated in response to various environmental
factors. Without models for the performance of the cells, the time of a batch
can be extended perhaps by a factor of 2–3, but without adequate feedback
control, true continuous bioprocessing is still a concept rather than a reality
[23].

6 Cell Separation
Once the bioreaction phase is over, it is typical to remove cells from the
spent or conditioned medium in order to begin purification of the product.
In mammalian and yeast culture, the product is typically secreted from the
cell, so the cell is considered part of the waste stream and need not be
recovered. In bacterial culture, the product may ultimately reside in an
intracellular compartment or an extracellular compartment depending on
how the recombinant gene is constructed. If the product is secreted, it is
desirable to have a cell separation method that does not lyse the cells. If the
cells are lysed, proteases are released that could degrade the product, and
the released DNA, RNA, lipids, and carbohydrates increase the purification
challenge downstream. Disk stack centrifugation has been the state of the
art for this step for more than 30 years but has recently become limited by
the high cell densities now achieved with mammalian cell culture and yeast
cell culture. Depth filtration is supplanting centrifugation as a gentler
separation methodology.

6.1 Centrifugation
Centrifugation is limited by shear force and solids concentration. A disk
stack centrifuge, which can intermittently or continuously discharge a
concentrated solids fraction, is only able to achieve a solids concentration
of about 50% wet weight. Above this solids concentration, the viscosity of
the solids fraction increases, and the solids cannot be discharged through
the nozzles used to regulate the flow. Yeast culture can easily achieve 50%
wet weight, so these cultures must be diluted prior to use of a disk stack
centrifuge. If dilution is used, yield is still a consideration, as the upper
limit of 50% wet weight will not be overcome. For example, if a 50% wet
weight harvest broth is diluted to 16% by adding 2.125 kg of water for
every kg of harvest broth, the maximum product that can be recovered is
81%, presuming recovery of 2.125 kg of liquid phase for every 1 kg of solid
phase and assuming that the 1 kg of solid phase includes 500 g of solids and
500 g of liquid. Unless in-line dilution is used, a tank that can contain
6,250 kg of diluted harvest broth and another one that can collect 4,250 kg
of liquid phase from the centrifuge will be needed. The need for large tanks
reduces the flexibility that is considered one of the advantages of single-use
technology.
Shear force is another consideration. In a typical disk stack centrifuge,
the disks spin at 3,000–10,000 rpm [24], producing high rates of shear at
the fluid inlets and outlets. Filtration shear rates are at least an order of
magnitude lower, which matters for mammalian cells that are fragile
enough to be lysed by bubbles [25]. This limits the applicability of
centrifugation to bacteria and yeast primarily, since cell lysis is a factor for
mammalian cells.

6.2 Depth Filtration


Depth filtration has been used as a method for collecting cells and other
solids. Depth filters are designed for high solids loads but do not provide an
absolute barrier to solids of a certain size. Rather, filtration is due to
adsorption or entrapment of cells in tortuous channels. It is useful to think
of depth filters as resembling cotton or glass wool, through which a solution
will be poured. Solid is captured by the random threads of the material, and
the deeper the bed of random threads, the more likely the solid is to be
captured. However, solids do eventually break through this type of filter.
Some depth filters are supplemented with filter aids, such as diatomaceous
earth, which provide more solids retention. Eventually the filters clog as a
solids cake begins to form at the surface of the filter medium, and the
filtration has to be terminated, or the filters changed out for new ones. It is
not uncommon for depth filters used as the primary means of cell separation
to process only 10–100 L/m2. Once loaded with solids, these filters are
discarded, as they cannot be regenerated. Although the filters themselves
are relatively inexpensive, the waste stream is considered biohazardous and
is expensive to dispose of. As the cell density in the bioreactor increases,
more area is needed for depth filtration, increasing cost.

6.3 Cross-Flow Filtration


Given the limitations of the two traditional means of providing cell
separation at harvest, other approaches are being tested. Not unlike the cell
retention devices described above, cross-flow filtration and sedimentation
are both being employed, and both suffer from limitations as described
above. Cross-flow filtration has high shear, and the membranes used are
subject to fouling. Cross-flow filtration also has a maximum solids
limitation and requires dilution for recovery of cells, although the system in
Fig. 2 overcomes some of the plugging by reversing flow every 30 s to
1 min.

6.4 Sedimentation
Sedimentation is slow and requires too much volume. In order to speed up
sedimentation, flocculants can be added to the harvest broth, such as
polyethyleneimine or diatomaceous earth. The ideal flocculant would have
high capacity for cell solids with low affinity for the product, a density
much higher than water, no extractable compounds, and minimal toxicity in
humans. Acid can also be used to aggregate some cells but would pose a
degradation risk for some products. Significant opportunity for solid/liquid
separation improvements exists for therapeutic protein production.

7 Downstream Processing
Downstream processing is often portrayed as a bottleneck in
biomanufacturing [26]. Chromatography and ultrafiltration have been the
work horses in downstream processing since they displaced differential
precipitation as a means of fine purification for biomolecules.
Chromatography is slow, with low capacity, and is typically not optimized
for throughput. Chromatography has also been difficult to adapt to
continuous operations. Ultrafiltration is usually not used for purification but
rather for the change of a buffer salt and pH and is not as rapid and
inexpensive as the alternatives of dilution and pH adjustment with acid and
base, and therefore it is avoided in general. However, a founding principal
in preparative biochemical purification is that removing water is expensive
and the balance between dilution and operating at higher concentrations is
not always appreciated. Challenges, principles, and design criteria are the
subject of several books and are briefly outlined below [24, 27, 28].

7.1 Chromatography and Adsorption


Chromatography for biotherapeutic molecules bears more resemblance to a
traditional adsorption /desorption step than to analytical chromatography,
although counterflow for product recovery is rarely employed. The
objective is to purify a single component from a multicomponent mixture,
rather than to separate each component from each other. For this reason, the
preparative chromatography feed stream should be thought of as having
three components: (1) the target product molecule, (2) those molecules that
bind more tightly to the resin, and (3) those molecules that bind less tightly
or not at all. This ternary mixture is the most complicated to purify since the
desired molecule has properties in between the two others [29].
There are two operating modes that are a special case of this general
one: affinity adsorption and flow-through operation. In affinity adsorption,
a ligand with high selectivity and avidity for the product is bound to the
chromatography resin, and in principal, there should be no molecules that
bind more tightly. There may, however, be “product-related impurities”
such as dimers or oxidation products that bind with the same selectivity and
avidity as the product itself.
Alternatively, some adsorption separations are made in “flow-through”
mode, in which the product does not bind to the resin at all, in which case,
there is no fraction of molecules that bind less well to the resin. Using the
resin to capture only contaminants is an excellent strategy, as contaminants
such as RNA and DNA are typically in very small amounts relative to the
product (parts per million to parts per billion) so a small amount of
adsorbent can be used relative to the product. This separation mode also
does not typically separate product-related impurities from product.
When the most general condition exists, that the separation is intended
to purify the product from molecules binding more and less strongly, the
separation must be carefully thought out and tested. The most efficient
separation is likely to occur when the resin binding capacity is saturated
with product although there are trade-offs in product purity when this
occurs. Consequently, multiple sequential steps may be used based on
“orthogonal” binding chemistries, i.e., ion exchange, reversed phase, or size
exclusion, where protein retention is based on different principles.
Examples of binding isotherms are shown in Fig. 4. Proteins and DNA
typically have a Langmuir isotherm form, with a very high slope at low
concentrations. At high concentrations, the resin is saturated and the
isotherm flattens horizontally. Under these conditions, the less strongly
binding components will largely be displaced from the resin. To get the
most binding from the resin, a length of unused bed (LUB) approach may
be used [24]. To minimize the LUB, dispersion must be minimized in the
column, and a condition where a sharp breakthrough curve would be
obtained is favored. A sharp breakthrough curve can be generated by
operating the unit operation at high protein concentration as above, away
from the linear portion of the binding isotherm. If gradients are utilized, it is
possible to encounter a number of nonideal phenomena, and these must be
accounted for in the selection of column operating conditions (i.e., gradient
slope, column load, mobile phase composition, protein concentration,
media chemistry, and particle size) [24, 27, 30].

Fig. 4 Different binding isotherms. Proteins and biotechnology products often have a Langmuir
shape with a very high slope at low concentration [24]

Dilution of the product prior to loading decreases the saturation of the


resin. As the concentration of the product goes from right to left on the x-
axis in Fig. 4, the concentration bound to the resin decreases. This moves
the operation down the binding isotherm and will lead to a more diffuse
breakthrough profile and a longer LUB. Operating at high concentration
will offset dispersion caused by mechanical and mass transfer effects that
are hard to overcome in protein purification [28]. Minimized dispersion
requires good packing and good mechanical column design with even flow
distribution. It also requires the smallest resin particle size practical for the
application and a flow rate that allows sufficient mass transfer [31].
Unfortunately, small particles are difficult to handle and pack in a
manufacturing environment, and the linear velocity that matches to the rate
of inter-particle diffusion for a protein is on the order of 1 × 10−8 cm/s,
which is not very practical. Operating at high protein concentration is the
most beneficial and easiest parameter to control for efficient adsorption
behavior.

7.2 Intensified Chromatography


While operating at high protein concentration could improve the efficiency
of adsorption significantly, decreasing mass transfer path lengths could also
increase the efficiency of adsorption and the throughput of downstream
processing. There have been at least three variations on this theme with
varied success, macroporous resins, membrane adsorption, and ordered
media.
Macroporous resins have been synthesized that have an outer diameter
on the order of 100 μm but have flow paths within the particles on the order
of 1 μm [32]. A type of this resin is sold under the brand name POROS and
is very popular for the separation of antibodies and other therapeutic
proteins. In principal, these resins should allow some amount of convection
within the pores of the particle, so mass transfer is not limited by pore
diffusion within the particle. The diffusion length would be closer to
0.5 μm, characteristic of a 1 μm particle, rather than the existing 50 μm
diffusion path length that accompanies a 100 μm particle. This inter-particle
convection is possible, but the pressure drop across one particle diameter is
the pressure required to move fluid through a bed of 100 μm particles, so
not sufficient to drive much convection through 1 μm pores. Additionally,
since the particles are macroporous, they have lower ligand densities and
consequently lower binding capacities on a volumetric basis. This might
mean that more resin is required to process the same amount of therapeutic
protein compared to a more conventional resin. Nevertheless, there is some
improvement in LUB in these resins, leading to their popularity.
Membrane chromatography was initially conceived of in the form of
parallel hollow fiber membranes with adsorbing ligands within the
membrane material [33]. The concept provided a low Reynolds number
methodology for decreasing the diameter of the flow path to increase the
rate of mass transfer. The thickness of the hollow fiber wall was ideally as
small as possible, making the largest resistance to mass transfer the transfer
of a molecule through the liquid phase to the interface with the ligand-
bearing solid phase. This methodology was reduced to practice and showed
promise. However, much like macroporous adsorption, the ligand density is
low. This technology is also difficult to scale, as the hollow fibers have to
be held tightly at each end and have a monodispersed inner diameter;
otherwise the widest fibers will have a higher flow rate at constant pressure
(according to Poiseuille’s law, flow increases as diameter to the fourth
power at constant pressure), and dispersion will be introduced that way.
The concept morphed to membrane chromatography, in which existing
filtration membranes are derivatized with ligands and the feed stream is
forced through the membrane’s tortuous flow paths [34]. This idea is
categorically different from the original idea which sought to keep the flow
path straight and narrow in order to get maximum mass transfer at
minimum momentum transfer. In today’s membrane chromatography, the
pressure drop is high, and the feed stream must be completely clear of
particulates or the membrane will become clogged. Furthermore, membrane
manufacturing processes are well adapted to make membranes wider, but
not deeper, so volumetric binding capacity is problematic. Because of this,
membrane chromatography is almost always operated in flow-through
mode, where small levels of impurity are scavenged from a concentrated
product stream. This is a very efficient method for removing DNA,
endotoxin, and RNA from a protein product, for example. This
methodology allows for an inexpensive single-use format as well.
Finally, the original idea for hollow fiber membrane chromatography
was extended into a more manageable form by utilizing a separations media
consisting of wound woven cloth [35, 36]. In this idea, a derivatized fabric
is tightly rolled on an axis and packed into a column. The directions of the
fibers in the cloth are both parallel and perpendicular to the direction of
flow, but not random as in a packed bed. Fibers on the order of 1 μm can be
used to minimize the mass transfer path length, but because the cloth is an
“ordered medium,” the pressure drop is a small fraction of the pressure drop
generated in a random medium. Furthermore, cloths can be made and
derivatized inexpensively and enclosed in thermoplastic, making this
configuration a reasonable candidate for a single-use application.
8 Single Use Downstream Processing
There is significant penetration of single-use technologies in downstream
processing. Plastic bags in portable superstructures are commonly used to
hold buffers and in-process intermediates. When working at high
concentration, smaller columns and buffer volumes can be used. However, a
true single-use adsorption system has not yet been developed, primarily due
to the high cost of the resins. Resin costs can be up to $10,000/L for affinity
resins, so their reuse is mandatory in an economical process. Prepacked
columns are available that are advertised as single use, but this practice is
only advised for very price-insensitive molecules, which will be very few as
biosimilar molecules become more conventional. Some of the
configurations mentioned above may be more amenable to single-use
modalities if their lower cost can be proven.

9 Continuous Downstream Processing


Continuous downstream processing is a field of active research. Simulated
moving bed chromatography has been adapted to biomolecule separations
and may be feasible for some extended batch size. However, feedback
control is not yet developed for this operation, so true continuous
processing will not be possible in the short term. Large gains in volumetric
efficiency and cost reduction have been reported using simulated moving
bed chromatography.

10 Conclusions
There are many challenges remaining for producing therapeutic
biomolecules at large scale for reasonable prices. Just a few have been
reviewed in this chapter, including:
Cell cultures with higher cell densities and protein production levels
Cell retention methodologies that allow the discharge of dead cells and
cell debris, while retaining viable cells
Solid/liquid separations with high yield and no cell lysis
High concentration/high intensity downstream processing
Adsorbent development with improved mass transfer properties
Acknowledgments
The authors wish to acknowledge support from the College of Engineering
from “Engineering Faculty Conversation on Future Manufacturing” and
Hatch Act Support 10677 and 10646, Purdue University.

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Insights on the Formulation of Recombinant


Proteins
Rita Ribeiro1, 2, Teresa Raquel Abreu1, 2, Ana Catarina Silva3, 4, João Gonçalves5
and João Nuno Moreira1, 2
(1) CNC – Center for Neuroscience and Cell Biology, Faculty of Medicine (Pólo
I), University of Coimbra, Coimbra, Portugal
(2) FFUC – Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal
(3) UCIBIO, UCIBIO, REQUIMTE, MEDTECH, Laboratory of Pharmaceutical
Technology, Department of Drug Sciences, Faculty of Pharmacy, University
of Porto, Porto, Portugal
(4) FP-ENAS (UFP Energy, Environment and Health Research Unit),
CEBIMED (Biomedical Research Centre), Faculty of Health Sciences,
University Fernando Pessoa, Porto, Portugal
(5) iMed.ULisboa – Research Institute for Medicines, Faculty of Pharmacy,
University of Lisbon, Lisbon, Portugal

João Nuno Moreira


Email: [email protected]

Abstract
Recombinant proteins are large and complex molecules, whose therapeutic
activity highly depends on their structure. Formulation of biopharmaceuticals
aims at stabilizing protein conformation, promoting its efficacy, and preventing
safety concerns, such as immunogenicity. Currently, the rational design of
formulations is possible due to the availability of several techniques for molecule
characterization and an array of both well-known and new excipients. Also, high-
throughput technologies and Quality by Design approaches are trending and have
been contributing to the advancement of the field. Still, there is a search for
alternatives that ensure quality of the medicines through its life cycle, particularly
for highly concentrated formulations, such as monoclonal antibodies. There is
also a demand for strategies that improve protein delivery and more comfortable
administration to the patients, especially with the arising of recombinant proteins
in the treatment of chronic diseases, such as autoimmune conditions or heart
diseases. In this chapter, current and future advancements regarding recombinant
protein formulation and its impact in drug development and approval will be
addressed.
Graphical Abstract

Keywords Formulation – Immunogenicity – Recombinant proteins – Stability

1 Introduction
Recombinant proteins represent an increasing fraction of the therapeutic arsenal
available nowadays. Relative to small molecular weight drugs, recombinant
proteins are larger and more complex active pharmaceutical ingredients, whose
efficacy and safety highly depend on the ability to recapitulate their structure in
the different stages of the protein lifetime. Additionally, challenges arise from the
development of formulations with high concentrations of protein, as in the case of
monoclonal antibodies, as well as for low-volume formulations (as demanded for
self-administration devices). The pursuit of a formulation that can stabilize the
active protein, from manufacture to administration, in an economically effective
way, is then a crucial aspect to consider in the development of biological
medicines. Herein, the formulation of recombinant proteins will be discussed,
addressing general concepts, current trends, and future perspectives.
2 Protein Structure
2.1 Molecular Structure
Proteins are large molecules composed of one or more chains of amino acid
residues, displayed in an order that is determined by a DNA sequence. The linear
polypeptide form of the protein corresponds to the protein’s primary structure.
Intermolecular interactions promote the arrangement of these chains into
secondary (alpha-helices and beta-sheets) and tertiary structures. The latter can be
assembled into quaternary structures, when two or more subunits interact. As
different amino acids have different side chains with unique chemical properties,
intra- and intermolecular interactions vary in strength, from weak forces
(hydrophobic, electrostatic, hydrogen, van der Waals) to strong covalent bonds
(disulfide bridges), adding to the complexity of the final structure [1].
If chemical and/or physical degradation occurs, protein may unfold, and
hydrophobic residues that would be hidden in the native state may become
exposed. Consequently, the molecule can remain unfolded or misfolded in a
thermodynamically non-stable state, interacting with other proteins and causing
aggregation and precipitation [2]. The presence of aggregates in biological
medicines has been associated with immunogenicity, loss of biological activity,
and renal failure [3]. Further in this chapter, mechanisms of protein degradation
will be addressed, as well as approaches to minimize aggregates formation and
assure the efficacy and safety of the final medicine.
Protein degradation can take place at different stages of the product’s life
cycle, from manufacturing and processing to storage, administration, and delivery
[4]. Because recombinant proteins are produced in cell-based systems,
purification and recovery steps are needed to isolate the product of interest from
destabilizing impurities. Long-term shelf-life (about 18–24 months for a protein-
based therapeutic) could promote degradation pathways (e.g., hydrolysis or
oxidation), even when the adequate storage conditions are maintained [5].
Different delivery systems are also to consider, since the materials and processes
used for their preparation and final presentation may impact protein stability [6].

2.2 Chemical Degradation


Chemical degradation involves modifications of covalent bonds of the protein,
such as deamidation, isomerization, hydrolysis, oxidation, and disulfide bond
shuffling [7]. It affects the primary sequence of the protein, which may lead to
significant changes in the final three-dimensional structure [3].
Deamidation is one of the most common forms of chemical degradation. It
consists in the hydrolysis of asparagine (Asn) to aspartic (Asp) and iso-aspartic
acid, or that of glutamine (Gln) to glutamic acid (Glu), via a succinimide
intermediate. Subsequently, a neutral amino acid residue is converted into a
negatively charged polar one [8]. The reaction is favored by neutral and basic pH
[9] and temperature above 25°C [10], as well as by the steric effect of adjacent
residues (mainly in the C-terminal) and their conformation. In this respect, the
higher the size and branching of the side chain, the lower the rate of deamidation,
with glycine (Xxx-Asn-Gly-Xxx) resulting in the highest extent of deamidation
[11].
Hydrolysis of a peptide bond can take place either under acidic or alkaline
conditions, or it can be enzymatically mediated. As a result, the polypeptide chain
breaks, leading to protein fragmentation. Some bonds are more susceptible to
hydrolysis, like Asn-tyrosine or Asn-proline [12].
Oxidation can be a consequence of several external factors, including
exposure to light, oxygen, transition metal ions (iron and copper [13]), or
formulation degradation products (e.g., hydrogen peroxide from polysorbate
autoxidation [14]). Amino acid residues such as tryptophan (Try) and histidine
(His) are more susceptible to oxidation due to their aromatic rings, while
methionine (Met) and cysteine (Cys) are vulnerable to oxidation as the result of
their highly reactive sulfur atoms [15]. The steric exposure of the reactive residue
and pH of the solution also influence the oxidation rate [16].
Cross-linking is an additional chemical modification involving the formation
of disulfide bonds, either from the formation of new Cys-Cys bonds or exchange
of pre-existing ones. This modification is favored with increasing pH values, as
thiolate ions are the major reactive species involved [17].

2.3 Physical Degradation


Physical degradation implies changes in the secondary, tertiary, or quaternary
structure of the protein, leading to aggregation, precipitation, or adsorption to
surfaces. Mechanisms of physical degradation usually involve unfolded or semi-
unfolded intermediates and are triggered by stress factors, such as mechanical
stress (stirring, pumping, filtration, and filling during manufacturing and shaking
during administration [18]), high temperatures (during shipping and storage), and
freeze/thaw cycles. High temperatures, i.e., temperatures close to the protein’s
melting point (value at which half of the protein population is unfolded and the
other half is folded [19]), lead to the formation of aggregates via non-covalent
interactions between the unfolded proteins [20]. Freeze/thaw cycles may cause
partitioning of the protein to ice-water interface, cryo-concentration, and change
in pH due to nonuniform buffer recrystallization, affecting protein stability [21].
Cold denaturation is not very common, as it takes place only below the freezing
point of water, when water molecules are ordered in a layer around the protein,
changing its thermodynamics and leading to destabilization [22]. These low
temperatures (usually below −20°C) are not achieved under regular
manufacturing, transport, or storage conditions. However, upon freeze-drying for
lyophilization purposes, the glass transition temperature usually goes below
−20°C, which increases the chances of cold denaturation.
Aggregation is one the most common forms of protein physical degradation.
The trigger of aggregation and the characteristics of aggregates depend on
protein’s environment: pH, temperature, ionic strength, concentration, and
presence of co-solutes and excipients [23]. This form of degradation is
particularly significant when dealing with monoclonal antibodies, as they are
usually formulated at high concentrations [24]. Moreover, large, multidomain
proteins are more prone to aggregate, because gentle conditions, such as exposure
to temperatures below their melting temperature (T m) [25], can be sufficient to
break the non-covalent interactions that usually maintain the several domains
properly organized, unfold the protein, and induce aggregation. On the other hand,
small, single-domain proteins only have their intramolecular forces disrupted
under more extreme conditions, such as pH below 3 or above 10, or temperatures
above 40°C [26]. Nowadays, it is possible to predict the extent and rate of
aggregation based on the physicochemical properties of the protein, upon
experimentally testing the effect of certain residues’ mutations [27, 28],
complementing with in silico prediction tools [29]. Recent developments on
statistical modeling and machine-learning enable the assessment of aggregation
potential of proteins using a high-throughput screening, and thus the selection of a
lead with the lowest risk of aggregation, in an early stage of development [30, 31].

2.4 Characterization of Proteins


The complex structure of recombinant proteins demands a thorough
characterization in early development to better control the impact on their stability
and biological activity. Several techniques of biophysical and biochemical
analysis have to be performed in order to determine, in a rational way, the best
conditions for drug stabilization through its whole life cycle. Information derived
from this early characterization will be further used to establish the specifications
to which the active substance, excipients, and final product must conform to be
considered acceptable by manufacturers and regulatory authorities. In this respect,
the International Conference on Harmonization (ICH) Q6B guideline unifies a set
of international analytical procedures and respective acceptance criteria regarding
biotechnological products, based on their molecular and biological characteristics
[32].
These methodologies are based in separation (chromatographic, SDS-PAGE)
and spectroscopic methods, complemented by calorimetric and light scattering
techniques. They assess protein size, conformation, hydrophobicity, and
hydrodynamic features [33, 34]. The detection of aggregates requires imaging
techniques, such as microscopy and light scattering that provide information to be
used in computer modeling predictive tools, giving insight in possible
degradation, aggregation, and immunogenicity. A review of computational tools
used to assess protein aggregation can be found elsewhere [29], while the most
commonly performed assays and respective measured properties are summarized
in Table 1.
Table 1 Methodologies and properties assessed for protein characterization (Adapted from Angkawinitwong
[35])

Method Technique Measurement Property assessed


Primary Secondary Tertiary Aggregates
structure structure structure
Chromatographic Ion exchange Charge variants X X
chromatography
Size exclusion Hydrodynamic size X
chromatography
High-pressure Degraded product X
liquid
chromatography
(HPLC)
Spectroscopic UV absorbance Protein concentration X X X
Raman Conformation X X
spectroscopy
Fourier- β-sheet, α-helix X X
transform
infrared
spectroscopy
(FTIR)
Near-/far-UV Thermal stability X X
circular
dichroism
Fluorescence Folding/unfolding, X X
hydrophobic surface
Optical density Turbidity X
(OD)
Calorimetric Differential Melting temperature X X X
scanning (T m), thermal
calorimetry unfolding/aggregation
(DSC)
Light scattering Static/dynamic Diameter and X
light scattering monodispersity of
monomers
Method Technique Measurement Property assessed
Primary Secondary Tertiary Aggregates
structure structure structure
Microscopic Transmission Particles X
electron
microscopy
(TEM)
Fluorescence Particles X
microscopy
Atomic force Particles X
microscopy
(AFM)
Mass Size, peptide X
spectrometry mapping, degradation
products
SDS-PAGE Molecular weight X
Analytical Molecular X
ultracentrifugation weight/shape

Finally, it is very important to do a functional characterization of the active


protein, according to its mechanism of action, in order to evaluate its
performance. Blotting, electrophoresis, chromatography, immunoassays (e.g.,
ELISA), and in vivo bioassays are examples of such methodologies [33].

3 Formulation of Proteins
Efficacy and safety of recombinant proteins is highly dependent on their unique
structure, which is why it is critical to develop a formulation with an optimum
qualitative and quantitative composition for each therapeutic. The choice of
excipients will depend on several factors, aiming at stabilizing the active protein
through the life cycle of the product, up to administration to the patient.
Excipients can promote protein stabilization by increasing thermodynamic
stability, either through strengthening protein-stabilizing forces or destabilizing
the unfolded state, forcing the refolding to the native conformation [36]. This is
possible because of their direct binding to the protein (e.g., stabilizers) or their
exclusion from protein surface and replacement by water molecules, i.e., protein
hydration (e.g., sugars) [37].
Aside from the intrinsic physical and chemical properties of the active protein
and excipients, some other aspects have to be considered when choosing the most
appropriate excipients. First, special attention to chemical and physical stability
issues during manufacturing is demanded. This requires a good understanding of
the critical attributes and processes that impact quality, from early development
[38]. The formulation approach intended is important, since liquid or lyophilized
formats require different processing [39]. The final concentration of protein is
another significant aspect to consider. As mentioned before, formulations with
highly concentrated protein (50–150 mg/mL) [40], as monoclonal antibodies, are
more susceptible to aggregation. It is also important to consider possible non-
specific adsorption to surfaces, as to the primary packaging or to the
administration device, especially for hydrophobic proteins [41]. The route of
administration must be regarded too, as adjustments in formulation may be
necessary to keep the required tonicity or osmolarity and pH for parenteral
administration [39].
All the components to be used in a formulation must be characterized, and
their compatibility with each other and with the therapeutic protein must be
evaluated. Depending on the concentration that is being used, the same excipient
may have different roles in different formulations, making it difficult, sometimes,
to categorize them. Herein a classification of the most common excipients used in
the formulation of recombinant proteins is presented (Table 2).
Table 2 Classification, role, and examples of commonly used excipients in the formulation of recombinant
proteins

Excipient class Role in Examples


formulation
Buffers pH stabilization Amino acid (histidine, methionine, glycine, arginine), non-amino
acid (phosphate, acetate, citrate), and salts (sodium hydroxide,
hydrochloric acid, phosphoric acid)
Osmotic agents Isotonicity Sodium chloride, potassium
maintenance
Stabilizers/bulking Protein Amino acids (histidine, glycine, arginine, glutamate) and sugars
agents stabilization and (mannitol, sucrose, trehalose)
formulation
support
Surfactants Solubility Sodium dodecyl sulfate and polysorbate 20, polysorbate 80
enhancement and
anti-aggregation
Preservatives Minimization of Benzyl alcohol, phenol, metacresol
bacterial
contamination
Antioxidants Minimization of Ascorbic acid, acetylcysteine, glutathione
oxidative stress
degradation
Adjuvants and Modulation of Aluminum salts, squalene-based oil-in-water emulsions,
immune immune response monophosphoryl lipid A
potentiators
3.1 Buffers
At a pH value at the protein isoelectric point, the net neutral charge of the protein
limits electrostatic interactions, thus favoring protein-protein interactions and
subsequent aggregation. Buffers are, therefore, an important component of protein
formulation in order to enable a pH of the protein solution below or above of the
corresponding isolectric point [42]. As therapeutic proteins are preferably
administered parenterally, on the choice of the most appropriate pH, the
physiological range (pH = 7.35–7.45) [43] necessary to avoid irritation or pain
during administration must be also considered. When balancing these aspects, pH
ranging between 3 and 11 may be considered acceptable [44]. Substances like
amino acid, as histidine, methionine, glycine or arginine, or non-amino acid,
phosphate, acetate or citrate, are examples of buffers used in protein formulation.
Acids and bases can also be used as buffers, either as pH modifiers or to form
salts in combination with other components of the buffer (e.g., sodium hydroxide,
hydrochloric acid, phosphoric acid). Along formulation development, the
presence of other excipients or the influence of certain processes that may
promote precipitation or crystallization of the buffer, altering the pH, should be
carefully assessed. This is the case of acetic acid that being volatile, its use in
lyophilized formulations is not indicated [39]. Interestingly, formulations with
high concentrations of protein may even dismiss the need of buffer, namely, when
they have buffering amino acids in their composition, such as aspartate,
glutamate, and histidine [45].

3.2 Osmotic Agents


In medicines for parenteral administration, the maintenance of the isotonicity of
the formulation in a range between 280 and 300 mOsm/L is required, to avoid
damage of the tissues (phlebitis or infiltration) or pain at the site of injection. This
is particularly difficult for formulations with a high concentration of protein. The
most common tonicifiers are sodium chloride and potassium chloride [39].
Careful choice of the agent is required as, for example, sodium chloride and water
react to form an eutectic mixture at −21°C. In this case, water crystallizes during
freeze-drying, and the concentration of sodium chloride increases until it
precipitates. For this reason, sodium chloride is added to the diluent for
reconstitution instead to the lyophilized product [46].

3.3 Stabilizers/Bulking Agents


The solvent surrounding the protein and the solutes interacting with it are very
important in maintaining the correct folded structure. The pharmaceutical
presentation of the protein-based dosage form, either in solution or lyophilized,
influences the nature of those interactions and impacts the choice of stabilizers
and bulking agents.
The most common stabilizers are amino acids (histidine, glycine, arginine,
glutamate) and sugars (mannitol, sucrose, trehalose). They stabilize the proteins
by direct interactions or solute exclusion (stabilizer amino acids are repulsed from
the protein surface, increasing the free energy of the unfolded state and favoring
folding in the native form) [47], but their specific mechanism is sometimes
difficult to assess due to the diversity and complexity of their side chains.
Mixtures of different amino acids (e.g., arginine and glutamic acid) [48] or
combinations of amino acids and sugars (e.g., histidine and sucrose or mannitol)
might enable a synergistic effect in the protein stabilization [49].
Bulking agents are incorporated in lyophilized formulations to increase
product mass (thus preventing its collapse), to aid in rehydration (facilitating
dissolution) and to improve product appearance (by providing mechanical
support) [50].
Due to their amorphous or crystalline physical state, sugars may act as
stabilizers or bulking agents, respectively. For example, sucrose maintains an
amorphous state after lyophilization, acting as a stabilizer, while mannitol is
capable of crystallizing and supporting the lyophilized cake, thus acting as a
bulking agent. Another case shows that, in a formulation of a human growth
hormone, when mannitol and glycine were used separately, both crystallized
during freeze-drying. However, when combined in the same formulation,
mannitol crystallized, while glycine remained amorphous. This partially
amorphous system offered the best protein stabilization [51]. Sugar
recrystallization should be avoided, since the conversion between amorphous and
crystalline states can compromise protein stability [52].

3.4 Surfactants
Surfactants are molecules whose mechanism of action will depend on their nature
(either ionic or nonionic), acting as solubility enhancers, anti-adsorption or anti-
aggregation agents. Upon competitive binding to interfaces like air-water or
container-water, surfactants prevent the non-specific adsorption of proteins
through binding to their hydrophobic residues. They also compete with other
proteins for the binding to protein surface, preventing aggregation [53]. Their
activity has a significant impact in highly concentrated formulations, which are
more prone to form aggregates.
Ionic surfactants are rarely used in protein formulations, due to their tendency
to denature proteins. Nonionic surfactants, such as sodium dodecyl sulfate and
polysorbate 20 or polysorbate 80, are efficient, less toxic, and less sensitive to the
presence of electrolytes [12]. Polysorbates are the more common surfactants
found in protein formulations, but their concentration must be selected carefully,
since they can undergo autoxidation and give origin to hydrogen peroxide, which
will not only compromise protein stability but also the safety of the product [14].

3.5 Preservatives
Preservatives are present in formulations to minimize microbial contamination.
They are particularly important in products with multiple doses, due to their
likelihood to contaminate in repeated usages. Examples of preservatives include
metacresol, phenol, and benzyl alcohol. The latter was even shown to stabilize the
partial unfolded state of a protein, inhibiting its aggregation [54]. In the case of
volatile and reactive preservatives, incorporation in lyophilized products is rather
performed in the solvent for reconstitution, than in the lyophilized cake [39]. The
addition of preservatives to a formulation may, however, impact protein stability
in a negative way. In this respect, it is proposed that preservatives interact with the
active proteins, destabilizing their weakest links (so-called hot-spots), thus
decreasing conformational stability and increasing propension to aggregation [55,
56]. This issue may be minimized upon deepening the understanding of the effect
of preservatives on proteins and by applying strategies enabling protein stability.
Besides, a case of binding of preservatives to a model peptide decreased their
antimicrobial effect, thus raising possible safety concerns [57].

3.6 Antioxidants
Proteins rich in methionine, cysteine, tryptophan, tyrosine, and histidine residues
are more susceptible to degradation due to oxidative stress. In these cases,
formulation with antioxidants is a requirement. Ascorbic acid and acetylcysteine
are examples of antioxidants often used in the formulation of proteins.
Glutathione also behaves as reducing agent, upon creating disulfide bonds with
cysteine residues of the protein. Replacement of oxygen by an inert gas in the vial
is a complementary measure to reduce oxidation [58]. Other mechanism of
oxidation prevention is the chelation of metal ions. In this regard,
ethylenediaminetetraacetic acid and calcium chloride have been used as
antioxidants [59].

3.7 Adjuvants and Immune Potentiators


Recombinant peptide or protein-based vaccines have the ability to induce immune
responses against a specific antigen. Their formulation often includes adjuvants
that help to modulate or even increase immune responses. Aluminum salts are the
most commonly used adjuvants. They act in the site of injection, adsorbing the
antigen, enabling its stability and uptake by antigen-presenting cells, and inducing
local pro-inflammatory reactions [60]. Other examples are MF59 and AS03,
squalene-based oil-in-water emulsions, that consist of oil nanoparticles suspended
in an aqueous phase via a surfactant. They have shown to be more effective than
aluminum in the avian H5 pandemic flu vaccines [61].
Currently, the objective is to go beyond antibody-mediated immunity and
accomplish long-lasting cellular responses, like those mediated by T helper and T
cytotoxic lymphocytes. This can be achieved by the activation of Toll-like
receptors (TLR) or NOD-like receptors (NLR) by immune potentiators, like
pattern-recognition receptors (PRR) agonists [62]. Monophosphoryl lipid A
(MPL) is a natural molecule that targets TLR4 and has been also used as an
adjuvant. Synthetic analogues have been studied, namely, in combination with
aluminum or emulsions [63].
A relevant formulation aspect relies on the assessment of the mechanism of
action of the antigen and of the adjuvants, as well as the interaction of these
components with each other, as this may impact both the efficacy and the safety of
the drug [64]. For example, aluminum salts can adsorb the antigen (with a
strength that is antigen-specific), destabilizing it and promoting protein
aggregation [65]. As for emulsions, they can lead to protein denaturation or
desorption, structural rearrangements, and inter-protein interactions, with
subsequent aggregation [66]. To minimize these effects, particle-based
formulations for stabilization and delivery of the adjuvant are under development,
such as nanotechnology-based delivery systems, virus-like particles, and
polymeric systems [67].

3.8 Trends in Formulation Approaches


An overview of recombinant protein medicines currently approved by European
Medicines Agency (EMA) and Food and Drug Administration (FDA) gives an
idea of the formulation approaches that are preferentially being followed. In
Europe, monoclonal antibodies comprise the major class of therapeutic proteins,
which, as mentioned earlier, implies the use of excipients appropriate for highly
concentrated products. Therefore, upon briefly running through the major classes
of excipients, buffers, surfactants, preservatives and tonicifiers arise as the major
components of current protein formulations [39]. In the United States, the
scenario is similar. Antibody therapeutics are also the highest selling class of
biopharmaceuticals, delivered by parental routes of administration and stored
either in solution or powder formats. Among these, the most commonly used
excipients fall into the buffers, bulking agents, and surfactants categories [68]. For
a more detailed and practical understanding of the formulations currently used in
commercialized drugs, the European Public Assessment Reports (EPAR) [69] of
the 10 top-selling biopharmaceutical products in 2017 [70] were analyzed, and
their excipient composition and pharmaceutical presentation were compiled in
Table 3.
Table 3 10 top-selling biopharmaceuticals’ formulations

Product Active Excipients Pharmaceutical


substance presentation

Humira® Adalimumab Mannitol, polysorbate 80, water for injection Solution for
injection

Enbrel® Etanercept Mannitol, sucrose, trometamol Lyophilizate for


reconstitution and
injection

Rituxan® Rituximab Sodium citrate, polysorbate 80, sodium chloride, sodium Concentrated
hydroxide, hydrochloric acid, water for injection solution, for
MabThera® dilution and
infusion

Remicade® Infliximab Sucrose, polysorbate 80, monobasic sodium phosphate, Lyophilizate for
dibasic sodium phosphate reconstitution,
dilution, and
infusion

Herceptin® Trastuzumab L-histidine hydrochloride monohydrate, L-histidine, Lyophilizate for


polysorbate 20, α,α-trehalose dihydrate reconstitution,
dilution, and
infusion

Avastin® Bevacizumab Trehalose dihydrate, sodium phosphate, polysorbate 20, Concentrated


water for injection solution for
dilution and
infusion

Lantus® Insulin Zinc chloride, metacresol, glycerol, hydrochloric acid, Solution for
sodium hydroxide, water for injections, polysorbate 20 injection
(when stored in vial)

Eylea® Aflibercept Polysorbate 20, sodium dihydrogen phosphate Solution for


monohydrate, disodium hydrogen phosphate injection
heptahydrate, sodium chloride, sucrose, water for
injection

Opdivo® Nivolumab Sodium citrate dihydrate, sodium chloride, mannitol, Concentrated


pentetic acid, polysorbate 80, sodium hydroxide, solution for
hydrochloric acid, water for injection dilution and
infusion

Neulasta® Pegfilgrastim Sodium acetate, sorbitol, polysorbate 20, water for Solution for
injection injection

3.9 Formulation of Biosimilars and Biobetters


A biosimilar is a biopharmaceutical that is highly similar to an already approved
biologic (reference medicine) in terms of quality, biological activity, safety, and
efficacy. The complexity of proteins associated with their different levels of
structural organization and high molecular weight determines a demonstration of
similarity between a biosimilar and the reference medicine, based on the
assessment of toxicity and efficacy in a clinical setting. This implies not only a
structural similarity between the active protein in the biosimilar and reference
medicine, but also the same posology and route of administration. Some
deviations from the reference product may be accepted, such as pharmaceutical
dosage form, formulation, excipients, or presentation, provided that they are
properly justified [71].
In fact, both EMA and FDA allow for advancements in formulation science to
be incorporated in the biosimilar presentation, admitting excipients to differ from
those of the reference. In this respect, any relevant effects of the revised
formulation on the stability, physiochemical, and functional characteristics of
biosimilars must be assessed [72]. However, as stated before, it is hard to collect
information on the safety and immunogenicity of biopharmaceuticals based only
in the characterization of the active molecule and nonclinical data. The same is
applied to their biosimilars. Therefore, clinical trials and post-marketing vigilance
are required by the regulatory authorities to ensure at least the same efficacy and
safety from the innovator medicine [73, 74].
Biobetters are the new players in biopharmaceutical development. They act
against the same target as an already marketed medicine and include changes in
the molecular structure or formulation. However, unlike biosimilars, these
alterations aim to improve pharmacokinetic or pharmacodynamic properties, to
decrease toxicity or immunogenicity, or to make administration more comfortable
or less frequent, i.e., biobetters are clinically superior than the respective
references either in efficacy, safety, or compliance, or both [75]. Biobetters are not
regulated yet, so they are treated as new drugs and can only enter the market after
a full application submission, which is both expensive and time-consuming.
Nonetheless, they have been used for biopharmaceutical companies that want to
extend the life cycle of drugs whose patent is expiring, as a way to manage
competition from biosimilars [76]. For example, in 2013, Roche obtained
marketing authorization for a subcutaneous formulation of Herceptin®, since
intravenous trastuzumab patent would expire in 2014. The new formulation
presented efficacy and safety profiles similar to the intravenous one and further
enabled administration of higher volumes. Along with the advantage of shorter
duration of treatment, saving time and resources for healthcare professionals, and
improving patient compliance [77], it became a concern for biosimilars of
intravenous trastuzumab developers. This case illustrates the importance of
formulation development beyond the drug’s marketing authorization.
4 Pharmaceutical Presentation
Even with an appropriate choice of excipients supporting the stability of the active
protein, the dosage form and drug packaging can have an impact in maintaining
the medicine’s quality throughout its life cycle. Therefore, pharmaceutical
presentation must be regarded during formulation development [78].

4.1 Lyophilized Therapeutic Proteins


Most biopharmaceutical products are stored in the form of aqueous-based
solutions or lyophilized powders [39]. As mentioned before, protein solubility
depends on several aspects, including pH, ionic strength, and associated
excipients. If stability and solubility can be achieved, a solution is preferred over a
suspension, since the latter is harder to process and handle. Suspensions have
increased chemical stability, but not necessarily higher physical stability, due to
the propensity to form agglomerates when exposed to mechanical stress [79].
Often, the liquid formulation does not provide the required long-term stability
of the protein and quality of the product, as the presence of water may promote
physical or chemical degradation. The dried state is thus the preferred form, and
the protein must be lyophilized.
Lyophilization is a process that comprises three sequential phases: freezing,
primary drying, and secondary drying. During the freezing step, temperature is
reduced below the eutectic temperature (T e) of the mixture (i.e., a temperature
that is lower than the melting point of each component of the formulation). Then,
during primary drying, crystallized water and water that is not bound to the
protein or excipients are removed by sublimation, with temperature still below the
T e, and under reduced pressure. Secondary drying allows the removal of water
that is bound to the protein and excipients, by slowly increasing the temperature,
although still below T e, and gradually rising the pressure [80]. The result is a
dried powder that contains the protein in a glassy phase, amorphous excipients,
and some residual water. It must have a uniform and elegant appearance [81] and
be rapidly dissolved in the reconstitution solvent.
This process of water removal from frozen solutions under reduced pressure
may damage the active protein. The incorporation of sugars and polyols (e.g.,
sucrose, trehalose, sorbitol) will mostly replace the hydrogen bonds between the
protein and the surrounding water molecules by hydrogen bonds created with the
excipients, stabilizing it thermodynamically and thus acting as lyoprotectants.
From a kinetic perspective, it is proposed that these sugars stabilize the active
molecule by forming an amorphous glass matrix, so they should not recrystallize
during storage [82].
Due to the crystallization of different solutes in the mixture at different stages
during freeze-drying, a differential precipitation of excipients may occur. Upon
precipitation of buffer components, a shift in the pH of the mixture may take
place, with negative consequences for protein stability. This is hardly detected as,
upon reconstitution, buffers will redissolve and the pH measured will be the
targeted one. Salts are used to minimize changes in pH and tonicity, but one must
be conscious in their choice, since pure solvent freezes first than the salts, leading
to an increase of their concentration and altering the ionic strength and
compromising the protein. Sugars like lactose and mannitol can also crystallize
and separate from the solution. They should be replaced by carbohydrates (e.g.,
sucrose) or amino acids (e.g., histidine or glycine) that present amorphous
characteristics [39]. During freeze-drying carbohydrates can also react with amino
groups of the protein, promoting the Maillard reaction and originating a yellow-
brown cake. To avoid this, nonreducing sugars like sucrose or trehalose may be
used [34].
When considering lyophilized biopharmaceuticals, the reconstitution step is of
major importance. Reconstitution solvent can interfere with protein stability.
Therefore, besides using sterile water as diluent, excipients like stabilizers and
preservatives may be added to maintain the quality of the drug until
administration. Reconstitution times may have an impact in protein stability,
especially for high-concentration therapeutics that are formulated with higher
concentrations of stabilizer excipients. This increases reconstitution time and
protein propensity for degradation. The incorporation of wetting agents, like
surfactants, in the reconstitution solvent is a possible solution [83].

4.2 Containers and Administration Devices


Packaging of biopharmaceuticals must accompany the chosen form of
pharmaceutical presentation in the maintenance of drugs’ stability during shelf-
life and administration, while providing ease of handling and improving patient
compliance. Biologics are stored in vials, pre-filled syringes or cartridges,
ampoules, or bags, according to the physicochemical properties of the
formulation, product volume, materials of the containers, and their intended
functionality [84]. There is a high potential of interactions between containers and
components of the formulation, especially due to phenomena of leaching
(migration of unwanted particles from the container to the medicine) and sorption
(diffusion of an ingredient to the container material, through mechanisms of
adsorption, absorption or permeation) [85], thus requiring a rigorous compatibility
assessment.
Pre-filled delivery devices are increasing in popularity, as they allow for the
patients to self-administer the drug in ambulatory, without the help of a healthcare
professional, improving compliance and reducing time and cost expenses with
specialized practitioners. This is relevant in a time of increasing development of
biopharmaceuticals to treat chronic diseases (diabetes, rheumatoid arthritis). They
also reduce the risk of contamination and waste-associated costs, since there is no
need to overfill the container [58]. When choosing the excipients and packaging
material for such devices, some aspects must be considered. For example, to be
administered in a syringe, liquid solutions must have low viscosity [40]. Also,
during transportation or handling, agitation may lead to the incorporation of air,
which could potentially originate new liquid-gas interfaces, and subsequently
protein unfolding.

5 Safety and Immunogenicity of Biopharmaceuticals


5.1 Quality Framework
Safety is a major concern regarding the use of biopharmaceuticals, and it can be
influenced by the quality of the medicine. In fact, unlike small drugs, recombinant
proteins are produced in living systems and are subjected to downstream steps of
processing and purification. It has been shown that the complexity of their
manufacturing may result in contaminants or impurities that affect the medicine’s
safety [86, 87]. Moreover, the physical and chemical features of therapeutic
proteins are hard to fully characterize, as well as their potential for aggregation
and precipitation. To add to the active protein’s intrinsic features, the
corresponding formulation can have a significant impact in the drug’s safety
profile. In fact, excipients are perceived as pharmacologically inactive substances,
but this concept is quite simplified, as the safety of a biopharmaceutical also
involves the quality and toxicity of its excipients [88]. For known excipients, it is
only required to prove quality to ensure safety, since they are well-characterized
and associated with market approval. For novel excipients, however, regulatory
authorities request preclinical studies to demonstrate the desired safety of the
compound on its intended formulation, which can be very expensive and time-
consuming [89]. This justifies a strict regulatory framework on the quality control
and toxicological data of the active protein and excipients [89].
The ICH provides guidance on the quality and safety of medicines. The ICH
Q5A-Q5E and the S6(R1) guidelines provide information on contaminants
evaluation, stability testing, and preclinical safety testing within the scope of
biotechnological products. The World Health Organization (WHO) also presents
recommendations for both manufacturers and regulatory agencies on the quality,
safety, and efficacy of biotherapeutic products, giving guidance on manufacture
and quality control of recombinant protein therapeutics, and on nonclinical and
clinical evaluation [90]. The Good Manufacturing Practice Guide for bulk
Pharmaceutical Excipients is harmonized across the different regulatory agencies
and helps manufacturers to evaluate the quality of raw materials, in order to
comply with Good Manufacturing Practices [91]. The European and the United
States pharmacopoeias also have a natural impact in formulation development and
excipients quality control. These documents support pharmaceutical companies on
the fulfillment of requirements set by the EMA’s regulatory guidance on
excipients [92] and the FDA’s Code of Federal Regulations (Title 21, Chapter I,
Subchapter C).
Despite the effort to assess all possible safety issues during development and
manufacturing, adverse events may occur when the final medicine is administered
to patients. Biologics adverse reactions have been classified based on the already
existing classification for small drugs adverse reactions (type A to E), having into
account mechanisms of action rather than clinical symptoms [93]. Thus, to
distinguish this classification from the one existing for chemical drugs, adverse
events can be categorized from type α to type ε, as immune stimulation,
immunogenicity (against therapeutic protein), immune deviation
(immunosuppression or autoimmunity), cross-reactivity (with similar molecule),
and non-immunological.

5.2 Mechanisms of Immunogenicity


Immunogenicity is a major issue in recombinant proteins’ safety and efficacy,
because of their high potential to elicit anti-drug responses [94]. The
immunogenic potential of biopharmaceuticals depends on a variety of factors,
such as structural features of the active molecule or excipients, presence of
impurities, route of administration, dose and duration of treatment, and patient
features [95].
Reactions against biologics can be a consequence of either breakdown of
immune tolerance to autoantigens (human homologous) or activation of the host’s
immune system to neoantigens [96]. The first mechanism is not yet well
understood, but it seems that repeated administration of therapeutic proteins that
are similar to endogenous ones, depending on their dose, may induce immune
responses and clinical adverse effects. This is exemplified by the development of
thrombocytopenia in patients who were administered with a pegylated
recombinant megakaryocyte growth and development factor. This therapeutic
protein shares the first 163 amino acids of endogenous thrombopoietin, which led
to the development of antibodies that cross-reacted and neutralized the
endogenous promoter of platelet production [97].
The second proposed mechanism of immunogenicity is related to the degree
of non-self of the active protein, glycosylation, and protein aggregates, all of
which can be examples of neoantigens (not previously recognized by the immune
system). The degree of non-self, i.e., of structures foreigner to one’s organism, is
related with the source of the protein: animal (e.g., bovine insulin is more
immunogenic than porcine insulin, which is more immunogenic than the human
recombinant protein [98]), bacterial, or plant origin (absence of posttranslation
modification mechanisms [99]). Glycosylation is one important characteristic that
influences protein three-dimensional conformation. When a glycan composition is
not recognized by the immune system, or affects protein structure, decreasing its
stability and solubility, immune reactions against the drug may be triggered.
However, in some cases, a glycosylation pattern different from the native protein’s
one can be beneficial, and it is even a modification proposed to decrease protein
immunogenicity, as it can improve solubility through protein-solvent interaction
or upon hiding antigenic sites [100].

5.3 Protein Aggregation


Protein aggregation has been considered the most important structural change
related to immunogenicity [101]. It is hypothesized that the immune system was
developed to recognize repeated patterns of proteins, sugars, or lipids present at
the surface of invading microorganisms and develop a humoral response upon
generating antibodies against the active site of the molecule. In the case of
therapeutic proteins, the generated antibodies are named anti-drug antibodies
(ADAs). This is likely the reason why multimerization of proteins is a key factor
in the development of an anti-drug immune response, along with protein
aggregates, regardless the size of the monomeric or aggregated form of the protein
[102]. Aggregates can be classified according to their size (submicron soluble
aggregates, insoluble particles up to 100 μm, and visible particles – above
100 μm), binding type (non-covalent or covalent) [3], or format (aggregates of
proteins in the native structure; denatured structure, aggregated in an irreversible
way; and covalently bound native and denatured proteins) [103]. There are no
regulatory limits regarding the size of aggregates in biopharmaceutical products,
but the United States [104] and the European [105, 106] pharmacopoeias
determine the recommended limits for injectables and methods to assess them.

5.4 Anti-drug Antibodies


When ADAs are produced in a conformation-dependent manner (instead of the
linear form of the epitope), they will alter the pharmacokinetics and
pharmacodynamics of the protein, therefore neutralizing its activity and
therapeutic efficacy [107]. Non-neutralizing antibodies did not used to be
considered clinically significant, as they do not neutralize the activity of the
protein. However, they can bind to it and form immune complexes that are rapidly
eliminated, thus altering the pharmacokinetics of the drug and affecting its
bioavailability [94]. Consequences of ADAs can range from clinically non-
relevant to severe responses, such as hypersensitivity, anaphylactic reactions
[103], or deficiency syndromes (thrombocytopenia or pure red cell aplasia [108]).
Thus, it is important to use ADA determination as a predictive marker of
immunogenicity during drug development or as an assessment tool after drug’s
approval [109]. Nonclinical studies have a low predictive value. Despite the
efforts, there is still the need to develop more accurate in silico models that can
predict protein-immune system interaction, as well as animal models that can
overcome the differences between human and animal immune systems and their
inevitable immune reactions against recombinant human proteins [110]. This
supports the requirement from the regulatory authorities to generate data on ADA
detection in clinical trials, as well as the incorporation of ADA detection methods
in the drug’s risk management plan [111]. EMA and FDA generated documents
that provide guidance and harmonization on the analytical methods to be used for
ADA detection [112–114].

6 Current Trends in Formulation and Future


Perspectives
The biotech industry has had a huge development, with great scientific and
technical improvements, that increased the efficacy, safety, and quality of
biopharmaceutical products. Nonetheless, challenges associated with the efforts to
better understand the structure-function relationship, rational design of suitable
formulations, engineering novel protein formats, avoidance of aggregates
formation and immunogenicity, or development of less invasive administration
routes, still remain.

6.1 Novel Excipients


The research on novel excipients is a significant aspect to consider in the
development of novel biotechnology-based drugs and delivery devices. Excipients
are considered novel when they are associated for the first time with a drug or
route of administration. Therefore, both new substances and existing compounds
may fit in this definition [92].
Novel excipients can bring numerous advantages: they can be used in smaller
concentrations even in highly concentrated solutions; they can decrease viscosity
and increase protein stability; they can improve manufacturing processes or allow
to differentiate a medicine. Therefore, they can have a central role in increasing
drug’s efficacy and safety and contribute to patient’s compliance [115]. Some
novel substances have been approved or are under development. Recombinant
human hyaluronidase has been recently used in products for subcutaneous
administration. It enhances protein bioavailability by creating an interstitial matrix
that promotes dispersion of the therapeutic protein [116]. Surfactants based on
trehalose fatty acid esters are being tested as stabilizers in shake-induced stress
conditions, reducing aggregate formation and adsorption to container surfaces.
These properties make them a plausible alternative to poly(ethylene glycol)-
derived surfactants that induce protein oxidation during static storage [117]. With
the increasing development of high-concentration antibody solutions, excipients
that reduce viscosity are needed. Combined use of arginine and glutamic acid
seems to reduce intermolecular attractions, thus decreasing aggregation [48].
Hydrophobic salts, which compete with hydrophobic protein residues in protein-
protein interactions, are also helpful in avoiding aggregation [118]. Cyclodextrins
are cyclic oligosaccharides that have been used as excipients before, but not in
parenteral protein formulations [119]. Their interest in recombinant protein
formulations has been rising since they form inclusion complexes of the drug,
increasing solubility and stability, and reducing aggregation [120].
Despite the awareness on the importance of novel excipients and the efforts
made to develop them, they are not frequently found in approved products. This is
because regulatory authorities have very strict requirements about the safety of
these new substances, making the evaluation of the application process very
complex, time-consuming, and expensive [121]. In fact, the experience associated
with human use of excipients approved by regulatory authorities determines that
any or only little additional toxicological data is needed. For novel excipients,
stricter requirements are demanded [121], such as extensive preclinical
evaluations, as well as data on the pharmacokinetics of the new excipient on its
intended formulation. In Europe, the dossier that needs to be submitted for the
application of a new excipient is similar to the one of an active substance. It
requires information on manufacturing, characterization, in process controls and
safety. It must be submitted together with the application for marketing
authorization, in accordance with the EMA Guideline on Excipients in the Dossier
for Application for Marketing Authorization of a Medicinal Product [92]. The
FDA elaborated a guidance on Nonclinical Studies for the Safety Evaluation of
Pharmaceutical Excipients that includes recommendations on how to obtain a
safety profile for these new entities [122].
One last concern regarding the regulatory requirements for new excipients is
that their development generates proprietary data that demands protection. As a
way of promoting the development of novel compounds by the pharmaceutical
companies, manufacturers of the United States of America and Japan follow an
Excipient Master File, which contains both open and confidential information on
quality and toxicity of the new excipients. By using this information, new
excipients can be approved apart from a marketing drug application, a situation
that is different at the present moment in Europe [121].

6.2 Alternative Routes of Administration


Therapeutic proteins have been formulated for parental administration, to allow
for maximum availability and minimum pre-systemic degradation. Intravenous
formulations are administered either in a single bolus dose or in a continuous
infusion. Subsequently, maximum bioavailability is rapidly achieved. However,
this pharmacokinetic profile may not be the desired one for all therapeutics. In
fact, for proteins with short blood half-lives, an absorption of the drug through a
prolonged period may be beneficial. One way to obtain the desired concentration-
time profile is to change the administration route from intravenous to
subcutaneous or intramuscular, which delay the absorption of the drug into the
blood circulation [123].
Nowadays, other routes of administration are being studied for
biopharmaceuticals, in search for a better convenience of administration
(noninvasive) for the patient, improving therapeutic adhesion and self-
administration in ambulatory [124]. This is especially relevant in a time where
more biopharmaceuticals to treat chronic diseases are available. The oral
administration provides the previously mentioned advantages. However,
bioavailability achieved by this route is very low, not only because of the nature
of the proteins themselves (polarity and high molecular weight) but also due to
protein degradation in the gastrointestinal tract and in first-pass hepatic
metabolism. Strategies to overcome these difficulties are being studied and
include the co-administration of absorption enhancers, which can act by
temporarily disrupting the intestinal barrier or serving as a carrier (e.g., calcium
chelators, surfactants, and bile salts), or protease inhibitors, which prevent
enzymatic degradation of the drug (e.g., aprotinin and puromycin) [125].
Inhalation and intranasal administration routes can be advantageous due to the
convenience of administration, the highly vascularized large surface area for
absorption and avoidance of first-pass metabolism. Exubera® was a recombinant
human insulin that, when inhaled, was absorbed more rapidly and had a shorter
half-life than subcutaneous insulin. This profile was actually similar to the one of
endogenous insulin after a meal [126]. However, the product was withdrawn from
market in 2008 due to the low bioavailability and local side effects. Intranasal
administration has the benefit of a direct delivery of the drug from the nasal cavity
to the brain, without being retained by the blood-brain barrier. This is particularly
interesting in therapeutics for central nervous system diseases, as exemplified for
some neurotrophins, neuropeptides, and cytokines in in vivo studies [127]. On the
other hand, intranasal administration is associated with more variability in
absorption, due to the physiological mucociliary clearance mechanism and
uncertain mucus secretion in cases of cold, allergies, or hay fever.
Transdermal administration also offers the advantage of bypassing hepatic and
gastrointestinal degradation and allows for a prolonged drug release. However,
bioavailability is limited due to proteins’ high molecular weight and hydrophilic
nature. Penetration enhancers like cell-penetrating peptides, ethanol, propylene
glycol, dimethyl sulfoxide or surfactants can be incorporated in the formulation to
change the permeability of the skin, facilitating drug absorption [128]. However,
these passive strategies of transdermal delivery are limited to peptides and small
molecular weight proteins. In this regard, permeation techniques like
sonophoration (application of low-frequency ultrasound), iontophoresis (use of
low-level electric current), and polymeric microneedles (either coated,
dissolvable, degradable, or bioresponsive, according to the type of drug-polymer
system desired) [129] have been used to allow transdermal delivery of proteins
like insulin, hormone-releasing factor, calcitonin, and parathyroid hormone [130].
Based on the advantages associated with alternative routes of administration
stated above, a new route of administration may be an interesting strategy to
extend the life cycle of a drug already marketed in an injectable formulation. In
this case, new nonclinical and clinical data would be required, to ensure
comparability of efficacy, safety, and tolerability between the two formulations
[131].

6.3 Other Strategies to Improve Protein Delivery


Manipulating the choice and concentration of excipients may not be enough to
achieve the desired pharmacokinetics of certain proteins. Therefore, other
strategies to improve drug delivery have been proposed. A controlled delivery
system enables the maintenance of drug therapeutic levels for an extended period,
decreasing the need of frequent administrations and thus improving patient
comfort and compliance. The components of the system must be nontoxic, non-
immunogenic, biodegradable, and/or biocompatible [132]. Options available
today include mechanical pumps (that can deliver the drug at a constant or pulsar
delivery rate), subcutaneous osmotic mini-pumps (mechanism based on a
difference of osmotic pressure between the interior of the pump and the outside),
and regulated approaches (like biosensor-pump combinations or even self-
regulating systems) [133]. Other strategies include the delivery of the therapeutic
drug encapsulated in matrix-type delivery systems. Examples of such approach
are nanoparticles and microparticles that prevent the contact of the protein with
proteases and other enzymes and, consequently, its degradation before reaching
the target site [134], or immobilization in hydrogel networks (beneficial in
maintaining protein three-dimensional structure) [135].
6.4 High-Throughput Characterization and Formulation Screening
Due to the complexity of recombinant proteins and the diversity of excipients now
available, drug characterization and formulation development are difficult and
time-consuming. The use of high-throughput screening for protein
characterization has been increasing, with the improvement of equipment such as
high-density microplates, microfluidics and more sensitive detectors [136], and
automatization of conventional technologies like spectroscopy, light scattering, or
imaging [137]. When applied to formulation development, high-throughput assays
give a large amount of information about protein stability, in a short period of
time, and with a reduced amount of sample, which is critical in an early
development stage [37].
Such assays should be preceded by pre-formulation studies, in order to
increase knowledge on the physical and chemical properties of the protein and
optimize the choice of experimental conditions to perform. These studies can go
from the aforementioned characterization methods to mathematical and computer
modeling tools. Mathematical models are an interesting source on the prediction
of the protein’s conformational dynamics and aggregation propensity [138, 139].
To assess protein structure, in silico characterization software is available. It is
mainly based on the knowledge provided from protein sequence and signature
databases (e.g., UniProt, Protein Data Bank). They combine data from amino acid
sequences and three-dimensional data, to give an insight on protein structure,
hydrophobicity, or even function [140]. For subsequent steps, several tools and
online services have been developed that allow a fast and efficient analysis of the
protein’s active conformation, protein-ligand interactions, and affinity data [141].
Forced degradation characterization must be also included in pre-formulation
studies, in order to detect and quantify possible degradation products [142]. It is
assumed that when exposed to stress conditions for a short period of time, such as
elevated temperatures, freeze-thawing, extreme pH, mechanical stress, or
oxidizing environments, the degradation profile of the drug will correlate with
degradation during the medicine lifetime [143]. This will enable the determination
of the best stabilizing conditions, selection of stability parameters for quality
monitoring and comparability, and prediction of degradation upon accidental
exposure to unrecommended conditions. Most common forced degradation
studies and analytical methods to assess degradation products have been reviewed
by Hawe et al. [144]. After data collection from pre-formulation studies, an
experiment can be designed, and a high-throughput platform (Fig. 1) can be put in
place, either for characterization assays or for selection of appropriate
formulation. There is a first step of sample preparation, using automated handling
systems that place the samples in microplates (96–384 wells), allowing for the
screening of multiple parameters simultaneously (concentration, pH, buffers,
dosage form, and storage temperature). This is followed by sample analysis,
usually with the help of specific software [145]. Several methodologies have been
performed in high-throughput platforms. Noninvasive methods are the ones that
allow for a recovery of the formulation, thus reducing waste. They include
techniques such as UV-Vis absorbance, fluorescence spectroscopy, and light
scattering. Invasive procedures should be executed after the noninvasive ones, to
make use of the same sample. Examples of such methodologies are calorimetry,
capillary electrophoresis, and mass spectrometry.

Fig. 1 Flow diagram of the high-throughput formulation development concept (Adapted from Martinus et al.
[145])

An example of a high-throughput platform for protein characterization that


has been successfully developed is that of an automated workstation for large-
scale protein identification, using reverse liquid chromatography and mass
spectrometry, followed by analysis using SEQUEST software [146]. Regarding
formulation development, Zhao et al. engineered an automated system that can
test up to 500 different samples through a variety of analytical techniques,
allowing for the screening of more formulations in a very short time, with the
same reliability than classical approaches [147].

6.5 Developability Assessment and Quality by Design


Developability assessment is the likelihood of a drug candidate to be successfully
developed into a commercial product [148]. It comprises a set of approaches that,
when implemented in early stages of development, enable a more rational choice
of lead candidates to be developed into optimal commercial products. These
strategies include pre-formulation studies, stability evaluation, interaction
between discovery and formulation design teams, and manufacturability
assessment [149]. Therefore, developability offers the chance to re-engineer the
lead molecule or to adapt the development process according to potential
undesired features discovered in early development. This decreases the risk of
failure in more resource-consuming and costly stages of drug development [150].
Developability assessment correlates with the implementation of Quality by
Design (QbD), another trend in biopharmaceutical development and manufacture
[151]. ICH guidelines Q8 and Q8(R2) define QbD as “a systematic approach to
development that begins with predefined objectives and emphasizes product and
process understanding and process control, based on sound science and quality
risk management” [152]. With a great understanding of the active protein and
excipients from pre-formulation studies and developability assessment, it is
possible to define, monitor, and adjust the parameters that contribute the most to
consistently deliver products with a defined quality. Within the QbD, the
parameters that are proven to impact protein stability, named Critical Quality
Attributes (CQAs), should be monitored throughout the manufacturing process,
such as type of excipients, concentration, or grade. Design of Experiments (DoE)
is another important element of QbD, defined as a tool that incorporates the
possibility to change the formulation attributes within a defined design space,
whose limit is justified based on solid scientific data. This qualitative and
quantitative flexibility allows for a rapid screen and optimization of a formulation
during manufacturing [153]. Any deviations in formulation composition made
within the defined design space are not considered changes in manufacturing,
which is very attractive from the regulatory point of view. Risk analysis can help
defining CQA’s upon, for example, using tools of prediction of aggregates
formation and immunogenicity propensity, which may impact the efficacy and
safety of the medicine [100]. In respect to the establishment of the range enabling
the maintenance of the protein quality (in terms of physical stability and activity),
a risk management plan must be developed to minimize the impact of any damage
that may occur, which is contemplated in the ICH Q9 Quality Risk Management
[154]. This plan must be defined by a multidisciplinary team that can correctly
assess the major technical and logistic aspects that can impact the drug (from
physical and chemical stability problems, to operation errors, or issues associated
with raw material suppliers) [155].

7 Conclusion
Protein-based therapeutics are complex products, whose stability from
manufacturing to patient administration is hard to achieve. From formulation
optimization to the development of delivery systems, several strategies have been
studied to try to overcome biopharmaceuticals stability limitations and safety
concerns or to improve product quality and patient compliance. Despite these
efforts, many challenges remain unsolved, like immunogenicity and safety
concerns, stability issues, and exploration of alternative routes of administration,
giving space for further research for different solutions and technical advances in
this field.

Acknowledgments
Rita Ribeiro is a student of the Ph.D. Program in Pharmaceutical Sciences from
the Faculty of Pharmacy, University of Coimbra, and a recipient of the fellowship
SFRH/BD/121935/2016 from the Portuguese Foundation for Science and
Technology. This work was funded by Portuguese National funds via FCT –
Fundação para a Ciência e a Tecnologia, I.P. – under projects Cancel Stem
(reference POCI-01-0145-FEDER-016390), CENTRO-01-0145-FEDER-000012
(HealthyAging2020), Euronanomed2 (FCT reference ENMed/0005/2015), and
CNC.IBILI (FCT reference UID/NEU/04539/2019).

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A. C. Silva et al. (eds.), Current Applications of Pharmaceutical Biotechnology, Advances in
Biochemical Engineering/Biotechnology 171
https://doi.org/10.1007/10_2019_116

Therapeutic Antibody Engineering and


Selection Strategies
Joana Ministro1, Ana Margarida Manuel2 and Joao Goncalves2
(1) LeanBioPro, Barcelona, Spain
(2) iMed – Research Institute for Medicines, Faculty of Pharmacy at
University of Lisbon, Lisbon, Portugal

Joao Goncalves
Email: [email protected]

Abstract
Antibody drugs became an increasingly important element of the
therapeutic landscape. Their accomplishment has been driven by many
unique properties, in particular by their very high specificity and selectivity,
in contrast to the off-target liabilities of small molecules (SMs). Antibodies
can bring additional functionality to the table with their ability to interact
with the immune system, and this can be further manipulated with advances
in antibody engineering.
The expansion of strategies related to discovery technologies of
monoclonal antibodies (mAbs) (phage display, yeast display, ribosome
display, bacterial display, mammalian cell surface display, mRNA display,
DNA display, transgenic animal, and human B cell derived) opened
perspectives for the screening and the selection of therapeutic antibodies
for, theoretically, any target from any kind of organism. Moreover, antibody
engineering technologies were developed and explored to obtain chosen
characteristics of selected leading candidates such as high affinity, low
immunogenicity, improved functionality, improved protein production,
improved stability, and others. This chapter contains an overview of
discovery technologies, mainly display methods and antibody humanization
methods for the selection of therapeutic humanized and human mAbs that
appeared along the development of these technologies and thereafter. The
increasing applications of these technologies will be highlighted in the
antibody engineering area (affinity maturation, guided selection to obtain
human antibodies) giving promising perspectives for the development of
future therapeutics.
Graphical Abstract
Keywords Anti-drug antibodies – Biosimilars – European market –
Immunogenicity – Product information – Recombinant drugs – Summary of
product characteristics – Therapeutic biologics

1 Antibody Overview
1.1 Antibody Discovery
When in the 1700s the first perceptions of the immunology field were being
explored through vaccination experiments, no one could imagine the impact
of antibodies in today’s society. In fact, immunity acquisition against a
previous encountered disease has been documented for many centuries [1].
The first reference to antibodies appeared in 1890 from Emil von Behring
and Shibasaburo Kitasato. In a breakthrough experiment, they treated
diphtheria-infected animals with serum from immunized animals [2]. The
potential of this therapy was immediately foreseen for human application,
and Behring was later awarded with the Nobel Prize.
The term Antikörper (antibodies) was introduced around 1900 by Paul
Ehrlich, who performed several studies about toxicity and immunology. He
is considered one of the fathers of modern immunology for his insights into
the antibody mechanics and for the suggested theory that side chains of the
antibodies react with antigens and bind them and subsequently travel
around the body in the blood [3]. In the 1940s, Linus Pauling showed that
the interaction between antibodies and antigens was more dependent on
their shape than on their chemical composition, confirming the lock-and-
key theory proposed by Ehrlich [4].
The modern era of antibody research started in 1975 with the
development of the hybridoma technology, the first mechanism to produce
monoclonal antibodies in large quantities [5]. Since then, antibodies have
become crucial in biotech and pharma industry, either for research purposes
or for high-profit therapies. The first monoclonal antibody for therapy was
approved in 1986 [6], and by December 2017, the FDA had already
approved 79 therapeutic antibodies, with much more currently under
evaluation in various phases of clinical trials [7, 8].

1.2 Characteristics and Structure of Antibodies


Today it is well-known that the recognition of foreign agents by
immunoglobulins (Igs) is a core function of the adaptive immune response
in mammals. Igs are glycoproteins produced by all vertebrates and can be
localized on the surface of B lymphocytes, known as B cell receptors
(BCR), or free in the blood or lymph, known as antibodies (Ab). If
produced by the same cell, these two molecules are identical except for a
small portion that allows membrane anchoring in BCRs and secretion in
Abs [9]. Since antibodies are soluble and can be secreted in large quantities,
they are easily obtainable and studied. They are Y-shaped molecules
consisting of two heavy and two light polypeptide chains, linked by
disulfide bonds, and divided into constant and variable domains (Fig. 1a).
They can be also divided in three equal-sized portions, two antibody
fragment (Fab) arms, containing the variable domains at both ends, and the
crystallizable fragment (Fc) domain. All domains are connected by a
flexible amino acid chain, called the hinge region [10, 11]. The Fab and the
Fc domains perform the two main functions of antibodies; while the Fab
recognizes a foreign element (or an antigen), through the variable domains,
the Fc induces an immune response by activating the complement system
and/or the antibody-dependent cellular cytotoxicity [12].

Fig. 1 General structure of antibodies. (a) A typical IgG molecule is composed of two heavy chains
(blue) and two light chains (pink), linked by disulfide bonds. CH and CL are constant domains of
heavy and light chain, respectively. VH and VL are variable domains of heavy and light chain,
respectively. CDRs stand for complementary determining regions and are responsible for antigen
binding. FRs are conserved regions that confer structure to the CDRs. The two antigen-binding
fragments (Fab) are linked to the crystallizable fragment (Fc) by the hinge region. (b) An IgG has
two antigen-binding regions, containing the VH and VL domains. Each variable region has three
CDRs, flanked by the FRs regions. Adapted from O’Kennedy et al. [20]
All antibodies are assembled in the same way. However, two classes of
constant light chains (κ and λ) and five classes of constant heavy chains (μ,
δ, γ, α, and ε) can be distinguished. Immunoglobulins (IgM, IgD, IgG, IgA,
and IgE) are named by their class of constant domains of the heavy chain.
Also called isotypes, these different constant regions determine the
functional properties of an antibody. Moreover, IgA has two sub-isotypes
(IgA1 and IgA2), and IgG has four sub-isotypes (IgG1, IgG2, IgG3, and
IgG4). IgM is the largest antibody and is mainly expressed in immature B
cells. IgD is expressed in naïve B cells and activates basophils and mast
cells upon exposure to an antigen. IgE is involved in allergic responses and
IgA in mucosal immunity. IgG is the most frequent isotype, accounting for
70–85% of total immunoglobulins in serum. Its stability, long half-life and
generally high affinity make IgG the most used isotype for therapeutic use
[13–15].

1.3 Antibody Paratope


For an antibody to exert its biological function, it must bind to a specific
target. The variable domains provide the contact to the antigens and
determine the specificity of each antibody. Each variable domain forms an
immunoglobulin fold that consists of a pair of β-sheets, linked by a
disulfide bond, and three hypervariable loops lying at the end of the
structure, known as complementary determining regions (CDRs). There are
three CDRs in each variable domain, and their hypervariable characteristic
allows diversification and the recognition of an almost unlimited number of
antigens. Variation of the amino acid sequences inside these loops provides
the major mechanism for the generation of the vastly diverse set of
antibodies and T cell receptors expressed by the immune system. While
CDR1 and CDR2 domains vary somewhat between different
immunoglobulins, CDR 3 differs very dramatically. This happens because
contrary to CDR1 and CDR2, CDR3 suffers imprecise rearrangements
during the process of V(D)J recombination (Sect. 1.4.1) [16]. The three
CDRs of each variable domain are distributed between four structural and
less variable regions, known as framework regions (FR). The FR regions
function as a scaffold to hold the CDRs in position to contact the antigen.
Even though sequence diversity is concentrated in the CDRs, the
framework regions are also diverse to some extent, which may be
advantageous in antibody function and stability to accommodate highly
diverse CDRs [17].
The variable regions of both chains interact to form a molecular site (the
paratope) that binds strongly to a part of the antigen (the epitope), and the
strength of this binding is called antigen-binding affinity (Fig. 1b). While
identification of paratopes is often done through identification of CDRs, not
all the residues within the CDRs bind the antigen. In fact, 3D analysis of the
antibody structure suggested that only 20–33% of the residues within the
CDRs participate in antigen binding. Nonetheless the residues that are
directly involved in the interaction with the antigen are, in general, the most
variable ones. The paratope is ultimately a function of the folding pattern of
the association of heavy and light polypeptide chains [18, 19].

1.4 B Cell Development and Activation


The antibody producing cells are called B cells or B lymphocytes. They
perform a core function in humoral immunity not only by secreting
antibodies but also by presenting antigens (antigen-presenting cells) and by
secreting cytokines [21]. In mammals, B cells start maturation in the bone
marrow, from hematopoietic stem cells, where they undergo a process
called somatic recombination or V(D)J recombination (see Sect. 1.4.1).
This process is responsible for the generation of the first level of antibody
diversity and comprises the rearrangement of immunoglobulin genes
through random recombinations [22]. While developing, B cells undergo
both a positive and negative selection, a process that assures that the B cell
receptor (BCR) is binding to a proper ligand and not to a self-ligand. In case
this would not occur, the cell would not receive the right signals to proceed
and would stop the development. B cells then migrate to peripheral
lymphoid organs, such as the spleen, where they become mature B cells
expressing both IgM and IgD; these cells are also called naïve B cells
before antigen exposure [23, 24].
Mature B cells circulate through the blood to the secondary lymphoid
organs where they contact with antigens that circulate in the lymph. The
activation of a B cell is triggered when the BCR binds to an antigen. This
event induces the cells to migrate to the interface between the follicle and T
cell zone, and it is at this stage that the B cells present the antigen to helper
T cells. At this point both cells integrate inputs to decide between death and
proliferation. B cells can then start to proliferate and form stable
connections with helper T cells near the interfollicular zone, where there is
a great number of dendritic cells. A few days after antigen exposure, cells
integrate more inputs to differentiate into either plasma cells (in order to
secrete large amounts of antibodies) or memory cells (responsible for a
secondary immune response) or to go to the germinal center for affinity
maturation [25]. In the germinal centers, cells divide quickly and
hypermutate the BCR V-regions in a process called somatic hypermutation.
They also undergo a process called class switch recombination, a
mechanism that changes the immunoglobulin isotype (see Sect. 1.4.2).
Depending on their BCR affinity for foreign and self-antigens, the cells may
be selected again for survival or death [9]. Some antigens can activate B
cells directly in the absence of helper T cell. The ability of B cells to
respond directly to these antigens provides a rapid response to many
important pathogens. However, as these antibodies are not subject to
affinity maturation, they are therefore less variable and less functionally
versatile than those induced with T cell help [26].
B cell development comprises a series of decision points where many
inputs are integrated influencing the cells’ fate. Evidence from cell
dynamics and integration of signals support the theory that more complex
principles of control also exist [27].

1.4.1 V(D)J Recombination


An efficient immune response absolutely requires genetic diversity at the
immunoglobulin gene locus. The first level of diversity consists in the
rearrangement of multiple immunoglobulin genes, in a process called V(D)J
recombination. It occurs in the bone marrow, during maturation, and before
antigen encounter. In mammals, immunoglobulin gene segments contain
more than 100 variable (V), 30 diversity (D), 6 joining (J), and 9 constant
(C) exons. Random choices of these genes encode different proteins,
generating in the order of a million different antibodies and matching an
enormous panoply of antigens. Rearrangements occur orderly (Fig. 2a). At
the heavy chain locus, D–J segments assemble first, followed by V segment
gathering, and, after transcription, the heavy variable regions are connected
to the constant regions by RNA splicing. At the light chain locus, D
segments are absent; thus only V–J exons are paired. CDR 1 and CDR 2 are
encoded by the V gene segments, while CDR 3 (the most variable and
exposed CDR) is generated as a result of the imprecise joining of the VDJ
or VJ exon. The antibody diversity is then further increased by the assembly
of different heavy and light chains [22, 24, 28].
Fig. 2 Immunoglobulin heavy chain diversification processes. (a) Random formation of diverse
V(D)J heavy combinations generates combinatorial diversity. Curved lines with arrows indicate gene
rearrangement events. The regions where somatic hypermutation accumulates are mainly the CDRs;
(b) schematic representation of protein–DNA complexes in V(D)J recombination. The 12-RSS and
23-RSS are represented as white and black triangles, respectively. Blue and yellow rectangles
represent the coding segments to be recombined, and the red rectangle represents the newly
generated nucleotide sequence at coding junction. Gray areas represent protein complexes; AID
activation-induced cytidine deaminase, C constant regions, D diversity segments, J joining segments,
S switch regions, TdT terminal deoxynucleotidyl transferase, V variable segments. Adapted from Di
Noia and Neuberger [39] and Mansilla-Soto and Cortes [31]
V(D)J recombination is activated by a V(D)J recombinase that consists
of two lymphoid specific proteins, RAG1 and RAG2, that cleave DNA
[29]. The recombinase recognizes conserved DNA sequences, as well as the
recombination signal sequences (RSS) that flank each V, D, and J segments.
In order to control this mechanism, recombination only occurs between
RSSs with different spacer lengths (the “12/23 rule”), which prevents
joining of two gene segments from the same group [30]. RAG proteins
introduce a pair of site-specific double-strand breaks between the coding
segments and the RSSs, creating a circular signal joint and a linear coding
joint. This cleavage generates hairpins at the extremities, which cannot be
directly ligated (Fig. 2b). The random opening of the hairpins by the
endonuclease protein Artemis at each extremity generates a combination of
different DNA ends that will strive for a mismatch between the two ends,
enabling therefore the formation of junctional diversity [31]. Thus,
junctional diversity arises by the imprecise fusion of V, D, and J segments
and can result in deletion and/or addition of nucleotides in the joint region.
Deletions occur through the activity of an exonuclease that is responsible
for trimming the DNA ends before ligation. In fact, 80% of human Ig genes
show junctional diversity caused by deletions. A less common process is
the addition of template nucleotides (P nucleotides) in the junction site [32].
Another source of junctional diversion is the addition of non-template N
nucleotides. These random nucleotide insertions are added to the DNA ends
by a lymphoid-specific enzyme, called terminal deoxynucleotidyl
transferase (TdT), in a template-independent manner, which can add up to
15 extra residues in the joint, further increasing junctional diversity [33].
Actually, the fact that this rearrangement process provides around 105–106
different antibody specificities in humans is in a great extend due to TdT
activity. An end-joining process strictly restricted to fully complementary
ends would be unable to ligate the coding joints. In contrast, a versatile but
conservative process, such as nonhomologous end joining (NHEJ), is able
to join these DNA ends and generate a highly diverse immune repertoire
while protecting against side genomic instability [34, 35]. During the
process of V(D)J recombination, the numerous combinations of multiple
gene segments and the junctional modifications brought about by their
joining result in a large repertoire of antigen receptors, thereby assuring the
hallmark diversity of the adaptive immune system [36].
1.4.2 Somatic Hypermutation and Class Switch Recombination
Despite the great diversity achieved by V(D)J recombination, the B cell
requirements for antigen targets exceed largely the information presented in
the cell genome. Thus, the cell needs to achieve larger repertoires by itself.
After encountering an antigen and moving to the germinal centers,
immunoglobulin genes suffer both somatic hypermutation (SHM) and class
switch recombination (CSR) [37].
In mice and humans, SHM causes mutations in the variable
immunoglobulin both in the light and heavy chains locus. The mutation rate
is about one million-fold higher than the spontaneous mutation rate in other
genes, and it is mainly due to single base substitutions, with occasional
insertions and deletions. The mutation activity of SHM starts around 150
nucleotides downstream of the IgV promoter, and it is extended for about
2 kb pairs [38]. All four bases can be mutated, but the targeting of
individual bases is, however, not random. Interestingly, certain regions,
especially those responsible for antigen binding, are intrinsically more
mutable than others. The result of these mutations may alter the specificity
or affinity of the encoded antibody, potentially enhancing the response to a
specific antigen [39, 40]. In the same B cell, the heavy chain is rearranged
down the chromosome through CSR or isotype switching (Fig. 2a). CSR is
the process by which CH domains encoding one isotype are exchanged for
another, thereby influencing the effector function of the resulting antibody.
In addition, CSR is accompanied by an increase in antigen-binding affinity
[41–43].
The mechanisms of SHM and CSR are not completely elucidated, but a
key factor of the two processes is well-known: the activation-induced
cytidine deaminase (AID) [44]. Upon B cell activation, AID expression is
upregulated and is associated with the cell transcription apparatus, gaining
access to single-stranded DNA regions. In most of the cases, AID
recognizes specific small sequences (hotspots), where it deaminates
cytosines and converts them to uracils, creating dU:dG mismatches. This
DNA lesion is then processed by mismatch repair (MMR) and base excision
repair (BER) pathways, resulting in point mutations [45, 46]. In the CSR
process, a protein machinery is recruited after AID deamination process
creating double-strand breaks in the switch regions (S) located upstream of
the constant regions. The DNA between the S-regions is subsequently
deleted from the chromosome, removing unwanted heavy chain constant
regions. The free ends of the DNA are rejoined by NHEJ yielding B cells
that express IgG, IgE, or IgA [41].
SHM and CSR are usually more evident after a second exposure to an
antigen and are responsible for the dramatically strong secondary immune
response. These events of affinity maturation work in combination to
generate an antibody repertoire estimated to be superior to 109 in humans
[47].

1.5 Monoclonal Antibodies and Their Recognition as Tools


Due to their high specificity and selectivity, antibodies have the potential to
be of great use for biochemical, diagnostic, and therapeutic purposes.
Antibodies are categorized into two groups, polyclonal or monoclonal.
Polyclonal antibodies (pAbs) are a heterogeneous mixture of antibodies
directed against various epitopes on the same antigen. These antibodies are
generated by different B cell clones and, as a consequence, are
immunochemically different, with different specificities and affinities.
While they are inexpensive and easy to produce, pAbs cannot be reliable
due to the high variability from batch to batch. In contrast, monoclonal
antibodies (mAbs) originate from a single antibody-producing B cell and
therefore only bind to one epitope of an antigen. Thus, while more
expensive to produce, mAbs have high specificity to a single epitope and
present batch-to-batch homogeneity [48].
The development of the hybridoma technology was a big hallmark in
the antibody field, allowing the relatively easy production of large
quantities of mAbs for diverse applications. These antibodies were
generated by fusing antibody-producing mouse cells with myeloma mouse
cells, resulting in the formation of immortal cell lines expressing a single
antibody with specificity for one particular epitope of an antigen. Once
produced, hybridomas can be cultured in vitro indefinitely [5].
Many of the current laboratory procedures use mAbs as tools to answer
basic research questions. They allow researchers to identify molecules not
seen by the naked eye, enabling conclusions about the target molecule and
pathway of interest. Routine techniques such as Western blot, flow
cytometry, immunohistochemistry, and enzyme-linked immunosorbent
assay (ELISA) all rely on antibody properties. MAbs have also become an
important component of many diagnostic techniques including detection of
infections, measurement of biological markers in blood, and recognition of
allergies, having increased the speed and accuracy of many tests [49]. Also,
radiolabeled antibodies can be used in the imaging diagnostic allowing, for
instance, differentiation between cancerous and non-cancerous cells [50].
With the ability to bind an almost unlimited number of targets with high
specificity and stability, mAbs were sought for therapeutic applications
since the first time they were developed in vitro. Therapeutic mAbs can act
through multiple mechanisms, such as blocking of targeted molecule
functions or disrupting a signaling pathway [51]. Nevertheless, it soon
became clear that antibodies were facing many problems for therapeutic
use, mainly due to their murine nature. Developments in molecular biology
made possible the creation of recombinant variants of mAbs that led to
optimized antibodies and ushered the age of antibody engineering [52, 53].

1.6 Antibody Engineering


1.6.1 Reduce Immunogenicity
The success of the first therapeutic antibody, OKT3 (muromonab), as a
treatment for transplant rejection, was not immediately followed by a series
of approvals as anticipated. Although very promising, patients treated with
antibodies from hybridoma technology often developed an immune
response that attenuated the half-life and the efficacy of the antibodies [54].
In order to reduce immunogenicity, numerous strategies have been pursued
for the humanization of animal antibodies, replacing constant regions
(chimeric antibodies) or all the non-specificity determining residues
(humanized antibodies) with corresponding human antibody sequences
[55]. Since the late 1990s, with the introduction of chimeric and humanized
antibodies, therapeutic antibodies have become one of the fastest-growing
classes of therapeutics in the biological drug market [56]. More recently, the
generation of transgenic mice expressing a repertoire of human heavy and
light chain genes, followed by the generation of human hybridomas, was
shown to be an effective way for the generation of human antibodies against
many antigens [57–59].

1.6.2 Antibody Formats


Besides increasing safety, antibody engineering allowed the development of
several antibody formats for different functions. Immunoglobulins are large
proteins (between 150 and 180 kDa) and have difficulty to penetrate tissues
or to target antigens in small areas such as enzyme active sites. In drug
development a heavy chain may not be desirable or even necessary [60].
Researchers have dismembered antibodies into their component parts,
taking advantage of the smaller sizes in order to develop new therapeutics
with exciting properties (Fig. 3). These “domain antibodies” come in
numerous formats: fragment antigen binding (Fab), single-chain variable
fragment (scFv), or VH and VL domains [61, 62]. Disadvantages of these
small domains comprise aggregation, poor solubility, and reduced half-life
in circulation. However, genetic engineering also allowed multimerization
of antibody fragments, and these formats can help to overcome the decrease
of affinity and stability from fragment domains. Moreover, this has also led
to the generation of multispecific antibodies, with the ability to recognize
different epitopes or different antigens [63–65]. The large panel of antibody
formats that has been developed reflects the strong interest for these
molecules. While in many cases the manufacturing remains challenging,
several antibody formats are currently in clinical trials with some already
approved for commercialization [61, 66].

Fig. 3 Examples of antibody formats. A variety of antibody fragments are represented including
Fab, scFv, and single domains VH and VL. Multimeric formats such as minibodies, bis-scFv,
diabodies, triabodies, and tetrabodies are also depicted. Adapted from Holliger and Hudson [61]

1.6.3 Target Delivery


Recently, mAbs are being exploited for modern drug delivery systems,
aiming to direct a therapeutic agent to a specific tissue or cell for enhanced
pharmacology. Antibody-based conjugates have opened up a whole new
field of clinical possibilities with several platforms emerging on the market,
with most promising applications in cancer therapy [60].
Without the toxicity associated with chemotherapeutic agents, antibody
therapy held a tremendous promise in the elimination of tumor cells.
However, only modest success has been achieved in patients with solid
tumors, with cell elimination not being as effective as expected [67].
Antibody-drug conjugates (ADCs) combine the targeting ability of an
antibody with the cell-killing activity of a cytotoxic drug [68]. The basic
ADC technology is composed of an antibody vehicle, the cytotoxic drug,
and a linker, and each ADC is constructed in a customized manner for the
specific antibody and drug combination. By the end of 2018, there were
four ADCs already approved for cancer therapy [69, 70].
Therapeutic efficacy can also be increased by coupling an antibody with
a nanoparticle conjugate. In the context of drug delivery, nanoparticles are
used to encapsulate a drug for enhanced drug release and reduced side
effects, similar to ADCs. While fused to nanocarriers, mAbs can be used for
targeting cell surface receptors resulting in enhanced intracellular drug
accumulation [71, 72].
The understanding that multiple factors might contribute for disease
progression led to the development of bispecific antibodies, in a large
variety of formats. Bispecific T cell engagers (BiTE®) have stood out from
the panoply of bispecifics due to their function in recruiting the host
immune system, more specifically T cells cytotoxic activity, to the cancer
cells. They consist of two different scFvs, one binding to T cells, usually
through the CD3 receptor, and the other to a tumor cell, through a tumor-
specific molecule. One example of a BiTE antibody construct is
blinatumomab (trade name Blincyto), approved in 2017 for relapsed or
refractory B cell precursor acute lymphoblastic leukemia [73–75].
Another innovative approach for antibody use is the chimeric antigen
receptors (CARs). CARs are engineered receptors, which combine
specificity (conferred by the antibody) with immune effector function of T
cells. In cancer therapy, T cells are removed from patients, and the receptors
are modified so that they become specific to the patient’s cancer. The
engineered T cells are then reintroduced into the patient, with the new
ability to recognize and kill the cancer cells [76, 77]. A CAR therapy has
been recently approved for use against acute lymphoblastic leukemia [78].
The development of creative antibody engineering technologies together
with the progress toward deciphering disease pathways has generated robust
interest, resources, and investments in antibody discovery.

1.7 Market Importance of Therapeutic Antibodies


Over the past 40 years, therapeutic antibodies have rapidly become the
leading product within the biopharmaceutical market. The global market for
monoclonal antibodies was valued at 85.4 billion dollars in 2015 and is
expected to reach a value of 138.6 billion dollars by 2024 [79]. The
growing health concerns and the increasing of diagnostic techniques have
boosted the demand for these therapeutics in recent years, particularly for
chronic diseases. Nevertheless, the spectrum of diseases potentially treated
by antibody therapies is massive, including cardiovascular, respiratory,
hematology, kidney, immunology, and oncology diseases. While antibodies
are very efficient, their cost-effectiveness has always been discussed due to
their high costs, estimated to be more than one billion dollars from
preclinical development to market approval. Consequently, therapeutic
antibodies are inaccessible to many patients in both developed and
developing countries. The development of biosimilar antibodies, which
show a similar quality, efficacy, and safety as the original antibody, may
help decrease the associated costs and provide alternative treatment options
[80, 81].

2 Antibody Libraries
2.1 Generation of In Vitro Antibody Libraries
As discussed before, the immune system has the incredible ability to create
a vast antibody repertoire, combining antibody gene segments
recombination and somatic hypermutation. Since the perception of the
several applications for antibody molecules, scientists have been trying to
mimic the vast diversity achieved inside B cells by constructing antibody
libraries in vitro [82].
It was only in 1989 when the antibody repertoire from the B cells of an
organism was recombinantly cloned in large combinatorial libraries that
mAb technology really exploded [83, 84]. Another hallmark for in vitro
antibody discovery occurred roughly 1 year after, with the development of a
display technology for the isolation of mAbs from these large collections of
recombinant antibody fragments [85]. Display technologies provided a way
to quickly select antibodies from libraries on the basis of the antigen-
binding behavior of individual clones and allowed to overcome the
limitations from toxicity and immune tolerance. Moreover, the continuous
development of automation techniques made it possible to identify
hundreds of different antibody leads against a single therapeutic target,
opening a new field for different kinds of libraries without the need for
immunization [86].
The most important parameter for in vitro antibody discovery is the
quality of the antibody libraries. Evolution has allowed the development of
a wide range of strategies for library generation that can differ in both
design and means of construction. They were first focused on affinity and
thus on library size and diversity, and, more recently, they are also focused
on biophysical properties and reduced immunogenicity. Based on the source
of the antibody repertoire, four types of libraries can now be identified:
naïve, immune, synthetic, and semisynthetic (Fig. 4) [87, 88].

Fig. 4 Different types of antibody libraries. (a) Naïve libraries are amplified from nonimmunized
donors. (b) Immune libraries are derived from immunized or immune donors. (c) Synthetic libraries
are based on computational in silico design and gene synthesis. (d) Semisynthetic libraries comprise
a combination of natural sources with in silico design. All libraries can be cloned in suitable vectors
to be used in display techniques. Adapted from Ponsel et al. [87]

2.1.1 Naïve Libraries


Naïve libraries are derived from B cells of nonimmunized donors. Although
other tissues can be used for B cell extraction, most naïve libraries are
originated from peripheral B lymphocytes, where they are not biased from
the process of affinity maturation that occurs in secondary lymphoid organs.
Moreover, IgM repertoire is often preferred because it closely reflects the
diversity following immunoglobulin gene rearrangement, without tolerance
or antigen selection [89]. mRNA isolated from B cells is used as template
for the amplification of the variable regions of antibody genes using
degenerate oligonucleotide primer sets. The amplicon is then cloned into a
suitable vector for display and selection against a desired target. One of the
primary limitations of naïve libraries is the fact that the immune system
deletes clones with affinity to self-antigens, in order to prevent the
development of autoimmune diseases, which may prevent the isolation of
antibodies with affinity for many important human targets. Despite the
uncertainty of its content, the main value of these kinds of libraries relies in
the large repertoires (of up to 1011 antibodies) that can be constructed and in
the fact that a single library can be used for the selection of antibodies
against several types of antigens, including toxins [90, 91].

2.1.2 Immune Libraries


Immune libraries are derived from B cells of immunized donors or
previously in contact with an antigen. The library construction is performed
using the same method, but, contrary to naïve, immune libraries are smaller
in size (normally 106–108) and are not well suited for the identification of
antibodies against a large panel of antigens. Nevertheless, the pre-exposure
of the host to an antigen results in the production of very specific and high-
affinity antibodies against the particular antigen, mainly due to affinity
maturation, which makes immunization a very suitable model for antibody
discovery. As human libraries can only be generated from B cells obtained
from disease-affected patients, several animal models are used for the
generation of this kind of libraries, including mice, rabbits, camelids, and
sharks [92, 93]. The latter two express antibodies that are only composed of
heavy chains. Thus, antigen binding is performed by only one single
domain referred to as VHHs in camelids and vNARs in sharks. Those
fragments are naturally highly stable and soluble, and it has been shown
that they can generate high-affinity binders [94, 95]. However, there are
some important limitations when considering immune libraries, including
the time required for animal immunization, the unpredictability of the
immune response to the immunized antigen, and the fact that a new library
must be generated for each desired antigen [89].

2.1.3 Synthetic Libraries


While naïve and immune libraries are based entirely on naturally occurring
sequence diversity and do not require human input during the generation of
sequence diversity, synthetic libraries are diversified according to design
and need a strategy for the sequence diversification. In this approach the
antibody diversity is designed in silico and then synthesized in a controlled
manner [96]. A key advantage of synthetic diversification is that the
composition of the CDRs and frameworks can be exactly defined and
controlled. Synthetic libraries prevent the natural biases and redundancies
of in vivo antibody repertoires and allow control over the sequences of
variable genes and the introduction of diversity [53]. From simple
degenerate oligonucleotide synthesis to physiochemically optimized library
design, many different strategies have been applied since the first reports of
synthetic antibodies, in 1992 [97]. Most of the constructed synthetic
antibody libraries maintain a single framework sequence and have their
diversity concentrated in the CDRs, which are generated by random
combinations of mono- and trinucleotide units [98–100]. Nonetheless, there
are a few synthetic libraries with multiple variable heavy and light chain
framework regions, such as the HuCAL libraries and Ylanthia library [101,
102]. Multiple framework sequences can accommodate more diverse CDR
structures, but it makes library design and construction more complex, and
the clones are not as uniform in their properties as in the case of single
framework libraries [103].
The increasing knowledge of sequence, structure, function, and
physicochemical properties of antibodies has allowed researchers to design
and construct highly functional and sophisticated synthetic antibody
libraries. However, it is still a very complex process to assemble synthetic
libraries, and intensive study regarding structure and folding needs to be
performed in order to guarantee functional antibodies [104].
2.1.4 Semisynthetic Libraries
In semisynthetic libraries there is a combination of naturally derived and
synthetically designed parts, and the ratio of both parts may vary in
different strategies [97]. They are often created to increase the natural
diversity while maintaining a good level of functional diversity. Such
libraries have been created, for example, by combining natural CDR
regions or by joining naturally rearranged and highly functional CDR 3
sequences with synthetic CDR 1 and CDR 2 diversity [105, 106].

2.2 Technologies for Antibody Selection


Antibody libraries need to be followed by a selection step in order to collect
the leads with the required affinity, specificity, and stability. In the immune
system, B cells link genotype to phenotype through presentation of the
expressed antibody on the surface, where only the positive selected cells
will succeed [107]. The most common selection methods also rely on the
display at cell surface of each antibody from a desired library. The antibody
display can be on the surface of phages, on eukaryotic cells, or even on
ribosomes following in vitro transcription. All of these methods consist of
antibody incubation with a relevant antigen followed by several rounds of
washing and recovery of the best binders. Display systems offer a number
of advantages for antibody discovery and optimization as they are
significantly fast and can be carried out in a high-throughput mode [108].

2.2.1 Phage Display


Phage display was the first display technology developed for antibody
discovery and still remains widely used due to its simplicity to present large
libraries. It was first described by George P. Smith, in 1985, when he
demonstrated the display of peptides by fusing a peptide of interest to the
gene III of a filamentous phage. This technology was further developed and
improved for display of antibodies mainly by the groups of Winter and
McCafferty, at the Laboratory of Molecular Biology (Cambridge, UK), and
by the groups of Lerner and Barbas, at The Scripps Research Institute (La
Jolla, USA) [85, 109, 110]. In phage display, the library DNA is usually
inserted in fusion with the gene of the phage coat protein pIII or pVIII.
After infection of bacteria, a progeny of phages is released, each phage
displaying an antibody on its surface while containing the antibody gene
inside. By immobilizing a desired target to the surface of a microtiter plate
well, it is possible to incubate the phage library, in which the phages
displaying antibodies with affinity for the target will remain bound while
others are removed by washing. Those that remain can be eluted, and this
resultant pool contains a phage mixture that is enriched with relevant
antibodies. This mixture is then amplified by infecting bacteria and
subjected to another incubation step. This repeated cycling is referred to as
“panning” (Fig. 5), and usually after three or four pannings, the DNA from
eluted phages is collected and sequenced [111].

Fig. 5 Sequence of events of phage display technology. Biopanning of a phage display library to
select antibody binders to an immobilized target. This cycle is usually repeated 3–4 times and
includes binding of the phage library to an antigen-coated surface, washing with the desired
stringency, elution of phages containing the antibody genes, amplification, and analysis. Reprinted
from Huang et al. [111], with permission from ASM

Phage display of antibody libraries has become a powerful method for a


fast selection of antibodies for therapy. It was used to select synthetic
human libraries, allowing fully human antibodies to be created in vitro
[112]. One of the most successful antibodies discovered by phage display
was adalimumab (HUMIRA), an antibody with affinity to human TNF-α,
the world’s first fully human antibody [113]. Nevertheless, phage display
also presents some drawbacks, as it cannot accommodate all antibody
formats and often requires reformatting to produce soluble and well-
expressed antibodies with properties compatible with efficient
manufacturing. Also, in most of the cases, antigens are not presented in
their native conformations, which decreases the likelihood of selecting
successful leads for therapeutic uses [114].

2.2.2 Ribosome and mRNA Display


In ribosome and mRNA display, a DNA library is transcribed and
transduced in vitro. Without the need to transform cells for library selection,
it is possible to achieve much higher library diversity. While in ribosome
display, the translated protein remains connected to the ribosome and to its
encoding mRNA for the selection step, in mRNA display, the mRNA is first
translated and then covalently bound to the protein it encodes, using
puromycin as an adaptor molecule. Also, a mutagenesis-based PCR can
introduce random mutations after each selection round, allowing
combination of selection with affinity maturation [115, 116].

2.2.3 Yeast Cell Display


In recent years, several cell-based screening technologies have emerged.
They rely on the multi-copy display of antibodies or antibody fragments on
a cell surface in a functional form followed by high-throughput screening.
Cell-based screening harbors the benefit of single-cell analysis and
characterization of individual library candidates [117]. The most common
method used for cell-based screening is flow cytometry, which allows the
analysis of hundreds of thousands of cells per minute according to their
size, granularity, and fluorescence properties. Selection of monoclonal
antibodies presented on the cell surface can be performed by antibody
expression level, by display strength, or by antigen affinity [118].
The yeast display technique was first described by the group of Wittrup,
where a protein of interest was displayed in fusion to a cell wall protein of
Saccharomyces cerevisiae [119]. Although not suitable for large libraries,
due to limitations on yeast transformation which limit the complexity of
these libraries [120, 121], yeast display has still the advantageous of linking
an eukaryotic secretory pathway, with the potential for the selection of
antibodies with improved folding characteristics, to the flow cytometry
system, for a high-throughput screening, making it still widely used. It is a
very effective method for the isolation of high-affinity antibodies against
labeled targets and provides the possibility to discriminate between
different affinities of distinct clones [122].

2.2.4 Mammalian Cell Display


Despite the clear advantages, all of the above platforms share the downside
of expressing antibodies in a nonnatural environment. As large and complex
proteins that possess several functional domains, antibodies can only be
efficiently expressed and assembled into functional forms during the
mammalian expression and secretory pathway. The display of antibodies on
mammalian cell surface often requires the fusion of a transmembrane
domain to the C-terminus. The ability to identify antibodies directly from
mammalian cells therefore enables selection of antibodies with desirable
properties from an early stage, such as high-level expression and stability
[123, 124]. Several groups have developed different mammalian display
strategies, from directly isolation of B cells specific for an antigen to more
complex approaches, as introduction of combinatory libraries through
lentivirus infection [125, 126]. Most of these systems, however, are
complex and time-consuming, which significantly hamper the application
of mammalian display [127].

2.2.5 In Vitro Compartmentalization


A next generation technology, called in vitro compartmentalization, intends
to substitute cells by water droplets for antibody production and display.
The water droplets are surrounded by an oil phase and contain all
components required for protein synthesis, as well as single genes that are
competent for transcription and translation. This system allows the co-
compartmentalization of DNA and protein, and this cell-like droplets (up to
1010 per ml) can therefore be used in flow cytometry-based selections of
large libraries, with a high degree of control over selection conditions and
stringencies [128, 129].
A multitude of discovery technologies are nowadays available to isolate
high-affinity antibody binders to nearly any given target. Promising
strategies are also emerging to improve the discovery process (Fig. 6). The
biggest challenge remains on how to include other important features into
the screening process as, for example, high folding and expression, low
tendency to aggregate, and correct glycosylation patterns. Various
innovative approaches are expected to appear in next years that will include
as many parameters as possible and, certainly, will increase the likelihood
of antibody molecules to successfully reach the market [117].
Fig. 6 General antibody discovery process and the emerging technologies. The most common
antibody discovery process starts with animal immunization with the target antigen. The variable
genes are isolated from the antibody-producing B cells of positive responders, and polyclonal
libraries are constructed and packaged into in vitro display technologies. The displayed libraries are
then selected and screened for binding to the target antigen. Positive binders are sequenced, recloned,
and further characterized in functional assays. Improvements in the discovery process include DNA
vaccines and virus-like particles (VLPs) to enhance immunogenicity, alternative animals to increase
repertoire diversity, fully synthetic libraries to eliminate the need for immunized animals, mammalian
cell display for a native presentation, high-throughput technologies to decrease discovery time, and
autocrine-based selection systems to bypass the target-binding process and directly select for
antibodies with a defined functional phenotype. Reprinted from Kennedy et al. [130], with
permission from Taylor & Francis publisher

2.3 Maturation Strategies for Directed Evolution


One of the greatest challenges in library generation when compared to in
vivo methods is the absence of somatic hypermutation (SHM), which
enables nature to create high-affinity antibodies. The in vitro occurrence of
spontaneous mutations is generally insufficient to obtain desired gene
variants on a practical time scale. Therefore, several genetic diversification
techniques can be used to direct and evolve antibody libraries toward a
defined goal, thereby accelerating the identification of desired proteins.
This bottleneck process is named directed evolution and can be applied in
vitro with the purpose of mimicking the process of natural selection [131,
132]. A single gene or a library is evolved by being subjected to
mutagenesis, screening, and amplification. Rounds of these steps are
repeated, using the best variants from one round as the templates for the
next round, in order to reach the best improvements [133].
Some methods based on introducing a certain degree of diversity into
selected, moderate affinity candidates are used and can be generally
differentiated between targeted and nontargeted diversification strategies
[134]. Nontargeted approaches, such as error-prone PCR and the use of a
mutator E. coli strain, can be used for random introduction of nucleotides
into the whole antibody. Error-prone PCR is based on the use of low-
fidelity polymerases as well as on modified reaction conditions, creating a
high error rate during amplification [135]. E. coli mutator strains are
conditional mutants that produce single-base substitutions with higher rates
than normal cells [136]. Chain shuffling is another method used for
nontargeted diversification, where one of the two antibody chains is
replaced by a repertoire while the other chain is kept constant [137].
In contrast, targeted strategies introduce diversity at predicted positions,
mainly at the ones contributing for antigen binding, the CDRs. CDR
walking, an approach where diversity is introduced in short amino acid
stretches, can provide up to a 420-fold increased affinity [138]. A more
recent approach for targeted modifications relies on programmable
nucleases, which can be easily customized to cleave DNA at almost any
desired site. MAGE, abbreviated from multiplex automated genome
engineering, takes advantage of a template repair where different loci on the
chromosome can be targeted simultaneously, generating a multiplex
genomic mutant library. This is achieved by homologous recombination of
artificial single-stranded oligonucleotides with flanking homology to the
target region. The oligonucleotides hybridize to the complementary exposed
strands during the process of replication [139].
Both target and nontargeted strategies are usually followed by stringent
selections with targeted strategies being more likely to improve affinity and
least likely to create problems with protein stability [87]. Besides, the
majority of mutations are usually deleterious, and so mutated libraries often
have most of the variants with reduced activity. Therefore, a high-
throughput selection and screening is important to find the rare variants
with beneficial mutations that improve the desired properties [140].
Another important feature of directed evolution is the genotype-phenotype
link. When functional antibodies are isolated, it is necessary that their genes
are also collected. In phage display, for example, this is performed by
compartmentalization of gene (inside the phage) and protein (phage
surface). The isolated gene sequences are then amplified by transforming
host bacteria or by PCR. The best pool of sequences are then used for the
next round of mutagenesis, and this repeated cycle can generate protein
variants adapted to the applied selection pressures (Fig. 7) [141].
Fig. 7 Key steps in the cycle of directed evolution. A diverse library of genes is translated into a
corresponding library of gene products and screened for functional variants in a manner that
maintains the correspondence between genotype (gene) and phenotype (protein). These functional
genes are replicated and serve as starting points for subsequent rounds of diversification and
screening or selection

In practical terms it is impossible to cover the entire possibilities of


mutations in a typical protein. The entire randomization of a decapeptide
would yield 1013 unique combinations of amino acids, which exceeds the
library size of all known library creation methods. Nonetheless, directed
evolution represents already a hallmark of protein development and is being
practiced for many academic and industrial purposes. The easy
manipulation of biological systems makes them very good substrates for
engineering and for redesigning new evolutionary approaches [142].

Over the last 40 years, monoclonal antibodies have stood out as


important scientific tools and powerful human therapeutics. Due to
their high specificity to the target, these proteins are extremely
important for innumerous therapeutic applications and are recognized
as a major drug modality in a variety of diseases. Until now, the
leading methodologies for therapeutic antibody generation,
immunization, and display approaches have been effective for the
generation of antibodies against various targets and have led to a
significant number of commercialization approvals.
Despite the moderate success, the common technologies for antibody
discovery are time-consuming and imply complex methods. Today,
there is a need to improve the antibody discovery process, creating
more efficient technologies. There is a need, for example, to avoid the
cost and time-consuming practice of humanization or de-
immunization, which could be surpassed by a system capable of
developing human antibodies. Also, it is a main concern to increase
the size and quality of antibody libraries. The higher the quality and
size of antibody libraries, the higher the likelihood of finding efficient
therapeutic leads. Furthermore, the most widely used display
technique, phage display, does not allow antibody and antigen
molecules to interact in their native conformation. Having the
possibility to select different antibody formats in their native
conditions would also be a big benefit, increasing the chances of
success in further assays. Finally, it would be more efficient if one
could select good candidates for high affinity and, at the same time,
optimized candidates for large-scale production.
Hybridoma technology and phage display have revolutionized the
field of antibody discovery by providing important approaches for the
isolation of monoclonal antibodies. However, the low efficiency of the
hybridoma isolation process and the limitations of the prokaryotic
expression-based technologies have been important technical
drawbacks difficult to overcome [143, 144]. Various in vitro
approaches have been developed in recent years to display full-length
IgG on the surface of eukaryotic cells, with the aim to render
discovery of therapeutic monoclonal antibodies more efficient, with
favorable biophysical properties [122, 123, 126, 145]. Despite the
improvements, these technologies are limited either by the size of the
antibody libraries that can be expressed or by the lack of a stable
genotype to phenotype link. With the increased number of antibodies
reaching the market, there is a need to develop new technologies that
stem from the limitations of current discovery platforms.
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Biochemical Engineering/Biotechnology 171
https://doi.org/10.1007/10_2019_105

Cytokines and Growth Factors


A. C. Silva1, 2 and J. M. Sousa Lobo1
(1) UCIBIO/REQUIMTE, MEDTECH, Laboratory of Pharmaceutical
Technology, Department of Drug Sciences, Faculty of Pharmacy,
University of Porto, Porto, Portugal
(2) FP-ENAS (UFP Energy, Environment and Health Research Unit),
CEBIMED (Biomedical Research Centre), Faculty of Health Sciences,
University Fernando Pessoa, Porto, Portugal

A. C. Silva
Email: [email protected]
Email: [email protected]

Abstract
Several cytokines have been used to treat autoimmune diseases, viral
infections, and cancer and to regenerate the skin. In particular, interferons
(INFs) have been used to treat cancer, hepatitis B and C, and multiple
sclerosis, while interleukins (ILs) and tumor necrosis factors (TNFs) have
been used in the management of different types of cancer. Concerning the
hematopoietic growth factors (HGFs), epoetin has been used for anemia,
whereas the colony-stimulating factors (CSFs) have been used for
neutropenia. Other growth factors have been extensively explored, although
most still need to demonstrate in vivo clinical relevance before reaching the
market.
This chapter provides an overview on the therapeutic applications of
biological medicines containing recombinant cytokines and growth factors
(HGFs and others). From this review, we concluded that the clinical
relevance of recombinant cytokines has been increasing. Since the 1980s,
the European Medicines Agency (EMA) and/or Food and Drug
Administration (FDA) have approved 89 biological medicines containing
recombinant cytokines. Among these, 18 were withdrawn, 24 are
biosimilars, and 18 are orphans.
So far, considerable progress has been made in discovering new
cytokines, additional cytokine functions, and how they interfere with human
diseases. Future prospects include the approval of more biological and
biosimilar medicines for different therapeutic applications.
Graphical Abstract

Keywords Cytokines – Growth factors – Hematopoietic growth factors –


Interferons – Interleukins – Tumor necrosis factors

1 Introduction
Biopharmaceuticals are generally recombinant therapeutic proteins obtained
by biotechnological methods, such as genetic engineering techniques (e.g.,
recombinant DNA technology) that use biological systems (e.g.,
microorganisms, cells, plants, or animals) to produce proteins similar to
those that occur naturally in the body. Sometimes biopharmaceuticals are
called biologicals or biological products, although this is a broader concept
that includes pharmaceutical substances produced or extracted from living
sources. Examples of biological products are vaccines, blood-derived
products, allergenics, somatic cells, gene therapy, and recombinant
therapeutic proteins [1–3].
The clinical use of recombinant proteins requires the manufacture of the
corresponding biological medicine, which is defined by the European
Medicines Agency (EMA) as a medicine containing an active substance
produced by a living organism [4]. In contrast, the Food and Drug
Administration (FDA) includes biological medicines in biological products
that may contain sugars, proteins, nucleic acids, or combinations of these
molecules, living cells, or tissues and are obtained from natural sources or
produced by biotechnology techniques or other innovative technologies [3].
Biological medicines have been used to treat medical conditions that do
not have other treatments or when they are the best therapeutic option for
the treatment of some diseases. Examples of these conditions include
autoimmune, cardiovascular, metabolic, dermatological, neurological
diseases, cancer, and eye and respiratory disorders [5]. Furthermore, several
biological medicines have been granted with the orphan designation, being
used for the treatment of rare life-threatening or chronically debilitating
conditions [6].
The advent of biological medicines brought a new era for therapeutics,
being the first biological medicine approved in the 1980s (recombinant
human insulin). To date, the expiration of some patents led to the
development of biosimilar medicines, which are medicines with the same
quality, safety, and effectiveness of an approved biological medicine, the
reference medicine [7, 8].
Biopharmaceuticals can be divided into different classes, including
monoclonal antibodies, cytokines, growth factors, hormones, blood
products, enzymes, vaccines, cells and tissues, and products of gene
therapy. In this chapter, an overview is given on the therapeutic applications
of biological medicines containing cytokines and growth factors
(hematopoietic growth factors and others).

2 Cytokines
Cytokines display a crucial role in the mediation of immune response,
controlling effector cells activity on the target cells. Immune regulation,
disease pathogenesis, and management of immune-mediated diseases are
among the activities of cytokines [9, 10]. These molecules are generally
proteins or glycoproteins that are secreted by cells in small quantities and
bind to other cell type’s specific receptors, triggering and modelling the
immune responses (Fig. 1). Leukocytes and other cells that interact with
hematopoietic cells produce most cytokines. These mediators are essential
to regulate immune and inflammatory responses, hematopoiesis, and wound
healing. Thereby, the administration of cytokines improves immune
responses against some viral infections and cancers and manages skin
regeneration [2, 10]. Most of these molecules are hydrophilic, being the
formulation of the respective medicines challenging. Notwithstanding, they
have high affinity to the binding receptors, usually are administered in small
doses and show a narrow therapeutic index. So far, several cytokines have
been identified, and some are used in clinics to treat cancer, autoimmune
diseases, and viral infections, for example, the colony-stimulating factors to
the treatment of neutropenia; the interferons to the treatment of viral
infections, neurodegenerative disorders, and cancer; and the interleukins
and tumor necrosis factor to the management of different cancers [11, 12].
In contrast, the overexpression of cytokines can induce diseases.
Accordingly, cytokine antagonists (e.g., cytokine-binding receptors, such as
monoclonal antibodies) have been used in clinic [10], but these products are
out of the scope of this chapter.

Fig. 1 Schematic representation of the general activity of cytokines

Table 1 shows examples of the different clinical applications of


interferons (INFs), interleukins (ILs), and tumor necrosis factors (TNFs)
and corresponding biological medicines.
Table 1 Examples of interferons (INFs), interleukins (ILs), and tumor necrosis factors (TNFs),
therapeutic indications, and marketed biological medicines

Recombinant Therapeutic indications Marketed medicines References


cytokine
Recombinant Therapeutic indications Marketed medicines References
cytokine
INF-α Various cancers; chronic hepatitis B Intron A®/Alfatronol®, [2, 11–23]
and C
Virtron®/Viraferon® a, Roferon
A®, Infergen® a, Rebetron®,
Pegasys®, PegIntron®,
Sylatron®, Alferon N®
INF-β Multiple sclerosis Betaferon®, Betaseron®, [2, 11–15,
24–28]
Avonex®, Rebif®, Plegridy®,
Extavia®
INF-γ Chronic granulomatous disease; Actimmune® [2, 11, 14,
osteopetrosis 15, 29]
IL-1 receptor Rheumatoid arthritis; Schnitzler’s Kineret® [2, 15, 30–
antagonist syndrome; Familial Mediterranean 32]
(anakinra) fever; mevalonate kinase
deficiency; TNF receptor-associated
periodic syndrome
IL-2 Renal carcinoma; melanoma Proleukin® [11, 12, 15,
(aldesleukin) 33]
IL-2 Cutaneous T-cell lymphoma Ontak® a [11, 15, 34]
(denileukin
diftitox)
IL-3 – Acute myeloid leukemia Orphan medicine [35]
diphtheria
toxin
IL-7 Progressive multifocal Orphan medicine [36]
leukoencephalopathy
IL-7 – Idiopathic CD4 lymphocytopenia Orphan medicine [37]
antibody
Pegylated IL- Pancreatic cancer Orphan medicine [38]
10
IL-11 Prevention of chemotherapy- Neumega® [2, 11, 15]
(oprelvekin) induced thrombocytopenia;
avoidance of platelet transfusions
after chemotherapy
IL-12 Acute radiation syndrome Orphan medicine [39]
TNF-α-1a Adjunct for surgery tumor removal; Beromun® [2, 12, 40]
(tasonermin) palliative care after irresectable soft
tissue sarcoma of the limbs
Recombinant Therapeutic indications Marketed medicines References
cytokine
NGR-human Malignant mesothelioma Orphan medicine (Zafiride®)a [41, 42]
TNF
Hepatocellular carcinoma Orphan medicine [43]

IL interleukin, INF-α interferon alpha, INF-β interferon beta, INF-γ


interferon gamma, NGR-human TNF human TNF coupled to cngrcg
peptide, TNF tumor necrosis factor
aMedicine withdrawn

2.1 Interferons (INFs)


Different cell types, showing diverse biological effects, including cellular
antiviral states, regulation of immunological and inflammatory responses,
cell growth processes, and apoptosis, produce INFs. Regarding these
activities, INFs have been used in clinical practice to promote immune
responses against infections and to treat autoimmune disorders and different
types of cancer [2, 15].
INF-α, INF-β, and INF-γ comprise the three human classes of INFs,
where INF-α and INF-β have similar amino acid sequences and bind to the
same cell receptors, originating identical biological activities, particularly,
against viral infections and antiproliferation of tumor cells. In contrast,
INF-γ has a distinct amino acid sequence and binds to different cell
receptors, having an activity related to anti-inflammatory and
immunological responses. Owing to their different biological effects, INF-α
and INF-β are also named type I interferons, and INF-γ is classified as type
II interferon. Type I has 12 subtypes or isoforms of INF-α and 1 subtype of
INF-β, while type II is only the INF-γ. Current INFs used in clinical
practice are recombinant, being typically produced in E. coli, since they do
not require posttranslational modifications to display the biological activity,
although some INF-β have been produced in CHO (Chinese hamster ovary)
cells [2, 15].
Concerning therapeutic applications of INFs, INF-α subtypes are the
most used. For example, INF-α-2a (Roferon A®) has been used to treat
hairy cell leukemia, Kaposi’s sarcoma, chronic myelogenous leukemia,
cutaneous T-cell lymphoma, chronic hepatitis B and C, follicular
lymphoma, renal cancer, and malignant melanoma [20]. INF-α-2b (Intron
A®/Alfatronol®) has been indicated to the treatment of multiple myeloma,
chronic myelogenous leukemia, chronic hepatitis B and C, carcinoid tumor,
hairy cell leukemia, follicular lymphoma, malignant melanoma,
condylomata acuminate, and Kaposi’s sarcoma [16]. IFN-α-n3 (Alferon
N®) has been used to treat condylomata acuminate [23]. Regarding other
INFs types, INF-β-1b (Betaferon®, Betaseron®, and Extavia®) and INF-
β-1a (Avonex®, Rebif®, and Plegridy®) have been used to the management
of multiple sclerosis [24–28], while INF-γ-1b (Actimmune®) is indicated to
the treatment of chronic granulomatous disease and osteopetrosis [29].
Furthermore, the use of recombinant INF-γ has been suggested to improve
atopic dermatitis treatment in patients with predisposition for skin
infections [44]. Pegylated INFs (i.e., INFs chemically linked to
polyethylene glycol – PEG) have also been marketed (Pegasys®,
PegIntron®, and Sylatron®) to increase molecules half-lives, improving
administration regimens [2, 11, 15, 18, 21, 22]. Table 1 shows examples of
the different clinical applications of INFs and their corresponding marketed
biological medicines.
Depending on the dose regimen, the administration of INFs usually
induces flu-like symptoms, since they promote the production of
endogenous INFs that are also produced during influenza virus infection.
These symptoms are mild and can be relieved with paracetamol.
Nonetheless, there are reports of more severe adverse effects for INF-α and
INF-β [2, 15].
To our knowledge, there are no marketed biosimilar medicines with
recombinant INFs, although some are expected in the near future. Recently,
Mufarrege et al. used of a multiplexed gene expression system, based on a
human monocytic cell line, to characterize the bioactivity of recombinant
INF-β. The researchers suggested the application of this system to compare
recombinant INF-β medicines (Rebif® and Betaseron®) with the bioactivity
of the respective biosimilar candidates [45].

2.2 Interleukins (ILs)


Interleukins (ILs) are a large group of cytokines comprising several
subtypes that are produced by different cells, such as macrophages,
eosinophils, vascular endothelial cells, fibroblasts, and keratinocytes. The
biological activities of ILs are complex and not totally understood and
include the regulation of normal and malignant cells growth and
differentiation and the management of immunological and inflammatory
responses. Similar to INFs, ILs bind to specific receptors on the surface of
neighborhood cells, stimulating the production of more ILs. The medical
use of ILs is limited, due to the lack on the knowledge of all biological
functions. Thereby, only IL-1, IL-2, and IL-11 are approved for clinical use
[2, 15]. Nonetheless, the therapeutic potential of other ILs subtypes has
been studied. For example, the use of IL-4, IL-10, and IL-11 to the
treatment of psoriasis and rheumatoid arthritis has been tested, but the
results of clinical trials were not satisfactory, since patients modestly
improved upon administration of the ILs [46]. Steen-Louws et al. proposed
the use of IL-4–10 fusion protein as an alternative to the administration of
isolated IL4 and IL-10, obtaining a synergistic therapeutic effect [47]. Tang
et al. performed a clinical study using a recombinant fusion protein
consisting of two IL-22 molecules linked to an immunoglobulin constant
region, which promotes tissue repair and suppresses bacterial infection. The
results showed that the tested recombinant fusion protein was well tolerated
and can be used to treat inflammatory diseases and organ failure, such as
alcoholic hepatitis [48, 49].
The efficacy of ILs as adjuvants for cancer immunotherapy has been
demonstrated in clinical trials. Naing et al. reported promising results from
a phase I trial using pegylated recombinant IL-10 to treat advanced solid
tumors, suggesting its potential for cancer immunotherapy [50, 51]. The use
of IL-12 to the treatment of melanoma and cutaneous T-cell lymphoma was
tested, being observed the occurrence of antitumor activity and undesired
toxicity effects [46]. Currently, the National Cancer Institute (United Sates)
is carrying out a phase I clinical trial to evaluate the efficacy of a combined
therapy with monoclonal antibody (pembrolizumab) and recombinant IL-12
to treat solid tumors. Herein, the therapeutic interest of using recombinant
IL-12 is related with its ability to destroy tumors, by obstruction of the
blood flow to the tumor and stimulation of the white blood cells that kill
tumor cells [52]. Coyne et al. carried out a clinical trial where it was
observed that the use of immune checkpoint inhibitors combined with
recombinant IL-15 (which promote the production of CD8+T-cells, natural
killer cells, and inflammatory cytokines) improved the antitumor immune
responses [53]. In other study, Miller et al. observed the clinical efficacy of
subcutaneous recombinant IL-15 to the treatment of refractory solid tumors
in cancer patients [54]. Francois et al. carried out a clinical trial using
recombinant IL-7 in oncologic and lymphopenic patients and observed an
increase on the CD4+ and CD8+ lymphocytes levels. From their findings,
these researchers proposed the use of recombinant IL-7 for the treatment of
sepsis [55].
Table 1 shows examples of the different clinical applications of ILs and
corresponding biological medicines. From our research, no commercially
available biosimilars containing recombinant ILs were found.
The two subtypes of IL-1 (IL-1α and IL-1β) bind to the same receptors
and induce similar biological activities. Among these, the main function is
the pro-inflammatory activity that originates the production of
inflammatory mediators. Furthermore, it has been described that IL-1 plays
a role in the activation of T-lymphocytes and hematopoietic cells
differentiation and growth. Besides, together with IL-6, IL-1 induces
hepatocytes acute-phase proteins. The extension of these effects depends on
the amount of IL-1 produced, being local for low quantities and systemic
for high concentrations. Clinical investigations related with the potential of
using IL-1 for treating several cancers and chemotherapy-induced bone
marrow suppression have been carried out, but the results were not
satisfactory, since no significant antitumor activity and toxic side effects
were observed. However, regarding the ability to mediate acute and chronic
inflammation, the modulation of IL-1 levels has been showing interesting
clinical results, in particular using IL-1 receptor antagonists. In this field,
there is one marketed medicine, anakinra (Kineret®), a non-glycosylated
recombinant IL-1 receptor produced in E. coli., which was first approved to
treat rheumatoid arthritis [2, 15]. Later, this medicine was approved to treat
periodic fevers and auto-inflammatory disorders at all ages, being the first-
line treatment for Schnitzler’s syndrome and second-line treatment for
Familial Mediterranean fever, mevalonate kinase deficiency, and TNF
receptor-associated periodic syndrome [30]. Moreover, anakinra is used in
combination with tocilizumab to the treatment of adult-onset Still’s disease
refractory to second-line therapy [31]. Thomas et al. are conducting a
clinical trial to assess whether the use of anakinra in combination with
corticosteroids improves outcomes in patients with acute severe ulcerative
colitis [56]. Recently, Zhu et al. performed in vivo experiments where it
was observed that the use of recombinant IL-1 provides a protective role
against myocardial ischemia-reperfusion injury in rats, suggesting its
potential for the treatment of this disorder [57].
IL-2 is the most studied type of ILs, being the first identified T-growth
factor and having a crucial role in immune response activation, tolerance,
and memory induction. IL-2 is produced by T-lymphocytes and stimulates
natural killer cells and antibody production by B-lymphocytes. The clinical
relevance of IL-2 is related with its immunostimulatory activity that
promotes body antitumor responses [2, 15]. Two medicines based in IL-2
have been approved, despite only one is currently marketed. Aldesleukin
(Proleukin®) is a recombinant IL-2 approved to treat renal carcinoma and
melanoma [33]; denileukin diftitox (Ontak®) is a recombinant engineered
fusion protein composed by diphtheria toxin fragments linked to IL-2,
which targets T cells and was used to the treatment of cutaneous T-cell
lymphoma, being discontinued by FDA in 2014 [11, 15, 34].
The use of high IL-2 concentrations originates cardiovascular, hepatic,
and pulmonary adverse effects, which limits the duration of treatments.
Besides, some adverse effects can be induced indirectly, promoting the
synthesis of other cytokines [2, 58]. Some recent works referred the
potential of using IL-2 to the management of other disorders. For example,
Humrich and Riemekasten showed that a low-dose of IL-2 is well tolerated
and influences positively the clinical course of patients with active systemic
lupus erythematosus. Nonetheless, phase II clinical trials are in progress to
confirm these preliminary results [58]. In other study, Zhang et al. proposed
the use of recombinant IL-2 as an adjunctive immunotherapeutic agent to
treat tuberculosis. The results of the performed experiments suggested that
the use of IL-2 increases the proliferation of CD4+ and natural killer cells
and improves sputum culture and smear conversion in patients with
pulmonary tuberculosis. Nonetheless, the real clinical efficacy of this use
needs to be demonstrated [59].
When activated by IL-1, bone marrow stromal cells and fibroblasts
produce IL-11. The latter acts as a hematopoietic growth factor (Sect. 3.1),
stimulating thrombopoiesis (i.e., platelet production) and the growth and
differentiation of bone marrow cells. Recombinant IL-11, called oprelvekin
(Neumega®), is produced in E.coli and indicated to the prevention of severe
thrombocytopenia and avoidance of platelet transfusions after
chemotherapy. Despite IL-11 is well tolerated, some adverse effects have
been reported, including edema, tachycardia/palpitations, dyspnea, and oral
moniliasis [2, 11, 15]. Kuo-Ming et al. demonstrated that the use of
pegylated recombinant IL-11 did not originate additional toxicities in
primates, when compared to the recombinant IL-11 alone. Therefore, these
researchers proposed the use of pegylated recombinant IL-11 as a safe long-
acting treatment, originating less fluid retention, which is most common
adverse effect [60].
EMA approved the market of some recombinant ILs as orphan drugs,
for example, pegylated IL-10 (in 2017) for pancreatic cancer [38]; IL-12 (in
2016) for acute radiation syndrome [39]; IL-7 (in 2014) for progressive
multifocal leukoencephalopathy [36]; IL-7 fused with antibody (in 2017)
for idiopathic CD4 lymphocytopenia [37]; and IL-3 fused with diphtheria
toxin (in 2015) for acute myeloid leukemia [35].

2.3 Tumor Necrosis Factors (TNFs)


Tumor necrosis factors (TNFs) comprise the subtypes α and β that induce
similar biological effects. Among these, the most studied is the TNF-α, also
named TNF, cachectin, macrophage cytotoxic factor, macrophage
cytotoxin, or necrosin. This cytokine is synthetized by activated
macrophages, although several cell types can produce it, for example,
natural killer cells, eosinophils, Kupffer cells, glomerular mesangial cells,
fibroblasts, B- and T-lymphocytes, polymorphonuclear leukocytes,
astrocytes, Langerhans cells, and brain microglial cells. The biological
effects of TNFs include immunological activation in response to the
presence of microorganisms (e.g., Gram-negative bacteria), regulation of
inflammation, necrosis of some tumor cells types, and mediation of several
disorders (e.g., septic shock, cachexia, and anorexia) [2, 61].
Table 1 shows examples of the different clinical applications of TNFs
and corresponding biological medicines. From our research, no
commercially available biosimilars containing recombinant TNFs were
found. The first clinical attractiveness of TNF-α was for cancer therapy.
Nonetheless, it has been observed that some tumors are not susceptible to
TNF, due to a reduced necrosis activity and occurrence of adverse effects
upon the administration of therapeutic doses. In contrast, some clinical
studies focused in the neutralization of the negative outcomes originated by
the overexpression of TNF. Examples of these adverse effects are induced
cachexia and tumor growth stimulation in cancer patients; tissue necrosis
and vascular leakage in patients with septic shock; inflammation in patients
with rheumatoid arthritis; and diabetes by induction of insulin resistance
after pancreatic cells death. The administration of anti-TNF monoclonal
antibodies or anti-TNF receptor reduces the severity of these effects.
Regarding the direct use of TNF in therapy, only one product (tasonermin –
Beromun®) is approved [2]. This medicine contains recombinant TNF-α-1a
that has been produced in E. coli and is indicated as adjunct for surgery
tumor removal, to prevent or delay amputation, or in a palliative situation,
for irresectable soft tissue sarcoma of the limbs in combination with
melphalan [2, 12, 40]. In 2008, EMA granted the orphan designation to
NGR-human TNF (human TNF coupled to cngrcg peptide) for the
treatment of malignant mesothelioma [42], being the respective medicine
(Zafiride®) withdrawn in 2017 [41]. Latter, in 2009, EMA approved NGR-
human TNF as an orphan medicine to the treatment of hepatocellular
carcinoma [43].
Li et al. reported the results of a retrospective trial where it was
observed that the intrapleural instillation of recombinant TNF-α controlled
the malignant pleural effusion and minimized invasive intervention in a
cohort group of lung cancer patients. However, more trials are required to
define the optimal therapeutic dose and confirm clinical efficacy [62].

3 Growth Factors
3.1 Hematopoietic Growth Factors (HGFs)
Growth factors are cytokines that stimulate cell proliferation,
differentiation, and/or activation. Among these, hematopoietic growth
factors (HGFs), which are glycoproteins that regulate the production and
maturation of blood cells (i.e., hematopoiesis), have been showing high
therapeutic potential [2, 63, 64].
HGFs with clinical relevance have been produced by DNA recombinant
techniques and include IL-11 (Sect. 2.2), recombinant erythropoietin or
epoetin and darbepoetin alfa, and the white cells factors: granulocyte
colony-stimulating factor (G-CSF) and granulocyte macrophage colony-
stimulating factor (GM-CSF) [2, 63, 65].
Erythropoietin is an uncharacteristic cytokine that acts as an endocrine
hormone and is produced by the kidneys, although the liver synthetizes a
small amount. Its main function is the stimulation and regulation of the
production of red blood cells (i.e., erythropoiesis), by a mechanism that
increases the number of cells able to differentiate in erythrocytes and
promotes the migration of mature red blood cells from the bone marrow to
the peripheral circulation. In addition, anemia-related tissue hypoxia
induces erythropoietin production by the kidneys. Besides, erythropoietin
improves body resistance to exercise and well-being and reduces the need
of blood transfusions in anemic patients. This hormone is present in low
concentrations in the urine of anemic patients, being initially isolated from
there for clinical use. Nonetheless, due to the small amount available by this
method, the erythropoietin currently used in therapy is produced by DNA
recombinant technique, in mammalian cells (e.g., CHO), being called
epoetin [2, 63].
The white cell factors, granulocyte colony-stimulating factor (G-CSF)
and granulocyte macrophage colony-stimulating factor (GM-CSF), play a
major role in the differentiation of neutrophils from hematopoietic stem
cells. G-CSF is a glycoprotein synthetized by different cells (bone marrow
stromal cells, macrophages, and fibroblasts) and has a biological activity
related to the proliferation of neutrophils and further activation of mature
cells (i.e., gain of specific functions). Moreover, G-CSF promotes the
proliferation and migration of endothelial cells and, when associated with
other HGFs, has a synergic effect on the differentiation of various
hematopoietic cells. In contrast, GM-CSF is a glycosylated polypeptide
produced by several cells (macrophages, T-lymphocytes, fibroblasts, and
endothelial cells), which induces the proliferation of neutrophils,
macrophages, eosinophils, erythrocytes, and megakaryocytes [2]. G-CSF
and GM-CSF have been used to treat neutropenia. Furthermore, they show
therapeutic effect against infections and some cancers and in the
management of bone marrow transplants. The approved G-CSF and GM-
CSF medicines are marketed under several trade names and are usually
produced in E. coli. Recombinant G-CSF include filgrastim, pegfilgrastim
(filgrastim linked to a PEG molecule), and lenograstim, while recombinant
GM-CSF comprise molgramostim and sargramostim [2, 63].
Table 2 shows examples of the different clinical applications of HGFs
and corresponding biological and biosimilar medicines.
Table 2 Examples of hematopoietic growth factors (HGFs), therapeutic indications, and marketed
biological and biosimilar medicines
Recombinant Therapeutic indications Marketed medicines References
HGFs
Epoetin alfa Anemia caused by several disorders Epogen®/Procrit®, [2, 8, 63,
66–69]
Eprex®/Erypo®,
Epoetin Alfa Hexal® a,
Abseamed® a,
Binocrit® a
Epoetin beta Anemia originated by chronic renal failure NeoRecormon®, [2, 63, 70–
or post-chemotherapy nonmyeloid 72]
malignancies Mircera®
Epoetin theta Eporatio®, Biopin® [73, 74]

Epoetin zeta Anemia caused by several disorders Silapo® a, Retacrit® a [8, 75–77]

Darbepoetin Anemia originated by renal failure or Aranesp®, Nespo® b [2, 63, 78–
alfa myelosuppressive chemotherapy 80]
G-CSF Neutropenia and avoidance of febrile Neupogen®, [2, 63, 81–
(granulocyte neutropenia in patients receiving 92]
colony- myelosuppressive chemotherapy, or Tevagrastim® a,
stimulating patients undergoing myeloablative Zarzio® a,
factor) or chemotherapy followed by bone marrow Ratiograstim® a,
filgrastim transplantation; reduction of severe
neutropenia effects in patients with Grastofil® a, Nivestim®
congenital, cyclic, or idiopathic a, Nivestym® a,
neutropenia
Accofil® a, Filgrastim
Hexal® a, Biograstim®
a,b, Filgrastim
Ratiopharm® a,b
G-CSF Amyotrophic lateral sclerosis Orphan medicine [93]
(granulocyte
Spinal cord injury [94]
colony-
stimulating
factor) or
filgrastim

N l t ® L ®
G-CSF Neutropenia Neulasta®, Lonquex®,
Recombinant Therapeuticand avoidance of febrile
indications Marketed medicines [2, 63, 95–
References
(granulocyte
HGFs neutropenia in patients receiving Granix®, Ziextenzo® a, 108]
colony- myelosuppressive chemotherapy, or Pelgraz® a, Pelmeg® a,
stimulating patients undergoing myeloablative
factor) or chemotherapy followed by bone marrow Udenyca® a, Fulphila®
pegfilgrastim transplantation; reduction of severe a, Ristempa® a,b,
neutropenia effects in patients with
Neupopeg® a,b,
congenital, cyclic, or idiopathic
neutropenia; increased drug residence time Efgratin® a,b, Cavoley®
in the body a,b

GM-CSF Acute respiratory distress syndrome Orphan medicine [63, 109]


(granulocyte
macrophage
colony-
stimulating
factor) or
molgramostim
GM-CSF Hematopoietic syndrome of acute radiation Leukine® [2, 63, 110]
(granulocyte syndrome
macrophage
colony-
stimulating
factor) or
sargramostim

aBiosimilarmedicine
bMedicine withdrawn

FDA approved the first recombinant erythropoietin in 1989


(Epogen®/Procrit® – epoetin alfa) to treat anemia in patients with chronic
renal failure that are unable to produce this hormone due to the loss of renal
function. Later, the clinical use of epoetin was extended to treat or avoid
anemia caused by other disorders, for example, to improve the production
of red blood cells in patients undergoing myelosuppressive chemotherapy
and bone marrow transplantation and those undertaking therapy for viral
infections, such as human immunodeficiency virus (HIV) and hepatitis C.
However, some adverse effects have been associated with the use of
Epogen®/Procrit®, including cardiovascular events, hypertension, seizures,
stroke, thrombosis or thromboembolism, tumor recurrence or progression,
and severe anemia [2, 63, 65, 69]. Later, several subtypes of epoetin were
approved for similar therapeutic indications. For example, epoetin beta
(NeoRecormon® and Mircera®) [70–72] and epoetin theta (Eporatio® and
Biopin®) [73, 74] were approved to treat symptomatic anemia originated by
chronic renal failure or post-chemotherapy nonmyeloid malignancies. Two
medicines containing darbepoetin alfa (Aranesp®, Nespo®) were approved
to treat anemia originated by renal failure or myelosuppressive
chemotherapy, although Nespo® was withdrawn in 2009 [78–80].
With the expiration of epoetin patents, some biosimilar medicines have
been developed and more are expected for the next years. Current approved
epoetin biosimilars are based in the reference medicine Epogen®/Procrit®
and include alfa epoetin (Alfa Hexal®, Abseamed®, and Binocrit®) and zeta
epoetin (Retacrit® and Silapo®). Among these, Retacrit® is the unique
approved by FDA, being all approved by EMA. Despite the cost reduction
of using biosimilar epoetin, some clinicians have been showing skepticism
regarding its similar therapeutic efficacy and still prescribe the reference
medicine [8, 111].
The most common indications of epoetin biosimilar medicines include
the treatment of anemia caused by several disorders, such as chronic renal
failure or kidney problems, chemotherapy treatments (reducing the need of
allogenic blood transfusions), and defective production of blood cells. In
addition, epoetin biosimilars are used to regulate normal blood levels before
surgery in patients that undergo autologous blood transfusion and to reduce
the need of allogenic blood transfusions in patients with moderate anemia
that undergo major surgery (e.g., hip or knee surgery) [66, 75, 76]. Epoetin
Alfa Hexal® and Silapo® have also been used in patients with risk of
developing acute myeloid leukemia that show low levels of erythropoietin
[66, 75]. Regarding Retacrit®, it has been used for the same medical
indications of the other epoetin biosimilars, and FDA also approved its use
for the treatment of anemia in HIV patients that have been treated with the
antiviral zidovudine [76, 77].
Filgrastim (Neupogen®) was the first recombinant G-CSF approved by
FDA, in 1991, to reduce neutropenia and avoid febrile neutropenia in
patients receiving myelosuppressive chemotherapy, except the ones with
chronic myeloid leukemia and myelodysplastic syndrome. This medicine
has also been used to reduce neutropenia period in patients undergoing
myeloablative chemotherapy followed by bone marrow transplantation,
which show high risk of prolonged severe neutropenia. Furthermore,
filgrastim reduces the incidence of severe neutropenia effects (e.g., fever‚
infections, and oropharyngeal ulcers) in patients with congenital, cyclic, or
idiopathic neutropenia. Later, in 2015, Neupogen® was indicated to treat
patients acutely exposed to myelosuppressive doses of radiation, designated
by hematopoietic syndrome of acute radiation syndrome, promoting the
recovery of neutrophils production by bone marrow cells and improving
body immunity. Nonetheless, the use of filgrastim has been associated with
the occurrence of some adverse effects, such as fever, pain, rash, headache,
cough, breath difficulty, and nose bleeding. In 2015, FDA authorized
Zarxio® to the same indications of Neupogen® [90, 91]. More recently,
FDA approved sargramostim (Leukine®), which is the recombinant form of
GM-CSF, to increase the survival rate of patients showing hematopoietic
syndrome of acute radiation syndrome [110].
EMA approved the use of pegfilgrastim since 2002, by means of
Neulasta® (pegfilgrastim) and Lonquex® (lipegfilgrastim), to the same
therapeutic indications of filgrastim. The PEG linkage to filgrastim
molecule increases the body circulation time of the drug, avoiding fast
elimination and, therefore, reducing the number of required administrations
[106, 107].
Two orphan designations have been attributed to filgrastim: treatment of
amyotrophic lateral sclerosis, protecting nerve cells from damages [93], and
reducing the effects of spinal cord injury, due to the capacity for protecting
spinal cord cells from death [94]. Molgramostim, a recombinant form of
GM-CSF, is approved to treat acute respiratory distress syndrome, due to
the ability to repair cells and eliminate microbes from the lungs, improving
the oxygen flow into the blood [109].
To date, there are eight approved filgrastim biosimilars from the
reference Neupogen® [112]: Filgrastim Hexal® [88], Accofil® [87],
Nivestim® [86], Nivestym® [92], Grastofil® [85], Ratiograstim® [84],
Zarzio® [83], and Tevagrastim® [82]. Concerning pegfilgrastim, there are
five approved biosimilars to the same indications of the reference Neulasta®
[106]: Fulphila® [95], Udenyca® [97], Ziextenzo® [99], Pelgraz® [100], and
Pelmed® [101]. Furthermore, some biosimilars have been withdrawn:
Biograstim® and Filgrastim Ratiopharm® for filgrastim [81, 89] and
Ristempa®, Neupopeg®, Efgratin®, and Cavoley® for pegfilgrastim [102–
105].
The clinical use of G-CSF has been explored in the management of
several disorders. For example, Affentranger et al. reviewed the clinical
trials that investigated the synergic therapeutic effect of using G-CSF
combined with erythropoietin in anemic patients with lower risk of
myelodysplastic syndromes and concluded that an improved efficacy can be
achieved [113]. In other revision, Hamel et al. suggested that the use of G-
CSF can accelerate the white blood cell count in patients with leukopenia
related to kidney transplant, despite more data is required to confirm this
evidence [114]. The benefits of using G-CFS in infertile women, after
embryo implantation, seem to play an important role in the clinical outcome
of assisted reproductive technology [115, 116]. Herrmann et al. carried out a
pilot study and observed that the use of G-CSF improved bone regeneration
in patients with large segmental defect, fracture non-unions, or insufficient
vascularization [117].

3.2 Other Growth Factors


Apart from the HGFs, other growth factors have been applied for different
therapeutic indications. For example, the recombinant human epidermal
growth factor (EGF) is used to treat diabetic foot ulcers (Heberprot-P®,
Regen-D® 150, and Easyef®), vascular ulcers and bed sores (Regen-D®
150) [118], and healing burns and donor site skin grafts (Regen-D® 60)
[119]. The EGF mechanism of action is related to the improvement of
keratinocytes proliferation and increase on the tensile strength of the new
skin. Current EGF-based medicines are topical, for cutaneous
administration or intralesional injection [120, 121]. Nonetheless, in 2018,
EMA released a decision on granting a waiver for all EGF approved
indications [122]. Several researches showed that the topical use of EGF in
individualized formulations (i.e., magistral formulations) is effective in the
management of diverse adult skin lesions, without showing adverse effects.
However, more studies are required to establish clinical protocols for this
application [123]. Furthermore, some products containing EGF for wound
healing are currently under clinical trials, being expected to be marketed
soon [124].
The platelet-derived growth factor (PDGF), in particular the isoform BB
(PDGF-BB), is used in the management of chronic wounds, regulating the
healing process. This growth factor is released by activated platelets at the
site of the damage and is a mitogenic and chemoattractant for mesenchymal
stem cells, promoting the initialization of the tissue repair process. It also
plays a role in angiogenesis [2, 121]. A single medicine containing human
recombinant PDGF-BB was approved for the treatment of chronic diabetic
ulcers (Regranex®) but was withdrawn by EMA [125, 126]. FDA approved
an injectable containing PDGF-BB (Augment®) for arthrodesis and ankle
hindfoot in patients that need supplemental graft material, being an
alternative to autografts [127]. In this sense, Sun et al. conducted a meta-
analysis to evaluate the efficacy of using recombinant PDGF-BB in
comparison to autologous bone grafts, in ankle and foot fusion patients.
From their study, the authors concluded that the use of this growth factor
avoids the problems associated with the autografts procedures (e.g., pain,
scarring, blood loss, and extra surgery time), although more studies are
required to confirm these evidences [128]. Human recombinant PDGF-BB
is used in dental therapy to treat periodontal defects, by means of an
osteoconductive matrix enriched with this growth factor (GEM 21S®)
[129].
Similar to PDGF-BB, the fibroblast growth factor 2 (FGF-2) or basic
FGF (bFGF) shows ability to improve wound healing, stimulating the
proliferation of epithelial and mesenchymal cells and promoting
neovascularization. Furthermore, the use of bFGF in the recovery of skin
burns is also effective. Fiblast® spray (trafermin) is the first marketed
product containing bFGF, which is indicated to the treatment of skin ulcers,
including leg and burn ulcers. Latter, this medicine was approved for dental
therapy to promote the regeneration of periodontal and bone tissues [121,
130]. EMA attributed the orphan designation to the fibroblast growth factor
19 (FGF-19) for the management of primary biliary cirrhosis, primary
sclerosing cholangitis and avoidance of liver damages, since this protein
decreases the production of bile acids [131, 132]. The keratinocyte growth
factor (KGF), palifermin or FGF-7, belongs to the FGF family. This growth
factor stimulates the epithelial cells proliferation, improving tissue
formation, and was approved by EMA (Kepivance®) for the treatment of
oral mucositis in patients undergoing chemotherapy and radiotherapy, being
withdrawn in 2016 [133]. Some researchers suggest the potential of KGF
for the management of cutaneous wounds, despite human clinical studies
are required to prove this evidence [121].
The vascular endothelial growth factor (VEGF) has been explored to
improve wound healing, since it plays a major role in the angiogenesis
initiation, improving the proliferation and migration of endothelial cells.
Nonetheless, to our knowledge, there are no available products in the
market. In this sense, Hanft et al. carried out randomized controlled trials to
evaluate the efficacy of using human recombinant VEGF in patients with
neuropathic diabetic foot ulcers. These researchers observed that the topical
application of VEGF originated an improved wound healing, reached in a
smaller period, when compared to the placebo [121, 134]. The orphan
designation was granted by EMA to the human recombinant VEFG to treat
amyotrophic lateral sclerosis, due to the ability of this growth factor to
stimulate the development of blood vessels. This medicine is for direct
brain administration, promoting the growth of blood vessels that irrigate
nerve cells, increasing their survival [135]. In contrast, some medicines
containing VEGF inhibitors are available and are used to treat age-related
macular degeneration and different cancers [136].
The placental growth factor (PlGF) is an angiogenic protein that
belongs to the VEGF family, which is highly produced during pregnancy
and is fundamental to the growth of placenta blood vessels. Thereby, PlGF
is fundamental to normal pregnancy and baby’s development and is a useful
clinical indicator to predict adverse outcomes. For example, low levels of
PlGF are associated with the occurrence of preeclampsia [137, 138]. EMA
granted the orphan designation to human recombinant PlGF to restore blood
PlGF levels, improving preeclampsia symptoms [139].
Transforming growth factors (TGFs) are a family of mitogenic
polypeptides that includes the subtype TGF-α, which has an activity similar
to EGF and induces angiogenesis. Some studies suggested that TGF-α
promotes the reepithelialization, but more experiments are required to
confirm this activity [2, 121]. TGF-β is another subtype that has been
showing an important role in the early stages of wound healing, which has
three isoforms (β1, β2, and β3). Isoforms β1 and β2 promote fibroblast
differentiation, contraction, synthesis of the extracellular matrix, and
scarring, while isoform β3 reduces scar formation [121, 140]. So et al.
carried out successful phase I and II clinical trials with human recombinant
TGF-β3 (avotermin), where observed a significant scarring reduction [141].
However, this product failed phase III trials and is not marketed [121].
Insulin-like growth factors (IGFs) family includes the subtypes I and II,
which are structurally similar to proinsulin. Thereby, the administration of
IGF decreases the levels of insulin and glucagon and increases the cellular
glucose uptake. Diverse activities have been attributed to IGFs, regarding
their ability to inhibit apoptosis and regulate cells growth and differentiation
[2]. Accordingly, high circulating levels of IGF-I have been associated with
the development and progression of different cancers [142], including
breast [143], prostate [144], and thyroid [145] cancers. In 2005, FDA
approved the use of IGF-I (mecasermin – Increlex®) to treat growth failure
in children with severe IGF-I deficiency or with growth hormone
insensitivity syndrome (alterations of the growth hormone gene that unable
the use of this hormone by the body). However, IGF-I should not be used
instead of growth hormone [146]. IGF-I was also approved by FDA to treat
amyotrophic lateral sclerosis [147], and EMA granted the orphan
designation to IGF-I for different therapeutic indications. For example, the
recombinant human IGF-I/insulin-like growth factor-binding protein-3
(IGF-I/IGFBP-3) was approved to treat type A [148] and B [149] extreme
resistance insulin syndrome, inherited extreme insulin resistance (or Rabson
Mendenhall syndrome) [150], primary growth hormone insensitivity
syndrome (or Laron syndrome) [151], and leprechaunism [152].
Nonetheless, all these medicines are withdrawn.
Neurotrophic factors are a large group of molecules that play an
important role in the development and survival of nerve cells [2]. Among
these, the nerve growth factor (NGF) has been the most studied, since it
regulates the development and survival of specific peripheral neurons and
basal forebrain cholinergic nuclei. Furthermore, NGF levels are high in
several anti-inflammatory and autoimmune disorders (e.g., chronic arthritis,
systemic lupus erythematosus, and multiple sclerosis), and a relation with
diabetic pathology was observed. Thereby, NGF constitutes a multifactorial
modulator of neuro, immune, and endocrine systems [153].
Regarding its ability to regulate the growth and survival of retina and
cornea cells, in 2013 EMA granted the orphan designation to NGF to treat
retinitis pigmentosa. This growth factor improves the survival of retina
cells, slowing the progression of the disease and preserving vision [154].
Latter, in 2015, EMA also attributed the orphan designation to NGF to treat
neurotrophic keratitis. The use of NGF improves the eye normal healing
process and repairs the damages to the cornea, which are typical of this
disorder [155]. In 2018, FDA approved the medicine Oxervate® containing
the recombinant human NGF (cenegermin) to treat neurotrophic keratitis
[156]. The use of NGF to improve the recovery of other ocular degenerative
diseases has been suggested. For example, Mesentier-Louro et al. observed
that the ocular application of NGF reduces the retinal ganglion cell
degeneration that occurs after optic nerve crush in adult rats, suggesting the
potential of this growth factor to treat optical neuropathies, such as
glaucoma [157]. Other researches have investigated the use of NGF for
different clinical applications. For example, Sacchetti et al. carried out a
phase IIa prospective, open label and multiple-dose clinical trial in 40
patients with dry eye disease, which received eye drops of recombinant
NGF during 28 days. The results showed that the use of NGF is safe and
effective to treat dry eye disease, although randomized clinical trials are
required to confirm these findings [158]. Aloe et al. reviewed the results of
the researches related with the implication of NGF in the induction and
progression of carcinogenesis that remains open to debate [159]. In
contrast, Denk et al. highlighted the clinical relevance of NGF antagonists
as effective analgesic drugs for the treatment of several conditions,
including osteoarthritis and back pain [160].
Table 3 shows examples of the clinical applications of diverse
recombinant growth factors and corresponding biological medicines.
Table 3 Examples of recombinant growth factors for different therapeutic indications and marketed
biological medicines

Recombinant growth factor Therapeutic indications Marketed References


medicines
EGF (epidermal growth factor) Diabetic foot ulcers Heberprot-P®, [120, 121]
Regen-D® 150,
Easyef®
Vascular ulcers and bed Regen-D® 150 [118, 121]
sores
Healing of burns and donor Regen-D® 60 [119, 121]
site skin grafts
bFGF/FGF-2 (basic fibroblast Skin ulcers, regeneration of Fiblast® spray [121, 130]
growth factor or fibroblast growth periodontal and bone tissues
factor 2) or trafermin
FGF-19 (fibroblast growth factor Biliary cirrhosis Orphan medicine [131]
19)
Primary sclerosing Orphan medicine [132]
cholangitis
IGF-I (insulin-like growth factor-I) Growth hormone Increlex® [2, 161]
or mecasermin insensitivity syndrome
Recombinant growth factor Therapeutic indications Marketed References
medicines
Amyotrophic lateral Iplex® a [2, 162]
sclerosis
IGF-I/IGFBP-3 (insulin-like growth Type A and B extreme Orphan medicine [148, 149]
factor-I/insulin-like growth factor- resistance insulin syndrome
binding protein-3)
Inherited extreme insulin Orphan medicine [150]
resistance
Primary growth hormone Orphan medicine [151]
insensitivity syndrome
Leprechaunism Orphan medicine [152]
PGF (placental growth factor) Preeclampsia Orphan medicine [139]
PDGF-BB (platelet-derived growth Ankle arthrodesis and/or Augment® [127]
factor-isoform BB) hindfoot when supplemental
graft is needed
Periodontally related defects GEM 21S® [2, 129]

Chronic diabetic ulcers Regranex® a [2, 125,


126]
NGF (nerve growth factor) or Retinitis pigmentosa Orphan medicine [154]
cenegermin
Neurotrophic keratitis Oxervate® [155, 156]

KGF (keratinocyte growth factor) or Oral mucositis Kepivance® a [133]


palifermin
VEGF (vascular endothelial growth Amyotrophic lateral Orphan medicine [135]
factor) sclerosis

aMedicine withdrawn

4 Conclusion
Among the most important activities of cytokines are the triggering of
immune responses against cancer and viral infections and the ability to
regenerate the skin. In this sense, numerous recombinant cytokines have
been used in clinical practice. For example, the INFs to the treatment of
several cancers, hepatitis B and C and multiple sclerosis, and the ILs and
TNFs for the management of different cancers. Concerning the HGFs,
epoetin has been used to treat anemia caused by diverse disorders, while
colony-stimulating factors have been used for neutropenia. Regarding other
growth factors, those used for wound management have been extensively
explored, although most still need to demonstrate clinical relevance in vivo
before reaching the market.
From this review, we concluded that the clinical relevance of
recombinant cytokines has been increasing. Since the 1980s, EMA and/or
FDA have approved 89 biological medicines containing recombinant
cytokines (INFs, ILs, TNFs, HGFs, and other growth factors). Among
these, 18 were withdrawn, 24 are biosimilars, and 18 are orphans. The
withdrawal of the medicines from the market has been requested by the
producers and is related to economic reasons, occurrence of toxicity or non-
efficacy events. Regarding biosimilars, only the HGFs epoetin and
filgrastim were approved. This can be explained by their antiquity over
other classes of recombinant cytokines, which allowed patent expiration.
Thus, the approval of more biosimilars containing recombinant cytokines is
expected for the next years. Orphan designation was granted mainly to non-
HGFs, for different clinical uses. However, HGFs, ILs, and TNFs also have
orphan medicines.
So far, considerable progress has been made in discovering new
cytokines, additional cytokine functions, and how they interfere with human
diseases. Future prospects include the approval of more biological and
biosimilar medicines for different therapeutic applications.

Acknowledgments
This work was supported by the Applied Molecular Biosciences Unit-
UCIBIO and FP-ENAS, which are financed by national funds from
FCT/MCTES (UID/Multi/04378/2019 and UID/Multi/04546/2019,
respectively).

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https://doi.org/10.1007/10_2019_111

Hormones, Blood Products, and Therapeutic


Enzymes
Ana Catarina Silva1, 2 , Cládia Pina Costa1, Hugo Almeida1,
João Nuno Moreira3, 4 and José Manuel Sousa Lobo1
(1) UCIBIO, REQUIMTE, MEDTECH, Laboratory of Pharmaceutical
Technology, Department of Drug Sciences, Faculty of Pharmacy, University
of Porto, Porto, Portugal
(2) FP-ENAS (UFP Energy, Environment and Health Research Unit), CEBIMED
(Biomedical Research Centre), Faculty of Health Sciences, Fernando Pessoa
University, Porto, Portugal
(3) CNC – Center for Neuroscience and Cell Biology, Faculty of Medicine (Pólo
I), University of Coimbra, Coimbra, Portugal
(4) FFUC – Faculty of Pharmacy, Pólo das Ciências da Saúde, Coimbra,
University of Coimbra, Coimbra, Portugal

Ana Catarina Silva


Email: [email protected]

Abstract
Therapeutic uses of biological medicines are diverse and include active substances
from different classes. This chapter provides an overview on the clinical
applications of biological medicines containing hormones, blood products, and
therapeutic enzymes. Currently, therapeutic hormones have 78 approved
medicines, including insulin and analogs, glucagon and analogs, growth hormone,
gonadotropins (follicle-stimulating hormone, luteinizing hormone, and human
chorionic gonadotropin), thyroid-stimulating hormone, and parathyroid hormone.
In contrast, recombinant blood products, and particularly blood factors,
anticoagulants, and thrombolytic agents, incorporate 49 approved biological
medicines. Regarding recombinant therapeutic enzymes, there are 22 approved
medicines. Among the referred biological medicines, there are six biosimilar
hormones, and no biosimilars have been approved for recombinant blood products
and therapeutic enzymes, which is unexpected.
Current investigations on recombinant hormones, recombinant blood products,
and therapeutic enzymes seem to follow the same directions, searching for
alternative non-injectable administration routes, development of new recombinant
molecules with improved pharmacokinetic properties and discovering new clinical
applications for approved medicines. These approaches are showing positive
results and new medicines are expected to reach clinical approval in the coming
years. Future prospects also include the approval of more biosimilar medicines.
Graphical Abstract

Keywords Anticoagulants – Blood factors – Glucagon – Gonadotropins – Growth


hormone – Insulin – Therapeutic enzymes – Thrombolytic agents

1 Introduction
The approval of recombinant human insulin as the first biological medicine in the
1980s marked a new era for medical therapy that brought together the
biotechnological and pharmaceutical industries. Biological medicines are often
based on recombinant proteins (also named as biopharmaceuticals) and produced
in living organisms, including microorganisms, cells, plants, and animals.
However, due to cost-effective manufacturing, some biological medicines contain
proteins extracted directly from natural sources. Thus far, the number of biological
medicines that gained clinical approval is impressive along with an increased
number of biosimilars approved. Current therapeutic applications of these
medicines are diverse and include active substances from different classes, such as
monoclonal antibodies, cytokines, growth factors, hormones, blood products,
enzymes, vaccines, and cellular therapies [1–5]. Almost all of these medicines are
of parenteral administration, as other routes pose a number of different biological
barriers that are yet to be overcome [6].
This chapter focuses on the clinical applications of biological medicines
containing hormones, blood products, and therapeutic enzymes. Therapeutic
hormones that were initially extracted from natural sources are currently produced
through biotechnological techniques (i.e., recombinant-DNA technology), and
include insulin and analogs, glucagon and analogs, growth hormone,
gonadotropins (follicle-stimulating hormone, luteinizing hormone, and human
chorionic gonadotropin), thyroid-stimulating hormone, and parathyroid hormone.
In contrast, some blood products are generated by cost-effective methodologies
from the natural source. Nonetheless, herein, only recombinant blood products,
and particularly blood factors, anticoagulants, and thrombolytic agents, will be
addressed. Similarly, some therapeutic enzymes are natural and others are from
recombinant origin and have several clinical applications. Each section of this
chapter briefly addresses current therapeutic uses of the respective recombinant
protein, and further discusses future applications.

2 Hormones
2.1 Insulin
Insulin is composed of 51-amino acids and produced by the β-cells of the islets of
Langerhans [7]. The major function of this hormone is regulating the blood
glucose levels, but it participates in other metabolic processes, promoting glycogen
synthesis in the liver and muscles, fatty acid synthesis in the adipocytes and
inhibiting the glycogenolysis and the gluconeogenesis [8–10]. Therapeutic use of
insulin is classically for the management of type 1 diabetes, which is an
autoimmune metabolic disorder characterized by a deficient or absent production
of insulin. Nonetheless, in some cases, insulin can be used for type 2 diabetes,
when is observed the production of ineffective insulin [8, 11–13].
Insulin manufacture started with the advent of recombinant-DNA technology.
Before that therapeutic insulin was purified from bovine and porcine pancreas.
Nonetheless, the use of animal-derived products presents disadvantages, including
immunogenicity, difficulties to obtain enough supplies, and risk of contamination.
Furthermore, the insulin amino acids sequence is different among the various
species [8, 9, 14–16].
Figure 1 shows a schematic representation of human insulin, which has a
dimeric structure formed by two polypeptide chains, A- and B-, linked by disulfide
bonds between A7–B7, A20–B19, and A6–A11. A-chain consists of 21 amino acids,
whereas B-chain contains 30 amino acids [9, 13, 16, 18, 19].

Fig. 1 Schematic representation of the human insulin. Ala alanine, Arg arginine, Asn asparagine, Cys cysteine,
Glu glutamic acid, Gln glutamine, Gly glycine, His histidine, Ile isoleucine, Leu leucine, Lys lysine, Phe
phenylalanine, Pro proline, Ser serine, Thr threonine, Tyr tyrosine, Val valine (adapted from [17])

First human recombinant insulin was approved in 1982 (Humulin®), being the
first drug produced by genetic engineering techniques in Escherichia coli. Since
then, several recombinant insulins have been marketed, mostly produced in
Escherichia coli, despite some are produced in Saccharomyces cerevisiae and
Pichia pastoris [8, 15, 19–21]. Moreover, the use of genetic engineering
techniques allowed the manufacture of different types of recombinant insulin
(insulin analogs), changing the amino acid sequences of the native human insulin.
These products have different pharmacokinetic and pharmacodynamic profiles and
are called fast-acting or short-acting, intermediate-acting, and slow-acting or long-
acting insulins. Fast-acting increases the blood insulin concentration more quickly,
while slow-acting enters the bloodstream more slowly, but has a longer duration,
helping to maintain basal levels of insulin in the blood. Intermediate acting insulin
with specific activity was also produced [8, 13, 14, 19, 22].
Table 1 shows examples of marketed biological and biosimilar medicines
containing recombinant human insulin and insulin analogs approved by the
European Medicines Agency (EMA) and/or the Food and Drug Administration
(FDA).
Table 1 Examples of approved biological and biosimilar medicines containing recombinant human insulin
and insulin analogs, and respective duration of action and structure

Duration of Recombinant Structure Marketed medicines References


action protein
Short-acting, Human Identical to native human Humulin®; Humulin®R; [23–33]
fast-acting, or insulin insulin
rapid-acting Novolin®R; Insuman®;
Actrapid®; Velosulin® BR;
Exubera®; Insulin human
Winthrop®; Afrezza®;
Solumarv®a, b
Insulin lispro Engineered insulin: Humalog®; Liprolog®; Insulin [34–38]
inversion of B28–B29 Lispro Sanofi®a; Admelog®a
proline–lysine sequence
Insulin aspart Engineered insulin: NovoLog®/NovoRapid®; [39–41]
B28proline is replaced by Fiasp®
aspartic acid
Insulin aspart NovoLog Mix® [42]
and insulin
aspart
protamine
Insulin Engineered insulin: B29 Apidra® [43, 44]
glulisine lysine is replaced by
glutamic acid and B3
asparagine is replaced by
lysine
Long-acting or Human Identical to native human Protaphane®; Monotard®; [45–47]
slow-acting insulin insulin
Ultratard®
Insulin Engineered insulin: A21 Lantus®; Toujeo®; [21, 48–56]
glargine asparagine is replaced by Suliqua®/Soliqua®;
glycine and B-chain
elongated by two arginines Abasaglar®a; Lusduna®a, b;
Semglee®a
Insulin Engineered insulin: 14 fatty Levemir® [57, 58]
detemir acids are covalently attached
to B29 lysine and B30
threonine is deleted
Duration of Recombinant Structure Marketed medicines References
action protein
Insulin Engineered insulin: B30 Tresiba®; Xultophy® [59–62]
degludec
threonine is deleted and B29
is coupled to
hexadecanedionyl-ƴ-L-
glutamate
Intermediate Human Identical to native human Actraphane®; Mixtard®; [63–66]
acting, dual- insulin insulin
acting, or long- Insulatard®
acting Insulin lispro Engineered insulin:
combined with Humalog® Mix; Liprolog [67, 68]
and insulin inversion of B28–B29
fast-acting lispro Mix®
proline–lysine sequence
protamine
Insulin aspart Engineered insulin: B28 NovoMix® [69]
and insulin proline is replaced by
aspart aspartic acid
protamine
Insulin Engineered insulin: B30 Ryzodeg® 70/30 [70, 71]
degludec and 29
insulin aspart threonine is deleted and B
is coupled to
hexadecanedionyl-ƴ-L-
glutamate; B28 proline is
replaced by aspartic acid

aBiosimilar medicine
bMedicine withdrawn

Apart from the medicines mentioned in Table 1, new insulins have been
introduced in some countries. For example, Afrezza® (pulmonary insulin) was
recently authorized in Brazil [72], and Glaritus® (insulin glargine) is only
approved in some Asian and African countries [73].
Concerning insulin biosimilar medicines, the firsts were approved by EMA in
2014 (Abasaglar®) [54] and by FDA in 2015 (Basaglar®) [74, 75]. Later, more
biosimilar insulins reached the market and some have been withdrawn (e.g.,
Solumarv® and Lusduna®) [33, 56].
In addition to regulating blood glucose levels, insulin also increases intestinal
cells growth. In this sense, the orphan designation was granted by EMA to
recombinant insulin for the treatment of short bowel syndrome, a condition where
the body cannot absorb nutrients and fluids due to a missing of part of the small
bowel. Thus, insulin can regenerate the intestine of patients with short bowel
syndrome, improving the disease symptoms [76].
2.1.1 New Insulin Formulations
Excluding Afrezza®, current approved insulin medicines are for subcutaneous
administration, but researchers have been looking for alternative non-invasive
routes, improving patient’s compliance [9, 77, 78]. The results seem promising and
there are several products under clinical studies. For example, MidaForm®
PharmFilm, a buccal film containing insulin, is under phase II clinical trials [78,
79]; and IN-105 or tregopil, which is an oral insulin analog (differs from insulin at
position B29) with improved stability against enzymatic degradation, is under
phase II/III clinical trials [80].
Regarding the subcutaneous administration of insulin, advances in
formulations have been made to improve the management of diabetes. In this
sense, insulin pumps for subcutaneous implantation have been used. These devices
contain a glucose sensor connected to an insulin delivery system that promotes an
optimal glycemic control. However, limitations to control the postprandial
hyperglycemia were observed. To circumvent this, human insulin rapid-acting
analogs have been included in implantable pumps. Currently, the only pump
available is the MIP 2007D that is marketed by Medtronic® in Europe. This
titanium pump is surgically implanted into the abdominal wall [81–83]. Artificial
pancreas or closed-loop system is the most promising insulin device, which
consists of a pump and an infusion system with an integrated glucose sensor and a
computer that analyzes the blood glucose level and adjusts the insulin flow rate [8,
78, 81, 84]. Recently, more accurate closed-loop devices, which allow the real-
time monitoring of interstitial glucose levels, by means of transcutaneous glucose
sensors (glucose-oxidase-based electrochemical sensors) and external insulin
pumps, have been successfully tested for the management of type 1 diabetes in
children. These systems contain an electronic device that measures the blood
glucose level and adjusts the insulin infusion rate, maintaining glucose within the
normal range [85].

2.1.2 Insulin Analogs Under Development


Several ultra-fast-acting insulin analogs, showing higher physiological rate than
native insulin and fast-acting insulin analogs, have been developed and some are
under clinical evaluation [78, 81]. For example, VIAject®/Linjeta® acts faster than
insulin lispro, due to the addition of ethylenediaminetetraacetic acid (EDTA) that
chelates zinc and solubilizes insulin hexamers [81, 86–88]. Biodel insulin (BIOD-
531) is a concentrated formulation of recombinant human insulin with improved
post-meal glucose control, compared to Humalog®, due to the presence of EDTA,
citrate, and magnesium that promote tissue dispersion of insulin [16, 89].
BioChaperone® Lispro contains insulin lispro and oligosaccharides that promote
insulin absorption. Clinical studies with this type of insulin showed an increase of
early insulin exposure in 168%, compared to Humalog®, and improved metabolic
profile, compared to NovoLog® and Fiasp® [90, 91]. LY900014 which is an ultra-
rapid insulin lispro that recently finished phase II clinical trials is waiting for
regulatory review [92]. Ultra-fast-acting insulin aspart, containing nicotinamide to
promote absorption and arginine as a stabilizer, showed earlier onset of action and
exposure, compared to insulin aspart [89, 93, 94].
Another approach used to improve the onset of human insulin analogs involves
combining the molecule with recombinant human hyaluronidase [78]. For
example, a study in 14 healthy volunteers showed ultra-rapid profiles with a faster
onset associated with reducing postprandial hyperglycemia for insulin analogs
linked to hyaluronidase [81, 95]. Similar results were observed in another study,
where was administered human insulin combined with hyaluronidase to 40 patients
with type 1 diabetes [96].
Currently, some companies are conducting clinical trials with new insulins for
subcutaneous administration in type 2 diabetes patients. For example, Eli Lilly is
evaluating LY3209590, which is an engineered insulin fused to an antibody Fc
domain that provides a long-acting basal profile, in comparison to glargine and
degludec insulins [97, 98].

2.2 Glucagon
Glucagon is a 29 amino acids polypeptide synthetized by the α-cells of the islets of
Langerhans and other related intestinal cells. Its major function is to prevent
hypoglycemia, promoting liver glycogenolysis and gluconeogenesis, increasing the
blood glucose level. Thereby, glucagon is frequently used to prevent hypoglycemia
caused by insulin administration in patients with type 1 diabetes [99, 100]. In
addition, glucagon precursors or glucagon analogs produced by intestinal
endocrine L-cells have been identified, including glucagon-like peptide 1 (GLP-1)
and glucagon-like peptide 2 (GLP-2). The first has an opposite activity to
glucagon, stimulating the β-cells proliferation with consequent insulin secretion
and reduction of the blood glucose. In contrast, GLP-2 stimulates intestinal growth
while slowing proximal bowel motility and secretion [101–103]. Similar to insulin,
initial therapeutic glucagon was obtained directly from porcine and bovine
pancreatic tissues, being GlucaGen® the first recombinant glucagon produced in
Saccharomyces cerevisiae approved by FDA in 1998 [8, 104]. Table 2 shows
examples of recombinant glucagon and glucagon analogs approved by EMA
and/or FDA.
Table 2 Examples of recombinant glucagon and glucagon analogs, therapeutic indications, and respective
approved biological medicines
Recombinant Marketed Therapeutic indications References
glucagon medicines
Glucagon GlucaGen®; Severe hypoglycemia in adult patients with [104, 105]
Glucagon for diabetes type 1 treated with insulin
injection™ Inhibition of the gastrointestinal tract motility for
radiologic examinations
Orphan medicine Noninsulinoma pancreatogenous hypoglycemia [106]
syndrome (excessive growth of pancreas cells with
insulin overproduction and hypoglycemia episodes)
Congenital hyperinsulinism (inherited disorder [107, 108]
where occurs a not needed increase of insulin)
Glucagon linked to [109]
human
immunoglobulin Fc
fragment
Liraglutide (GLP-1 Saxenda® Adjunct to chronic weight management for obese [110, 111]
receptor agonist) and overweight patients undergoing diet and
physical exercise

Victoza® Adjunct to diet and exercise to improve glycemic [112, 113]


control (stimulate insulin production) in patients
Semaglutide (GLP-1 Ozempic® with type 2 diabetes [114, 115]
receptor agonist)
Dulaglutide (GLP-1 Trulicity® [116, 117]
receptor agonist)
Teduglutide (GLP-2 Revestive®(orphan Short bowel syndrome [118, 119]
analogue)
medicine)/Gattex®
Apraglutide (GLP-2 Orphan medicine [120]
analogue)
GLP-2 linked to [121]
human
immunoglobulin Fc
fragment

GLP-1 glucagon-like peptide 1, GLP-2 glucagon-like peptide-2

The pharmacokinetic and pharmacodynamic characteristics of dasiglucagon, a


glucagon analog, were evaluated in comparison to GlucaGen®, in children with
type 1 diabetes. The results showed increased blood glucose levels for the patients
treated with dasiglucagon. From these findings, the authors suggested the use of
dasiglucagon for hypoglycemia rescue therapy [122]. However, to the best of our
knowledge, there are no marketed medicines available, although the EMA
approved this indication in 2018 [123].

2.2.1 New Glucagon Formulations


A glucagon nasal powder formulation was successfully evaluated in phase III
clinical trials for use in severe hypoglycemic episodes and is currently waiting for
regulatory review. This new medicine is indicated for emergency situations, being
the first needle-free treatment for hypoglycemia [97, 124]. A liquid glucagon
rescue pen showed positive results in phase III clinical trials, where all the patients
achieved optimal blood glucose levels up to 30 min after the administration.
Thereby, this new device was suggested as an effective alternative for ready-to-use
in severe hypoglycemia in type 1 diabetes patients, as compared to the currently
marketed injectable glucagon [125].

2.3 Growth Hormone


Somatotropin, growth hormone (GH) or human growth hormone plays a major role
in the regulation of body growth, cell metabolism, and circadian rhythm. This
peptide hormone has 109 amino acids and is secreted by the hypothalamus anterior
pituitary gland, in a process regulated by the GH-releasing factor or somatorelin
(stimulatory peptide) and the GH-release inhibiting hormone or somatostatin
(inhibitory peptide) [8, 126, 127].
GH biological effects include increase of bones, cartilage, and muscles growth,
stimulation of protein synthesis, anti-insulin and lipolytic effects, and improved
renal function. GH can act directly, binding to specific cell receptors, or indirectly,
binding to liver receptors that promote different growth effects in the body. The
latter is mediated by the insulin growth factor-1 (IGF-1), which controls the
secretion of GH [8, 126, 127].
Therapeutic use of GH started in the 1950s, being this hormone obtained
directly from cadaveric human pituitaries, which presented limitations of safety
and available amounts. Only in the 1980s, by means of recombinant DNA
technology, was produced the first recombinant GH in Escherichia coli [8, 127,
128].
Clinically approved GH is usually indicated for the treatment of children with
short stature caused by different conditions, including GH deficiency, Prader–Willi
syndrome, Turner syndrome, homeobox-containing gene deficiency, or idiopathic.
In addition, GH is used for the management of growth failure in children with
chronic renal insufficiency, in children born small for gestational age, metabolism
regulation in short bowel syndrome, acquired immunodeficiency syndrome
(AIDS)-related cachexia, and for replacement therapy in adults with GH
deficiency. There is also a widespread illicit use for athlete body building [8, 127,
129]. Table 3 shows examples of EMA and/or FDA approved medicines
containing GH or somatotropin, where it can be seen that, despite all contain
recombinant GH, the therapeutic indications of each medicine are not the same.
Table 3 Examples of approved biological and biosimilar medicines containing recombinant growth hormone
(GH) or somatotropin and respective therapeutic indications

Recombinant Marketed medicines Therapeutic indications References


GH
Growth Humatrope® Children with short stature associated with GH [130]
hormone or deficiency, Turner syndrome, homeobox-
somatotropin containing gene deficiency, or idiopathic and born
small for gestational age

Norditropin®; Tev- Long-term treatment of GH deficiency [131–137]


Tropin®; Somatropin
Biopartners®a;
Valtropin®a, b; Saizen®
(orphan medicine)

Genotropin®; Zomacton®; Children with short stature associated with GH [138–141]


deficiency, Turner syndrome, Prader–Willi
Omnitrope®b syndrome, homeobox-containing gene deficiency,
or idiopathic and born small for gestational age

Nutropin AQ® Children with short stature associated with GH [142, 143]
deficiency, idiopathic short stature, Turner
syndrome, and chronic kidney disease

Zorbtive® (orphan Short bowel syndrome in patients receiving [144]


medicine) nutritional support

Serostim® HIV patients with wasting or cachexia [145, 146]

aMedicine withdrawn
bBiosimilar medicine

2.3.1 New Growth Hormone Formulations


New approaches in the development of medicines containing recombinant GH
have focused on increasing the drug circulating half-life, reducing the required
number of administrations from daily up to weekly or monthly [147]. Examples of
formulations under clinical studies include: TransCon (prodrug containing GH
bounded to a linker carrier) for weekly administration, which is in phase II (adults)
and III (children) [148]; MOD-4023 that is a carboxy-terminal peptide-modified
GH for weekly administration, which is in phase III (adults) [149]; GX-H9 (hybrid
Fc-fused to GH) for twice-monthly administration that is in phase II (adults and
children) [150]; Somapacitan (albumin-binding GH) for weekly administration that
is in phase III (adults) [151].

2.4 Gonadotropins
Gonadotropins comprise a family of hormones secreted by gonadotrope cells of
the anterior pituitary, including the follicle-stimulating hormone (FSH), luteinizing
hormone (LH), and human chorionic gonadotropin (hCG). FSH and LH play a
major role in the reproductive function regulation and sexual characteristics, while
hCG has a central role during pregnancy. These dimeric hormones are formed by
one α-polypeptide subunit of 92 amino acids and one β-polypeptide subunit with
111 FSH, 121 LH, and 145 hCG amino acids [8, 152, 153].
In men, FSH is responsible for sperm production, targeting the testis Sertoli
cells and regulating the early stages of spermatogenesis. In women, FSH
stimulates the ovarian follicle maturation, participates in the synthesis of estrogen
and glycosaminoglycans, and regulates the reproductive function. LH stimulates
the women ovulation of mature follicles, and (together with FSH) participates in
the conversion of androgens to estrogens. In men, LH is involved in the
testosterone production and sperm maturation. In contrast, the hCG is produced by
pregnant women, having an important function during the early phase of
pregnancy [8, 154, 155].
Gonadotropin hormones have been used in assisted reproductive therapies and
in the treatment of several women infertility disorders. For example, to induce or
stimulate ovulation for support of natural conception or intrauterine insemination,
and to induce multifollicular growth required for in vitro fertilization procedures.
In men, FSH and hCG stimulate sperm synthesis and are used for the management
of hypogonadotropic hypogonadism. There is also an illicit use of hCG by athletes
to stimulate testosterone production. First therapeutic FSH and LH were extracted
from women menopausal urine, while hCG was obtained from the urine of
pregnant women [8, 152, 155, 156]. Owing to the increase on the number of
fertility treatments, the quantities of urinary gonadotropins available have been
scarce. For this reason, recombinant gonadotropins (or gonadotropins analogs)
have been produced in mammalian cell lines, such as Chinese hamster ovary
(CHO) cells, among others. Nonetheless, urinary gonadotropins remain in clinical
use [155, 156]. Table 4 shows examples of recombinant gonadotropins approved
by EMA and/or FDA, respective therapeutic indications, and biological and
biosimilar medicines.
Table 4 Examples of approved biological and biosimilar medicines containing recombinant gonadotropins
(FSH follicle-stimulating hormone, LH luteinizing hormone, hCG human chorionic gonadotropin) and
respective therapeutic indications

Recombinant Marketed medicines Therapeutic indications References


gonadotropins
Follitropin alfa Gonal-f®; Women: induction of ovulation and pregnancy in [157–160]
(FSH) anovulatory infertile patients; development of
Ovaleap®a; multiple ovulatory follicles in patients undergoing
Bemfola®a
Recombinant Marketed medicines Therapeutic indications References
gonadotropins
Follitropin beta fertility treatments; patients with severe
Puregon®/Follistim®; deficiencies of LH and FSH [161–163]
(FSH)
Fertavid® Men: stimulation of spermatogenesis in
congenital or acquired hypogonadotropic
hypogonadism
Follitropin delta Rekovelle® Development of multiple ovulatory follicles in [164]
(FSH) women undergoing fertility treatments: in vitro
fertilization or intracytoplasmic sperm injection
Corifollitropin alfa Elonva® [165]
(long-acting FSH)
Lutropin alfa (LH) Luveris® Induction of ovulation in women with severe LH [166, 167]
and FSH deficiencies
Follitropin Pergoveris® Women with FSH and LH deficiencies: induction [168]
alfa/lutropin alfa of ovulation (FSH) and eggs release (LH)
(FSH/LH)
Choriogonadotropin Ovitrelle®/Ovidrel® Women treated with LH and FSH to stimulate [169, 170]
alfa (hCG) ovaries: trigger ovulation and development of the
corpus luteum to support pregnancy
Women undergoing fertility treatments (in vitro
fertilization)
Anovulatory or oligo-ovulatory women

aBiosimilar medicine

From Table 4, it can be seen that only recombinant FSH has approved
biosimilar medicines, although a recombinant hCG biosimilar was recently
evaluated to induce ovulation in patients undergoing intrauterine dissemination.
The results demonstrated clinical equivalence to Ovitrelle®, suggesting the
potential of using this biosimilar as an alternative to the reference medicine [171].
Concerning the clinical applications of gonadotropins, there is an open field for
therapeutic uses, including the treatment of polycystic ovary syndrome,
management of ovary and prostate cancers, prevention of postmenopausal
symptoms (avoidance of bone loss and weight gain), and as contraceptives [156].

2.4.1 New Gonadotropins Formulations


Recombinant gonadotropins have been used clinically in fertility treatments, by
means of injectable formulations. Nonetheless, some limitations have been pointed
to these medicines, regarding the necessity of performing multiple administrations,
lack of stability, and adverse effects, such as ovarian hyperstimulation syndrome.
Researches have been focused in the development of long-acting recombinant
gonadotropins with improved stability, achieving appropriate pharmacokinetic
profiles and minimizing the number of administrations [156]. Several studies
showed that corifollitropin alfa, a long-acting FSH analog, reduces the drawbacks
of the in vitro fertilization treatments (Table 4) [172, 173]. In this sense, other
strategies have been studied to develop more long-acting recombinant FSH
molecules. Examples include the addition of glycosylated peptides or polyethylene
glycol – PEG (PEGylation) to the gonadotropin molecule, and fusion of the
gonadotropin with an immunoglobulin Fc domain (fusion protein) [153, 156].

2.5 Other Recombinant Hormones


In addition to insulin, glucagon, growth hormone, and gonadotropins there are
other recombinant hormones under clinical use, which are the thyroid-stimulating
hormone (TSH) and the parathyroid hormone (PTH). TSH or thyrotropin has a
molecular structure that resembles gonadotropins, containing one β-subunit and
one α-subunit. TSH is produced by the anterior pituitary and targets the thyroid
gland, stimulating its functions, including iodine uptake, production of the iodine
containing thyroid hormones (iodothyronines) triiodothyronine (T3) and thyroxine
(T4), and promotion of thyroid growth. Furthermore, TSH participates in the
prevention of thyroid cells apoptosis and in thyroid ontogenesis. Recombinant
TSH produced in CHO cells has been used for the diagnostic of thyroid cancer and
for the detection of thyroid remnants in post-thyroidectomy patients [8, 174]. PTH
or human PTH is an 84 amino acids polypeptide that regulates extracellular
phosphate and calcium metabolism. In bone, PTH has a catabolic effect,
stimulating osteoblasts for bone formation. In kidneys, PTH promotes the
synthesis of vitamin D that increases the intestinal calcium absorption and inhibits
the renal phosphate reabsorption. Thereby, recombinant hPTH produced in
Escherichia coli was approved for the management of osteoporosis in women
post-menopausal and in men and for chronic hypoparathyroidism [8, 175]. Table 5
shows examples of recombinant TSH and PTH approved by EMA and/or FDA,
respective therapeutic indications, and biological and biosimilar medicines.
Table 5 Examples of approved biological and biosimilar medicines containing recombinant thyroid-
stimulating hormone (TSH) and parathyroid hormone (PTH) and respective therapeutic indications

Recombinant Marketed medicine Therapeutic indications References


TSH and
PTH
Thyrotropin Thyrogen® Thyroid cancer: detection of thyroid tissue left after [176, 177]
alfa (TSH) surgery; elimination of remaining thyroid tissue (in
combination with radioactive iodine) in patients
who removed the thyroid gland
Teriparatide Forsteo®/Forteo®; Osteoporosis in postmenopausal women and in [178–181]
(PTH) men with increased risk of fracture
Movymia®a/Terrosa®a
Recombinant Marketed medicine Therapeutic indications References
TSH and
PTH
PTH Natpara®/Natpar®(orphan Hypocalcemia control in patients with chronic [182, 183]
medicine) hypoparathyroidism

aBiosimilar medicine

In addition to Table 5, other formulations containing recombinant PTH have


been investigated. For example, TransCon PTH has successfully completed phase I
clinical trials to treat hypoparathyroidism [184].
Current approved medicines containing PTH are for subcutaneous
administration, which reduces patient compliance. Nasal and transdermal routes
have been suggested as alternative routes for the administration of PTH in
osteoporosis treatments. The results of in vitro studies are promising, although in
vivo studies are required to confirm this application [185, 186].

3 Blood Products
Blood products of therapeutic interest are proteins that can be extracted directly
from the natural source, i.e., from the blood red and white cells, platelets, and
plasma. However, due to the large amount required for clinical use and some
safety concerns, several therapeutic blood proteins have been produced by genetic
engineering techniques, including recombinant blood factors, anticoagulants, and
thrombolytic agents [187, 188]. Concerning the scope of this chapter, we focus
only in recombinant blood products.

3.1 Blood Clotting Factors or Coagulation Factors


Blood clotting factors (or blood factors) have been used for the treatment of
hemophilia, which is a rare bleeding inherited disorder that reduces the normal
blood clotting process and causes severe consequences. People with this disease
bruise very easily and show difficulty in blood coagulation after trauma, increasing
the bleeding time. There are 12 different blood clotting factors and several
cofactors that play a key role in the blood coagulation process, particularly, in the
blood coagulation cascade. A genetic defect in the expression of blood factors (or
anti-hemophilic factors) originates hemophilia, which is divided into subtypes A,
B, and C. In hemophilia A (or classical hemophilia that occurs in about 90% of the
cases) the blood clotting factor VIII is deficient or abnormal, whereas in
hemophilia B the blood clotting factor IX is deficient or abnormal [188–191]. Both
blood factors VIII and IX play vital roles in the coagulation cascade, being
essential for the conversion of prothrombin into thrombin. Afterwards, thrombin
converts fibrinogen to fibrin, a primordial fibrous protein of the blood coagulation
process. Hemophilia C is a rare type that is characterized by a deficient or
abnormal blood clotting factor XI [191–193].
Hemophilia treatment is performed by supplying the impaired blood clotting
factor, which was firstly obtained directly from the blood donors. However, this
process showed disadvantages related to the risk of virus infection that originates
severe diseases, such as AIDS, hepatitis C and B, and others. Therefore,
recombinant blood clotting factors, produced by DNA recombinant techniques in
CHO cells, are used for the management of hemophilia [188, 189, 191]. In
addition, some companies are conducting clinical trials with gene therapy products
for the treatment of hemophilia A and B, which may be better than the chronic
treatments with weekly injections of recombinant blood clotting factors. Examples
of such companies are: BioMarin® [194], Sangamo Therapeutics [195], and
uniQure [196].
Table 6 shows examples of recombinant blood products (blood factors,
anticoagulants, and thrombolytic agents), therapeutic indications, and names of the
respective approved biological and biosimilar medicines, by EMA and/or FDA.
Table 6 Examples of recombinant blood products (blood clotting factors, anticoagulants, and thrombolytic
agents), therapeutic indications, and respective approved biological and biosimilar medicines

Recombinant blood Marketed medicines Therapeutic indications References


products
Blood clotting factors
Eptacog alfa (factor Novoseven®; Novoseven RT®; Hemophilia A and B; factor VII [197, 198]
VIIa) deficiency; Glanzmann’s
Niastase®; Niastase RT® thrombasthenia
Octocog alfa (factor Advate®; Bioclate®; Helixate Hemophilia A [199–204]
VIII)
FS®; Kogenate FS®;
Kovaltry®; Recombinate®;
Iblias®
Turoctocog alfa NovoEight®; Zonovate® Hemophilia A [199, 205]
(factor VIII)
Lonoctocog alfa Afstyla® Hemophilia A [199, 206]
(factor VIII)
Susoctocog alfa Obizur® Hemophilia A [207]
(factor VIII)
Rurioctocog alfa Adynovi® Hemophilia A [208]
pegol (factor VIII)
Damoctocog alfa Jivi® Hemophilia A [209]
pegol (factor VIII)
Recombinant blood Marketed medicines Therapeutic indications References
products
Efmoroctocog alfa Eloctate®/Elocta® Hemophilia A [199, 210]
(factor VIII linked to
Fc fusion protein)
Moroctocog alfa Refacto AF®; Xyntha® Hemophilia A [199, 211]
(factor VIII)
Simoctocog alfa Nuwiq®; Vihuma® Hemophilia A [199, 212,
(factor VIII) 213]
Pegylated factor VIII Adynovate® Hemophilia A [199]
(factor VIII linked to
PEG)
Vonicog alfa (von Veyvondi® von Willebrand disease [214]
Willebrand factor)
Eftrenonacog alfa Alprolix® (Orphan medicine) Hemophilia B [210, 215]
(factor IX linked to
Fc fusion protein)
Nonacog alfa (factor BeneFix® Hemophilia B [216]
IX)
Nonacog gamma Rixubis® Hemophilia B [217]
(factor IX)
Nonacog beta pegol Refixia® Hemophilia B [218]
(factor IX linked to
PEG)
Albutrepenonacog Idelvion® (Orphan medicine) Hemophilia B [219, 220]
alfa (factor IX linked
to albumin)
Factor IX IXINITY®, RIXUBIS® Hemophilia B [197]

Andexanet alfa ANDEXXA® Reversal of anticoagulation for [221, 222]


(factor Xa, patients treated with rivaroxaban and
inactivated-zhzo) apixaban
Catridecacog (factor TRETTEN®; NovoThirteen® Congenital factor XIIIa subunit [223, 224]
XIIIa) deficiency
Anticoagulants
Lepirudin Refludan®a Heparin-induced thrombocytopenia [225, 226]
type II and thromboembolic disease
Desirudin Revasc®/Iprivask®a Prevention of thrombosis in patients [2, 227,
undergoing hip or knee replacement 228]
surgery, and for pulmonary embolus
Antithrombin alfa ATryn® Prevention of thromboembolic events [229, 230]
in congenital antithrombin deficiency
Drotrecogin alfa Xigris®a Reduce risk of blood clots during [2, 231]
sepsis by inhibition of clotting factors
Va and VIIIa
Recombinant blood Marketed medicines Therapeutic indications References
products
Thrombolytic agents
Alteplase (tissue Actilyse®; Cathflo® Activase® Management of acute myocardial [232, 233]
plasminogen infarction
activator)
Reteplase Ecokinase®a; Retavase®; [234–236]
Rapilysin®
Tenecteplase Metalyse®; Tenecteplase [237–239]
Boehringer Ingelheim Pharma
GmbH Co. KG®a; TNKase®

aMedicine withdrawn; PEG polyethylene glycol

Recombinant human coagulation factor VIIa or eptacog alfa is a vitamin K-


dependent glycoprotein that activates factor IX and factor X, which are essential
for hemostasis. Thus, eptacog alfa is involved in the clotting process initiation and
controls bleeding disorder, being used for the treatment of patients with
hemophilia A and B, congenital factor VII deficiency, and Glanzmann’s
thrombasthenia (a rare bleeding disorder) [198, 240, 241]. Recently, Biron-
Andreani and Schved revised the pharmacodynamics and pharmacokinetics data of
eptacog beta, a new type of recombinant factor VIIa produced in the milk of
transgenic rabbits. From their search the authors concluded that, when compared to
eptacog alfa, eptacog beta requires a lower dose to obtain the same in vivo effect,
being a less expensive alternative for the management of hemophilia [242].
Recombinant human coagulation factor VIII is used for the treatment of
patients with congenital factor VIII deficiency (hemophilia A). Biological and
biosimilar medicines available are shown in Table 6 and include octocog alfa,
turoctocog alfa, lonoctocog alfa, susoctocog alfa, simoctocog alfa, rurioctocog alfa
pegol, and damoctocog alfa pegol [200–209, 212, 213, 241, 243–248].
Efmoroctocog alfa is a recombinant factor VIII-Fc fusion protein used as an
antihemorrhagic agent for the treatment and prophylaxis of acute bleeding
episodes in patients with hemophilia A. The addition of the Fc protein (B-domain)
to the recombinant factor VIII extended the drug half-life, compared to the
recombinant factor VIII alone [191, 192, 210, 241, 247]. Pegylated factor VIII
contains the human recombinant factor VIII covalently linked to a PEG molecule,
which increases the circulation half time, improving therapeutic efficacy [249,
250].
Vonicog alfa is a recombinant von Willebrand factor used to control bleeding
in patients with von Willebrand disease (an inherited bleeding disorder), when
desmopressin is not effective [214, 251]. The hemostatic efficacy of using vonicog
alfa alone or in combination with recombinant factor VIII, in patients with severe
von Willebrand disease that are undergoing surgery, was evaluated in a phase III
clinical study. Researchers observed that the use of vonicog alfa alone originated
hemostasis 6 h after the administration and the effect lasted from 72 up to 96 h.
From these findings the authors concluded that, according to each patient risk
factors, the treatment of von Willebrand disease should be performed with vonicog
alfa alone or in combination with recombinant factor VIII [252].
Eftrenonacog alfa is a recombinant fusion protein containing the human factor
IX covalently linked to the constant region (Fc) domain of the human
immunoglobulin (IgG1), which is used for the treatment and prophylaxis of
bleeding episodes in patients with hemophilia B. This molecule restores the levels
of factor IX, promoting normal blood coagulation. Adding the Fc domain to factor
IX increases its half-life, improving the bioavailability and, consequently, the
therapeutic efficacy [193, 215, 241]. Albutrepenonacog alfa is a recombinant
fusion protein that comprises human factor IX linked to albumin, which is
indicated for patients with hemophilia B for the control and prevention of bleeding
episodes [219, 220]. Nonacog alfa, nonacog gamma, and nonacog beta pegol are
also recombinant human factor IX used for the treatment of hemophilia B [188,
197, 216–218].
FDA approved the recombinant coagulation factor Xa, inactivated-zhzo, or
andexanet alfa to reverse the effects of factor Xa inhibitors, apixaban and
rivaroxaban, when an anticoagulation effect is required [222]. Some studies
suggested the use of andexanet alfa for reversing the effect of edoxaban, which is
another factor Xa inhibitor, but this indication has not been approved yet [221,
253].
Patients with deficient or abnormal coagulation factor XIII can be treated with
recombinant human factor XIIIa, also known as catridecacog [223, 224, 241, 254].
Recently, Sottilotta et al. performed a clinical study where was observed that the
use of catridecacog is effective for continued prophylaxis and for conducting major
surgical procedures in patients with congenital factor XIII deficiency [255].

3.2 Anticoagulants
Thrombus formation occurs by changes in the blood coagulation process within
the vessels and causes serious health problems or even death. Anticoagulants are
able to extend the coagulation cascade length and have been used to treat and
prevent embolic events or thrombotic disorders, avoiding the changes in the blood
clotting process [188, 256].
Heparin, dicoumarol, warfarin, and hirudin are the most studied anticoagulants.
Heparin was first extracted from the liver, but current marketed medicines contain
enoxaparin sodium (low molecular weight heparin) obtained by alkaline
depolymerization of the heparin benzyl ester that is extracted from porcine gastric
mucosa. This heparin derivative activates the antithrombin III and, thus, inhibits
the clotting factors Xa and IIa, being used to prevent and treat vein thrombosis,
acute coronary syndromes, pulmonary embolism and for the prophylaxis of
ischemic complications (angina and myocardial infarction). EMA and FDA
approved biological (Lovenox®, Clexane®, Clexane T®, Clexane Forte®,
Klexane®, Qualiop®, Enoxaparin Sanofi®, and Enoxaparine Sanofi®) and
biosimilar (Inhixa® and Thorinane®) medicines containing enoxaparin sodium to
prevent and treat conditions associated with blood clots. These include deep vein
thrombosis (usually in the legs), unstable angina (related to impaired blood flow to
the heart), and certain types of myocardial infarction or heart attack [241, 257–
261]. When compared to recombinant biological medicines (Table 6), heparin and
derivatives are less expensive. Nonetheless, disadvantages have been pointed,
related to the risk of severe side effects, including bleeding and thrombocytopenia
[262].
Dicoumarol and warfarin are obtained by chemical synthesis and will not be
referred, because they are not biological medicines. Hirudin is a natural small
polypeptide that exists in the saliva of bloodsucking parasites (Hirudo medicinalis)
and has anticoagulants properties, binding and inhibiting thrombin [188, 256, 263,
264]. Regarding the small amount of hirudin available for removal from the
natural source, lepirudin (Table 6), a recombinant hirudin variant-1, has been
produced in Saccharomyces cerevisiae, and is indicated for preventing thrombus or
clot formation. Nonetheless, this medicine is not currently available [188, 225,
226, 265, 266]. El-Mowafi et al. conducted a clinical trial where was observed that
topically administered recombinant hirudin gel is effective and safe for the
management of symptomatic hematomas [267]. Similar products have been
produced by recombinant techniques. For example, desirudin produced in
Saccharomyces cerevisiae was approved by EMA and FDA (Table 6) as a selective
thrombin inhibitor that prevents deep venous thrombosis (in patients undergoing
elective hip or knee replacement surgery) and decreases the risk of pulmonary
embolus, but is not currently available [188, 227, 228].
Antithrombin is a plasma native inhibitor of the coagulation process that binds
to thrombin (factor IIA), factor IXa and Xa, and interrupts the coagulation cascade.
Antithrombin alfa (Table 6) was the first biological medicine produced in
transgenic animals, being obtained from the milk of genetically modified goats
[188, 268, 269]. Antithrombin alfa is used for the prevention of thromboembolic
events in patients with congenital low levels of antithrombin [230]. Drotrecogin
alfa (Table 6) is similar to the activated human protein C that inhibits the clotting
factors Va and VIIIa and reduces the blood coagulation process, and was approved
to avoid excessive blood clotting during severe sepsis, but is not currently
available [188, 231].

3.3 Thrombolytic Agents


Thrombolytic agents or fibrinolytics convert zymogen plasminogen (glycoprotein
synthesized by the kidneys) into plasmin, which is an active enzyme that promotes
the proteolytic degradation of fibrin, dissolving blood clots. There are several
recombinant thrombolytic agents used for the treatment of thromboembolic
disease, including alteplase, reteplase, tenecteplase, and urokinase (Tables 6 and 7)
[188, 191, 269, 300, 301].
Table 7 Examples of recombinant enzymes and respective approved biological medicines, therapeutic
indications, and mechanism of action

Recombinant Marketed medicines Therapeutic Mechanism of action References


enzymes indications
Alfagalsidase Replagal®; Fabry’s disease: Catalyzes the [270–272]
(alpha-galactosidase absent/insufficient hydrolysis of
A) Fabrazyme® alpha-galactosidase A globotriaosylceramide,
that breaks down the reducing the body
globotriaosylceramide, accumulation
which is a fatty
compound that
accumulates in the
body and affects the
nervous system,
vascular endothelial
cells, and major organs
Alteplase (tissue Actilyse®; Cathflo® Acute ischemic stroke Converts plasminogen [232, 235,
plasminogen or acute myocardial into plasmin, 273, 274]
activator) Activase® infarction dissolving the blood
clots responsible for
the blockage of the
coronary arteries
Recombinant Marketed medicines Therapeutic Mechanism of action References
enzymes indications
Asparaginase (L- Kidrolase®; Oncaspar®; Acute lymphoblastic Catalyzes the [273, 275–
asparaginase) leukemia hydrolysis of L- 277]
Spectrila® asparagine into L-
aspartic acid and
ammonia
Asparagine is
fundamental for
cancer cells
proliferation and a
reduction on its blood
levels originates death
of cancer cells.
Normal cells are able
to produce asparagine
and, thus, are not
affected by the
administration of
asparaginase
Pegylated Erwinia Erwinaze ® (orphan Acute lymphoblastic Catalyzes the [273, 278,
chrysanthemi L- medicine) leukemia in patients hydrolysis of L- 279]
asparaginase who have developed asparagine into L-
hypersensitivity to aspartic acid and
native E. coli derived ammonia
asparaginase Asparagine is
fundamental for
cancer cells
proliferation and a
reduction on its blood
levels originates death
of cancer cells.
Normal cells are able
to produce asparagine
and, thus, are not
affected by the
administration of
asparaginase
PEGylation reduces
the clearance of
asparaginase from the
body
Asparaginase (L- Graspa®a Acute lymphoblastic Catalyzes the [280]
asparaginase) leukemia hydrolysis of L-
asparagine into L-
aspartic acid and
ammonia
Asparagine is
fundamental for
cancer cells
proliferation and a
reduction on its blood
Recombinant Marketed medicines Therapeutic Mechanism of action References
enzymes indications
Orphan medicine Pancreatic cancer and levels originates death [281, 282]
acute myeloid of cancer cells.
leukemia Normal cells are able
to produce asparagine
and, thus, are not
affected by the
administration of
asparaginase
Contains asparaginase
encapsulated in
erythrocytes for
increasing its
circulation time and
protection against
blood degradation and
immunological
reactions
Dornase alfa Pulmozyme ® Cystic fibrosis: Catalyzes the cleavage [2, 273,
(deoxyribonuclease retention of purulent of the phosphodiester 283]
– DNase) and viscous secretions linkages of the mucus
that affect lung DNA. This process
function and cause reduces mucus
infections viscosity and
promotes secretions
clearance
Elosulfase alfa (N- Vimizim® (orphan Mucopolysaccharidosis Enzyme replacement [284, 285]
acetylgalactosamine- medicine) type IVA: lack of N- therapy, breaking
6-sulfatase) acetylgalactosamine-6- down
sulfatase, which breaks glycosaminoglycans
down that stop building up
glycosaminoglycans. in cells
Accumulation of
glycosaminoglycans
causes short bones,
difficulty moving and
difficulty breathing,
clouding vision and
hearing loss
Recombinant Marketed medicines Therapeutic Mechanism of action References
enzymes indications
Galsulfase (N- Aryplase®/Naglazyme® Mucopolysaccharidosis Enzyme replacement [273, 286,
acetylgalactosamine (orphan medicine) type VI: lack of N- therapy. Galsulfase is 287]
4-sulfatase) acetylgalactosamine 4- taken up by cells into
sulfatase that breaks lysosomes and
down catalyzes the cleavage
glycosaminoglycans. of sulfate ester from
Body accumulation of terminal N-
glycosaminoglycans acetylgalactosamine 4-
originates sulfate residues of
macrocephaly, short glycosaminoglycan
body, difficult moving, chondroitin 4-sulfate
impaired vision and and dermatan sulfate,
hearing loss, reduced which increases the
pulmonary function, catabolism of
cardiac abnormalities, glycosaminoglycans
etc.
Idursulfase Elaprase® (orphan Hunter syndrome or Enzyme replacement [288, 289]
(iduronate-2- medicine) mucopolysaccharidosis therapy. Cleaves the
sulfatase) type II: disease caused terminal 2-O-sulfate
by insufficient levels of moieties from
the enzyme iduronate- glycosaminoglycans
2-sulfatase. Patients dermatan sulfate and
are not able to degrade heparan sulfate,
glycosaminoglycans, increasing the
which accumulate in catabolism of
cells, originating glycosaminoglycans
difficulty breathing and
difficulty walking
Laronidase (α-L- Aldurazyme® Non-neurological Enzyme replacement [290, 291]
iduronidase) symptoms of therapy. Catalyzes the
mucopolysaccharidosis hydrolysis of the
type I: α-L-iduronidase terminal α-L-iduronic
enzyme deficiency that acid residues from the
promotes the body glycosaminoglycans
accumulation of dermatan sulfate and
glycosaminoglycans, heparin sulfate, which
causing several increases the
dysfunctions, including catabolism of
enlarged liver, difficult glycosaminoglycans
moving, reduced lung,
and heart and eye
diseases
Recombinant Marketed medicines Therapeutic Mechanism of action References
enzymes indications
Imiglucerase Cerezyme® Type 1 and 3 Gaucher Enzyme replacement [292, 293]
(glucocerebrosidase) disease: lack of therapy. Catalyzes the
glucocerebrosidase or hydrolysis of
acid beta-glucosidase, glucosylceramide to
an enzyme responsible glucose and ceramide,
for the degradation of stopping it building up
the fatty waste product in the body
glucosylceramide,
which builds up in the
liver, spleen, and bone
marrow. Typical
disease symptoms
include enlarged spleen
and liver, anemia,
thrombocytopenia, and
bone disease
Velaglucerase alfa VPRIV® (orphan Type 1 Gaucher [294, 295]
(glucocerebrosidase) medicine) disease:
glucocerebrosidase
deficiency that affects
the liver, spleen, and
bones. Originates liver
malfunction, skeletal
disorders, bone lesions,
neurological
complications, anemia,
and low blood platelet
count
Rasburicase (urate Elitek®; Fasturtec® Acute hyperuricemia: Catalyzes enzymatic [296, 297]
oxidase) high levels of uric acid oxidation of uric acid
in the blood. Avoid to allantoin, which is
kidney failure in easily eliminated by
patients undergoing the kidneys
chemotherapy
Velmanase alfa Lamzede® (orphan Mild to moderate Enzyme replacement [298, 299]
medicine) alpha-mannosidosis: therapy. Degradation
congenital absence of of glycoproteins,
the enzyme alpha- avoiding tissue
mannosidase that accumulation of
breaks down oligosaccharides
glycosides. Body
accumulation of
oligosaccharides
originates failure of
body functions, such as
breathing and
movement

aMedicine withdrawn
Alteplase (Tables 6 and 7) is a human recombinant tissue plasminogen
activator used for the management of acute myocardial infarction or acute stroke
[232, 302]. Tenecteplase (Tables 6 and 7) and reteplase (Tables 6 and 7) are also
recombinant forms of the human tissue plasminogen activator that have been
indicated for the treatment of patients suspected or after having a heart attack. This
enzyme acts by dissolving the clots formed inside the vessels, improving the blood
flow into the heart. Compared to the other thrombolytic agents, reteplase has been
showing better clinical results, due to its longer half-life [234–239, 303].

4 Therapeutic Enzymes
Concerning some of their properties, enzymes play an important role in the
pharmaceutical field. For example, in diagnostic assays, due to the high affinity
and specificity to bind targets, and for the management of several diseases and
disorders. Some therapeutic enzymes are extracted from the natural source, while
others have been produced through recombinant-DNA techniques. The latter have
been originating higher yields, regarding the possibility of producing larger
quantities of pure enzymes [188, 273, 304]. Nonetheless, when therapeutic
enzymes can be easily withdrawn from the natural source, the production of
similar recombinant enzymes is not required, which reduces costs of the final
product. Examples of therapeutic enzymes obtained from natural sources include:
asparaginase derived from Escherichia coli (Elspar®), which is used for the
management of acute lymphoblastic leukemia [305]; collagenase Clostridium
histolyticum (Xiapex®) extracted from the bacterium Clostridium histolyticum,
which is used to break up collagen in patients suffering from Dupuytren’s
contracture and Peyronie’s disease [306]. Digestive enzymes including lipases,
proteases, and amylases (pancrelipase) that are used to treat pancreatic
insufficiency, caused by cystic fibrosis or chronic pancreatitis (Creon®,
Pancreaze®, Zenpep®, Pertzye®, Viokace®), are extracted from pig pancreas [307–
311]. Nonetheless, concerning the aim of this chapter, only recombinant
therapeutic enzymes are described.
Therapeutic enzymes obtained through biotechnological processes are widely
used for several clinical applications (Fig. 2), often in enzyme replacement
therapy, when the native enzyme is lacking, and as adjuvants to the management of
severe diseases and disorders. Table 7 shows examples of EMA and/or FDA
approved medicines containing recombinant enzymes, respective therapeutic
indications, and mechanism of action.
Fig. 2 Main clinical uses of therapeutic enzymes [273, 304]

5 Conclusion
From this review, we can conclude that biological medicines are currently a well-
established therapeutic area, enabling the treatment of various incurable diseases.
Concerning the three different therapeutic groups addressed in this chapter, the
hormones are the ones with highest number of marketed products (78 approved
medicines), where 38 are insulin and analogs, 9 are glucagon and analogs, 12
contain GH, 12 contain different gonadotropins, 1 contains TSH, and 6 incorporate
PTH. Furthermore, insulin has 4 biosimilars, 2 of which have been withdrawn and
1 has orphan designation. In contrast, no biosimilar glucagon was approved, which
is surprising, as the first recombinant glucagon was approved in 1998. The same is
observed for GH, which has only the first biosimilar medicine approved. Two
orphan designations and 2 withdrawn medicines were noticed for GH, while
gonadotropins have 2 approved biosimilars.
There are 49 approved biological medicines incorporating recombinant blood
products, where 35 are blood factors (2 orphan designations), 5 are anticoagulants
(3 withdrawn), and 9 are thrombolytic agents (2 withdrawn). None biosimilar has
been approved for recombinant blood products, which is unexpected since they
reached the market in the 1990s. Similarly, from the 22 approved therapeutic
enzymes, none is a biosimilar, 3 are orphan medicines, and 1 was withdrawn from
the market.
Current investigations on recombinant hormones, recombinant blood products,
and therapeutic enzymes seem to follow the same directions, searching for
alternative non-injectable administration routes, development of new recombinant
molecules with improved pharmacokinetic properties and discovering new clinical
applications for the approved medicines. These approaches are showing positive
results and new medicines are expected to reach clinical approval in the coming
years. Future prospects also include the approval of more biosimilar medicines.

Acknowledgments
This work was supported by the Applied Molecular Biosciences Unit-UCIBIO and
FP-ENAS, which are financed by national funds from FCT/MCTES
(UID/Multi/04378/2019 and UID/Multi/04546/2019, respectively).

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Advances in Vaccines
Helen H. Mao1 and Shoubai Chao1
(1) CanSino Biologics Inc., Tianjin, China

Helen H. Mao (Corresponding author)


Email: [email protected]

Shoubai Chao
Email: [email protected]

Abstract
Vaccines represent one of the most important advances in science and
medicine, helping people around the world in preventing the spread of
infectious diseases. However, there are still gaps in vaccination programs in
many countries. Out of 11.2 million children born in EU region, more than
500,000 infants did not receive the complete three-dose series of diphtheria,
pertussis, and tetanus vaccine before the first birthday. Data shows that
there were more than 30,000 measles cases in the European region in recent
years, and measles cases are rising in the USA. There are about 20 million
children in the world still not getting adequate coverage of basic vaccines.
Emerging infectious diseases such as malaria, Ebola virus disease, and Zika
virus disease also threaten public health around the world. This chapter
provides an overview of recent advances in vaccine development and
technologies, manufacturing, characterization of various vaccines,
challenges, and strategies in vaccine clinical development. It also provides
an overview of recently approved major vaccines for human use.
Graphical Abstract
Keywords Characterization – Dengue vaccine – Ebola vaccine –
Recombinant technology – Shingles vaccine – Vaccine clinical trials –
Vaccine development – Vaccine manufacturing – Viral vaccine

1 Introduction
Vaccines represent one of the best advances in science and medicine,
helping people around the world in eliminating and preventing the spread of
infectious diseases. Although many human vaccines have been developed
and are in use, infectious diseases are still threats to people’s health,
especially during epidemic outbreaks. During the 2003 SARS outbreaks in
Asia, there were more than 8,000 cases, and more than 800 deaths occurred,
and the economic impact of SARS exceeded US$50 billion, according to
the World Health Organization (WHO) report [1]. During the largest Ebola
outbreak in West Africa in 2014–2015, more than 28,000 cases were
reported, and there occurred more than 11,000 fatalities. It is estimated that
Guinea, Liberia, and Sierra Leone have endured more than US$2 billion
loss in economic growth as a result of Ebola virus disease outbreaks [2].
Based on the experiences from West Africa Ebola epidemic outbreaks, the
WHO published a prioritized list of 11 pathogens that likely to cause
outbreak situations, including Ebola virus, Lassa virus, Marburg virus,
MRSA, Zika virus, etc. [3]. Some of these epidemic infectious disease
vaccines are currently under development. As pointed out by the WHO, it
will be a common goal for all countries to provide equitable access to high-
quality, safe, affordable vaccines and immunization services throughout the
life course.

1.1 Most Effective Tools in Controlling Infectious Diseases


Vaccines have a long list of achievements in the past. Vaccines have been
tremendously beneficial in protecting individuals and communities from
serious infectious diseases and reducing healthcare costs around the world.
In developed countries, vaccines are more readily available to infants,
children, and adults. In the European region, more than 90% of children
receive at least basic vaccination during infancy [4]. In China, overall
vaccination rates for basic vaccines that are recommended to infants and
children exceeded 90%, according to data published by the Chinese Center
for Diseases Control and Prevention (CCDC) [5].
In the USA, the Advisory Committee on Immunization Practices
(ACIP) recommends routine vaccination programs, and each vaccine is
approved by the regulatory agency US FDA. The immunization schedules
are published by US CDC for different age groups including infants and
children, adolescents, and adults. For example, the immunization schedule
for infants from birth to 24 months includes vaccines against 14 potentially
serious illnesses. US CDC data from the 2017 National Immunization
Survey-Child (NIS-Child) database show that the vaccination rates are
higher than 80% in the USA nationally [6].
Huge progress in vaccination has been made in the last 30 years. Now
about 85% of children worldwide (about 116 million) receive essential,
lifesaving vaccines, protecting them from infectious diseases including
measles, diphtheria, tetanus, pertussis, hepatitis B, and polio. As
vaccination rates increase from about 20% in 1980 to about 85% in 2017
(as shown in Fig. 1), the numbers of cases for measles and pertussis have
decreased significantly, according to the WHO report [7]. This represents
dedication and hard work by all, including public health workers,
researchers, pharmaceutical industries, policy makers, parents, and
communities.
Fig. 1 Number of pertussis and measles cases and vaccine coverage % of measles, polio, and DTP3

However, there are still gaps in vaccination programs in many countries.


Out of 11.2 million children born in the EU region in 2012, more than
500,000 infants did not receive the complete three-dose series of diphtheria,
pertussis, and tetanus vaccine before the first birthday. Data shows that
there were more than 30,000 measles cases in the European region in recent
years [4]. According to preliminary WHO data, measles increased by
around 300% globally in the first 3 months of 2019, compared with the
same time last year, with sizable increases in all regions of the world [7].
The reasons for children not getting their vaccines are diverse for different
regions in the world; the major reasons are lack of access to vaccination
services, and with Sub-Saharan Africa region that has the lowest coverage
and the greatest burden of cases. There are about 20 million children in the
world that are still not getting adequate coverage of basic vaccines.
As indicated in European Vaccine Action Plan 2015–2020 (EVAP) [4],
EVAP’s goal is to guide countries in the European region toward their joint
vision of a region free of vaccine-preventable diseases. It establishes six
goals which include sustaining polio-free status, eliminating measles and
rubella, controlling hepatitis B infection, meeting regional vaccination
coverage targets at all administrative levels throughout the region, making
evidence-based decisions on introduction of new vaccines, and achieving
financial sustainability of national immunization programs.

1.2 Vaccine Development Life Cycle


Vaccine development involves many stakeholders and multiple disciplines
including sciences, medicine, public health, regulatory agencies,
manufacturers, healthcare professionals, vaccine safety professionals, and
consumers [8]. In many countries, the government agencies play important
roles in vaccine innovation, development, and commercialization. For
example, in the USA, the National Institutes of Health (NIH) conducts and
supports basic research, translational research, and clinical evaluation to
identify new vaccine targets and to advance new vaccine candidates through
product development pipelines. The regulatory agency US FDA is involved
in vaccine review and licensing, regulatory sciences, manufacturing
inspection, and post-licensure safety monitoring. The US Centers for
Disease Control and Prevention (CDC) identifies, controls, and prevents
infectious diseases through surveillance, detection and response, vaccine
use recommendations, vaccine purchasing and service delivery, health
communications, and post-marketing vaccine safety and effectiveness
monitoring. Vaccines are highly regulated products and require extensive
safety monitoring.
In the USA, vaccines are regulated by the FDA Center for Biologics
Evaluation and Research (CBER) and Office of Vaccines Research and
Review (OVRR), where the authority resides in Section 351 of the US
Public Health Service Act and the Federal Food, Drug, and Cosmetic Act.
CBER conducts thorough review of laboratory, manufacturing, and clinical
data to ensure the safety, efficacy, purity, and potency of the vaccine
products. Figure 2 illustrates the typical process of vaccine development
and licensure.
Fig. 2 Stages of vaccine review and regulation

1.3 Economics of Vaccine Development


Vaccine developments are long, expensive processes with high financial
risks. It may take more than 10 years and more than US$1 billion to
develop a new innovative vaccine. In fact, cost data of developing new
vaccines is usually scarce. According to Dimitrios Gouglas [9], the average
cost of successfully developing an epidemic infectious disease vaccine from
preclinical to Phase 2a is estimated to be US$84–112 million (excluding the
cost of facilities). Substantial investments are needed to develop the
vaccines for these new targets. Innovation is the key to the future
development of new vaccines to combat infectious diseases [10].
Although the cost of vaccine development is high, the benefits of
vaccines are also significant. The development and licensure of
pneumococcal conjugate vaccine (PCV) is a good example. The bacteria S.
pneumoniae is the leading cause of pneumonia mortality globally and
accounted for more deaths than all other causes (etiologies) combined in
2016. Most of these deaths occur in countries in Africa and Asia. Each year
Streptococcus pneumoniae causes approximately 3,300 cases of meningitis,
100,000–135,000 cases of pneumonia requiring hospitalization, and six
million cases of otitis media annually in the USA [11]. Pneumococcal
conjugate vaccine 7-valent (PCV7) was approved in year 2000 for its use in
the USA. It was designed to cover the seven serotypes that account for
about 80% of invasive infections in children younger than 6 years of age. In
2007, the WHO published a position paper recommending all countries to
include PCV as part of the routine infant immunization schedule. PCV7 and
later PCV13 (13-valent pneumococcal conjugate vaccine) have been widely
used in the world. There are 142 countries having put PCV13 into national
immunization programs.
Following the introduction of the pneumococcal conjugate vaccines in
the USA (PCV7 in 2000 and PCV13 in 2010), there are about 90%
reduction of pneumococcal diseases. Invasive pneumococcal disease
decreased from 100 cases per 100,000 people in 1998 to 9 cases per
100,000 in 2015, according to the data published by US CDC [6]. Currently
there are two approved PCV vaccines in the USA, including PCV10
developed and marketed by GSK and PCV13 (Prevnar 13) developed and
marketed by Wyeth Pharmaceuticals (Pfizer).

1.4 Promoting Innovation


Vaccine industries have achieved tremendous successes in the development
of human vaccines that are currently in use. There are more than 200
vaccine clinical trials ongoing with more than 120 vaccine candidates for
more than 40 infectious disease targets [12]. However, for the remaining
targets, there are many significant challenges in developing new vaccines
for these targets. The remaining infectious disease (ID) vaccine targets
include those diseases affecting large populations such as respiratory
syndrome virus (RSV) disease, human immunodeficiency virus (HIV),
malaria, etc. and emerging infectious diseases such as Lassa, Marburg,
Ebola, MERS, and Zika listed in the WHO vaccine pipeline tracking sheet.
As the development of new vaccines against these remaining targets faces
significant challenges, the vaccine policy makers and the industries need to
work together and encourage innovations through collaboration and support
[13, 14].
To continue promote innovations in vaccine research and development,
policy makers and regulatory agencies are recognizing the evolving
development in sciences and medicine and using new approaches in vaccine
review and approval (fast-track approach), as well as in the clinical trial
design (demonstrated in the Ebola clinical trials in Africa during the 2018–
2019 outbreaks) [15].
2 Manufacturing of Vaccines
2.1 General Considerations
Vaccine manufacturing is a complex process. Vaccines take a long time to
manufacture, ranging typically from 6 months to 36 months. During the
manufacturing process, more than 50% of production time is usually
dedicated to the quality control tests. During a vaccine manufacturing
process, several hundred quality control tests may be required before
releasing a batch of vaccine products. The ability to manufacture at the
commercial scale is also very important.
New vaccine commercialization is a complex and costly process [16].
Licensing of a new vaccine is based upon the demonstration of safety and
effectiveness (through clinical evaluation) and the ability to be
manufactured in a consistent manner (through process development and
validation). The critical path toward developing safe and effective vaccines
includes the following:
Speedy development of new technologies
Well-designed and well-operated facilities
Improved manufacturing methods and scale-up
Improved analytical evaluation tools and reference standards
Streamlined preclinical and clinical evaluations
Improved international cooperation
Effective vaccine review and release process
Improved product safety monitoring programs
In the early phases of vaccine development and manufacturing, the
following information are critical for addressing product safety aspects:
Source (biological seed or cell bank) characterization
Raw materials including biologically derived materials (such as cell
culture, media, etc.)
Initial scalable process development
Initial product characterization
Testing/qualification/clearance of impurities, contaminants
Process control especially for safety of the products (e.g., sterilization,
virus clearance where applicable)
During the clinical development stages, the following chemical and
manufacturing control (CMC) activities are important, and gradually
phased-in approaches are usually taken:
Process and product characterization
Formulation development
Raw material qualifications and supply management
Component characterization
Process and analytical method qualification
Specification development
Stability studies
Manufacturing process scale-up and development
Process control and validation
Packaging development and label design
Cold-chain establishment and vendor qualification
During the vaccine development and manufacturing process, applying
current good manufacturing practice (cGMP) is very important to ensure
product safety, efficacy, and consistency. It is highly recommended that
cGMP be in effect for manufacture of products used in clinical studies –
starting from Phase 1. One should follow the general approaches and
principles that are broadly applicable, tailoring cGMP applications to
specific product, process, and facilities by assessing potential risks and
taking appropriate actions (risk-based approach).

2.2 Vaccine Technologies Development


A wide range of technologies have been used in developing successful
vaccines, including:
Live attenuated bacteria and viruses (e.g., BCG, MMR, etc.)
Inactivated bacteria and viruses (e.g., whole-cell pertussis, IPV, etc.)
Proteins (e.g., diphtheria and tetanus toxoids)
Polysaccharides (PneumoVax)
Conjugated polysaccharides (e.g., meningococcal conjugate vaccine,
PCV13)
Viruslike particles (VLPs)
Recombinant proteins
Application of mRNA
Adjuvant development and applications
Many vaccines involve pathogens; thus there are special requirements
for facility and equipment design depending on the biosafety levels. The
manufacturing methods include traditional egg-based influenza
manufacturing or newly developed cell culture-based bioreactor
manufacturing. Traditional egg-based processes use millions of eggs each
year and a large number of manual handlings during manufacturing and
testing. Advances in automation of these steps have greatly enhanced the
process consistency and reduced the potential contamination from human
intervention. Fermentation processes often use stainless steel fermenters for
the manufacturing of many current bacterial vaccines on the markets. Cell
culture-based production processes are used in many viral vaccine products.
The investigational new vaccine candidates often use single-use bioreactors.
The advantages of single-use bioreactors include faster and lower cost of
initial installation, faster development cycle, and quicker delivery of clinical
trial materials. The disadvantages include higher cost of operations
(disposable bioreactors) and potential issues with mechanical strengths in
the joints and ports of the bioreactors.
A typical vaccine manufacturing and product release process include the
following steps:
1.
Culture process: Selected bacterial strains or virus seeds or cells grow
in appropriate culture media through fermentation or cell culture to
generate the desired antigens.
2.
Inactivation: Pathogens from the above culture are usually inactivated
using chemical agents (e.g., formalin) or heat (e.g., 65°C).
3.
Harvesting: Remove cells and cell debris from the product stream.
Antigens are separated from the cells through filtration and/or
centrifugation.
4.
Purification: Impurities are removed from the harvest through
purification methods such as ultrafiltration, chromatography, etc. The
antigens are also concentrated during purification processes.
5.
Detoxification of toxins: The pathogenicity is suppressed, and the
immunogenicity is maintained in the product.
6. Bulk drug substance: The desired antigen is collected and stored
under controlled temperatures for future use.
7.
Formulation: Target antigens are assembled together with excipients
to make final formulation.
8.
Filling process: The final formulation is filled with automated filling
machine under aseptic conditions into glass vials or prefilled syringes
or other packaging containers.

9.
Outer packaging: The filled vials or syringes are packaged into the
secondary containers for future storage and shipping.
10.
QC testing: Quality control laboratories conduct the quality control
tests of the intermediates and the final product.
11.
QA release: Quality assurance confirms that the product has been
manufactured and tested according to approved specifications and
procedures and can be released.
12.
Final release for clinical trials: For clinical trials conducted in EU
region, a qualified person (QP) releases the final product batch into
the clinical trial usage.
13.
Final release for distribution: For commercial vaccine products, many
countries require that the National Regulatory Agency (NRA) release
the final product for distribution into the market, such as in the USA,
European Union, and China.
14.
Product shipping: Most vaccine products currently are stored and
shipped under cold-chain management, typically under 2–8°C, with
very few products shipped under −60°C.
15.
Product monitoring: After the product is released into the markets,
product safety monitoring for serious adverse events (SAEs) is
tracked and reported back to the manufacturers and to the regulatory
agencies accordingly.

2.3 New Trends in Manufacturing of Vaccines


In recent years, there are new trends in the manufacturing of vaccine
products, and these include:
1.
Use of recombinant technology
It is more often that vaccine constructions are derived from recombinant
technology, such as Shingrix vaccine, adenovirus-based Ebola vaccine
(Ad5-EBOV), and rVSV-ZEBOV Ebola vaccine [17, 18]. Recombinant
technology is used in a new recombinant pertussis vaccine development
which provides higher product yield and less impurities.
2.
Single-use technology
In recent years, single-use technology has been used more and more
often in many new vaccine development and manufacturing processes. The
advantages of using single-use technologies in vaccine development and
manufacturing include:
Minimizing potential contamination
No need for equipment cleaning between batches
No need or less requirement for cleaning validation
Less initial capital costs
Fast and easy installation
The disadvantages of single-use technologies include the following:
Need mechanical strength to avoid component breakup.
Installation of testing probes.
Mixing may not be as good as in stainless steel tanks.
Potential leachable and extractable materials from the bags.
Scalability depending on the bioreactor design.
More suitable for viral products than bacterial products.
3.
Continuous manufacturing
Recent advances in disposable manufacturing technologies and process
analytical technologies (PAT) have made the continuous manufacturing
possible. The application of continuous process in antibody manufacturing
has been reported in a number of conferences by companies such as WuXi
Biologics and Amgen. The development of purification technologies has
enabled integration of continuous processes from upstream through to
downstream to final bulk drug substances manufacturing. Application of
continuous manufacturing in vaccines has gained attention from nonprofit
organizations such as Bill and Melinda Gates Foundation. It is reported
(private conversation) that application of continuous manufacturing could
result in a much lower cost of goods. The goal is to make vaccines
affordable to everyone in the world. This will greatly improve the
accessibility and affordability of vaccines in less developed nations,
especially for GAVI (Global Alliance for Vaccines and Immunisation)
countries.
4.
Use of animal-free components
The raw materials and media used for vaccine fermentation and
purification processes are mostly animal component-free to avoid the
potential BSE/TSE risks, especially for final products.

2.4 Vaccine Manufacturing Challenges


One of the big challenges in the manufacturing of vaccine products is that
materials used in Phase 3 clinical trials are typically manufactured in a
commercial-scale facility. Many different technology platforms are used for
various vaccines. It is difficult to standardize facilities and equipment.
Unique facility and equipment are usually necessary for each vaccine or for
each family of vaccines. There is a long lead time for building up a new
commercial-scale manufacturing facility (typically 3–5 years), and large
capital investment is often required.
The successful scale-up requires deep understanding of the processes
and conducting well-designed experiments to complete process scale-up. As
demonstrated in the assessment of safety and immunogenicity of two
different lots of diphtheria, tetanus, pertussis, hepatitis B, and haemophilus
influenzae type b vaccine manufactured using small- and large-scale
manufacturing process, successful scale-up was completed and proved
through clinical trial results [19].
During the manufacturing of biological products including vaccines, it
is often considered that process is product, and the process/product is
tightly linked with clinical experiences and outcomes. Major changes in
manufacturing facilities or manufacturing processes may require regulatory
approval or even conducting additional clinical trials. Thus for vaccine
manufacturing, once a process is confirmed, it is usually not changed unless
there is a strong reason to make a major change. This situation makes
process improvements for existing vaccines challenging.
Another manufacturing challenge lies in the large-scale process
validation. It is an expensive exercise to produce full-scale process
validation lots to meet regulatory filing (e.g., line-specific real-time stability
requirements for fill and finish in the USA). In addition, vaccines are
biological products that are typically with low fill volume but very high
throughput (with million to tens of million doses annually); thus it is
challenging to run product filling lines under aseptic conditions for a long
period of time. Employee training and/or utilizing isolation technology is
important. Moreover, live viral vaccine (such as FluMist, measles vaccine)
or live bacterial vaccine (such as BCG vaccine) requires dedicated fill-and-
finish facility to avoid potential contaminations.
In addition, as current vaccines are typically temperature sensitive, thus
they need cold-chain storage under 2–8°C or even lower temperatures
(−20°C or −60°C). This generates significant challenges for handling in-
process holding and final product storage as well as supply chain
management. Large cold rooms are required in the manufacturing areas to
meet the cold-chain storage requirements. Consistent electrical power
supplies to these cold storage areas are essential to maintain the cold
temperature control all the time. Most vaccine producers maintain
emergency backup power supply in the event of power outages; the power
supply can be switched to the backup power supply quickly.
Another significant challenge is the unpredictability of demand for
seasonal vaccines (e.g., flu vaccines) and vaccines for emergency use (such
as Ebola vaccine). The vaccine manufacturers will need advanced
procurement and significant lead time to prepare raw materials, facilities
and equipment, quality control, and human resources for the production of
these vaccines.

3 Characterization of Vaccines
Vaccines, unlike other pharmaceutical products, are often perceived as
being not well characterized due to their complex structures and properties.
The implications are that Phase 3 clinical trials need to be conducted using
clinical trial materials made in large-scale manufacturing facilities to ensure
the consistency of Phase 3 clinical materials with the commercial products.
The other approach is to conduct clinical bridging studies to demonstrate
equivalence of Phase 3 clinical trial materials with commercial materials.
The potential impact of this requirement is twofold: (1) delay in final
facility readiness and (2) requirement of large capital investment in
facilities much ahead of time. Thus it is very important to perform
characterization of vaccine products as early as possible, to avoid or
minimize costly changes later in the Phase 3 clinical trial stages.
In general, vaccines are very heterogeneous in structure. As greater
characterization of vaccines becomes more prevalent, it may be possible to
connect structural changes in the vaccine components with changes in
potency and toxicity. This, in turn, may provide a better understanding of
how certain vaccines function and interact with the immune system.
Information gained in this area will undoubtedly improve the effectiveness
and safety of future vaccines.
Vaccines can be divided into three major categories: live vaccines,
killed or attenuated vaccines, and component (subunit) vaccines. The
component vaccines are generally the more easily characterized. They
usually consist of a relatively small number of immunogenic components.
The live or killed/attenuated vaccines include complex biological
components such as attenuated or killed viruses and intact bacteria or
multiple bacterial components. Advances in proteomics make the
characterization of even these difficult vaccines more manageable.

3.1 Characterization of Bacterial Seeds


Characterization of vaccine generally involves analysis of bacterial strains,
virus seeds, cell banks, intermediates, bulk drug substance, formulated final
bulk, and finished product. Various physical, chemical, and biological tests
are used for the analyses of these different biological materials. The purpose
of these characterization tests is to ensure product quality and consistency.
For genetically modified bacterial strains used for vaccine
manufacturing, the following tests need to be performed in addition to
generally accepted characterization tests of bacterial strains:
Genotype verification by PCR
Verification of linear plasmid DNA
Gene sequence verification
Verification of flanking sequence
Table 1 is an example of quality tests on a pre-master seed for a
genetically modified bacterial seed (recombinant pertussis vaccine under
development) for GMP material manufacturing.
Table 1 Quality control tests of a genetically modified seed lot

Items Methods Results


Culture LB medium No growth
characteristics culture
Bordet-Gengou Small, round, smooth, convex, silver-gray, opaque colonies and
medium culture without abnormal colonies
Biochemical Glucose No utilization of glucose
tests biochemical
medium
Nitrate peptone No reduction of nitrate
water medium
Urea medium No urease reaction
Citrate medium No use of citrate as a carbon source and nitrogen source
Semisolid No flagellar power
nutrient agar
medium
Morphology Gram stain Gram negative
Serological Serum Serum agglutination: positive
tests agglutination
test
Phenotype ELISA Positive for target antigen
tests
Genotype PCR and (1) The results of the recovered PCR products showed that the
tests restriction restriction enzyme map of the original strain is consistent with
digestion the size of the theoretical sequences. (2) The sequence is identical
to the sequence of target strain
OD600 Culture in flask 3.1

Target protein ELISA >5


expression
(μg/mL)
Items Methods Results
Skin necrosis Intradermal Positive
test injection of
bacterial
suspension
(rabbit model)

3.2 Characterization of Viral Seeds, Cell Banks, and Other


Biological Materials Used in Viral Vaccine Manufacturing
Cell-based processes are used in viral vaccine development and
manufacturing. The characterization of both viral seeds and cell lines is
required. In addition, biological raw materials used in manufacturing
processes should also be characterized with clear source of origin with
potential risks of introduction of adventitious agents or viruses into the
process and product stream. For viral product, US FDA guidance
“Characterization and Qualification of Cell Substrates and Other Biological
Materials Used in the Production of Viral Vaccines for Infectious Disease
Indications” has clearly outlined the requirements for characterization of
cell substrates, cell banking, viral seeds, vaccine intermediates, and
biological raw materials at different stages of vaccine development [20].
For viral subunit vaccine product manufacturing, the most important
factor is to prevent contaminations from adventitious materials and other
contaminants in all stages of processes. It is important to demonstrate viral
clearance through process validation.
However, live attenuated viruses, whole inactivated virus, or viruslike
particles often cannot be purified as rigorously as viral subunit vaccines.
Unlike the production of protein products, it is not possible to introduce
validated viral inactivation or removal steps to mitigate these risks.
Addressing these issues requires application of GMP principles and
approaches in development programs at a very early stage, focusing on the
history, purity, and stability of the viral seeds, cell banks, and biological raw
materials.
For viral vector-based vaccine development, it is now common for a
viral vector to be rescued from synthesized plasmids, allowing for
traceability of the viral vector and full sequencing to be performed of the
plasmids and resulting viral vector. Studies can also be performed to
demonstrate genetic stability of vectors at an early stage of development.
For manufacturing cell lines, it is essential that the origins are known with
clear history and that they are free from adventitious agents and that
generation of cell banks for both process development and production meet
GMP requirements. In addition, it is very important to maintain segregation
in the processes throughout development programs to prevent potential
contamination of viral stocks and cell banks.
Although the production of viral vectors poses a number of technical
challenges in cell culture, recovery, characterization, and analytical
perspective, but with recombinant viral vector systems, where the virus is
essentially used as a delivery vehicle and the manufacturing approach is
independent of the genes it carries, a platform process can be developed.
Therefore, the application of a platform can be developed for the production
of those vaccines based on viral vectors, such as adenovirus, adeno-
associated virus (AAV), and lentivirus.
For cell substrates (cell lines) intended for vaccine manufacturing, the
following characterization tests or documentation needs to be provided:
Cell substrate properties such as plasmid sequence, phenotype, and
expression of antigens
Source of the cell line including species of origin and the tissue type
Donor’s medical history and the results of tests performed on the donor
for the detection of adventitious agents
Culture history of the cell line, including methods used for the isolation
of the tissues from which the line was derived
Passage history, medium used, and history of passage in animals
Documentation of the history of human-derived and animal-derived
materials used during passage of the cells
Documentation of any genetic material introduced into the cell substrate
Identity test, cytogenetic characteristics
Results of all available adventitious agent testing
Growth characteristics
Expression characteristics
Susceptibility to adventitious agents
Generation of cell substrate
Long-term storage conditions
Stability of cell lines
Based on the above thorough understanding of the cell substrates, the
cell banking systems with primary cell bank (PCB), master cell bank
(MCB), and working cell bank (WCB) can be established.
The passage history and derivation history of viral seeds intended for
vaccine manufacturing should also be well documented, including:
Sourcing of each biological starting material (e.g., plasmids, parental
viruses)
Donor screening, testing, and donor medical history
Any manipulation of the viral phenotype, such as cold adaptation and
development of temperature sensitivity
Any attenuation of virulence and genetic manipulations such as
reassortment or recombination
Nucleic acid sequences
Growth characteristics on production cell substrate
Genetic markers
Long-term storage conditions
Viability during storage
Genetic stability through production
Absence of adventitious agents
Other biological raw materials such as serum and other biologically
sourced raw materials should also be controlled to prevent adventitious
material contaminations.

3.3 Advances in Vaccine Characterization


The continuous development of safe, effective, and innovative vaccines
around the world calls for new technologies, not only in the vaccine
discovery areas but also the new technologies for characterization of
vaccines.
The traditional methods of vaccine characterization rely on the study of
physical-chemical properties using methods such as differential scanning
colorimetry (DSC) and thermogravimetric analysis (TGA), pH, various
stress conditions (agitation, freeze-thaw, etc.) based on particulate
formation, and methods of quantitating protein content as well as elemental
composition. While these methods are capable of determining whether or
not the end product is consistent with previous batches, they are unable to
detect small changes that can result in a vaccine with reduced or even lost
immunogenicity. Not all changes to the structure of the vaccine components
have physical consequences, but many of them result in reduced vaccine
effectiveness. Most of these techniques lack sensitivity when it comes to
detecting small changes in the structure of the vaccine components that can
cause them to fail during use. Some changes can cause severe side reactions
even in small quantities.
TGA and DSC are used to analyze the denaturing point of the vaccine
protein or nucleotide. These tests generally give indirect indications of
changes in the vaccine with time and stress. Changes in protein sequence or
modifications can substantially affect denaturing kinetics, but these
techniques provide no way to correlate these changes with actual changes in
the structure of the molecule.
Appearance and pH are used to monitor major changes in the
composition of the vaccines and are relatively insensitive to these changes.
Other physical characteristics that affect vaccine function include particle
size and particle size distribution. Clumping of the vaccine antigen can
degrade the function of the vaccine and can cause unwanted side effects.
Specific tests for quantitating proteins or oligonucleotides, such as
elemental analysis and total protein content (bicinchoninic acid or BCA)
tests, can provide vital quality control data for troubleshooting
manufacturing problems, but they are of limited value in analyzing
degradation of the vaccines since elemental composition changes from
degrades represent only a small percentage of the overall elemental
composition. In addition, most protein degrades will still be identified as
proteins in a total protein analysis. The conditions used for these assays also
break up clumped proteins or oligonucleotides and are insensitive to most
changes caused by minor structural modifications of these molecules.
Besides the traditional physical and chemical tests for vaccines,
advanced technologies such as high-resolution mass spectrometry (MS) and
nuclear magnetic resonance (NMR) spectroscopy, chromatography, and
polymerase chain reaction (PCR) are used in the new vaccine development.
ICP-MS, GC-MS, HPLC-MS, etc. are the examples of MS used for vaccine
characterization. ICP-MS can be used for quantitatively measurement of
metals in the vaccine intermediates and finished product. GC-MS and
HPLC-MS can be used to determine the molecular weight of vaccines and
detect if there are changes in the molecular structure. The mass
spectrometry method can detect the structure change of functional proteins
and lipids in vaccine with a high degree of accuracy, which is often linked
with vaccine immunogenicity. Other biophysical tools such as florescence
spectroscopy, light-scattering spectroscopy, etc. are also used for
macromolecule characterization.

4 Clinical Aspects of Vaccine Development


Like other pharmaceutical products, the development of new vaccines will
include clinical testing phases. Through well-controlled clinical trials, data
will be collected to demonstrate that the vaccine product has an effect on
clinical endpoint or a surrogate endpoint that is reasonably likely, based on
clinical, serologic, epidemiologic, therapeutic, pathophysiologic, or other
evidence, to provide clinical benefits.

4.1 Design of Clinical Trials for Vaccines


Generally, clinical trials for vaccines include clinical trials from Phase 1 to
Phase 3 and Phase 4 post-approval. Phase 1 clinical trial is to test the initial
safety and tolerability of the candidate vaccine; Phase 2 clinical trials test
the safety, immunogenicity, and dose ranges; and Phase 3 clinical trials are
for additional safety data, immunogenicity, and efficacy.
Prior to initiating Phase 3 clinical trials, it is recommended that the
vaccine developer has continuous dialogue with regulatory agencies to
discuss study details such as disease prevention or treatment; study sites;
subject selection; choice of control group; trial design including endpoints,
case definitions, diagnostic tests, dose selection, dosing schedule, study
duration, concomitant vaccinations, and medications; and safety
assessments to ensure that they meet the intended goals for clinical trial and
product licensure. Vaccines are approved for licensure from regulatory
agencies if clinical trial data show that the product is safe, pure, and potent
and that the manufacturing facility meets the standards designed to assure
that the vaccine product continues to be safe, pure, effective, and non-
inferior when administered with concomitant vaccines.
From regulatory point of view, the proof of effectiveness of a vaccine
would consist of controlled clinical investigations that are adequate and
well-controlled studies, unless waived as not applicable to the vaccine
product or when an alternative method is adequate to substantiate
effectiveness, such as using serological response data where a previously
accepted correlation with clinical effectiveness exists [21].
The effectiveness may be proven by a well-designed, single clinical
study. In the case of preventive vaccines, one adequate and well-controlled
clinical trial may be supported by compelling animal challenge/protection
models, human serological data, passive antibody data, or pathogenesis
information.

4.2 Consistency Lots


Phase 3 vaccine clinical trials usually use the same scale of manufacturing
and make clinical trial materials in the intended commercial manufacturing
facilities. This requirement has made it challenging for the vaccine
developers, as the facilities need to be ready long before the vaccine can be
approved for commercial launch.
Unlike other therapeutic biological products, the efficacy of Phase 3
clinical trials for vaccines usually needs to demonstrate the protection of
healthy people from a particular disease in the target population. Thus
depending on the disease burden of a particular candidate vaccine, the
Phase 3 efficacy clinical trial can be very large, enrolling several thousand
or more healthy volunteers. For example, the recently approved Shingrix
vaccine had Phase 3 clinical trials with more than 17,000 people enrolled.
Thus Phase 3 clinical trials for preventive vaccine are usually very lengthy
and costly. It is estimated that the cost of Phase 3 clinical trials of
preventive vaccines may exceed US$100 million [9].

4.3 Vaccine Immunogenicity


Vaccine immunogenicity is often tested in the clinical trials. For example,
during the clinical trials conducted for Ad5-EBOV Ebola virus disease
vaccine, the Ebola-specific antibody responses against the vaccine-matched
2014 Zaire-Makona glycoprotein (GP) were assessed with enzyme-linked
immunosorbent assay (ELISA) method, and anti-adenovirus type-5
neutralizing antibody titers were detected with a serum neutralization assay
before and after vaccination. GP-specific IgG titers are the important data
for the immune response of the vaccine. Anti-Ad5 neutralizing antibody
detection was used to analyze the vector immunity and the preexisting
immunity influence on the vaccine’s immunogenicity. Specific T-cell
response was quantified by enzyme-linked immunospot (ELISpot) assay
(IL-2, IFN-γ, and TNF-α), and IFN-γ, tumor necrosis factor-α (TNF-α), and
interleukin-2 (IL-2) in peripheral blood mononuclear cells when phase I and
phase Ib clinial trials were conducted in China. Another phase 2 clinical
trial was conducted in Sierra Leone in 2015 [22–24]. The study results
demonstrated that one shot of intramuscular injection with Ad5-EBOV
vaccine could elicit strong humoral and cellular immune response in three
clinical trials. Ad5-EBOV vaccine produced a considerable GP antibody
response and GP-specific T-cell response in healthy African participants in
China after 14 days [22]. GP antibody response peaked at day 28 and lasted
more than 6 months. With a booster at the sixth month, the GP antibody
response can last more than 12 months. The pre-exsiting immunity to Ad5
can be overcome with selected vaccine dosage.
Another example is Shingrix vaccine. Shingrix is a recombinant,
subunit vaccine designed to restore VZV immunity in individuals who are
at increased risk of developing Shingles due to age or immunodeficiency
[25]. VZV gE is the most abundant envelope glycoprotein, predominantly
expressed on the surface of virus-infected cells. The VZV gE protein plays
a critical role in virus infectivity since it is involved in virus entry and cell-
to-cell spread, harboring sites for N- and O-linked glycosylation. The
subunit vaccine elicits stronger virus-specific CD4+ T-cell response as well
as antibody B-cell response to gE, compared to the currently used live
attenuated vaccine (Zostavax®). Evidence indicates that the gE protein
induces both neutralizing antibodies and T-cell responses. The gE antigen
component of Shingrix is derived from a VZV strain that was isolated from
a patient with severe varicella disease. This antigen is a recombinant
truncated form of VZV gE. The recombinant protein is then produced in
Chinese hamster ovary (CHO) cells that were genetically modified to
express the VZV gE gene [26].
There are many vaccine clinical trials ongoing for various vaccine
candidates, and these are published in the website either at clinicaltrial.gov
or at clinicaltrialsregister.eu.

4.4 Combination Vaccines


A combination vaccine consists of two or more live organisms, inactivated
organisms, or purified antigens either combined by the manufacturer as a
single product or mixed immediately before administration. It is intended to
prevent multiple diseases or to prevent one disease caused by different
strains or serotypes of the same organism. Meningococcal conjugated
vaccine MCV (A, C, Y, W135), DTcP, and Prevnar 13 are good examples of
combination vaccines.
The clinical studies are designed to demonstrate the safety,
immunogenicity, and efficacy of combination vaccines through randomized,
controlled studies. The efficacy of each component should be demonstrated
in clinical studies. Ideally, clinical trials will be prospective, randomized,
and controlled. Endpoints used to evaluate efficacy in these trials can range
from disease incidence to a well-established correlation of protection [27].

4.5 Special Populations


Vaccine development and clinical trials generally take stepwise approaches
starting from adults to children. For routine vaccines aiming for pediatric
applications, clinical trials in children’s population groups are normally
required, such as meningococcal vaccine, PCV vaccine, DTcP vaccine, etc.
However, for some global infectious diseases such as malaria or Ebola virus
disease, children suffer the same or even greater risks as their immune
systems are not as strong as those of adults. It was reported that during the
Ebola outbreaks in the Democratic Republic of the Congo, about one third
of the reported cases were from children or adolescents under 18 years of
age [28]. Thus it is important to include children and adolescents in the
vaccine development programs for global infectious diseases unless a
waiver is granted by the regulatory agencies. In some cases, if the course of
the disease and the effects of the drug are sufficiently similar in adults and
pediatric patients, the regulators may conclude that pediatric effectiveness
can be extrapolated from adequate and well-controlled studies in adults,
supplemented with other information obtained in pediatric subjects, such as
immune response studies, as pointed out in the US FDA guidance [29].
For vaccine clinical trials including pregnant women in the population
groups, reproductive toxicity studies need to be completed prior to
including them.

4.6 Accelerated Approval


For certain global infectious diseases such as tuberculosis, malaria, and
human immunodeficiency virus/acquired immunodeficiency syndrome
(AIDS) that are serious and/or life-threatening, accelerated approval may be
granted using a surrogate endpoint or a clinical endpoint other than survival
or irreversible morbidity for a vaccine that provides meaningful clinical
benefits for preventing the spread of infectious diseases or therapeutic
benefits to patients over existing treatments [29].
For some special vaccine products, approval may be granted based on
evidence of effectiveness from studies in animals when human efficacy
studies are not ethical or feasible [30]. In such cases, after approval, a
sponsor must conduct post-marketing studies, such as field studies, to verify
and describe the biological product’s clinical benefit and to assess its safety
when used as indicated in circumstances where such studies are feasible and
ethical. One example of applying “animal rule” is the approval of anthrax
vaccine by the US FDA [31].

5 Overview of Recent Approved Vaccines


In recent years, several new vaccines are approved in different countries
around the world. Brief descriptions are given in the following sections.

5.1 Shingrix Vaccine


Shingles causes painful rash of fluid-filled blisters and sometimes causes
chronic pain. Shingles is a virus that results from reactivation of the
varicella zoster virus (type 3 herpes zoster), the virus that causes chicken
pox. Chicken pox is the initial infection, and shingles is the reactivation of
the virus years later. Shingles may be developed at any age but most
common for people aged over 50 years. Shingles causes substantial pain
that can interfere with activities of daily living and reduce the quality of
life. The estimated average overall incidence of shingles is about 3.4–4.8
per 1,000 person years which increases to more than 11 per 1,000 person
years in those aged over 80 years in Europe, according to Robert W.
Johnson’s study [32]. Shingles is the third most common cause of chronic
neuropathic pain in the USA, with an estimate of approximately 500,000
cases yearly.
Vaccination is an effective way to reduce the incidence of shingles. Two
vaccines against shingles have been approved by the US FDA. Merck
developed zoster vaccine live (ZVL, Zostavax), and it has been in use since
2006 for people 60 years and older.
The second vaccine for shingles has been developed by GSK Company.
Shingrix is a recombinant zoster vaccine with adjuvant and is indicated for
prevention of herpes zoster (shingles) in adults aged 50 years and older. It
has two doses; the second dose is administered 2–6 months after the first
dose [33].
Since its approval in 2017, Shingrix has obtained tremendous success in
marketplace in the USA and internationally, as the sales of Shingrix
exceeded US$1 billion in 2018. It is recommended by ACIP as the
preferred shingles vaccine for people aged 50 years and older.
Shingrix is a suspension for injection supplied as a single-dose vial of
lyophilized varicella zoster virus glycoprotein E (gE) antigen component
reconstituted with the accompanying vial of AS01B adjuvant suspension
component. After reconstitution, a single dose of Shingrix is 0.5 mL. The
gE antigen is obtained by culturing genetically engineered Chinese hamster
ovary (CHO) cells, which carry a truncated gE gene, in media containing
amino acids, with no albumin, antibiotics, or animal-derived proteins. The
gE protein is purified by several chromatographic steps, formulated with
excipients, filled into vials, and lyophilized. The adjuvant suspension
component is AS01B, which is composed of 3-O-desacyl-4′-
monophosphoryl lipid A (MPL) from Salmonella minnesota and QS-21, a
saponin purified from plant extract Quillaja saponaria Molina, combined in
a liposomal formulation. The liposomes are composed of dioleoyl
phosphatidylcholine (DOPC) and cholesterol in phosphate-buffered saline
solution containing disodium phosphate anhydrous, potassium dihydrogen
phosphate, sodium chloride, and water for injection. After reconstitution,
each 0.5 mL dose is formulated to contain 50 μg of the recombinant gE
antigen, 50 μg of MPL, and 50 μg of QS-21. Each dose also contains 20 mg
of sucrose (as stabilizer), 4.385 mg of sodium chloride, 1 mg of DOPC,
0.54 mg of potassium dihydrogen phosphate, 0.25 mg of cholesterol,
0.160 mg of sodium dihydrogen phosphate dihydrate, 0.15 mg of disodium
phosphate anhydrous, 0.116 mg of dipotassium phosphate, and 0.08 mg of
polysorbate 80. After reconstitution, Shingrix is a sterile, opalescent, and
colorless to pale brownish liquid. Shingrix does not contain preservatives.
Each dose may also contain residual amounts of host cell proteins (≤3.0%)
and DNA (≤2.1 picogram) from the manufacturing process [33].
Shingrix has gone through multiple clinical trials. Overall, 17,041 adults
aged 50 years and older received at least 1 dose of Shingrix in 17 clinical
studies. The safety of Shingrix was evaluated by pooling data from 2
placebo-controlled clinical studies (Studies 1 and 2) involving 29,305
subjects aged 50 years and older who received at least 1 dose of Shingrix
(n = 14,645) or saline placebo (n = 14,660) administered according to a 0-
and 2-month schedule. Both studies were conducted in North America,
Latin America, Europe, Asia, and Australia. In the overall 4 population, the
majority of subjects were white (74.3%), followed by Asian (18.3%), black
(1.4%), and other racial/ethnic groups (6.0%), and 58% were female. The
safety profile was acceptable. Phase 3 clinical trials demonstrated that
Shingrix has been shown to be highly efficacious with VE against shingles
of 97.2% in subjects over 50 years of age and 91.3% in subjects over
70 years of age [34].
Shingrix is currently under significant shortage in the USA and around
the world. This supply issue is a common problem with many successful
vaccines. When the products do well because of their effectiveness, vaccine
developers are struggling to supply. It is good to address these supply issues
and how to mitigate it ahead of time. GSK announced in April 2019 that it
plans to expand its manufacturing facility in Montana with a capital
investment of US$100 million.

5.2 Ebola Vaccine: Ad5-EBOV


Ebola viruses (EBOVs) are enveloped, non-segmented, negative-stranded
RNA viruses belonging to the family Filoviridae. They are known to cause
lethal hemorrhagic fever in humans and nonhuman primates with a
mortality rate of 40–90% [35, 36]. Ebola virus disease (EVD), formerly
known as Ebola hemorrhagic fever, is a severe, often fatal illness in
humans. EBOVs are transmitted among humans through close contact with
infected blood, bodily fluids, or tissues; moreover, the intentional release of
EBOVs would probably result in mucosal infection by small-particle
aerosol dispersion.
From 1976 when the Ebola virus was first discovered to June 2019,
there have been more than 30 epidemic outbreaks in Africa causing tens of
thousands of deaths. The species of Ebola viruses were mainly Zaire and
Sultan types, and the outbreak sites were concentrated in Central Africa, in
countries such as Congo, Sultan, Uganda, and South Africa. The 2014
outbreak in West Africa has been one of the worst Ebola epidemics. On
August 8, 2014, the WHO director general declared this outbreak a Public
Health Emergency of International Concern [37].
Many methods have been used to develop EBOV vaccines. Some of the
vaccine candidates have been tested in nonhuman primates (NHP) and
showed good protection. These vaccine candidates include inactivated
vaccine, subunit vaccine, non-replicated virus vector vaccine, and replicated
virus vector vaccine. Most of them were developed to protect against Zaire
ebolavirus. In the past a few years, the protective mechanism of vaccine has
also been studied, and the results showed that the antibody protection is
essential.
A recombinant Ebola virus disease vaccine (Ad5-EBOV) has been
successfully developed jointly by Beijing Institute of Biotechnology and
CanSino Biologics Inc. (CanSinoBIO). The preclinical research of the
recombinant Ebola virus disease vaccine (adenovirus type-5 vector) (Ad5-
EBOV) was initiated in 2006. The key technology in vaccine preparation
and evaluation was based on the Ebola GP antigen. A significant gene
sequence variation of the GP antigen in the Zaire Ebola virus strain has
been identified in the 2014 Ebola epidemic outbreaks in West Africa. To
ensure the effectiveness of the vaccine, Ad5-EBOV vaccine was designed
according to the 2014 Ebola virus genotype. The vaccine candidate was
tested in animal models to confirm the immunogenicity, safety, and efficacy.
Challenge studies in guinea pigs and NHPs (cynomolgus monkeys)
conducted in biosafety level 4 (BL-4) lab in Public Health Agency of
Canada also confirmed that the vaccine candidate is 100% effective in
protecting guinea pigs and NHPs from Ebola virus infection.
CanSinoBIO started the development of recombinant Ebola virus
disease vaccine at the end of 2014 as the Ebola virus disease outbreaks
occurred in West Africa. The HEK293 cell line used for Ad5-EBOV
production was licensed from National Research Council (NRC), Canada.
The production process has been scaled up successfully. All key materials,
intermediates, and final product (including virus seeds, cell banks, purified
bulk, and final product) are QC tested and released internally. The entire
production process for EBOV vaccine is animal component-free. The
process of large-scale production of adenoviruses has been well
characterized and optimized. Several vaccines and biological products have
been developed using the Ad5 vector-based technology [38]. The platform
technology applied to Ad5 vector-based production has been demonstrated
by Ad5-EBOV to be robust and scalable and with high productivity.
In 2015, CanSino obtained a clinical trial permit from the Chinese Food
Drug Administration (NMPA, formerly CFDA) and from the Government
of Sierra Leone. The Phase 1 and Phase 2 clinical trials conducted in China
and Sierra Leone involved a total of 681 subjects, and the clinical trial
results indicated that the Ad5-EBOV vaccine is safe and immunogenic. The
immune response level in human is comparable with that of rVZV-ZEBOV
vaccine developed by Merck. Clinical trial results demonstrated that Ad5-
EBOV is well tolerated with good safety profile in tested subjects between
ages 18 and 60. The GMTs of anti-GP antibody peaked around 28 days after
vaccination regardless of the dose levels. Satisfactory immune response can
be reached at dosage of 8 × 1010 VP per dose. Pre-existing immunity to
Ad5 vector can be overcome by proper dose selection (8 × 1010 VP/dose),
and the conversion rate is 100%. Ad5-EBOV response is fast and long-
lasting, and these features could offer help in Ebola virus disease outbreak
situation.
In October 2017, the new drug registration application (NDA) for
recombinant Ebola virus vaccine (adenovirus type-5 vector) was approved
by the Chinese Food and Drug Administration (CFDA). The manufacturing
facilities, including QC testing center, are fully validated and in operation at
CanSinoBIO.

5.3 Meningococcal Subgroup B Vaccine (MenB)


Currently approved meningococcal ACWY conjugate vaccine
(MenACYW135) is given to preteens and teens at age 11 or 12 years with a
booster dose at 16 years to protect against serotypes A, C, Y, and W135. In
2014–2015, another meningococcal subgroup B vaccine (Bexsero and
Trumenba) [39, 40] were approved for adolescent and adult population aged
10–25 years who are at increased risk of meningococcal B diseases.
Bexsero is an FDA-approved vaccine to prevent invasive disease caused by
Neisseria meningitidis serogroup. It is not recommended for routine
vaccination at this point.
In fact, meningitis subgroup B infection is very rare in the USA. Among
all 11–23-year-old adolescents and young adults in the USA, between 50
and 60 cases are reported annually, with 5–10 deaths. According to the
CDC, there were 7 outbreaks on college campuses from 2009 to 2013, with
41 cases and 3 deaths [41].
Bexsero was developed by Novartis and is now marketed by the GSK
Company. It is a sterile suspension of three recombinant proteins and
meningococcal outer membrane vesicles (OMV). The recombinant proteins
neisserial adhesin A (NadA), neisserial heparin binding antigen (NHBA),
and factor H binding protein (fHbp) are individually produced in
Escherichia coli and purified. The OMV component is produced from N.
meningitidis strain NZ98/254 (expressing outer membrane protein P or A
serosubtype P1.4). The antigens are adsorbed onto aluminum hydroxide.
Each 0.5 mL dose of Bexsero is formulated to contain 50 μg each of
recombinant proteins NadA, NHBA, and fHbp, 25 μg of OMV, 1.5 mg
aluminum hydroxide, 3.125 mg sodium chloride, 0.776 mg histidine, and
10 mg sucrose at pH 6.4–6.7 according to the product insert [39]. Bexsero
is a suspension for intramuscular injection in 0.5 mL single-dose prefilled
syringes. Two doses are given at least 1 month apart from the first dose.
Another approved meningococcal group B vaccine is Trumenba®
developed by Pfizer, initially approved in 2014. Trumenba is a bivalent
meningococcal group B vaccine that contains two factor H binding proteins
(fHBP) from Neisseria meningitidis (N. meningitidis) serogroup B. fHBP is
a conserved, outer membrane lipoprotein and a virulence factor that
contributes to the ability of the bacteria to avoid host defenses.
Trumenba is a sterile suspension of two recombinant lipidated factor H
binding protein (fHBP) variants, one from each of the two antigenically
distinct fHBP subfamilies subfamily A and subfamily B (A05 and B01,
respectively). These proteins are also known as rLP2086 proteins. The
proteins are individually produced in Escherichia coli and subsequently
purified. Each 0.5 mL dose of Trumenba is formulated to contain 60 μg of
each fHBP variant subtype (120 μg total protein), 0.018 mg of polysorbate
80, and 0.25 mg of Al3+ as AlPO4 in 10 mM histidine-buffered saline at
pH 6.0, according to the product insert [40]. Trumenba is approved for use
in individuals from 10 through 25 years of age.
Later in 2017, Trumenba vaccine was approved for three doses schedule
(a dose administered at 0, 1–2, and 6 months) by US FDA. Additional
clinical trial results also demonstrated that it is safe to co-administer
Trumenba vaccine with meningococcal (groups A, C, Y, and W135)
polysaccharide diphtheria toxoid conjugate vaccine and tetanus toxoid,
reduced diphtheria toxoid, and acellular pertussis vaccine, adsorbed in
persons 10 years to less than 13 years of age.
5.4 HPV Vaccine
Human papillomavirus (HPV) is a sexually transmitted virus. It is passed
through genital contact or by skin-to-skin contact. HPV infection causes
benign and malignant dysplastic anogenital disease in men and women.
Nearly 100% of cervical cancers and 90% of anal cancers are caused by
oncogenic HPV types.
GARDASIL 9, human papillomavirus 9-valent recombinant vaccine,
was developed by Merck and initially approved by US FDA in 2014. Prior
to the licensure of GARDASIL 9, Merck’s 4-valent HPV vaccine,
GARDASIL, was licensed in 2006. GARDASIL protects against disease
caused by HPV types 6, 11, 16, and 18. GARDASIL 9 includes the original
four HPV types in GARDASIL, plus an additional five types, HPV 31, 33,
45, 52, and 58. GARDASIL 9 is indicated in girls and women aged 9–
26 years for the prevention of cervical, vulvar, vaginal, and anal cancer
caused by human papillomavirus (HPV) types 16, 18, 31, 33, 45, 52, and 58
and genital warts (condyloma acuminata) caused by HPV types 6 and 11.
GARDASIL 9 is also indicated in boys and men aged 9–26 years for the
prevention of anal cancer caused by HPV types 16, 18, 31, 33, 45, 52, and
58 and genital warts (condyloma acuminata) caused by HPV types 6 and
11.
A 0.5 mL dose of GARDASIL 9 contains recombinant viruslike
particles (VLPs) of the major capsid (L1) protein of HPV types 6, 11, 16,
18, 31, 33, 45, 52, and 58 adsorbed on preformed aluminum-containing
adjuvant (amorphous aluminum hydroxyphosphate sulfate or AAHS). The
amounts of HPV type L1 protein in each dose are as follows:
30 μg/40 μg/60 μg/40 μg/20 μg/20 μg/20 μg/20 μg/20 μg, respectively. It is
available as a suspension in 0.5 mL single-dose vials or prefilled syringes,
for intramuscular administration in two doses at months 0 and 2–6 month or
three doses at months 0, 2, and 6 according to product insert [42].

5.5 Dengue Vaccine


Dengue infection is caused by dengue virus which includes four known
serotypes (dengue virus 1, 2, 3, and 4), all transmitted primarily by Aedes
aegypti mosquitos, as well as other members of the Aedes mosquito family.
Annually, an estimated 390 million dengue infections occur worldwide, of
which approximately 100 million are associated with clinical
manifestations, 500,000 with hospitalization, and 20,000 with death [43].
Dengue disease is a major public health concern in more than 128 countries.
It is endemic in Asia, the Pacific area, Africa, and Latin America with the
four dengue virus serotypes found in tropical and subtropical regions,
including some European territories. Dengue is endemic in the US
territories of American Samoa, Guam, Puerto Rico, and the US Virgin
Islands [44–46].
A dengue tetravalent vaccine is developed by Sanofi Pasteur Inc. and is
approved by US FDA in May 2019. DENGVAXIA is a live, attenuated,
tetravalent, chimeric virus vaccine, containing the replication genes and the
capsid gene from the attenuated yellow fever [17D] virus and the Pre-M
and ENV genes from each of the four serotypes (CYD). Each CYD virus is
purified from Vero cells.
The indication of DENGVAXIA vaccine is for the prevention of dengue
disease caused by dengue virus serotypes 1, 2, 3, and 4 in individuals 9
through 16 years of age with laboratory-confirmed previous dengue
infection and living in endemic areas. Previous dengue infection can be
assessed through a medical record of a previous laboratory-confirmed
dengue infection or through current serotesting. The safety and
effectiveness of the vaccine were determined in three randomized, placebo-
controlled studies involving approximately 35,000 individuals in dengue-
endemic areas, including Puerto Rico, Latin America, and the Asia-Pacific
region. The vaccine was approximately 76% effective in preventing
symptomatic, laboratory-confirmed dengue disease in population 9 through
16 years of age who previously had laboratory-confirmed dengue disease.
DENGVAXIA has already been approved in 19 countries and has been
approved by the European Union.
DENGVAXIA is supplied as a vial of lyophilized powder containing
each of the four virus components that is reconstituted at the time of use
with the supplied sodium chloride diluent (0.4% NaCl). After
reconstitution, each 0.5 mL dose of DENGVAXIA is formulated to contain
4.5–6.0 log10 CCID50 of each of the CYD virus components. The
reconstituted vaccine is administered subcutaneously in three doses at 6-
month intervals (at day 0, month 6, and month 12) according to product
insert [47].

5.6 EV71 Vaccine


Enterovirus 71 (EV71) is one of the major causative agents of outbreaks of
hand, foot, and mouth disease or herpangina in Asia. An EV71 (Enterovirus
71) vaccine has been developed by Sinovac Biotech (China) using Vero cell
and EV71 C4 subgenotype. The Phase 2 study of inactivated vaccine (Vero
cell) against EV71 virus has been completed in December 2011 in China.
The purpose of the Phase 2 clinical study was to demonstrate the safety and
immunogenicity of EV71 vaccine in preventing hand, foot, and mouth
disease caused by EV71 in a total of 10,000 healthy infant volunteers aged
from 6 to 35 months old. The data from Phase 1 and 2 clinical studies
suggested that the inactivated EV71 vaccine had clinically acceptable safety
and good immunogenicity for healthy Chinese infants. A Phase 3 clinical
trial was conducted in China in 2014 with 10,007 healthy infants and young
children (6–35 months of age). The vaccine efficacy against EV71-
associated hand, foot, and mouth disease or herpangina was 94.8%. Vaccine
efficacies against EV71-associated hospitalization and hand, foot, and
mouth disease with neurologic complications were both 100%. In the
immunogenicity subgroup (1,291 children), an anti-EV71 immune response
was elicited by the two-dose vaccine series in 98.8% of participants at day
56. An anti-EV71 neutralizing antibody titer of 1:16 was associated with
protection against EV71-associated hand, foot, and mouth disease or
herpangina [48–55]. EV71 vaccine was approved by the Chinese Drug
Administration (formerly CFDA) in 2017.

5.7 HBV Recombinant Vaccine


Hepatitis B virus infection is a serious public health issue. More than 250
million persons are infected with hepatitis B virus (HBV) worldwide.
Approximately 887,000 deaths worldwide were reported in 2015, mostly
due to chronic hepatitis B and resultant end-stage liver disease and/or
hepatocellular carcinoma [56, 57].
A new HBV vaccine, recombinant, with adjuvant (Heplisav-B) has been
developed by Dynavax Technologies Corporation for preventing hepatitis B
virus infections. The product contains 20 μg recombinant hepatitis B
surface antigen with 3,000 μg adjuvant 1018, which is a novel cytosine
phosphoguanine (CpG)-enriched oligodeoxynucleotide (ODN)
phosphorothioate adjuvant. The indication and usage are for immunization
against infection caused by all known subtypes of hepatitis B virus in adults
18 years of age and older. The product Heplisav-B (rHBsAg-1018 ISS),
recombinant hepatitis B surface antigen (rHBsAg), subtype adw, is
produced in yeast cells. The dosage contains two 0.5 mL doses
administered 4 weeks apart.
Heplisav-B is supplied as single-use vials of 0.5 mL volume. Each
0.5 mL dose contains 20 μg HBsAg, 3,000 μg CpG 1018 adjuvant, 8 mM
sodium phosphate, 154 mM sodium chloride, 0.01% w/w polysorbate 80,
and pH 7.0 buffer. The vaccine does not contain preservatives. The shelf
life of the final container product is 36 months at 5 ± 3°C from the date of
manufacture according to the product insert [58–60].
The Heplisav-B vaccine was approved by the US FDA in 2017. This
was the first hepatitis B vaccine approved in the USA for the last 25 years.

6 Vaccines Under Development


Many vaccines are under development around the world; below are a few
examples of those vaccines under development.

6.1 rVSV-ZEBOV Ebola Vaccine


Although rVSV-EBOV has not been officially approved for licensing yet, it
is in the process of applying licensure both with US FDA and with EMA in
Europe. It has obtained fast-track review and breakthrough status. It has
completed Phase 3 clinical trials and demonstrated its safety and efficacy
[61].
In July 2015, clinical results of the Ebola ça Suffit ring vaccination
Phase 3 cluster-randomized trial of the rVSV-ZEBOV vaccine in Guinea
were obtained and published in Lancet. On the basis of interim analysis, the
trial showed 100% vaccine efficacy, with 75.1% vaccine effectiveness at the
cluster level, including herd immunity of unvaccinated members of clusters.
In March 2016, an rVSV-ZEBOV expanded access and compassionate
use trial was conducted, named as “Ring vaccination with rVSV-ZEBOV
under expanded access in response to an outbreak of Ebola virus disease in
Guinea,” and the interim results were published in Lancet. The ring
vaccination strategy was used between Ebola and the contacts. A total of
1,510 individuals were vaccinated in four rings in Guinea, including 303
individuals aged between 6 years and 17 years and 307 frontline workers.
The results show that a ring vaccination strategy can be rapidly and safely
implemented at scale in response to Ebola virus disease outbreaks in rural
settings.
Although there are a lot of progress made in recent combat to Ebola
outbreaks in Congo, and the data published by Gsell and Camacho [62]
regarding the ring vaccination results demonstrated 100% efficacy of rVSV
vaccine, it is worrisome that a survivor of Ebola virus disease previously in
Guinea in the 2014 outbreak had Ebola virus after 531 days and caused
another round of Ebola infections among the people he was in contact with.
We need much better tools to monitor the situation [63].
In addition, despite significant progress in the characterization of the
response to vaccination, correlation of protection (CoP) against Ebola virus
infection has not been established in humans [64]. This holds true even for
rVSV-ZEBOV, the most advanced Ebola vaccine candidate and the only
one with demonstrated efficacy in humans so far when this chapter is
published. This is especially challenging in emergency outbreaks settings,
and major efforts would be required to ensure that samples and data are
collected from the clinical trial participants. During emergency outbreaks, it
is very difficult to track those people and to collect samples for further
testing. Integration of data from preclinical and clinical vaccine studies
together with data from disease survivors will thus be essential to identify
Ebola vaccine correlates of protection. The information generated for
rVSV-ZEBOV may help identify the CoP of the other Ebola vaccine
candidates, although these may also be different.

6.2 RSV Vaccine


Respiratory syncytial virus (RSV) is an important cause of viral lower
respiratory tract illness in infants and children globally, but no vaccine is
currently approved to protect these vulnerable populations. RSV is
transmitted by direct and indirect contact with nasal or oral secretions and
causes repeat infections throughout life and significant disease in pediatric
and elderly populations [65–70]. The pathogen is an enveloped, non-
segmented, single-stranded, negative-sense RNA pneumovirus belonging to
the family Paramyxoviridae.
Many development programs for RSV vaccine are ongoing, some of
them are in the preclinical stage, and 16 RSV candidate vaccines are in
clinical development [71, 72]. RSV candidate vaccine developed by
Novavax is currently in Phase 3 clinical trial stage. RSV vaccine
development had a setback recently as Novavax Phase 3 efficacy trial for
RSV F vaccine in maternal immunization did not reach its Phase 3 primary
efficacy endpoint (www.novavax.com. Accessed 12 Apr 2019). 4,636 third-
trimester pregnant women were enrolled in this large clinical trial. The
clinical trial results demonstrated effectiveness in severe RSV cases. So far
there is no approved RSV vaccine on the market yet. The development
work is continuing, and more work remains to be done.

6.3 Malaria Vaccine


In 2013, there were an estimated 584,000 deaths and 198 million clinical
illnesses due to malaria, the majority of which is in sub-Saharan Africa.
Development of malaria vaccine has been ongoing for more than 40 years.
The fact that malaria is caused by parasites that makes the development of
effective vaccine against malaria more difficult than the development of
vaccines against bacteria and viruses [73–78].
There is no vaccine approved on the market for prevention of malaria at
present time. Clinical trials for malaria vaccines are ongoing.

6.4 Other Vaccines Under Development


Other vaccines currently under development include 15-valent and 20-
valent pneumococcal conjugated vaccines, HIV vaccine, TB booster
vaccine, Zika vaccine, Lassa vaccine, Middle East respiratory syndrome
coronavirus (MERS-CoV) vaccine, chikungunya vaccine, Nipah virus
vaccine, and so on [78]. Development of mRNA vaccines has achieved
significant progresses in recent years [79].

7 Challenges We Face
There are many challenges in providing adequate vaccination around the
world. Firstly, vaccine hesitancy is still a problem in many countries
including the USA, the UK, and also China [80]. The vaccine hesitancy
does not include situations where vaccine uptake is low because of poor
availability, e.g., lack of vaccine, lack of offer or access to vaccines,
unacceptable travel/distances to reach immunization clinics, poor vaccine
program communication, etc. Vaccine hesitancy reflects concerns about the
decision to vaccinate oneself or one’s children. Vaccine hesitancy and
refusal, a growing trend in recent years, hinders the elimination and
eradication of diseases. As of March 2019, there are multiple measles
outbreaks in the USA, and parts of New York City declared emergency due
to measles outbreak.
Although concerns about vaccine safety can be linked to vaccine
hesitancy, it is only one factor that may be related to hesitancy; many other
factors also contribute to the issue. Vaccine hesitancy and refusal factors
may include:
Compulsory nature of vaccines
Coincidental temporal relationships to adverse health outcomes
Unfamiliarity with vaccine-preventable diseases
Lack of trust in corporations and public health agencies
In some areas, people are allowed to not participate in vaccination
programs for religious reasons. In some other areas, people have limited
access to education, and they do not know much about the benefits of
vaccines. As a result, they refuse vaccination.
Overall, vaccine hesitancy is a complex and rapidly changing global
problem that requires ongoing monitoring. Each country and each region
need to come up with specific strategies to address the issue.
Another challenge is the resurgence of pertussis diseases. Despite the
vaccination of DTP to infants and children over three decades, the
occurrence of pertussis in adolescent and adult population is under
recognized. Although the adolescents and adults do not have significant
clinical symptoms when pertussis bacteria infect them, they can transmit the
disease to infants and young children around them. Studies in European
region (2019 ECDC Report on Pertussis) found that young adolescents
older than 15 years old had the highest infection rate and young infants less
than 6 months old had the most severe infections. The study results
demonstrated that the majority of cases above the age of 30 years were
unvaccinated. These data stress the need to refine vaccination strategies
with the final aim of protecting infants. In the USA and many European
countries, immunization of Tdap for adolescents and adults is
recommended in the national immunization programs. Thus it is
recommended that Tdap immunization of adolescents and adults should be
implemented in China and other countries that do not have Tdap
immunization for adults and adolescents.
In summary, vaccines have made tremendous contributions to the
society so far. However, we have more work to do to continue developing
safe and effective vaccines to control the infectious diseases and help
people around the world to live a healthy and long life [81–83].

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https://doi.org/10.1007/10_2019_117

Human Pluripotent Stem Cells:


Applications and Challenges for
Regenerative Medicine and Disease
Modeling
Cláudia C. Miranda1, Tiago G. Fernandes1, M. Margarida Diogo1 and
Joaquim M. S. Cabral1
(1) iBB - Institute for Bioengineering and Biosciences and Department of
Bioengineering, Instituto Superior Técnico, Universidade de Lisboa,
Lisbon, Portugal

M. Margarida Diogo
Email: [email protected]

Abstract
In recent years, human pluripotent stem (hPS) cells have started to emerge
as a potential tool with application in fields such as regenerative medicine,
disease modeling, and drug screening. In particular, the ability to
differentiate human-induced pluripotent stem (hiPS) cells into different cell
types and to mimic structures and functions of a specific target organ,
resourcing to organoid technology, has introduced novel model systems for
disease recapitulation while offering a powerful tool to provide a faster and
reproducible approach in the process of drug discovery. All these
technologies are expected to improve the overall quality of life of the
humankind. Here, we highlight the main applications of hiPS cells and the
main challenges associated with the translation of hPS cell derivatives into
clinical settings and other biomedical applications, such as the costs of the
process and the ability to mimic the complexity of the in vivo systems.
Moreover, we focus on the bioprocessing approaches that can be applied
towards the production of high numbers of cells as well as their efficient
differentiation into the final product and further purification.
Graphical Abstract

Keywords Bioprocessing – Disease modeling – Human pluripotent stem


cells – Organoids – Regenerative medicine

1 Introduction
The possibility of producing virtually any type of cell of the human body
from human pluripotent stem (hPS) cells has introduced a myriad of
possibilities regarding their applications in innovative fields such as
regenerative medicine, disease modeling, and drug discovery.
Besides the self-renewal capacity and differentiation ability associated
with stem cells in general, hPS cells provide the opportunity of generating
every cell type derived from the three embryonic germ layers – endoderm,
ectoderm, and mesoderm [1]. Importantly, the breakthrough discovery that
somatic cells can be reprogrammed back to a pluripotent stem cell state,
awarded with the Nobel Prize of Physiology or Medicine in 2012, has
introduced the opportunity of using these cells in personalized medicine
approaches, both including regenerative medicine and drug discovery [2].
The opportunity of generating organoids from hPS cells amplifies the
relevance of this stem cell source within the fields of disease modeling and
drug discovery [3]. Besides allowing the recapitulation of the inherent
differentiation of embryo-like cells into complex and organized structures
that are found within a normal embryonic development process, disease-
specific hPS cell-derived organoids also provide a unique opportunity of
understanding the underlying mechanisms of a particular disease and how
they will affect the final phenotypic characteristics [3]. On the other hand,
organoid technology opens the door for new methods of drug screening that
may reduce the need to resort to animal models that do not fully represent a
human response and that are associated with ethical issues [4].
Stem cell bioprocessing includes the main strategies for scaling up or
scaling out of stem cell expansion, differentiation, and purification for the
upcoming use of stem cells or their derivatives in the fields of regenerative
medicine, disease modeling, and drug screening. To fulfill the previously
described applications, scalable processes that involve the use of
laboratory-scale (e.g., spinner flasks) and fully controlled bioreactors for
the production of clinically relevant numbers of cells are currently under
development and optimization. So far, several robust and efficient
bioprocesses have been developed including the xeno-free scalable
expansion of hPS cells while retaining their pluripotent potential [5] as well
as the directed differentiation of human induced pluripotent stem (hiPS)
cells into cardiomyocytes [6]. Moreover, scale-out approaches that envisage
novel methodologies for drug screening and testing processes have been
described and hopefully will be integrated in the drug discovery pipeline in
a near future [7].
In this chapter, we address the main challenges and opportunities
regarding the application of hPS cell derivatives towards regenerative
medicine, disease modeling, and drug screening, and we revise the main
bioprocessing tools that are necessary to take full advantage of the potential
of hPS cells.
2 Human Pluripotent Stem Cells: The Tool
2.1 Methods for Isolation and Derivation of hES and hiPS
Cells
The properties of human pluripotent stem cells confer them a remarkable
potential towards biomedical and pharmaceutical applications. Embryonic
stem (ES) cells were firstly isolated from mouse embryos, and their
pluripotent potential was demonstrated by showing that these cells gave rise
to teratocarcinomas when grafted into adult mice [8]. Later, in 1998, it was
also possible to isolate ES cells from human embryos [1]. Soon – 4 to
5 days – after the fertilization of an egg in human embryos, there is the
formation of the blastocyst, a hollow sphere composed of an outer layer of
cells, the trophectoderm, and an inner cell mass, from where embryonic
stem cells are isolated [9]. These cells are known for their pluripotency, as
in vitro they can give rise to any type of cell originated from the three
embryonic germ layers (ectoderm, mesoderm, and endoderm).
Nevertheless, they are unable to form a whole new individual, since the
placenta is derived from the trophectoderm and therefore cannot be formed
[1]. The pluripotency of human ES (hES) cells can be assessed by embryoid
body formation, directed differentiation into cells of the three embryonic
germ layers, and teratoma formation assays [1].
Despite their enormous potential in cell therapy, drug discovery, and
disease modeling, the requirement of destroying a human embryo for
obtaining hES cells raises ethical concerns, namely, the ending of a
potential human life. The ethical implications and reduced availability of
hES cells led to search for alternative paths to obtain human pluripotent
stem cells. Several methods were explored, the first ones being nuclear
transfer [10] and cell fusion [11] which in spite of creating pluripotent stem
cell lines retained the ethical concerns and the impossibility of clinical use,
respectively. Soon after that, it was discovered another technology for
pluripotency induction, capable of bypassing these two problems, along
with the possibility of reducing the risk of immune rejection, hiPS cells,
obtained by direct reprogramming of somatic cells by defined transcription
factors [12]. Furthermore, hiPS cell technology offers the possibility of
developing personalized medicine approaches, since hiPS cells can be
induced from somatic cells of a specific patient.
The technique of nuclear transfer requires a somatic cell and an
unfertilized oocyte from which the nucleus was removed. It consists in the
transfer of the DNA content of the somatic cell into the oocyte, where the
union will occur, leading to the creation of an “embryonic-like” cell [13].
However, this process still maintains the ethical concerns associated with
hES cells, due to the origin of a cell that could theoretically form a human
being.
Cell fusion, as the name indicates, involves the fusion between two
cells, generally an embryonic stem cell and a somatic cell. The fusion
comprises that the reprogramming factors that exist in the cytoplasm of
embryonic stem cells will induce the somatic nucleus towards a pluripotent
stem cell state, expressing Oct4, Sox2, and Nanog. However, their
tetraploid nature prevents their use in clinical applications [13–15].
In the past few years, scientific research was directed towards bypassing
the ethical concerns associated to human embryonic stem cells. Takahashi
and Yamanaka [12] discovered in 2006 that terminally differentiated cells
could be reprogrammed back to a state of pluripotency, therefore achieving
an embryonic-like state [16]. This process was firstly performed using
mouse embryonic fibroblasts, but 1 year later the process of cell
reprogramming was also validated for human cells, by using adult human
fibroblasts to generate human iPS cells for the first time [2]. In this study,
four transcription factors were shown to be essential for the generation of
iPS cells, those being Oct3/4, Sox2, c-Myc, and Klf4. These four factors
were introduced into the somatic cells by viral vectors. One of the most
surprising results of this work was that Nanog, a transcription factor present
in ES cells, was not found to be essential in the reprogramming process. It
is important to notice that the optimal duration of transgene expression will
depend on the type of somatic cell that will be reprogrammed, but still, once
the cell achieves the pluripotency hallmark, the endogenous expression of
the factors is sufficient to maintain the pluripotent state [17].
Human iPS cells are identical to hES cells in terms of colony and cell
morphology, gene expression, and in vitro differentiation ability. At the
same time, these cell types share the expression of surface pluripotency
markers SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81, as well as the
transcription factors Oct3/4, Sox2, and Nanog [2, 18]. Despite the great
similarity observed between hES and hiPS cells regarding morphologic
characteristics and signaling pathways, there are some differences that
distinguish these two types of cells. Firstly, there are some indications that
differential promoter binding of the reprogramming factors leaves a
signature in the gene expression of hiPS cells [19]. Also, hiPS cells exhibit
a different methylation pattern when compared to hES cells. Moreover, it
has also been argued that hiPS cells may retain somatic mutations acquired
before reprogramming, which would make the reprogrammed cells
genetically different from its embryonic equivalents. In addition, it was
previously shown that hiPS cells have the ability of forming teratomas with
an efficiency of 100% when transplanted subcutaneously or intratesticularly
into immunocompromised mice, whereas this percentage decreases for 81
and 91% with hES cells, with subcutaneous and intratesticular implantation,
respectively [20].

2.2 Safety and Efficiency of hiPS Cell Reprogramming


The production of hiPS cells without genomic integration of
reprogramming factors is one key feature that facilitates their approval
mainly for regenerative medicine applications. In fact, methods based on
genomic integration can cause insertional mutagenesis and potentially
transform the cells towards a malignant pathway [21].
Although the first report of derivation of hiPS cells was based on
retroviral vectors that encoded key reprogramming factors with
reprogramming efficiencies averaging 0.02% [2], several different
approaches have also been reported that avoid integration of the genetic
material in the genome. Amid integration-free methods, episomal
transfection has demonstrated the ability to generate iPS cells in a
technically simple and reproducible manner [22, 23]. Despite that the
reprogramming efficiencies of non-integrating methods tend to be lower
when compared to viral vectors, averaging 0.01%, a recent study
demonstrated that there are no significant differences in terms of DNA
methylation pattern, marker expression levels, or developmental potential in
hiPS cells derived using different non-integrating techniques [24]. Another
way to evade introducing genetic material into the genome consists in the
direct delivery of synthetic RNAs, modified to prevent antiviral response
mechanisms from the cells, to reprogram somatic cells back to a pluripotent
state with an efficiency of more than 2%, which translates to a
reprogramming efficiency of two orders of magnitude higher comparing to
viral methods [25].
Although hiPS cells represent an alternative for overcoming the ethical
issues often associated with hES cells, they still retain the risk of forming
teratomas if not efficiently differentiated. A safer approach may reside in
using direct reprogramming to derive a somatic cell from another somatic
cell type or from an induced tissue-specific stem (iTS) cell. These strategies
allow bypassing of the pluripotent state, while at the same time reduce the
duration of the periods of hiPS cell derivation and differentiation from
several months to a few weeks [26]. As a good example of direct
reprogramming from a somatic cell type to another, the reprogramming of
mouse fibroblasts into cardiomyocytes was performed by overexpression of
cardiac-specific factors Gata4, Mef2c, and Tbx5 [27]. The derivation of
functional neurons from fibroblasts using lentiviral vectors to induce the
expression of Ascl1, Brn2, and Myt1l was also reported [28]. Still, a major
drawback of direct reprogramming into somatic cells relies on the reduced
proliferative capacity of the derived cells, as well as a lack of population
diversity, which in the end may compromise the regenerative medicine
potential of the cells [29]. Another alternative relies on the use of iTS cells,
which are derived by transfection of somatic cells with a plasmid that
harbors the genes for the four pluripotency induction factors in combination
with tissue-specific selection genes [30]. Using this process, mouse
pancreatic and liver iTS cells were derived by using transient
overexpression of reprogramming factors and combination with Pdx1+ or
HNF4α+ cell selection, respectively. These cells expressed genetic markers
for endoderm and pancreatic or hepatic progenitors and were able to further
differentiate into insulin-producing cells and hepatocytes.

2.3 Epigenetic Memory in hiPS Cells


Induction of pluripotency of somatic cells also involves epigenetic changes
in the DNA. Therefore, there is the need for specific DNA methylation and
methylation/acetylation of histones that will allow the required genes for
pluripotency to be expressed [17]. Although DNA methylation patterns are
dynamic throughout development and cell differentiation, some of these
patterns can be retained as a form of epigenetic memory.
The DNA methylation of Oct3/4 and the modification of histones
observed by Takahashi and Yamanaka [12] were the first suggestion that
iPS cells were caught in an intermediate epigenetic state between the initial
somatic cell and an ES cell state. Later, Bar-Nur et al. observed that hiPS
cells derived from pancreatic islet β cells had increased potential to
differentiate into insulin-producing cells [31]. After analyzing the open
chromatin structure, it was possible to determine that this hiPS cell line
retained a methylation pattern and an open chromatin structure at key β-cell
genes, introducing the hypothesis that methylation patterns in hiPS cell
lines are influenced by their tissues of origin. Therefore, the epigenetic
memory associated with the reprogramming process was also shown to
improve the efficiency of differentiation into mature cells of the same
lineage of the donor cell.
Nevertheless, the method of iPS cell generation can also influence the
epigenetic memory of the cells. Kim et al. compared iPS cells derived by
transcription factor-based treatment and by nuclear transfer [32]. While
transcription factor-based reprogramming demonstrated some residual DNA
methylation patterns characteristic of the somatic donor cells,
reprogramming by nuclear transfer yielded DNA methylation signatures
that closely resemble the ones of ES cells [32]. Still, in transcription factor-
based approaches, the predisposal of iPS cells to favor differentiation
towards the same lineage of the donor cell could be bypassed by sequential
differentiation and reprogramming of iPS cells, as well as with treatment
with chromatin-modifying drugs [19].

3 Human Pluripotent Stem Cells and


Regenerative Medicine
3.1 Clinical Challenges and Legislation
Given that regenerative medicine products provide a high-risk-high-reward
approach, some countries, including the United States and Japan, have
begun to review the laws of delivery of new regenerative medicine-based
products or even create new laws to fulfill the gaps in legislation.
Since late 2014, there are two laws in Japan that provide a fast track for
product approval for the commercialization of cell therapy products within
the country. Therefore, companies are allowed to receive a conditional
marketing approval and commercialize cell-based products while clinical
trials proceed to later stages. The first law, Act on the Safety of
Regenerative Medicine (Law No. 85/2013), states the guidelines that allow
the acceleration of clinical application and commercialization of innovative
regenerative medicine products, covering clinical research and medical
practice. The second law, Pharmaceuticals and Medical Device (PMD) Act
(Law No. 84/2013), introduces a specific regulatory framework for
regenerative medicine products, providing a provisory marketing approval
of 7 years after exploratory clinical trials have demonstrated safety and
basic efficiency data. During this period, companies are required to
continue to submit new clinical trials data to Japan’s Pharmaceuticals and
Medical Devices Agency and, in the end, apply for final marketing approval
or withdrawal of the product. In this Act, regenerative medicine products
include cell and gene therapy products, as well as tissue-engineered
products.
In the United States, the Food and Drug Administration (FDA)
approved a directive – the 21st Century Cures Acts – in order to accelerate
the process of giving access to innovative products to patients in need and
to reduce the regulatory hurdles for FDA approval. In this Act, there is also
a pathway to accelerate FDA approval and market entry of regenerative
medicine therapies, including cell therapies and other human tissue
products, known as the regenerative medicine advanced therapies (RMAT).
Among the main challenges of hPS cell-based product applications in
cell therapies, the high cost of the cell manufacturing process remains as
one of the main obstacles to tackle [33]. For example, the costs of the
preclinical studies of the first clinical trial using hES cell-derived
oligodendrocyte progenitors to treat spinal cord injury were estimated to be
200 million dollars [34].

3.2 Cell Sources


Although hiPS cells offer the possibility of patient-specific cell therapies,
the reprogramming, differentiation, and purification steps may require
months of processing before reaching clinically relevant cell numbers with
high quality and functionality. Thus, allogeneic therapy using universal
hiPS cells from healthy donors can be used instead of customized hiPS
cells. In fact, hiPS cells from donors with homozygous human leukocyte
antigen (HLA) who match ∼20% of the Japanese population at major HLA
loci have been generated and banked and potentially provide an excellent
alternative to autologous stem cell-based therapy [23]. It was already
demonstrated that upon hiPS cell-derived dopamine neurons engraftment in
nonhuman primates, major histocompatibility matching reduces the
immunological response, through suppression of immune cells in the site of
the graft and increases the survival of the grafted neurons [35].

3.3 Current Clinical and Preclinical Trials Involving hPS Cell-


Derived Products
Most of the clinical trials that have employed hPS cell-based products are
directed towards treatment of ophthalmologic, neurological, and
neurodegenerative diseases. Furthermore, there is a clear observation that so
far there has been more resourcing to hES cells than to hiPS cells for
differentiation into the final cell product (Tables 1 and 2). One of the major
debates concerning the clinical applications of hPS cell-based products
resides in whether to transplant mature cells with full functionality or
progenitor cells that still have some plasticity and may integrate better
within the host tissues. Several examples of both scenarios have been
reported, including the transplantation of fully differentiated cells, such as
dopaminergic neurons [35] or cardiomyocytes [36], or the transplantation of
dopaminergic [37], oligodendrocyte [38] or pancreatic [39] precursor cells.
Table 1 hES cell-based products in clinical trials

Disease Company/sponsor ID Status Country


AMD Southwest Hospital, NCT02749734 Phase I China
China
AMD Chinese Academy of NCT02755428 Phase I China
Sciences
AMD Chinese Academy of NCT03046407 Early phase I China
Sciences
Dry-form AMD Astellas Institute for NCT02463344 Follow-up of USA
Regenerative NCT01344993
Medicine
Dry-form AMD Cell Cure NCT02286089 Phase I/II Israel/USA
Neurosciences
Dry-form AMD CHA Biotech NCT01674829 Phase I/II Korea
Dry-form AMD Astellas Institute for NCT01344993 Phase I/II USA
Regenerative
Medicine
Dry-form AMD Regenerative Patch NCT02590692 Phase I/II USA
Technologies
Wet-form AMD Pfizer NCT01691261 Phase I UK
Disease Company/sponsor ID Status Country
Macular degenerative Astellas Institute for NCT03167203 Phase I/II USA
disease Regenerative
Medicine
Outer retinal Federal University of NCT02903576 Phase I/II Brazil
degenerations São Paulo
Parkinson’s disease Chinese Academy of NCT03119636 Phase I/II China
Sciences
Severe heart failure Assistance Publique – NCT02057900 Phase I France
Hôpitaux de Paris
Spinal cord injury Asterias NCT02302157 Phase I/II USA
Biotherapeutics
SMD CHA Biotech NCT01625559 Phase I Korea
SMD Astellas Institute for NCT02941991 Follow-up of UK
Regenerative phase I/II
Medicine
SMD Astellas Institute for NCT01469832 Phase I/II USA/UK
Regenerative
Medicine
SMD Astellas Institute for NCT01345006 Phase I/II USA
Regenerative
Medicine
SMD Astellas Institute for NCT02445612 Long-term USA
Regenerative follow-up of
Medicine NCT01345006
Type 1 diabetes mellitus ViaCyte NCT02239354 Phase I/II USA/Canada
Type 1 diabetes mellitus ViaCyte NCT02939118 Observational, USA
follow-up of
NCT02239354
Type 1 diabetes mellitus ViaCyte NCT03162926 Phase I USA/Canada
Type 1 diabetes mellitus ViaCyte NCT03163511 Phase I/II USA
and hypoglycemia
unawareness

AMD age-related macular degeneration, SMD Stargardt’s macular


dystrophy
Table 2 hiPS cell-based products in clinical trials

Disease Company/sponsor ID Status Country Type of


therapy
Disease Company/sponsor ID Status Country Type of
therapy
AMD Moorfields Eye NCT02464956 Observational UK Autologous
Hospital NHS
Foundation Trust
Wet-AMD RIKEN UMIN000011929 Interventional Japan Autologous
Graft-vs- Cynata Therapeutics NCT02923375 Phase I Australia/UK Allogenic
host disease
Neovascular Kobe City Medical UMIN000026003 Interventional Japan Allogenic
AMD Center General
Hospital
Thalassemia The Third Affiliated NCT03222453 Not China Autologous
Hospital of applicable
Guangzhou Medical
University

AMD age-related macular degeneration, SMD Stargardt’s macular


dystrophy, Not applicable trials without FDA-defined phases, including
trials of devices or behavioral interventions

The first clinical trial involving a hPS cell-based product dates back to
2014 and was started by Geron Corporation, in the United States. This
study was directed towards spinal cord injury and relied on a preclinical
trial that used hES cell-derived oligodendrocyte precursor (OP) cells to
remyelinate rat spinal cords [40]. In fact, it was demonstrated that OP cells
were able to not only survive but also migrate and further differentiate into
oligodendrocytes, restoring motor function. Currently, this trial is in phase
I/II by Asterias Biotherapeutics, with the main objective of optimizing the
injected cell dose.
Neurodegenerative diseases are potentially one of the main targets for
iPS cell therapies. Parkinson’s disease (PD), which is characterized by a
neurological dysfunction based on the loss of dopaminergic neurons, was
the first condition to be tackled using iPS cell-based therapy. In 2011, the
first evidence that dopaminergic progenitor cells derived from hES cells
were able to engraft and mature upon injection was attained using a rat
model [41]. In addition, the mature dopaminergic neurons survived for long
periods, retaining the ability to form synaptic connections and restoring
motor functions that were lost due to PD [42].
A preclinical trial in monkeys was initially run to confirm the safety and
efficacy of a treatment for PD resorting to hiPS cells [37]. In this study,
dopaminergic precursor cells were derived from hiPS cells and injected into
the putamen of macaque monkeys. For that, hiPS cells were firstly directed
towards midbrain dopaminergic progenitor cells, and then cells expressing
CORIN – a floor plate marker – were sorted in order to obtain a more
purified population of cells expressing FOXA2 and Tuj1, markers for floor
plate and immature neurons, respectively. In the end, this approach yielded
more than 85% of FOXA2+Tuj1+ cells, while more than 15% of the cells
expressed a marker of midbrain dopaminergic neurons (NURR1).
Furthermore, no pluripotent Oct4+ cells were detected, as well as neural
rosette-forming cells (Sox1+Pax6+), that might also contribute to tumor
formation. Upon transplantation, implanted cells were able to survive for at
least 2 years without harmful effects in the body and were able to even
integrate with monkey’s brain cells. In October 2018, the first clinical trial
comprising the implantation of hiPS cell-derived dopaminergic precursor
cells has been initiated in Japan. In the first approach of the procedure
performed in a single individual, 2.4 million cells were implanted into 12
different sites known to contain dopamine activity, and, if no complications
arise in the first 6 months, the procedure will be repeated. Until the end of
2020, there is the hope to confirm the safety and efficacy of the technique
by treating six more patients with PD, with the final goal to make the
therapy available by 2023 [43].
A rat model was used for validation of a preclinical trial to address
radiation injury to the brain, a clinically important need for cancer
survivors. The goal was to use hES cell-derived oligodendrocyte precursor
(OP) cells that upon engraftment into the forebrain had the ability to
remyelinate the brain and salvage cognitive deficits [38]. Also, upon
injection of 250,000 O4+ cells in the cerebellum, there was an improvement
in motor tasks of the rats, while there was no indication of teratoma
formation. Further efficacy and safety data in preclinical animal studies has
already demonstrated that OP cell distribution is limited to the spinal cord
and that there were no adverse clinical observations [44], supporting the
initiation of a Phase I/IIa clinical trial in the United States.
It was recently announced that in 2019 the first clinical trial to tackle
spinal cord injury using hiPS cell-derived neural precursors will be held in
Japan [45]. The preclinical trial has taken place in 2012, during which it
was demonstrated that the procedure, involving the injection of neural
precursors 2–4 weeks after the injury, promoted regeneration of the spinal
cords and increased the mobility in monkeys [46]. In the clinical trial that
will take place, two million neural precursors will be injected within the
same timeframe of injury, starting with a group of four patients.
Tissue engineering approaches combining the use of cells with
scaffolds, biomolecules, or devices promise an evolution in the regenerative
medicine field. Generation of pancreatic progenitor (PP) cells from hPS
cells has promised to establish an almost unlimited source of islet cells for
transplantation in diabetic patients [39]. Furthermore, these cells have
demonstrated the ability of treating diabetes upon transplantation into mice
[47]. ViaCyte, a biotechnology company, has been conducting a series of
clinical trials in the past years that are currently on Phase I/II (Table 1).
Their products comprise an islet-like cell population differentiated from
hES cell-derived PPs, which is microencapsulated within a permeable
device [48]. Upon transplantation into the patient, the islet-like cells can
further mature in vivo and generate glucose responsive β-like cells.
Although heart disease represents the main cause of death worldwide, it
was not until 2018 that the first clinical trial results started to emerge. The
main aim of these therapies involves the replacement of cells lost due to
acute heart failure and employs strategies such as cell sheets containing hPS
cell-derived cardiomyocytes [49] or fibrin patches comprising hPS cell-
derived endothelial and smooth muscle cells [50]. Recently, a clinical trial
for the treatment of severe ischemic left ventricular dysfunction has
demonstrated the safety of hES cell-derived cardiovascular progenitor
(CVP) cells. In this safety study, a combination of hES cell-derived CVP
cells embedded in a fibrin patch was delivered to patients during a coronary
artery bypass, and it was demonstrated that this product improved the
patient’s symptoms while avoiding the formation of teratomas or the
presence of arrhythmias, a common complication of cardiac cell therapy
[51]. Importantly, a preclinical trial that tested the efficiency and safety in
primates was already performed [36]. Here, it was observed that hiPS cell-
derived cardiomyocytes were able to survive up to 12 weeks post-
implantation and, importantly, were able to connect electrically with host
cardiomyocytes and thus improve cardiac contractile function. This study
will be one of the bases for the initiation of a small clinical trial led by the
cardiac surgeon Yoshiki Sawa, which is supposed to start in early 2019. In
this trial, allogeneic hiPS cell-derived cardiomyocyte sheets comprising 100
million cells each will be implanted onto the hearts of 3 patients suffering
from advanced heart failure.

4 Bioprocesses for hPS Cell Expansion,


Differentiation, and Purification
A bioprocess for production of hiPS cell derivatives, starting with the
collection of donor somatic cells and ending with the delivery of a purified
hiPS cell-derived product, is a long pathway that can take up to a few
months. Most of these bioprocesses still rely on culturing cells under 2D
conditions, in the absence of control over culture parameters, such as pH
and oxygen, and without automation, which can lead to operator variability
and, consequently, low reproducibility and higher risks of contamination
[52]. Therefore, to fulfill the potential of hPS cells in regenerative
medicine, there is the need to develop robust and scalable platforms that can
produce large numbers of high-quality hPS cells and/or hPS cell-derived
products.
Most of the cells within the human body, upon in vitro culture, depend
on the adhesion to a substrate to survive and proliferate. Under static
conditions, this dependence can be addressed by coating tissue culture
plates with the appropriate ECM molecules. However, in scaling up to
dynamic culture systems, there is the need to increase the surface-to-volume
ratio of the adhesion substrate and, at the same time, provide a system that
can be used under dynamic conditions to afford a homogeneous
environment to the cells. This can be attained using microcarriers. Although
the use of solid microcarriers has been extensively studied for hPS cell
expansion and differentiation either in spinner flasks [5, 53–55] or vertical
wheel bioreactors [56], their use towards regenerative medicine applications
requires an additional separation step. This limitation can be potentially
overcome by the use of dissolvable microcarriers, which allow the
integrated cell expansion and harvesting inside the bioreactor while
avoiding the use of filtration procedures [54]. Nevertheless, although 2D
culture can sustain the growth of almost all cell types, it fails to recapitulate
in vivo conditions, as cells in vivo are usually surrounded either by other
cells or ECM. Thus, 3D aggregate-based culture provides a better
recapitulation of the in vivo microenvironment while avoiding the need for
matrices that are typically required for cell adhesion, either in adherent or
microcarrier cultures, hence reducing the number of components within the
culture. Thus, in this chapter we will focus on this culture format for
scalable expansion and differentiation of hiPS cells.
The first described method for aggregate generation was the hanging
drop, which relies on the spontaneous aggregation of cells contained in
droplets hanging from a surface [57]. By controlling the cell number and
volume in each droplet, the size of the outcoming aggregate can be
adjusted. Another technique relies on the seeding of V-shaped well plates,
allowing generation of one aggregate per well [58]. This method can be
adapted, in order to be reduced to a microscale level, where the droplets are
formed in microwells, allowing the formation of smaller aggregates [59].
Moreover, the simplest technique involves the plating of a single-cell
suspension into static or dynamic non-adherent surfaces, leading to
spontaneous aggregation of the cells [60]. Cell density is an important
factor, since the more cells are in the suspension, higher the probability of
starting to form aggregates [61]. In addition, manually dissociated clumps
can be passed through a mesh filter and fragmented into smaller aggregates
[62]. Microfabrication methods can be used to produce microwell arrays
made from a PDMS stamp in agarose that comprises an inverted pyramid
shape where a cell suspension is seeded and, by centrifuging the plate
containing the cells, will be entrapped in the microwell and generate size-
controlled aggregates [63].
Scaling up of hPS cell culture exposes the cells to fluctuations in
physicochemical parameters and to the hydrodynamic shear stress inside the
bioreactors [64]. Importantly, homogeneity of size and morphology of hPS
cell aggregates can be improved using forced aggregation in microwells
[63], and the negative effect of shear stress under dynamic conditions can
potentially be controlled by cell encapsulation [65]. Nevertheless, the use of
dynamic suspension cultures also leads to increased spheroid homogeneity,
as well as increased cell yields [66, 67]. However, it has to be considered
that increased agitation speeds leads to an augmentation of shear forces,
thus compromising the viability of the cells [61]. Polysulfated compounds
have confirmed their capacity of reducing cell aggregation and promoting
cell survival by reducing apoptosis [68]. Dextran sulfate has recently been
demonstrated to improve aggregate size homogeneity on hPS cells without
compromising their pluripotency, which will improve their efficiency in
terms of expansion [69].
An important highlight on the scale-up of cell culture resides in the
reduction of the overall cost of the process. For example, it has been
demonstrated that scaling up of the production of β cells from hPS cells to
provide insulin for type 1 diabetes would be able to reduce the cost per
patient from $430,000 to $160,000 [70].

4.1 hPS Cell Expansion as 3D Aggregates


An initial approach towards expansion of hPS cells as suspension
aggregates included the use of mouse embryonic fibroblast (MEF)-
conditioned medium (MEF-CM), which was supplemented with bFGF and
ROCKi [71], but this strategy carried the risk of contamination from
xenogeneic products. A number of systems using animal-free conditions
have emerged since, including human foreskin fibroblast-CM, which was
used to scale up hPS cell expansion with a yield of eightfold in 7–10 days
[72]. Amit et al. developed one of the first protocols for hPS cell expansion
as suspension aggregates using serum-free media supplemented with
interleukins, bFGF, and ROCKi [73]. This system was able to attain a 25-
fold increase in 10 days, while maintaining the pluripotency and
differentiation potential of the cells. At the same time, Steiner et al.
developed a system that employed ECM-rich culture medium in
combination with bFGF, activin A, and a serum replacement [74]. Since
then, several defined culture systems have emerged and established major
improvements in terms of robustness and reproducibility (Table 3), as well
as compliance with good manufacturing practices (GMP) for eligibility for
future clinical trials.
Table 3 Scalable approaches for hPS cell expansion as suspension aggregates

Vessel Feeding Seeding Expansion Ref


strategy density
Type Volume Media Yield
(cells/mL)
(mL)
Spinner flask 45 Repeated 4–5 × 105 E8 4.5-fold in [75]
batch 4 days
50 Repeated 3 × 104 to DMEM/F12 + KOSR+ 25-fold in [60]
batch bFGF+IL6RIL6 10 days
1 × 106
Vessel Feeding Seeding Expansion Ref
strategy density
Type Volume Media Yield
(cells/mL)
(mL)
50 Repeated 0.33–1 × 106 mTeSR1 Sixfold in [76]
batch 4–7 days
50 Repeated 0.3 × 105 DMEM/F12 + Glutamax Eightfold in [72]
batch + bFGF 7–10 days
50 Repeated 1 × 106 mTeSR1 Twofold in [61]
batch 7 days
60 Repeated 2.5 × 105 StemPro SFM Fourfold in [77]
batch 4 days
100 Repeated 1.8 × 104 mTeSR1 25-fold in [78]
batch 6 days
100 Batch 2 × 104 mTeSR1 11–12-fold [79]
in 6 days
BioLevitator 30 Fed batch 0.3 × 105 mTeSR1 20-fold in [80]
4 days
30 Fed batch 0.3 × 105 E8 14-fold in
4 days
3D hollow 17 Perfusion 2.9 × 106 mTeSR1 100-fold in [81]
fiber 15 days
bioreactors
Controlled 100 Repeated 4–5 × 105 mTeSR1 Fourfold in [82]
bioreactor batch 7 days
125 Perfusion 5 × 105 mTeSR1 Sixfold in [83]
7 days
150 Perfusion 5 × 105 E8 6.7-fold in [84]
7 days

Culture of hPS cells as suspension aggregates in chemically defined


conditions using commercially available culture media, such as mTeSR [61,
76, 78–80, 82, 85–87], StemPro [77, 88], and E8 [75, 80], has been
extensively reported, leading to a maximum 25-fold increase in cell number
after 6 days [78]. As another improvement to this culture system, single-cell
inoculation and consequent aggregation of hPS cells have been used instead
of passaging cells as aggregates, resulting in a higher level of expression of
pluripotency markers [73, 76, 87]. 3D aggregate culture systems have been
performed using several scalable culture platforms, including shaking flasks
[60, 86], spinner flasks with a wide range of working volumes [61, 72, 73,
75–79, 82], and bioreactors with no impellers, using a gentle tube rotation
[80], or using 3D hollow fibers [81]. In this type of systems, factorial
design was used to maximize cell productivity, by allowing simultaneous
optimization of the different culture parameters, including the agitation
speed, and inoculation density. This mathematical tool allowed an increase
in cell number of 12-fold after only 6 days of culture [79]. The feeding
regimen of the reactor also plays an important role, with the use of
perfusion yielding a final cell density of 2.85 × 106 cells/mL, which
represents an increase of 47% in cell yield when compared with batch
cultures [83]. Furthermore, a recent approach by Manstein et al. describes
the use of four parallel bioreactors that allows the generation of two million
hPS cells using chemically defined medium under a perfusion feeding
regimen during 7 days [84].
Importantly, the quality of hPS cells cultured in bioreactors and other
scalable systems is closely monitored in terms of maintenance of the
expression of pluripotency markers, assessment of pluripotent potential by
multilineage differentiation or ability to generate teratomas in
immunocompromised mice, and karyotypic analysis [75, 79].

4.2 hPS Cell Differentiation as 3D Aggregates


Although hPS cell-derived products represent an amazing opportunity for
regenerative medicine, there is still the need to produce clinically relevant
numbers of cells, which can attain up to 109 cells per patient [89]. The
development of strategies for production of lineage-differentiated
derivatives from hPS cells on a large scale has been one of the foci in the
field of stem cell bioprocessing in the last years (Table 4). The use of small
molecules and chemically defined and xeno-free culture media has
increased the efficiency and robustness of differentiation protocols.
Table 4 Scalable approaches for hPS cell directed differentiation as suspension aggregates

Culture type Differentiation Ref


Type Directed lineage Yield (%)
Spinner flask Neural progenitors ~80 [90]
Neural progenitors ~62 [91]
Cardiomyocytes 27 [77]
Hematopoietic progenitors 25–80 [75]
Culture type Differentiation Ref
Type Directed lineage Yield (%)
Hematopoietic progenitors 5–6 [67]
Cardiomyocytes 90 [6]
Cardiomyocytes 80–99 [88]
Islet cells ~90 [95]
Rotary orbital shaker Cardiomyocytes >60 [86]
Pancreatic progenitors 7–32 [94]
Controlled bioreactor Cardiomyocytes 85 [86]
Cardiomyocytes ~80 [119]

In the case of neural induction, it was determined that an average


aggregate diameter of approximately 140 μm was optimal to efficiently
generate neural progenitor (NP) cells from hiPS cells [90]. Furthermore,
after only 6 days of induction using a dual SMAD inhibition protocol using
small molecules – 10 μM SB431542 and 100 nM LDN193189 – nearly 14
million Pax6+ NP cells were successfully derived from hiPS cells, in
spinner flasks, under chemically defined conditions with efficiencies
averaging 62% [91].
As previously mentioned, the use of 3D conditions has numerous
advantages comparing to adherent monolayer conditions. In the case of
cardiomyocyte derivation from hPS cells, 3D conditions have demonstrated
to promote an earlier mesendoderm lineage differentiation, as well as a
faster maturation of the cardiomyocytes, either structurally or functionally
[92].
Several groups have reported different methodologies that were able to
generate large numbers of cardiomyocytes [6, 77, 85, 88] by culturing hiPS
cells under 3D conditions in stirred bioreactors. The majority of these
protocols are based in a Wnt signaling pathway modulation, as established
by Lian et al. [93], which consists in an early Wnt signaling activation at
early stages of differentiation by using a GSK3 inhibitor such as
CHIR99021, followed by an inhibition of this signaling pathway by using
IWP to specify cardiac differentiation of the mesendoderm-directed cells.
Using this protocol for Wnt signaling modulation, hPS cell cardiac
differentiation using a fully controlled stirred bioreactor has led to the
production of up to 40 million cardiomyocytes with up to 95% of efficiency
[85, 86].
Chen et al. highlighted the importance of size control of hPS cell
aggregates before cardiac induction [88]. Here, an average hPS cell
aggregate diameter of 200 ± 20 μm was shown to be less susceptible to
changes in CHIR99021 concentrations, which could affect the efficiency of
the differentiation protocol. Therefore, the optimized protocol was
translated to a 1 L scale and was able to produce up to 96% of cardiac
troponin T (cTnT) positive cells at a density of 1.4 ± 0.4 × 106 cells per mL.
Furthermore, Fonoudi et al. developed a modified protocol that also
used the initial inhibition of the Wnt signaling pathway but that, in the
inhibition phase, also inhibited the TGF-β pathway and activated the sonic
hedgehog (SHH) pathway [6]. Here, an optimal hPS cell initial aggregate
diameter of 175 ± 25 μm promoted spheroid beating efficiency averaging
almost 100% and was able to derive up to 90% of cardiomyocytes in only
10 days at a 100 mL bioreactor scale.
A four-stage protocol to differentiate pancreatic progenitors (PPs) from
hPS cells was implemented under suspension conditions using ultra-low
attachment plates and rotary agitation [94]. The first step involved the
specification into definitive endoderm, by using a medium supplemented
with activin A and Wnt3A for 1 day, following another day without Wnt3A.
Then, KGF and TGF-β RI kinase inhibitor IV were used to promote
primitive gut tube, and TTNPB, KAAD-cyclopamine, and Noggin
supplementation favored posterior foregut formation. Finally, pancreatic
and endocrine progenitors were generated by further supplementation with
Noggin, KGF, and EGF. In the end, it was possible to obtain functional
glucose-responsive, insulin-secreting cells. Nevertheless, this protocol still
employs the use of fetal bovine serum in the first stages of differentiation,
which will need to be replaced before considering this approach towards
clinical applications. A similar protocol was translated to a 30 mL spinner
flask, promoting the generation of islet-like cells with an efficiency of more
than 90% of PDX1+ cells that, upon engraftment in mice, were able to
revert hyperglycemia [95].

4.3 Purification of hPS Cell-Derived Products


The major risk of the application of hPS cell-derived products resides in the
possibility of teratoma formation due to an uncontrolled proliferation of a
small number of pluripotent cells that remain in the final cell therapy
product. In order to prevent this, clinical-scale purification of hPS cell
derivatives for cell-based therapies needs to be addressed [96, 97].
Fluorescent-activated cell sorting (FACS) is probably the most widely
used and more accurate method for purification of cells. This technique
involves labeling the target cells with an antibody linked to a fluorescent
molecule and has demonstrated efficiencies of separation above 95% [98].
For example, Kikuchi et al. included a sorting step at day 12 of neural
induction of hPS cells that allowed the selection of more than 90% CORIN-
positive cells, which would further differentiate into midbrain dopaminergic
neurons [37]. In this study, on day 26 of differentiation, there were less than
1% of cells expressing pluripotency markers. FACS sorting of O4-positive
oligodendrocyte progenitors led to an enriched cell population containing
more than 93% of these cells that upon animal engraftment did not originate
teratomas [38].
A widely used example of a target antigen for cell purification is SSEA-
1, because this surface marker is absent from hPS cells [99]. Since the
efficiency of the differentiation protocol of hES cells into CVPs only led to
approximately 64% of efficiency, Menasche et al. integrated within the
production pipeline a purification step taking advantage of this marker [51,
100]. The purification strategy consists in the depletion of hPS cells in
culture by magnetically labeling cells with SSEA-1 and then using
magnetic activated cell sorting (MACS) to select SSEA-1-positive cells,
which had lost their pluripotency. Furthermore, the final product, which was
required to contain less than 0.1% of undifferentiated cells, was also tested
for the expression of Nanog.
For the treatment of Stargardt’s macular dystrophy (SMD) and dry-age-
related macular degeneration (AMD), hES cell-derived retinal pigment
epithelium (RPE) cells were purified by exposure to type IV collagenase
followed by manual isolation of RPE using a glass pipette [101]. Still, long-
term safety studies in preclinical models have not detected any uncontrolled
cell proliferation, indicating that no teratomas were generated upon
implantation of hES cell-derived RPE [102]. An approach to address spinal
cord injury by using hES cell-derived OP cells has demonstrated that a final
product containing up to 5% of undifferentiated hES cells did not lead to
teratoma formation [103]. However, the OP cell differentiation protocol has
an efficiency that leads to less than 1% of hES cells in the final product.
Metabolic purification of hPS cell-derived products has also been
focused in the last few years, mainly due to its reduced cost and high
technical feasibility, which relies on the supplementation of the culture
medium with components that are already present in most culture media
formulation. Furthermore, these methods can be easily integrated within
bioreactor culture systems without the need for cell singularization,
contrary to methods like FACS or MACS. One good example of the
application of these methods is for hPSC-derived cardiomyocyte
purification. In fact, the two main substrates for energy production by
cardiomyocytes are glucose and fatty acids, but cardiomyocytes can also
use lactate as an energy source in anaerobic metabolism [104]. In the first
report of hPS cell-derived cardiomyocyte purification using a metabolic
approach, a glucose-depleted and lactate-supplemented culture medium was
capable of purifying the cardiomyocyte population up to 99%, both in
mouse and human cells [105]. Still, glucose remains the main substrate for
primary ATP production in immature cardiomyocytes, whereas fatty acids
play an important role on the energy demand of mature cardiomyocytes. It
has been demonstrated that glucose depletion associated with fatty acid
supplementation is capable of reducing the number of hPS cells in the final
product while increasing the maturation of hPS cell-derived cardiomyocytes
[106].
Contrary to hPS cells, hPS-differentiated cells have mechanisms that
prevent excessive uptake of L-alanine – an amino acid present in the human
body – which would lead to cell swelling and, consequently, death. A novel
approach towards selective elimination of hPS cells relies on the use of high
concentrations of L-alanine to deplete the culture from pluripotent stem
cells, and that can be a cost-effective alternative for purification of hPS cell-
derived products to use in cell therapies [107].

4.4 Bioengineering Strategies for the Improvement of hPS


Cell-Derived Organoids
Organoids are complex structures that can be generated from hPS cells or
from organ-specific progenitor cells and that are capable of differentiating
into multiple cell types while self-organizing themselves in a structure
similar to the represented organ and, at the same time, recapitulating some
of the organ functions, such as neural activity or contraction [108].
The application of bioprocessing strategies is crucial towards
overcoming the limitations often associated with organoids. One of the
main limitations resides in the heterogeneity among different organoids
often associated with poor efficiency of differentiation of hPS cells into a
specific lineage. It was already demonstrated that the initial hPS cell
aggregate diameter is critical for the optimal differentiation into a specific
cell lineage [90, 109]. For that, strategies that employ the use of microwells
to control hPS cell aggregate size have proven effective to direct hPS cells
into cardiomyocytes [63]. Arora et al. demonstrated that the passage from a
spheroid containing hindgut progenitor cells into a pre-organoid is favored
by the size and morphology of the spheroid [110]. Here, the sorting of
aggregates with diameters greater than 75 μm favored the further
development into intestinal organoids in 3.8-fold when compared with non-
sorted populations.
As the size of the organoids increases, mass transport to the inner areas
of the organoid becomes critical, since diffusional limitations often arise in
spheroids with diameters greater than 300 μm [111]. The use of agitated
systems, such as spinner flasks, that enhance the mixing of nutrients and
promote gas transfer has led to a rapid development of brain organoids [3,
112]. Nevertheless, in the absence of medium agitation, a vascularized liver
organoid has already been developed, by combining hPS cell-derived
hepatic endoderm with mesenchymal stem cells and human umbilical vein
endothelial cells [113].
Another limitation of organoid technology relies on organoid
encapsulation, since many of the processes developed so far depend on the
embedding of organoids in a supporting extracellular matrix that helps
maintaining the organoid in a favorable microenvironment for growth and
maintenance [3]. Still, mechanical properties of the substrate in which the
organoids are embed also play an important role. Although intestinal
organoids present self-organization, embedding the organoids within a
collagen gel allowed the inner cystic structures to align, generating
macroscopic hollow tubes composed of multiple cells, a process that was
not observed without this matrix [114]. A bioprocessing approach that
employs the use of a two-fluidic electrostatic co-spraying technique has
tackled the issue of large-scale production of encapsulated organoids [115].
Here, the organoids are encapsulated in a Matrigel core-shell that promotes
cell growth and structural support and an outer alginate shell that protects
the capsule from shear stress in suspension culture inside bioreactors.
Lastly, the main challenge associated with organoids and other tissues
derived from hPS cells remains in the display of fetal-like characteristics
[116], which may not adequately model adult diseases. Human intestinal
organoids derived from hPS cells have demonstrated to resemble human
fetal intestine instead of adult tissue [117], but upon in vivo transplantation
are able to further mature and become more adult-like intestinal tissue. For
cardiac derivatives generated from hPS cells, physical conditioning using
electromechanical stimuli over a period of 4 weeks in culture was sufficient
to accelerate cardiomyocyte maturation into adult-like human cardiac tissue
[118].

5 Human Pluripotent Stem Cells and Disease


Modeling
The ability to generate disease-specific hiPS cells has brought the
opportunity of unveiling disease mechanisms that until recently were
unknown. In the past few years, and mainly by using a combination
between hiPS cells and organoid technologies, a myriad of disease models
have been developed, from neurological [3, 120] to cardiac [121] or gut
[122] diseases.

5.1 Modeled Diseases


Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder where
loss of motor neurons in the spinal cord and motor cortex induces
progressive paralysis and, ultimately, death [123]. One of the first reports
using hiPS cell technology consisted in the generation of ALS-specific hiPS
cell lines that efficiently differentiated into motor neurons [124]. Still, only
recently a model that incorporates 3D skeletal muscle bundles with light-
sensitive channelrhodopsin-2-induced motor neuron spheroids derived from
ALS patients was developed [125]. Using this platform, it is possible to
analyze muscular contraction through light activation of the motor neurons.
When ALS-specific motor neurons were used instead of healthy cells, there
was less muscular contraction, together with more motor neuron
degradation and decrease in their viability.
Second to Alzheimer’s disease, Parkinson’s disease (PD) is the most
common chronic progressive neurodegenerative disorder, characterized by
loss of dopaminergic neurons. Although patient-specific PD hiPS cells were
already derived [126], since the onset of PD occurs on average at 50 years
of a human life, additional cues – such as exposure to oxidative stress or
neurotoxins – are needed to replicate the pathological mechanisms of PD in
vitro.
Spinal muscular atrophy (SMA) is a genetic disorder based on the
mutation of the SMN1 gene and is characterized by selective degeneration
of lower α-motor neurons [127]. Derivation of hiPS cells from a child with
SMA followed by neural differentiation into motor neurons demonstrated
deficits in either cell number or size when compared with a control from a
parent [128].
Rett syndrome, a neurological disease commonly caused by mutations
in the MECP2 gene of the X chromosome, is related with impairment of
motor function and autistic-like behavior [120]. Using patient cells and
hiPS cell technology, it was possible to recapitulate an altered process of
neurogenesis in cells of these patients, as well as the presence of an inferior
number of neurons and less complex neurites [129].
Familial dysautonomia, or Riley-Day syndrome, is a fatal autosomal
recessive disease caused by a mutation in the IKBKAP gene and
characterized by the degeneration of sensory and autonomic neurons [130].
Neural induction of patient-specific hiPS cells has demonstrated that in
vitro these cultures present neural crest precursors with lower Tuj1+ neuron
differentiation potential, in addition to defects in migration behavior [131].
Schizophrenia affects around 1% of the worldwide population and
possesses a hereditary genetic component in almost 85% of the cases [132].
It was shown that neural differentiation of hiPS cells from schizophrenic
patients led to decreased neural connectivity and decreased number of
neurites and synaptic protein levels while presenting a normal
electrophysiological behavior and spontaneous calcium transient activity
[133].
Heart diseases have also been addressed as a target for disease modeling
using hiPS cells. Recapitulation of familial long QT syndrome was
achieved by generating hiPS cell-derived cardiomyocytes from patients,
recreating electrophysiological features of the disorder, such as prolonged
action potentials [121].
Dilated cardiomyopathy occurs in 1 of 250 adults and is characterized
by progressive left ventricular dilation and, eventually, heart failure [134].
This disease may arise from mutations that truncate the sarcomere protein
titin, which was verified by derivation of disease-specific hiPS cells that,
upon cardiac differentiation, resulted in the generation of cardiomyocytes
with sarcomere deficiency, combined with decreased responses to stress, as
well as to activation from growth factors [135].
Another example of a heart disease modeled using hiPS cell technology
consists in Duchenne muscular dystrophy (DMD), which is characterized
by the progressive weakness of the skeletal and cardiac muscle [136].
Cardiomyocytes derived from patients with DMD displayed dystrophin
deficiency when compared with controls from healthy donors. Furthermore,
disease-specific cardiomyocytes demonstrated higher levels of apoptotic
markers, including increased levels of cytosolic Ca2+ or caspase-3, since
these cells are more sensitive to stress-induced damages [137].

5.2 Organoids and Disease Modeling


Neural differentiation of hPS cells has been thoroughly investigated, since
neuroectoderm represents the default differentiation pathway upon
withdrawal of pluripotency maintenance culture conditions [138]. Brain
organoids derived from hPS cells were the first complex 3D models that
were able to recapitulate certain aspects of in vivo brain development such
as progenitor zone organization and the organization of outer radial glial
stem cells [3]. Using Matrigel as an extracellular matrix to support the
process of self-organization, it was possible to obtain brain organoids that
exhibited cortical self-organization, as well as specification of hindbrain
and forebrain regions. Since then, other brain-specific organoids have been
recreated using specific morphogen gradients, including the cerebellum
[139] and the optic cup [140], as well as organoids from other tissues, such
as the lung [116] or even liver buds [113]. Furthermore, it was recently
demonstrated that brain organoids from different regions could be fused to
mimic interactions between brain regions. The coculture of brain organoids
with dorsal and ventral forebrain identities led to the generation of a model
of a dorsal-ventral axis that demonstrated the migration of CXCR4-
dependent GABAergic interneurons from ventral to dorsal forebrain [141].
The combination between hiPS cell differentiation and organoid
technology has opened the possibility of using patient-specific cells to
recreate disease mechanisms in vitro using more relevant models of in vivo
tissues, as certain structural characteristics of a disease can also be
replicated in a 3D structure. Several disease models have been developed
using organoids, ranging from neurological diseases [3, 142–144], using
brain organoids, to heart conditions, using cardiac organoids [121, 135], or
cystic fibrosis [145], using lung organoids, as listed in Table 5.
Table 5 Disease modeling using hiPS cells and respective observed in vitro characteristics

Modeled disease Model characteristics Ref


Alagille syndrome • Impaired chloride transport [144]
Autism • Normal early neuronal differentiation [143]
• Imbalance in inhibitory GABAergic neurons over glutamatergic
Cystic fibrosis • Impaired forskolin-induced swelling [145]
Dilated cardiomyopathy • Sarcomere insufficiency [135]
• Impaired responses to mechanical and β-adrenergic stress
Familial adenomatous • Increased cell proliferation [122]
polyposis • Increased nuclear localization of β-catenin
Familial dysautonomia • Defects in neural differentiation [131]
Familial long QT • Prolonged action potential in cardiomyocytes [121]
syndrome
Hirschsprung’s disease • Impaired organization of enteric nervous system [146]
Microcephaly • Premature neuroepithelial differentiation [3]
• Aberrant glial orientation
• Smaller areas of differentiated tissue
Miller-Dieker syndrome • Poor neurite growth [142]
• Apoptosis in the ventricular zone
• Impaired neuronal migration
Rett syndrome • Impaired neurogenesis [120]
• Reduced neuronal migration
Schizophrenia • Decreased neural connectivity, neurite formation, and synaptic [133]
protein expression
Spinal muscular atrophy • Motor neuron differentiation [128]

In the field of neurological diseases, the study of microcephaly was the


first that was addressed using hiPS cell-derived brain organoids [3]. Using
fibroblasts from a patient with microcephaly, caused by truncating
mutations in CDK5RAP2, it was possible to generate a patient-specific hiPS
cell line that, under pluripotency maintenance conditions, behaved similar
to a hiPS cell control line. Still, upon induction to neuroectoderm as a 3D
structure, a smaller neuroepithelial tissue with fewer progenitor zones and
an increased premature neuronal outgrowth was observed.
Autism spectrum disorders affect 1 in every 60–70 children and have as
main symptoms behavioral deficits in social interaction, communication,
and interests, along with repetitive behaviors [147]. Using patient-specific
hiPS cell-derived neural organoids, normal neuronal differentiation was
observed, while there was an imbalance in the number of inhibitory
GABAergic neurons over glutamatergic neurons, which suggests an
underlying mechanism for this neurological disease [143].
Miller-Dieker syndrome is a genetic neurological disorder caused by
large heterozygous deletions of human band 17p13.3, and it is characterized
by a major absence of cortical folding of the brain, leading to microcephaly,
mental retardation, and intractable epilepsy [148]. Using brain organoids
derived from patient-specific hiPS cells, Bershteyn et al. demonstrated that
there were apoptotic areas in the subventricular zone, poor neurite growth,
and impaired neural migration [142].
The enteric nervous system (ENS) of the gastrointestinal tract is crucial
for the functions of this organ including motility, secretion, and blood flow
[149]. The development of a normal intestinal enteric nervous system has
been recapitulated by merging hiPS cell-derived neural crest cells and
human intestinal organoids, which were able to mediate contractile waves
[146]. Using this model, it was possible to recapitulate Hirschsprung’s
disease, characterized by congenital lack of enteric ganglia [150], and to
verify that in vitro there was an impaired neural crest and ENS development
[146].
A model of vascular disease based on blood vessel damage induced by
diabetes was recently developed [151]. Here, endothelial cells were derived
from hiPS cells, and, using a basement membrane, blood vessel organoids
were created. Furthermore, upon transplantation to mouse models and
exposure to a diabetic environment, there was thickening of vascular
basement membrane, characteristic of diabetic vasculopathy.
Cystic fibrosis (CF) is a genetic disease that affects several organs, such
as the liver and intestine, but mostly the lungs. Contrary to other lineage
specifications from hPS cells, differentiation protocols towards cells of the
respiratory track tend to be complex. Still, directed differentiation of hPS
cells into NKX2.1+ airway progenitor cells followed by low Wnt signaling
activation led to the generation of lung organoids that contained cells from
the goblet, basal, and secretory lineages [145]. Furthermore, using hiPS
cells derived from CF patients, it was possible to recapitulate key features
of the disease, including impaired forskolin-induced swelling.

5.3 Disease Modeling Using CRISPR/Cas9


Despite the several examples of genetic diseases modeled using hiPS cell
technology (Table 5), a new challenge that emerges is how to clearly
discriminate if the differences observed in the phenotype are due to the
mutation or to the individual’s genetic background. A state-of-the-art
approach relies on the use of genetic manipulation tools that correct the
mutation of a given clone, creating an isogenic hiPS cell line. The most
used tool is the clustered regularly interspaced short palindromic repeats
(CRISPR) modified with two components – Cas9, which is the enzyme
responsible for DNA cleavage, and guide RNA, which binds to Cas9 and
pairs with the desired DNA site [152]. The CRISPR/Cas9 technology
allows a footprint-free gene modification and at the same time has the
advantage of being a simpler manipulation procedure comparing to other
gene editing techniques such as zinc finger nucleases [153]. In addition to
this strategy, CRISPR/Cas9 can also be used for the insertion of a mutation
to recreate a mutated phenotype.
Huntington disease (HD) is caused by a CAG repeat in HTT and results
in impaired neural rosette formation and deficits in mitochondrial
respiration. Using a HD-specific hiPS cell line and a corrected isogenic
hiPS cell line, it was possible not only to differentiate the cells into
forebrain neurons but also to rescue phenotypic abnormalities in the
isogenic hiPS cell line [154]. Several other genetic diseases, like
amyotrophic lateral sclerosis [155], Alzheimer’s disease [156], and β-
thalassemia [157], among others, have been studied using this approach. On
the other hand, CRISPR/Cas9 has also been used to introduce the
CCR5del32 mutation in hiPS cells, which originated monocytes resistant to
HIV infection [158].
Despite all the apparent advantages, CRISP/Cas9 technology has been
thoroughly debated, since recent reports have demonstrated that this
methodology may not be as specific as initially presented, since significant
mutations near the target site have been detected [159].

5.4 Drug Discovery


Both organoids and hiPS cell-derived cells are currently part of a paradigm
change in the industry of drug screening and development. Apart from the
use of such technologies for studying the response of in vitro human tissues
to specific drugs, the possibility of attaining personalized treatments is also
possible due to hiPS cell technology.
We have previously reported the use of hiPS cell-derived NPs to predict
the effect of commonly used compounds, such as valproic acid (VPA),
during the early stages of the process of neurodevelopment [7]. Exposure of
cells to VPA during the initial stage of brain tissue formation, replicated in
vitro using hiPS cell spheroids, has led to disorganized neural rosette
structures, decreased cell viability, and delayed neuronal differentiation.
As previously mentioned in this chapter, using a combination of 3D
skeletal muscle bundles and light sensitive spheroids of motor neurons
derived from hiPS cells of an ALS patient, it was possible to analyze
muscular contraction based on light activation of the motor neurons [125].
This platform was used to test the efficacy of drug candidates to treat ALS,
such as rapamycin and bosutinib. In the presence of these drugs, the levels
of caspase-3/7 decreased, indicating fewer cell death, which may suggest a
neuroprotection mechanism of such drugs.
Another application of hiPS cell-based products relies on the validation
of drugs to inhibit viral infection of neural tissues. Derivation of NP cells
from hiPS cells has also been addressed to screen for compounds that block
Zika virus infection and, at the same time, clear the virus from already
infected NP cells [160].
Acute myeloid leukemia (AML) is an oncologic disease that is caused
by a translocation in the MLL gene and that compromises normal
hematopoiesis, by uncontrolled proliferation of myeloid progenitor cells.
iPS cells derived from AML patients were able to differentiate into
hematopoietic progenitors, while still retaining their malignant
characteristics [161]. Importantly, this study has demonstrated that clonal
differences in different hiPS cell lines derived from patients with AML can
represent a powerful tool to understand drug sensibility of individual hiPS
cell-derived subclones.
Although the efficiency of a drug is important, other aspects comprised
within the drug metabolism process cannot be neglected. Intestinal drug
absorption is important for the oral drug delivery system, and, for that,
using hiPS cell-derived epithelial cells, a model of prediction of drug
absorption was developed [162]. The derived epithelial cells displayed
similar characteristics to their in vivo counterparts, such as the presence of
thigh junctions, metabolic enzymes, and drug transporters, that closely
mimic the mechanisms of oral drug absorption, which makes this platform a
powerful tool towards the prediction of drug absorption in vivo.
Organ-on-a chip platforms provide invaluable tools for drug screening,
since they combine the opportunity of recapitulating the multicellular
architectures of an organoid with the possibility of testing different drugs on
a high-throughput scale. The derivation of multi-organ platforms from hPS
cells and their application for drug screening have already been reviewed in
Miranda et al. [4]. Still, we highlight the development of a multi-organ
platform that incorporates three different modules containing organoids
from the liver, heart, and lung [163]. This device has the great advantage of
allowing to analyze an individual response to a drug for each organoid or to
combine the response between the organoids from different tissues, which
gives a more complete preview of the in vivo behavior. Here, the effect of
bleomycin – a chemotherapeutic drug used to treat lung cancer – was
studied in separated organ modules and in an interconnected manner. Using
an isolated module setting, as predictable, there was an increase in
inflammatory markers in the lung module and no cardiotoxic effect in the
separate heart module. Still, in a three-organ setting, it was possible to
observe a decrease in heart rate due to inflammatory factor-driven
cardiotoxicity, which highlights the importance of a combined response
between different organs in order to efficiently predict cell behavior upon
drug exposure.
Overall, although organ-on-a-chip platforms provide an opportunity
towards drug discovery applications, most of them still uses primary cell
sources and immortalized cell lines. Despite the fact that primary cells
retain similar characteristics, such as metabolic profiles and functional
properties to the original tissues, they tend to lose these properties when
cultured in vitro for prolonged periods of time [164]. On the other hand,
immortalized cell lines frequently fail to recapitulate the original tissue and
are known for the accumulation of karyotypic abnormalities that may
compromise their response to stimuli [165]. Therefore, hPS cell derivatives
can address some of these issues, adding the possibility of generating
disease- and patient-specific cells that promote a more realistic approach to
drug screening applications.

6 Conclusions and Future Perspectives


The advancement in hiPS cell technology has been impressive since the
first derivation of these cells, providing great opportunities in the fields of
regenerative medicine, disease modeling, and drug screening. Here, we
summarized recent progresses in the biomedical applications of hiPS cells
as well as the bioprocessing challenges that have to be surpassed to fulfill
these applications, namely, to allow their translation either into clinical
settings or for study of disease mechanisms or towards drug discovery.
Some limitations still need to be addressed prior to a complete shift
towards these systems. First, there is the need to keep developing robust
protocols for hiPS cell lineage specification, since even protocols that
employ the use of defined culture medium and small molecules are not
always reproducible. Moreover, there is the need to develop robust, safe,
and scalable protocols for the large-scale production of these derivatives
with high quality and functionality. Second, even though patient-specific
hiPS cell lines can be derived, the individual hiPS cell clones may display
some variability. Furthermore, although isogenic hiPS cell lines are
considered state of the art, it must be considered that the single-cell cloning
procedures may result in clones that possess different properties from the
original cells.
Currently, most of the hPS cell-derived organoids represent only some
aspects of the target organ, displaying a limited number of cell types [166],
which can hinder a correct structural organization and thus function of the
tissue. In the future, it will be necessary to introduce protocols that will
differentiate hPS cells into a representative number of cell types, in order to
accurately represent the physical interactions and organization between
cells. Future approaches also include further maturation of organoids, since
current organoids obtained from hPS cells represent an early developmental
stage, often compared with fetal tissues [167]. It is expected that hiPS cell-
based disease models using organoids will improve preclinical drug
screening and accelerate candidate therapies into clinical trials.
Incorporation of organoid technology combined with organ-on-a-chip
technology will accelerate the readouts of each experiment, while
maintaining a humanized response in comparison with the currently
available animal models.

Acknowledgments
C.C.M. acknowledges support from FCT – Fundação para a Ciência e a
Tecnologia (CEECINST_IJ3/IST-ID). This work was further financially
supported by FCT Portugal through iBB, Institute for Bioengineering and
Biosciences (UID/BIO/04565/2013), and from Programa Operacional
Regional de Lisboa 2020 (Project No. 007317), PTDC/EMD-
TLM/29728/2017 and PTDC/EQU-EQU/29653/2017 projects, and project
PRECISE – Accelerating progress toward the new era of precision
medicine (PAC-PRECISE-LISBOA-01-0145-FEDER-016394,
SAICTPAC/0021/2015).

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https://doi.org/10.1007/10_2019_118

Addressing the Manufacturing Challenges


of Cell-Based Therapies
Miguel de Almeida Fuzeta1 * , André Dargen de Matos Branco1 * ,
Ana Fernandes-Platzgummer1, Cláudia Lobato da Silva1 and
Joaquim M. S. Cabral1
(1) Department of Bioengineering and iBB-Institute for Bioengineering
and Biosciences, Instituto Superior Técnico, Universidade de Lisboa,
Lisboa, Portugal

Cláudia Lobato da Silva


Email: [email protected]

* Contributed equally

Abstract
Exciting developments in the cell therapy field over the last decades have
led to an increasing number of clinical trials and the first cell products
receiving marketing authorization. In spite of substantial progress in the
field, manufacturing of cell-based therapies presents multiple challenges
that need to be addressed in order to assure the development of safe,
efficacious, and cost-effective cell therapies.
The manufacturing process of cell-based therapies generally requires
tissue collection, cell isolation, culture and expansion (upstream
processing), cell harvest, separation and purification (downstream
processing), and, finally, product formulation and storage. Each one of
these stages presents significant challenges that have been the focus of
study over the years, leading to innovative and groundbreaking
technological advances, as discussed throughout this chapter.
Delivery of cell-based therapies relies on defining product targets while
controlling process variable impact on cellular features. Moreover,
commercial viability is a critical issue that has had damaging consequences
for some therapies. Implementation of cost-effectiveness measures
facilitates healthy process development, potentially being able to influence
end product pricing.
Although cell-based therapies represent a new level in bioprocessing
complexity in every manufacturing stage, they also show unprecedented
levels of therapeutic potential, already radically changing the landscape of
medical care.
Graphical Abstract

Keywords Bioreactor – Cell therapy – Hematopoietic stem/progenitor cells


(HSPC) – Manufacturing – Mesenchymal stem/stromal cells (MSC) –
Process engineering

Authors “Miguel de Almeida Fuzeta and André Dargen de Matos Branco”


contributed equally.

1 The Landscape of Cell-Based Therapies


1.1 Hematopoietic Cell Transplantation: A Landmark Cell-
Based Therapy
The development of cellular therapies began with the establishment of
hematopoietic cell transplantation (HCT). Early work in murine models led
to the observation that supralethal radiation could be survived if affected
mice were infused with a bone marrow (BM) graft [1]. BM aspirates
containing hematopoietic stem/progenitor cells (HSPC) were able to
migrate to the affected BM after radiation-derived myeloablative treatment
and reconstitute the entire hematopoietic system.
After proving to treat radiation injury, BM transplantation was
considered as a possible treatment for leukemia. In 1956, Barnes and
colleagues were able to infuse normal BM grafts on leukemic mice as a
proof of concept [2]. Knocking out a murine hematopoietic system also
meant eliminating its blood-related malignancies. Transplants of healthy
grafts would then repopulate the BM and form a new hematopoietic system.
By transposing this knowledge to humans, Thomas and colleagues were the
first to successfully perform HCT in human acute leukemia patients, paving
the way toward showing the feasibility of cell therapies [3].
HCT development demonstrated that a main and contemporary threat in
cell therapies, known as rejection, could be overcome. Indeed, early on,
Medawar and colleagues were crucial in identifying rejection as an
immunologic response by observing the progressive degradation of skin
grafts [4]. In 1953, Billingham and colleagues described a breakthrough
that brought down the inevitability of rejection. The concept of acquired
tolerance was described when lack of rejection was seen in mice that were
previously infused with donor cells during fetal development, originating
chimeras [5]. HCT was able to surpass rejection through acquired tolerance
with elimination of the existing immunological response by radiation pre-
conditioning.
Rapidly after the discovery of acquired tolerance, another major
concern related with cell therapies arose. Billingham and Brent observed
that even with spleen or BM cells infused to induce tolerance, skin grafts
continued to fail as the age of the recipients increased [6]. The reason
behind this adverse reaction was another immunological response, brought
upon by the actual graft. Active immune cells from the donor that
accompanied the grafts were not tolerant toward the recipient.
Consequently, these would launch a hostile immune response mirroring
rejection mechanisms. However, this reaction is limited to HCT, not
occurring in solid organ transplantation, where the only mechanism of
rejection is the attack triggered by the recipient’s immune system against
the transplanted organ. Initially termed runt disease, since affected mice
would not develop into their adult life, this immune-based effect is
presently known as graft versus host disease (GVHD) [7].
Rejection and GVHD possess similar modes of action, being based on
an immunologic response. Circumventing these complications in a
transplant scenario meant finding the basis of immunological identity,
namely, genes encoding for the human leukocyte antigens (HLA) [8]. With
the development of HLA typing, transplants could be performed between
HLA-matched patients, with the hope of avoiding rejection and GVHD.
This led to the first successful HLA-matched HCT between two siblings
performed by Gatti and colleagues in 1968 to treat severe combined
immunodeficiency (SCID) [9], thus demonstrating the potential of HCT to
treat illnesses other than cancer.
HCT was the first cell therapy to be adopted in a clinical setting,
ultimately gaining widespread acceptance for treatment of genetic or
oncological disease with therapeutic success. The implementation of most
current cell therapies can be traced back to knowledge gained by the
establishment of HCT (Fig. 1). For instances, the field of solid organ
transplantation, which is very limited by tissue tolerance, has also benefited
from HCT experience [10]. Joint transplantation of organs with their
respective BM graft was able to cause acquired tolerance toward the solid
organ and overcome HLA mismatch [11]. Gained knowledge of the
potential and limits of allogenic and autologous approaches has facilitated
therapeutic objective delineation in skin grafts. Using an allogenic source
for the production of dermal and composite substitutes (e.g., Dermagraft®
and Apligraf®) has limited grafts to serve only as a temporary barrier that
promotes wound healing, without any permanent engraftment [12, 13]. On
the other hand, autologous skin grafts (e.g., EpiCel® and PermaDerm®)
integrate the skin of the patient while continuously promoting wound
closure, reducing scar tissue formation, and mitigating an inflammatory
microenvironment [14, 15].
Fig. 1 HCT as a platform for the development of cell-based therapies. Pioneering experience in
using cells as therapeutic agents laid the foundation for the diversification of cell therapies. HCT has
been challenged with most of the crucial obstacles of cell-based therapies, from technical hurdles due
to handling, isolation, or infusion of cells to unprecedented biological concepts (e.g., rejection)
transversal to any therapy involving cells as therapeutic agents. Success in solid organ transplants
was reliant on understanding compatibility between patients. Identifying the main agents of the
immunological system was imperative to the development of immunotherapies. Also, induced
pluripotent stem cell technology gained substantial impact since it showed that lack of compatibility
could be overcome by creating autologous sources for any type of cell. HCT hematopoietic cell
transplantation

Besides GVHD, HCT also demonstrates a graft vs. leukemia (GVL)


effect against residual malignant cells that defied conditioning treatments.
This behavior has been associated with lymphoid constituents of the
transplant, which has heightened interest in adoptive T-cell therapy. Donor
lymphocyte infusions (DLI) of specific T-cell subsets or, more recently,
enhancement of their antitumoral capabilities through chimeric antigen
receptor T (CAR-T) cell technology are approaches that have shown great
success in both treating cancer and mitigating GVHD in patients [16, 17].
Instead of tackling donor compatibility, rejection and GVHD could be
avoided altogether. Induced pluripotent stem cell (iPSC) technology has
opened up the possibility of using patients as cell sources for their own
therapies. Although very promising and prompting considerable investment,
this technology is yet at its infancy, with very few clinical trials to date and
still requires significant maturation before moving to clinical practice [18].
Nonetheless, its potential for cell therapies and disruptive nature are
founded on compatibility issues learned from HCT.
HSPC are the most studied cells in clinical trials for multiple innovative
applications [19], with over 3,500 clinical trials taking place worldwide, the
vast majority in the USA followed by Europe (Fig. 2a) (clinicaltrials.gov,
accessed on 29 May 2019, using the search term “hematopoietic stem cell
OR hematopoietic progenitor cell”). Neoplasms by histologic type, immune
system diseases, leukemia, lymphoproliferative disorders, and
immunoproliferative disorders are the top five conditions with the highest
numbers of undergoing clinical trials worldwide.
Fig. 2 Worldwide distribution of clinical trials obtained from “clinicaltrials.gov” on 29 May 2019,
using the terms “hematopoietic stem cell OR hematopoietic progenitor cell” (a) and “mesenchymal
stem cell OR mesenchymal stromal cell” (b)

1.2 Mesenchymal Stem/Stromal Cells as a New Paradigm for


Paracrine Cell Therapy
Following initial developments in HCT, the BM was once more the source
for the discovery of yet another promising stem cell population, named
mesenchymal stem cells (MSC) by Caplan in 1991 [20]. The foundations
for the discovery of these stem cells can be traced back to the nineteenth
century, when studies on the BM transplantation to heterotopic anatomical
sites resulted in de novo generation of ectopic bone and marrow [21, 22].
However, it was only later that the work of Tavassoli and Crosby clearly
provided evidence of an inherent osteogenic potential associated with the
BM [23]. In the 1960s–1970s, Friedenstein and colleagues isolated and
characterized a subpopulation of adherent spindle-shaped cells from mouse
BM that were responsible for the previously described osteogenic potential
[24, 25]. Moreover, they also demonstrated that BM cell suspensions could
generate colony-forming unit-fibroblasts (CFU-F). These cells were then
later designated as “mesenchymal stem cells” and shown to have
multilineage differentiation potential, including the osteogenic, adipogenic,
and chondrogenic lineages [20, 26].
Over the following decades, questions were raised over the usage of the
term “mesenchymal stem cells,” and alternative nomenclatures have been
proposed by different authors. This is due to unfractionated plastic-adherent
marrow cells being quite heterogeneous and current data being insufficient
to characterize them as stem cells. In order to address the inconsistency in
the nomenclature and account for the biological properties of these cells,
the International Society for Cellular Therapy (ISCT) proposed that plastic-
adherent cells described as mesenchymal stem cells should be termed
multipotent mesenchymal stromal cells, maintaining the acronym MSC
[27].
The controversy in the appropriate nomenclature is accompanied by the
inconsistency between investigators on the set of characteristics that define
MSC. Laboratories have developed different methods of isolation and
expansion, as well as different approaches to characterize these cells. Thus,
an appropriate comparison between studies may be difficult to achieve. In
order to address this issue, the ISCT has proposed minimal criteria to define
human MSC: (1) adherence to plastic; (2) expression of CD73, CD90, and
CD105 and lack of expression of CD14 or CD11b, CD79α or CD19, CD34,
CD45, and HLA-DR; and (3) osteogenic, adipogenic, and chondrogenic
differentiation potential under standard culture conditions [28]. Similarly,
minimal criteria for the definition of adipose tissue (AT)-derived
stromal/stem cells have also been recently established by a combined panel
from ISCT and the International Federation for Adipose Therapeutics and
Science (IFATS) [29].
MSC present additional characteristics that make them attractive for
therapeutic purposes, other than their ability to give rise to different
mesenchymal phenotypes. The secretion of a broad range of bioactive
molecules, such as growth factors, cytokines, and chemokines, renders them
with immunomodulatory and trophic activities, acting both in a paracrine
and autocrine manner [30, 31]. MSC trophic activity relies on bioactive
factors that assist in repair and regeneration processes. MSC are able to
inhibit scarring (fibrosis) and apoptosis, promote angiogenesis, and support
growth and differentiation of progenitor cells into functional regenerative
units [30, 31].
The panoply of beneficial effects ascribed to MSC has made them the
second most studied cells in clinical trials, immediately after HSPC [19],
with over 900 clinical trials taking place worldwide, receiving a special
focus in China, Europe, and the USA (Fig. 2b) (clinicaltrials.gov, accessed
on 29 May 2019, using the search term “mesenchymal stem cell OR
mesenchymal stromal cell”). MSC are promising candidates for the
treatment of a wide range of diseases, which is clearly observed from the
great diversity of conditions targeted in clinical trials. Musculoskeletal
diseases, immune system diseases, wounds and injuries, central nervous
system diseases, and vascular diseases are the top five conditions with the
highest numbers of undergoing clinical trials worldwide.
Many other cell types are being studied in clinical trials including
lymphocytes, dendritic cells, hepatocytes, and endothelial cells [19].
Nonetheless, a special focus will be given to HSPC and MSC throughout
this chapter.

1.3 Clinical Application and Challenges of Cell-Based


Therapies
Since 2009, 12 cell-based therapies have been approved and received
marketing authorization in the European Union (EU) and the USA
combined (Table 1) [32–34]. The first successfully approved product was
ChondroCelect, from TiGenix, despite being withdrawn in 2016 due to
commercial reasons. This product consisted in autologous cartilage cells
expanded ex vivo to treat knee cartilage defects. Holoclar (Chiesi
Farmaceutici) was the first approved stem cell product (2015), consisting in
ex vivo expanded autologous human corneal epithelial cells containing stem
cells to treat severe limbal stem cell deficiency. Other approved products
include the first CAR-T cell therapies for liquid cancers, Kymriah
(Novartis) and Yescarta (Kite Pharma), approved in 2017 in the USA and in
2018 in the EU, and more recently, Alofisel (Takeda Pharma) that consists
in expanded allogeneic AT-derived MSC to treat perianal fistulas in patients
with Crohn’s disease.
Table 1 Cell-based therapies that received MA in the USA and EU by September 2018 [32–34]

Product (MA Product description Therapeutic Date


holder) indication approved
Alofisel Expanded allogeneic mesenchymal adult Perianal fistulas in 2018 (EU)
(Takeda Pharma stem cells extracted from adipose tissue patients with Crohn’s
A/S) disease
Yescarta (Kite Autologous T cells genetically modified by Large B-cell 2018 (EU)
Pharma) retroviral transduction to encode an anti- lymphoma 2017
CD19 chimeric antigen receptor (CAR) (USA)
Kymriah Autologous T cells genetically modified Acute lymphoblastic 2018 (EU)
(Novartis) using a lentiviral vector to encode an anti- leukemia; large B-cell 2017
CD19 CAR lymphoma (USA)
Spherox Spheroids of human autologous matrix- Knee cartilage defects 2017 (EU)
(co.don AG) associated chondrocytes
Strimvelis Autologous CD34+ cells transduced with Severe combined 2016 (EU)
(Orchard an engineered retroviral vector encoding immunodeficiency
Therapeutics) the human adenosine deaminase sequence
Zalmoxis Allogeneic T cells genetically modified to Control mechanism for 2016 (EU)
(MolMed) express a truncated form of the human low graft-versus-host
affinity nerve growth factor receptor and disease after
the herpes simplex I virus thymidine kinase hematopoietic cell
transplantation
Holoclar Ex vivo expanded autologous human Severe limbal stem cell 2015 (EU)
(Chiesi corneal epithelial cells containing stem deficiency
Farmaceutici) cells
Provenge Autologous peripheral-blood mononuclear Metastatic prostate 2013 (EU)
(Dendreon) cells activated with prostatic acid cancer (withdrawn
phosphatase granulocyte-macrophage from EU in
colony-stimulating factor 2015)
2010
(USA)
Product (MA Product description Therapeutic Date
holder) indication approved
Maci (Vericel) Autologous cultured chondrocytes Knee cartilage defects 2016
(USA)
2013 (EU)
(suspended
in EU in
2014)
GINTUIT Allogeneic cultured keratinocytes and Mucogingival 2012
(Organogenesis) fibroblasts in bovine collagen conditions (USA)
Laviv (Fibrocell Autologous fibroblasts Severe nasolabial fold 2011
Technologies) wrinkles (USA)
ChondroCelect Autologous cartilage cells expanded ex Knee cartilage defects 2009 (EU)
(TiGenix) vivo expressing specific marker proteins (withdrawn
in 2016)

MA marketing authorization, USA United States of America, EU European


Union

Due to their uniqueness, cell therapies have earned their own category
in regulatory agencies with special directives concerning approval
candidature. Cell-based therapies are considered advanced therapy
medicinal products (ATMP), defined by the European Medicines Agency
(EMA) as medicines for human use that are based on genes, tissues, or
cells, offering groundbreaking new opportunities for the treatment of
disease and injury [33].
In spite of the establishment of guidelines and regulations applying to
cell therapies, a number of unresolved issues remain, making the regulatory
path toward clinical approval a challenge [35]. Certain important
requirements often lack in clarity, and regulation is not specific enough.
This results in products where the appropriate classification is not entirely
certain [36, 37]. Furthermore, discrepancies between regulatory agencies
from different countries hinder companies trying to reach the market at an
international level [37]. The challenging regulatory environment contributes
to the need to endure over long time periods before reaching the market.
Often, cell products only gain market access 15–20 years after the company
was founded [37]. Nevertheless, the regulatory environment is gradually
improving. In order to continue this path and make wise development
choices, it will be crucial to promote a cross talk between scientists,
companies developing cell therapies, and regulators [37].
Although this millennium has been marked with considerate advances,
with regulatory victories for several ATMP, cell therapy development has a
long and considerable track record. Recent success is due to much effort in
the past uncovering and understanding all the obstacles that stood between
the establishments of therapeutic options based on cells. HCT was decisive
as a vehicle of problem-solving and thus has deserved its recognition as a
foundation for cell therapy development [10].
With multiple cell-based therapies already reaching the market, one of
the most pressing issues will be addressing the challenges in manufacturing
these products. Most cell-based therapies are costly and target widespread
medical conditions. The robust and scalable cell manufacturing for the cost-
effective delivery of safe and potent cell-derived ATMP (either with
autologous origin (i.e., cells from the patient) or allogeneic) relies on
process engineering tools to understand the impact of cellular features
(biological, biochemical, etc.) on cell product function and performance and
how process variables influence the critical quality attributes of the cell
product. In general, the manufacturing process of cell-based therapies
consists of several stages: tissue collection, cell isolation, culture and
expansion (upstream processing), cell harvest, separation and purification
(downstream processing), and finally product formulation and storage (Fig.
3). The main advances made in the field and future challenges will be
addressed in this text, for each of the manufacturing stages.

Fig. 3 Manufacturing process for cell-based therapeutic products


2 Source and Isolation of Cells for Therapeutic
Use
From a manufacturing perspective, cell-based therapies have transformed
cells and tissues themselves into a bioprocess raw material. Consequently,
securing their supply is an unprecedented initial challenge in a production
pipeline, differing from previously established engineered cell factories. For
instance, cell retrieval from human tissues can be problematic. Although the
appropriation of biological waste can minimize this issue, some cell sources
may be very difficult to reach, while potentially posing health risks for a
donor or patient. Thus, management of this supply chain has a level of
complexity that is very case specific, depending on the cellular component
of the therapy [38].

2.1 Sources and Tissue Collection


Cell-based therapies depend primarily on obtaining the appropriate material
from which cells with possible therapeutic application can be isolated. So
far, multiple human tissues have been used as sources to obtain cells with
therapeutic potential [39].
Home to the hematopoiesis process, the BM harbors multiple cell types
that closely interact together, forming the so-called BM hematopoietic
niche, encompassing bone, osteoblasts, osteoclasts, HSPC, MSC,
macrophages, blood vessels, and extracellular matrix (ECM) [40].
However, the harvesting of BM requires an invasive procedure, allowing a
relatively small cell yield, which declines with donor age [41, 42]. For
example, HSPC (CD34+) frequency from BM aspirates after leukapheresis
is only 1.5% [43], and MSC frequency in a BM aspirate is only 0.001–
0.01% [26].
Mobilized peripheral blood (PB) is an alternative source of HSPC.
Donors are treated with granulocyte-colony stimulating factor (G-CSF) that
temporarily shifts HSPC from extravascular BM sites into the circulating
blood. This allows a painless and less invasive harvesting procedure
compared to BM aspiration. Hematopoietic recovery from mobilized PB
transplantation is similar to BM [44, 45]. However, the risk of GVHD is
lower with BM transplantation compared with PB [44, 45], and the
frequency of CD34+ cells in the PB is only 0.5%, which is lower than in
BM [43].
AT obtained from subcutaneous tissue represents an abundant source for
isolating MSC reliably using simple techniques. Liposuction, the technique
generally used for harvesting AT, has the advantage of being less invasive
than BM aspiration and is associated with high MSC isolation yields [46].
Specifically, liposuction allowed a yield of stromal vascular cells of
0.5 × 106 to 0.7 × 106 cell/g AT, and between 0.4 and 1.9% of the cells were
able to adhere and proliferate in culture [46]. Moreover, liposuction
material is considered medical waste, thus being an attractive alternative
source. The expansion potential, differentiation capacity, and
immunophenotype of MSC derived from AT are nearly identical to those
isolated from BM [42].
Neonatal tissues, such as the umbilical cord and placenta, are promising
alternative sources to adult ones. The umbilical cord is a rich source of
HSPC [47, 48] and has been shown to be a rich source of MSC [49]. For
example, about 0.6 million MSC were obtained per gram of umbilical cord
[50]. Harvesting the umbilical cord requires a painless and noninvasive
procedure. The umbilical cord is considered medical waste and is usually
discarded after birth, thus being an attractive alternative source. Within the
umbilical cord, HSPC are collected from the umbilical cord blood (UCB).
A large-scale study of 126,341 red blood cell-depleted UCB units in the
USA inventory revealed that the median frequency of CD34+ cells was
0.34% [51]. MSC on the other hand can be isolated from the UCB as well
as from the Wharton’s jelly, the connective tissue surrounding umbilical
vessels [52]. Most studies are performed with MSC derived from Wharton’s
jelly, which is commonly referred as the umbilical cord matrix (UCM).
Umbilical cord-derived MSC expand at a higher rate when compared to
BM- and AT-derived MSC [42, 53]. Furthermore, UCB enables a better
repopulation efficiency upon HCT, when compared to BM and mobilized
PB, as assessed through quantitative in vivo severe combined
immunodeficiency (SCID)-repopulating cell assay [54], despite the limited
cell number per unit, which has set UCB particularly suited for pediatric
patients.
Possibly due to their broad definition, MSC have been successfully
isolated from a number of tissues other than the previously mentioned,
including synovial membrane [55], placenta [56], dental pulp [57], brain,
liver, kidney, lung, muscle, thymus, and pancreas [58].
Notably, cells show different therapeutic capacity depending on the
source they were isolated from. For example, MSC isolated from BM, AT,
and UCM revealed different ability to suppress PB natural killer and B and
T cells, when co-cultured with phytohemagglutinin-stimulated PB
mononuclear cells [59].

2.2 Isolation of Target Cell Populations


Depending on the nature of a specific cell therapy, assuring source
availability and succeeding in tissue collection may be enough to proceed to
the following bioprocessing stage. For minimally manipulated cell
products, such as HCT, heterogeneous populations are isolated and directly
infused into the patient. However, newer and more advanced cell therapies
are becoming ever more population specific. Thus, bulk populations that
normally result from harvesting procedures need funneling techniques that
isolate a desired cell type [60].
Still, the most commonly used method to isolate MSC is very simplistic,
relying solely on the ability that MSC have to adhere to plastic surfaces
[28]. After tissue collection, cells are plated on polystyrene-based tissue
culture flasks. MSC will adhere to the plastic surface, while contaminating
cells, such as the ones from hematopoietic lineages, are washed away after
medium change and passaging [61, 62]. Typically, when MSC are obtained
from tissues such as UCM, AT, or synovial membrane, these can be either
enzymatically digested using collagenase solutions [46, 62, 63] or simply
plated directly onto plastic surfaces as explants [64–66].
More sophisticated techniques can be used to isolate specific cell
populations following tissue collection, typically relying on affinity-based
and centrifugation-based separations. Although affinity-based separation
has gained significant momentum in cell therapy manufacturing, classical
centrifugation techniques are still part of typical bioproduction processes.
When blood or marrow samples are used (e.g., BM, PB, and UCB), a first
isolation step with density gradient centrifugation, using a polymeric
solution (e.g., ficoll or percoll), separates the mononuclear cell (MNC)
fraction from other constituents such as plasma and erythrocytes [61]. A
considerable part of cell therapies are centered on hematopoietic
subpopulations (e.g., CAR-T cells, regulatory T cells (Treg), monocytes, and
HSPC), whose mentioned isolation consists of centrifugation approaches
for separation and extraction of MNC.
Several Sepax (originally developed by BIOSAFE, now GE Healthcare)
cell processing systems have brought a fully closed and automated
centrifugation unit to cell therapy production pipelines [67]. More advanced
centrifugation platforms combine different physical forces to achieve higher
isolation recovery and purity. Terumo BCT has established a continuous
centrifugation system (Elutra®) that joins centrifugal forces with
counterflow [68]. Using blood, initial centrifugation separates the MNC
layer from plasma and erythrocytes. Fluid moving in counterflow separates
the buffy coat into different fractions. By achieving cell population
separation based on size and density, these platforms are able to reach much
higher resolution in separation [69]. Monocyte isolation from peripheral
blood mononuclear cells using this technology managed to concentrate
monocytes in a single elutriation fraction with a recovery of 78% and a
purity of 62%. Nevertheless, in combination with a post-elutriation density
gradient, monocyte purity rose to 91% without affecting the initial recovery
[69].
Cell isolation through affinity is an ever-growing alternative due to its
separation criteria being based on biological instead of physical
characteristics. Cell population immunophenotype is commonly used to
isolate specific cells from their original sources, such as HSPC (CD34+
selection) [70, 71] and MSC (Stro-1+ selection) [72, 73]. Typically
mediated by antibody-antigen interactions, fluorescence-activated cell
sorting (FACS) and magnetic-activated cell sorting (MACS) occupy leading
roles in affinity-based separation. Through fluorescent-labeled antibodies,
FACS is able to separate cell populations based on their surface marker
expression. This technology allows for multiple marker selection with high
selectivity due to single-cell analysis [74]. In combination with cell
separation, multiparametric studies can be performed simultaneously,
allowing for identity and quality control.
MACS shares the same separation criteria as FACS (i.e.,
immunophenotype) but achieves cell sorting with antibodies coupled to
magnetic particles. While these techniques are not novel, direct and
thorough comparison has only recently been established [75]. Whereas
FACS dominates selectivity and subpopulation purity, the respective cell
sorter is not inherently prepared for a clinical setting. An expensive
hardware system combined with lack of parallelization, sterility issues, and
time-consuming protocols are some constraints that contribute against its
translation. Due to its column-based system, MACS is able to separate cells
at a much faster rate with possibility for parallel operation and is
compatible with current good manufacturing practice (cGMP) guidelines.
Closed versions of MACS (e.g., CliniMACS Plus® by Miltenyi Biotec and
CTS™ DynaMag™ by Thermo Fisher) have been developed for clinical-
scale cell isolation [76, 77]. The trade-off between purity and recovery has
burdened many isolation techniques and protocols. Natural killer cells
purified by MACS were able to achieve a 95% purity while only reaching a
median recovery of 37% [76]. Nevertheless, lack of bead detachment from
cells after isolation is a significant drawback for MACS as a cell therapy
bioprocessing unit. Besides particle contamination during the production
process, epitope restrain by antibody-magnetic particle complexes can
affect cell performance and undermine therapeutic value.
Interestingly, both techniques are complementary and therefore
selection of cell sorting technique is application dependent. Still, for cell
therapy process development, there is a clear tendency toward MACS due
to previously mentioned arguments. Recently, efforts have been made by
both sides to overcome their limitations.
New platforms for FACS that include disposable microfluidic cartridges
provide optimism for its adaptation into production pipelines. WOLF
(NanoCellect Biomedical) and On-chip Sort (On-chip Biotechnologies) are
two cell sorters that use this concept to overcome typical FACS limitations.
The microfluidic cartridges allow for a closed circuit that includes the
optical path and sterile sorting containers, minimizing contamination risks.
Still, these systems have intrinsically low sorting rates that combined with
demanding clinical cell dose targets make their use unrealistic [74].
Development of the MACSQuant Tyto (Miltenyi Biotec) system has
been the latest contribution toward FACS adoption into the cell therapy
bioproduction process. With sorting speeds reaching 30,000 cells/s,
MACSQuant Tyto outmaneuvers its microchip competitors by a factor of
100 [74].
Although not possessing an established clinical platform, vortex
actuated cell sorting (VACS) has come forward as a potential challenger for
MACSQuant Tyto. This technology uses the same principles for cell sorting
as FACS but possesses a different sorting mechanism. The valves or
deflection plates used to separate distinctive cell populations are substituted
by a microfluidic thermal vapor bubble actuator that deflects cells due to
the formation of an inertial vortex in the flow. Designed by Cellular
Highways, the prototype sorter named Highway 1 is currently being
developed, with sorting rates reaching 43,000 cells/s. It combines full
automation with closed circuits, possibility for multiplexing and a
respectable sorting speed [78].
On the other hand, strategies for MACS improvement regarding
disruption of cell-magnetic particle complexes have also been explored,
focusing on the interaction between antibody molecules and magnetic
particles. Thermo Fisher’s Dynabeads® FlowComp™ combine streptavidin-
coated beads with biotin-conjugated antibodies, enabling a post-isolation
bead removal mechanism [79]. STEMCELL Technologies established their
own product, termed Releasable RapidSpheres™, that possess a tetrameric
antibody complex which assures cell and particle interaction [80].
Following cell isolation, a mild dissociation agent cleaves the tetrameric
complex, releasing the magnetic particles. Successful particle removal will
also help alleviate regulatory issues over end product safety. Consequences
of nano- and microparticle contaminations are still debatable, with magnetic
bead internalization being a validated concern.
Instead of improving existing techniques, novel approaches for affinity-
based cell isolation have also been investigated. Since cell therapies possess
more stringent safety criteria, delivering cells without any by-products due
to bioprocessing is crucial. Therefore, antibody removal after affinity
separation is of considerable interest.
Traceless affinity cell selection (Fab-TACS®) is an innovative
technology that explores a reversible antibody-antigen interaction. Antigen-
binding fragments (Fab) are combined with a short peptide tag (Strep-
tag®II) with significant affinity toward a derivative of streptavidin (Strep-
Tactin®). By having a Strep-Tactin®-coated agarose matrix, desired cells are
retained in a Fab-TACS® column [81]. Using biotin analogs, cells are eluted
from the column due to interaction competition. Since the antibody
fragments have low affinity toward the selected antigen, Fab release occurs,
leaving the isolated cells without any separation by-product or trace. This
technology has been translated into an automated commercial device
(FABian® by IBA Lifesciences) [81].
A combinatorial approach for cell isolation has been explored by
Akadeum Life Sciences. Instead of improving or developing new
techniques, an innovative method has been devised that brings
centrifugation and affinity-based separation together. Buoyancy-activated
cell sorting (BACS™) brings several above-mentioned concepts together in
a novel manner [82]. Biotinylated antibodies are introduced in a cell
suspension to target undesired cells (negative selection). Glass-shelled
microbubbles coated with streptavidin are mixed to capture antibody-tagged
cells. These microbubbles are separated from the remaining cell populations
through centrifugation by flotation [82]. Purity and recovery values higher
than 80% have been reported for this novel technique [83].
Isolation of a target cell population can have different impact depending
on a specific cell therapy, with products ranging from bulk and
heterogeneous populations to very selective subpopulations with a defined
phenotype. Adequate selection of a separation method is also dependent on
the prioritization of opposing purification concepts, such as purity and
recovery [38].

3 Cell Production
The relatively low frequency of cells with therapeutic potential within the
native tissues, followed by harvesting procedures and eventually successive
isolation steps, yield a substantially low number of cells in the end.
Therefore, in order to use these cells in a clinical context, it is usually
required additional steps of manipulation and propagation ex vivo, which
depend on choosing the appropriate culture medium conditions,
physicochemical parameters, and culture platforms [84].

3.1 Culture Medium Formulation


The maintenance and propagation of animal cells in vitro require a cell
culture medium, supplying nutrients and inorganic salts, as well as
providing the appropriate physicochemical conditions. Generally, medium
components include glucose (carbon source), amino acids (nitrogen source),
vitamins (cofactors), inorganic salts (maintains electrolyte balance), sodium
bicarbonate (buffer, to maintain pH at 7.4), sodium chloride (adjusts
osmotic pressure), antibiotics (prevent microorganism contamination),
phenol red (visual pH indicator), and growth factors and hormones (growth
stimulation) [85, 86]. These components are provided in commercially
available basal medium formulations, such as Eagle’s medium and
derivatives (e.g., Dulbecco’s Modified Eagle’s medium (DMEM),
Minimum Essential Medium Eagle alpha (αMEM)), medium from Roswell
Park Memorial Institute (RPMI), and several other well-established media,
which have been subject of improvement over the years [86].
Generally, the addition of certain molecules to basal medium
formulations is required. For cell therapies based on hematopoietic lineages,
cytokines play an important part as a culture medium component. These
soluble factors have a great role in influencing cellular signaling pathways.
In the context of HSPC expansion, factors present in the medium prevent
cell differentiation while promoting the self-renewal capability of cells.
Stem cell factor, thrombopoietin, granulocyte-colony stimulating factor,
fms-like tyrosine kinase 3 ligand, and interleukin-6 are some cytokines
currently used in expansion protocols for the manufacturing of UCB
expanded grafts under study in several clinical trials [87–89].
Culture medium formulations usually require the addition of a protein-
rich supplement, containing growth and adhesion factors. The most
commonly used culture supplement is animal serum, especially fetal bovine
serum (FBS). Serum is a source of amino acids, proteins, vitamins,
carbohydrates, lipids, hormones, growth factors, and inorganic salts [86].
Moreover, it enhances cell adhesion, improves the pH-buffering capacity of
the medium, and helps reduce shear stress during cell manipulation.
However, it presents significant disadvantages such as being ill-defined,
wide batch-to-batch variability, risk of contamination with virus and prions,
and ability to transmit xenogeneic antigens, leading to increased
immunogenicity of cultured cells, thus limiting FBS application in the
clinical setting [90, 91]. Besides the cell biological perspective, ethical
concerns and animal welfare issues arise from the use of animal serum, as
serum collection causes animal suffering [90]. Furthermore, the global
supply of FBS is declining over the years, and this tendency is expected to
continue [90]. This will eventually result in a FBS supply that will not be
able to meet the increasing demand. Therefore, given its disadvantages,
using FBS for cell culture of clinically applied cell products is discouraged
and should be avoided. By complying with current international guidelines
and regulatory frameworks [92–94], there is need for developing alternative
culture supplements.
In the last decade, the development of serum-/xeno(geneic)-free (S/XF)
culture formulations (i.e., without serum or animal origin components) has
been a priority for the field of cell therapies. Although these media
represent a valuable alternative to FBS, as they are more consistent and
standardized, they still contain a cocktail of growth factors, proteins, and
hormones derived from human serum or even plant hydrolysates, classified
as chemically undefined [95]. Chemically defined, animal component-free
media, on the other hand, consist exclusively of well-defined and
characterized components and entirely free of animal (including human)
derived products. These include purified recombinant proteins and synthetic
bioactive molecules [95].
One of the well-established supplements used in S/XF media, proposed
as an alternative to FBS, is human platelet lysate (hPL). As early as the
1980s, hPL-supplemented medium was found to support proliferation of
established cell lines and primary fibroblasts [96, 97]. hPL is usually
prepared from fresh blood or platelet concentrates, containing bioactive
molecules such as growth factors, adhesion molecules, and chemokines,
which originate primarily in the α-granules of platelets [98]. Preparation of
hPL from platelet concentrates can be achieved either by repeated
freeze/thaw cycles, sonication, induced platelet activation by addition of
thrombin or CaCl2, or solvent/detergent treatment [98].
Blood banks routinely prepare pooled allogeneic platelets from human
blood donations. When these are not used for transfusion, they are used for
further manufacturing into hPL, thus allowing a steady supply for
manufacturing an allogeneic “off-the-shelf” hPL product for cell culture
[98, 99].
Multiple studies have demonstrated hPL-supplemented media to be
efficient for the isolation and expansion of MSC from various origins [100–
102], cultured both in static and dynamic systems [103, 104], already with
several ongoing and completed clinical trials (clinicaltrials.gov). Moreover,
it has been shown that both allogeneic and autologous hPL-supplemented
media allow improved cell proliferation when compared to FBS-containing
media [100, 102, 105, 106]. The main differences in hPL protein content
compared to FBS are the higher content of immunoglobulins and the
possible presence of fibrinogen and other coagulation factors, when hPL is
produced without thrombin activation [98].
In addition to its application for MSC expansion, hPL has been
evidenced as an efficient growth medium supplement for ex vivo expansion
of other cell types such as human gingival fibroblasts [107], chondrocytes
[108], osteocytes, myocytes and tenocytes [109], as well as endothelial cells
[110], indicating its potential applicability in multiple areas of cell therapies
and regenerative medicine. Although hPL is considered safer than FBS by
the scientific community and regulatory agencies, it still poses some
constraints, such as the risk of transmission of human diseases by known or
unknown viruses, ill-definition, and the possibility of triggering immune
responses [111]. Nonetheless, hPL products derived from pooled units and
produced in large scale are already commercially available [99] and seem to
be the most promising alternative to FBS supplementation in cell culture
medium in the near future. Moreover, novel gamma-irradiated hPL products
have been developed toward pathogen reduction. Results showed that
gamma radiation allowed 4 log10 reduction of viral titer with low impacts
on the potency for cell expansion [112].
There are other commercially available S/XF media that have been
successfully applied for cell culture. StemPro® MSC SFM (Life
Technologies) and MesenCult™-XF (STEMCELL™ Technologies) are two
chemically undefined S/XF media that have been used to successfully
expand MSC from different sources [113–116].
Considering the expansion of human HSPC, most current protocols are
targeting the use of serum-free medium formulations supplemented with
cytokines. StemSpan™ H3000 (STEMCELL™ Technologies) is a S/XF
medium with human-derived components and has been used for expansion
of HSPC [117]. Although being chemically undefined, containing human-
derived components, these media formulations represent an improvement
for cell culture, due to better definition and lower batch-to-batch variability
when compared to FBS- and hPL-supplemented media.
The ideal candidate for production of clinical-grade cell-based therapies
would be a chemically defined, animal component-free media (including
human), composed exclusively of well-defined factors that could replace
serum and serum-derived products. These include synthetic bioactive
molecules and purified recombinant proteins [95]. There are multiple
factors that can be combined in order to replace serum, such as growth
factors (e.g., EGF, FGF, TGF), hormones (e.g., growth hormone, insulin),
carrier proteins (e.g., albumin, transferrin), lipids (e.g., cholesterol, fatty
acids), transition metals (e.g., Se, Fe, Cu, Zn), vitamins, adhesion factors
(e.g., fibronectin, laminin), polyamines, and reductants (e.g., 2-
mercaptoethanol) [86]. The number of possibilities that result from the
combination of these components is enormous, making the selection of the
most appropriate ones and their respective concentration in a medium
formulation an extremely difficult task. For that purpose, design of
experiments is possibly the best strategy to find the optimal concentration
of each component in a culture medium, especially considering likely
interactions between the components [86]. Consequently, S/XF media,
especially chemically defined culture media, are often cell type specific.
The chemically defined medium TheraPEAK™ MSCGM-CD™
(Lonza) has been used for the expansion of MSC [118]. Successful
expansion of T cells was also achieved using chemically defined S/XF
media, relying, for instance, on the CTS™ Immune Cell Serum
Replacement supplement [119, 120].
In order to disseminate the development and application of serum-free
media for cell culture, “FCS-free Database,” a freely accessible serum-free
media database, is available online (https://fcs-free.org/), providing an
overview of FBS-free media for cell culture.

3.2 Physicochemical Parameters


Besides biochemical factors such as nutrient/metabolite concentration and
growth factors, physicochemical parameters such as pH, temperature,
osmolality, and oxygen tension are equally important for the maintenance of
animal cell cultures. The optimal values for each of these physicochemical
parameters will differ depending on the cell product, which poses an
additional challenge in cell manufacturing.
Most cell lines grow successfully at pH 7.2–7.4. However, the optimum
culture pH depends on the intended application. For example,
differentiation of human MSC into osteoblasts can be improved by
changing the pH of culture medium from normal to alkaline medium [121].
The differentiation of erythroid progenitors can also progressively increase
as pH is increased from 6.95 to 7.4 and 7.6 [122]. Usually the pH is
controlled in cell culture by using the CO2/ buffer system. Cells are

typically cultured in humidified incubators with gas phase CO2 at 5% and


sodium bicarbonate as a medium additive. The CO2 dissolved in the
aqueous phase stays in equilibrium with , adjusting the pH [86].
The optimal temperature to cultivate human and warm-blooded animal
cells is 37°C. However, cell culture at different temperatures may be
advantageous for certain purposes. For example, culturing MSC at 32°C
decreased the accumulation of oxidative damage and improved their
osteogenic differentiation ability, when compared to 37°C [123].
Although different cells have different optimal osmolality values, most
cells grow well in the range between 290 and 310 mOsm [124, 125]. As
previously mentioned (Sect. 3.1), the osmolality is mainly defined by the
sodium chloride content in the medium.
Most animal cell cultures are performed at an atmospheric oxygen level
(21% O2). However, the oxygen concentration in most tissues is lower than
the atmospheric one, due to gas transfer phenomena. Therefore, mimicking
the in vivo oxygen concentration might have a positive impact in cell
culture as well as on the therapeutic potential of the cultured cells. One
canonical example would be the BM, which is characterized by a hypoxic
environment, with oxygen concentration ranging in the interval between 1
and 6% [126, 127].
In light of this observation, several studies were performed by exposing
MSC to hypoxic conditions. Hypoxic conditions were found to have an
advantage for MSC expansion as well as in terms of differentiation [128–
130]. A study performed by Oliveira and colleagues revealed that both BM
MSC and AT MSC cultured in hypoxic conditions experienced an
immediate and concerted downregulation of genes involved in DNA repair
and damage response pathways [131]. Moreover, it revealed that AT MSC
reacted to hypoxic environment more slowly than BM MSC, as different
characteristics of each cell niche (e.g., degree of vascularization, oxygen
tension, cell-cell interactions) determine distinct sensitivities to hypoxia ex
vivo [131]. Likewise, hypoxia (5% O2) enhanced the proliferation of UCB-
derived HSPC, when compared to normoxia (21% O2), as well as allowing
a better preservation of bone marrow repopulation in SCID mice [132]. In
another study, the ex vivo expansion of UCB-derived HSPC in co-culture
with BM MSC was maximized in a 10% O2 atmosphere, when compared to
other hypoxia conditions and normoxia levels [133].
Besides the need to establish the most appropriate physicochemical
conditions for a certain cell-therapy manufacturing process, maintaining
these parameters at the correct values throughout culture is equally
important. In traditional culture systems, these parameters are often
observed, but rarely controlled, thus decreasing the robustness of the
manufacturing process. This issue will be addressed in more detail in Sect.
3.5.

3.3 Scalable Culture Vessels


Whether isolated cell populations need to undergo differentiation or
expansion, appropriate cell culture vessels and systems are necessary.
In terms of complexity, at the rear end of cell culture technology are
simple plasticware containers. Different geometries make up a broad
collection of vessels in order to cover any cell type and their projected
application. Petri dishes, T-flasks, roller bottles, and multiwell plates all
incorporate cell culture plasticware and are typically made of polystyrene
that is previously treated either chemically or physically in order to gain
hydrophilic functional groups (e.g., ketones, aldehydes, hydroxyl, and
carboxyl groups) [134]. Indeed, surface treatment has a dramatic impact on
adherent cell culture, with proper cell adhesion being a main concern.
Unfortunately, when using S/XF culture media, cell adhesion can be
compromised due to deficiency in serum-derived adhesion factors [135].
Commercially enhanced plasma treatment plasticware (e.g., CellBIND® by
Corning Life Sciences) and xeno-free surface coatings (e.g., CELLstart™
by Thermo Fisher Scientific and Synthemax® by Corning Life Sciences)
have been developed to address this issue [136, 137].
Besides allowing gas exchange through the cap region and having
excellent optical clarity, commonly used vessels are seriously limited
regarding any type of monitoring and control. Conventional plasticware as
culture flasks also lack an agitation mechanism, not being able to assure
fully homogenized cell cultures. Since their design was directed mainly
toward research purposes, manufacturers quickly identified scalability
issues for large-scale production. Advanced and scalable culture systems
based on plasticware were created to avoid laborious and unsustainable
scale-out.
Although very simplistic, plastic malleable bags have a consolidated
place in cell culture. Being integrated in basic plasticware, they offer a
simple closed system solution which is critical for manufacturing under
cGMP. However, limited culture control and poor agitation severely limit
their application in optimized processes. Nevertheless, therapies based on
hematopoietic cells (e.g., tumor-infiltrating lymphocytes, CAR-T, and
HSPC) have relied on these platforms for cell culture, reaching human use
in clinical trials [87, 138, 139].
Multilayered flasks (e.g., Nunc™ Cell Factory™ System by Thermo
Fisher Scientific) were designed to increase culture area while reducing
volumetric footprint of using multiple individual flasks. Additionally,
closed versions with perfusion mechanisms of these flasks were also
developed to overcome the open nature of conventional flasks. Large-scale
expansion of MSC in serum-free conditions was achieved using
HYPERStack system (Corning Life Sciences), yielding an average cell
density of 2 × 104 cell/cm2, corresponding to a fourfold increase in total cell
number after 4 days [140]. Proprietary gas permeable films improve gas
diffusion, which do not compromise cell viability in high-density adherent
cultures of tightly packed multilayered flasks. Flask potential has been
pushed further with the commercialization of the CellCube® by Corning
Life Sciences, a closed system comprising of densely packed thin individual
surfaces with continuous medium supply in laminar flow, reaching
85,000 cm2 (39 cm × 25 cm) for adherent cell culture [141]. The Xpansion®
multiplate system designed by Pall Corporation takes advantage of the same
concept, aside from assuming a cylindrical geometry with capacity for up to
122,400 cm2 of culture surface. Xpansion®-50 was used for large-scale
expansion of human periosteum-derived stem cells for the treatment of
bone defects, achieving a final cell density of 1.75 × 104 cell/cm2,
corresponding to a 3.9-fold change in total cell number after 7 days, and
presenting a final recovery efficiency of 45% [142].
Roller bottles have also been optimized for large-scale manufacturing of
cells. Cord blood-derived MNC were isolated and expanded in multiple
500 mL roller bottles with rotation assured by a bottle roller [143]. Further
improvement led to the design of RollerCell™ by Cellon, a system capable
of simultaneously holding 40 roller bottles with automated robotic
processors for cell handling. RollerCell™ comparison with CellCube® for
cell line production yielded similar results [144].
Although planar systems have evolved to closed and scalable systems
with possibility for dynamic regimen through continuous fluid flow (e.g.,
CellCube® and Xpansion®), bioreactors have been the ultimate objective
for cell therapy manufacturing, seeing that they incorporate monitoring and
control, reduce process footprint, and minimize cell handling.
Incorporating highlighted challenges of a cell-centered process requires
platforms capable of dealing with parameter complexity to deliver a safe
and reproducible cell-based product. Table 2 enumerates current cell-based
therapies in clinical trials that involve bioreactors in their production
process. Innovative bioreactor designs have come forward to challenge
more classical versions.
Table 2 List of clinical trials using bioreactors for cell based-therapies

Study name Type of Cells Condition Phase


bioreactor
Extracorporeal Immune Support System EISS- Human Severe sepsis Phase 1
(EISS) for the Treatment of Septic Patients immune- donor and septic and phase
(EISS-1)a cell granulocytes shock 2
bioreactor
device
Safety of Intramuscular Injection of PluriXTM Placental Critical limb Phase 1
Allogeneic PLX-PAD Cells for the 3D adherent ischemia
Treatment of Critical Limb Ischemiaa bioreactor stromal cells
system
Expansion of Invariant NKT Cells for a Bioreactor NKT cells Allogeneic Not
Cell Immunotherapeutic Approach hematopoietic available
Allowing the Control of GvHD and stem cell
Preserving the Graft Versus Leukemia (HSC)
Effect After Allogeneic HSC transplantation
Transplantationb
Laryngo-tracheal Tissue-Engineered Stem-cell Autologous Tracheal Not
Clinical Transplantationc seeded stem cells diseases applicable
bioartificial
tracheal
scaffold

Clinical trials were obtained from “clinicaltrials.gov,” on 21 March 2019,


using the term “bioreactor” and selected for cell therapy applications
Clinical trial status: acompleted; bnot yet recruiting; cunknown

Stirred tank bioreactors (Fig. 4a) maintain widespread use, with their
simpler and more standardized geometry. With extensive experience in
what concerns the production of traditional biopharmaceuticals, much
knowledge regarding these bioreactors has been transposed to cell-based
therapies. These systems have mechanical impellers that are responsible for
appropriate mixing and assuring dynamic flow. High compatibility with
monitoring probes and respective modules has made culture control an
intrinsic part of this bioreactor. Internal sparging mechanisms allow for
efficient gas transfer, although shear stress associated with bubbling can be
an issue to sensitive cells [145]. Exhaustive knowledge on fluid profiles
based on computational fluid dynamics (CFD) models have given
significant predictive control on culture estimates.

Fig. 4 Schematic representations of bioreactor configurations that can be potentially used in the
manufacturing of cell-based therapies: (a) stirred tank bioreactor, (b) packed bed bioreactor, (c)
hollow fiber bioreactor, (d) wave bioreactor, and (e) Vertical-Wheel™ bioreactor

While being naturally prone for suspension cultures [146], adherent cell
culture has been adapted through microcarrier development. These
spherical particles provide the surface area for cell adhesion to occur. A
broad variety of materials, porosity levels, and surface coatings have been
developed to fulfill specific cell needs. The high variety of microcarriers
has been extensively reviewed [147, 148].
Of notice, we have pioneered in the development of clinical-grade
expansion of MSC of different human sources (i.e., BM and AT) in scalable
microcarrier-based bioreactors using S/XF culture components, achieving
the production of 1.1 × 108 and 4.5 × 107 cells for BM MSC and AT MSC,
respectively, after 7 days of culture [149]. Building on this platform, we
have concentrated efforts in maximizing cell productivity by changing
different culture parameters. We have previously optimized feeding and
agitation regimes and performed microcarrier screening [114]. Furthermore,
we have successfully incorporated an alternative MSC tissue source (i.e.,
UCM) [150] and have implemented a different bioreactor configuration
with a vertical agitation design (Vertical-Wheel™) (Sect. 3.4) [104].
The scalability potential of stirred tank bioreactors for cell-based
therapies has been embodied by development of MSC expansion processes.
While initial studies restricted their culture scale to spinner flasks, Rafiq
and colleagues managed to scale up MSC expansion to a 5 L stirred tank
bioreactor, achieving a cell concentration of 1.7 × 105 cell/mL,
corresponding to over a sixfold expansion in total number of cells [151].
Subsequently, Lawson and colleagues pushed the scalability of stirred tank
MSC culture forward by successfully expanding human MSC in a 50 L
bioreactor, being able to produce 177 clinical doses (70 million cells/patient
assuming a 70 kg patient) in a single run [152]. In contrast to the above-
mentioned scale-up, with contributions of multiple groups to ever-
increasing culture dimensions, Schirmaier and colleagues were able to
perform an entire stepwise scale-up of AT MSC expansion from spinner
flasks to 35 L cultures, yielding 1 × 1010 cells at the end (35 L scale) [153].
Consequently, both adherent and suspension cultures are firmly
established for cell culture in stirred tank bioreactors. Commercial versions
of stirred tank bioreactors include the Celligene® series by Eppendorf and
the Finesse series by Thermo Fisher Scientific.
Mammalian cells are known to be more shear sensitive which
stimulated efforts to develop non-abrasive environments during cell culture.
Packed bed bioreactors (Fig. 4b) provide a fixed chamber where
microcarriers or scaffolds are located [154]. Adhered cells that populate the
chamber have translational movements restricted, thus being able to better
mimic solid tissue presence. Their constrained movement also promotes
structured organization and cell-cell interaction, leading to high-density
cultures. Low-velocity fluid flow guarantees dynamic culture without
causing shear damage to cells. Culture medium has access to the chamber
providing necessary nutrients and removing metabolites. Diffusion
limitations or nutrient deficiency can occur due to 3D culture organization.
Furthermore, significant cellular organization can result in beneficial
biological outcomes but will normally complicate cell extraction and
subsequent downstream processes. Expansion of MSC in a 2.5 L CelliGen®
bioreactor (New Brunswick Scientific) with Fibra-Cel® (Eppendorf) disks
demonstrated large-scale manufacturing potential for packed bed
bioreactors, achieving 9.2 × 107 cells after 9 days of culture, corresponding
to a 9.2-fold increase in total cell number [155].
Increasing available area for cell culture while protecting cells from
harsh conditions has inspired innovative bioreactor designs. Hollow fiber
bioreactors (Fig. 4c) fulfill those requirements by joining thousands of
hollow fibers. These fibers are made of thin and porous material that
provide a selective passage of nutrients. Culture medium recirculates
through the fibers producing interesting tangential flow, mimicking
vasculature to some extent [154]. However, significant quantity of fibers
originates successive diffusion barriers that cause concentration gradients
for nutrients, signaling factors, or gases. Similar to packed bed bioreactors,
cell extraction processes are challenging to perform due to high cell
interaction and difficulty in reaching cells uniformly inside the bioreactor.
With unprecedented tight regulatory measures, the field of bioreactors
has moved toward disposable and single-use versions. In order to avoid
clean-in-place (CIP) and steam-in-place (SIP) procedures and assure
contamination-free product quality, conventional stainless steel or other
reusable bioreactors are being substituted by plastic single-use bioreactors
(SUB). They reduce cross-contamination and can be combined with limited
monitoring probes. Disposable technology has been able to successfully
adapt existing geometries, such as the Mobius series by EMD Millipore for
stirred tank bioreactors and the Quantum® bioreactor by Terumo BCT for
hollow fiber bioreactors. The latter bioreactor has been validated with
adherent AT MSC, BM MSC, periosteum-derived MSC, and neural stem
cells [156–160]. However, novel designs, such as the wave bioreactor (Fig.
4d) and the Vertical-Wheel™ bioreactor (Fig. 4e), have also shown that
there is space for bioreactor innovation that integrate single-use technology.
Recently, an overview of SUB and their applicability toward cell therapy
have been investigated [161]. It was observed that SUB designs have
evolved, currently integrating well-known principles of mass transfer and
mixing. Their versatility and single-use nature align with cost reduction and
demanding regulatory guidelines associated with cell therapies. However,
culture monitoring remains a challenge, and long-term bag stability must be
assured.
Numerous bioreactor designs exist for performing cell culture;
nevertheless selecting the correct culture vessel with an appropriate
scalability strategy is the actual challenge for the manufacture of cell
therapies. Achieving parallelization of individual units (scale-out) tends to
be more associated with autologous therapies, while increasing bioreactor
size and maintaining culture conditions (scale-up) is more adequate for an
allogeneic production. A compromise between scalability and optimal
culture conditions is deemed necessary.

3.4 Agitation
One of the crucial factors for successful cell expansion is culture medium
homogenization. Bioreactors require sustained agitation of the culture
system, in order to allow an appropriate mass transfer of nutrients and
oxygen to the cells, as well as a removal of waste products derived from
cell metabolism. For that purpose, cells must be maintained in suspension
homogeneously, independently of whether the cells are cultured freely in
suspension, as cellular aggregates or adherent to microcarriers/scaffolds.
However, agitation may have an impact on cellular physiology, due to
increased shear stress. In this context, shear stress can be defined as the
force component acting tangentially to a material, due to fluid motion [162].
Therefore, in bioreactor processing, cells are exposed to shear stress
originating from fluid agitation. Shear stress has been described to have a
significant impact on cell phenotype, which can be either negative or
beneficial depending on the final application. In fact, it has been long
established that animal cells in general are sensitive to shear stress, which
compromises their viability above certain levels [163–165]. Additionally,
agitation affects HSPC surface marker expression, including cytokine
receptors [166] and CD34 [167], which impacts cell expansion by enriching
specific HSPC populations in culture. On the other hand, shear stress has
been demonstrated to induce osteogenic differentiation of BM MSC
through increased expression of osteogenic factors such as bone
morphogenetic protein-2 (BMP-2), bone sialoprotein (BSP), and
osteopontin (OP) [168] and also resulted in increased intracellular Ca2+
levels [169]. Shear stress also improved the angiogenic potential of human
AT MSC through stimulation of vascular endothelial growth factor (VEGF)
secretion [170].
The importance of agitation and the impact it has on culture outcome
have led to the development of new technologies and bioreactor
configurations that specifically target this issue. Wave bioreactors (Fig. 4d)
are suitable for the manufacturing of shear-sensitive cells. Their agitation
through rocking motion prevents the use of an impeller exerting high shear
forces directly in the cells. Very low level of shear stress was found in wave
bioreactors compared to classical stirred tank reactors [171]. Wave
bioreactor implementation for culture of suspension cells, with emphasis to
hematopoietic lineages, is well-known [172, 173].
In the same line, Vertical-Wheel™ bioreactors (Fig. 4e), developed by
PBS Biotech, incorporate a vertically rotating wheel, allowing a more
efficient mixing than the traditional horizontal stirring solutions. By
allowing lower agitation rates, they are able to minimize shear stress
effects. The vertical mixing allows a higher mass transfer rate and more
homogenous and gentle particle suspension, favorable for anchorage-
dependent cells on microcarriers [174]. Moreover, this technology is fully
scalable, being available at working volumes that range from 60 mL up to
500 L. Vertical-Wheel™ bioreactors have been successfully applied in
microcarrier-based cell culture systems for the expansion of MSC from
multiple sources [104, 175], as well as for human iPSC [176].
In summary, agitation can modulate culture conditions and have a
significant impact on the characteristics of expanded cells. Different
agitation rates and configurations can be used to influence the cell culture
outcome. An appropriate balance needs to be found at an agitation rate that
allows adequate mass transfer for cell growth, without compromising cell
integrity or stem cell fate due to excessive shear stress. Different bioreactor
technologies and configurations are available to fine-tune cell culture
agitation for each specific application.

3.5 Culture Monitoring


Monitoring of culture conditions is essential in any cell manufacturing
process. An ideal continuous gathering of information from every
bioprocess stage would allow for real-time informed decision-making,
complete control and oversight, extensive knowledge of the whole
manufacture process, and model estimation with response simulation.
Naturally, any cell therapy manufacturing process would hugely benefit
from such observational power, especially due to inherent complexity of
being based on living organisms. The dynamic nature of cells is a
significant source of instability for process control [177].
Monitoring has been exposed to ever-increasing difficulties associated
with more advanced processes and products. Cell therapies have turned the
spotlight toward cells, and monitoring has not been able to fully respond to
newfound needs. However, regulatory agencies have implemented
guidelines in order to stimulate the improvement of monitoring tools,
establishing the process analytical technology (PAT) framework [178, 179].
It highly recommends design, incorporation, and control of innovative
analytical tools for continuous improvement of cell therapy manufacturing.
In order to ensure product quality and facilitate monitoring variable
selection strategies and prioritization, critical process parameters (CPP)
must be identified and closely followed.
As previously mentioned, cell therapies possess conventional
physicochemical process parameters (e.g., pH, temperature, and agitation
speed). Nevertheless, introduction of cells has brought a significant amount
of unprecedented process parameters, whose nature revolves around
cellular-based concepts. For a cellular product, assurance of cell viability
and cellular fitness involves controlling a microenvironment based on
complex nutrient formulations and dissolved gas concentrations. Besides
parameters related with cellular well-being, complexity in cell therapy
production monitoring is associated with information on cell state,
including phenotype and functionality. Identity of a cell population during
production must meet desired standards and quality control, thus surface
marker expression, transcriptomics, and metabolic profile are also
important monitoring targets. Concisely, having cells as therapeutic
products has considerably extended the list of CPP in both length and
complexity, forcing PAT to advance and to try to develop novel tools at an
unprecedented and unmanageable rate [177].
With the identification of process parameters present in cell therapy
production that can be difficult to measure, versatility in monitoring might
facilitate early development of new PAT tools. Measurement of process
parameters should preferably be performed in situ, avoiding lag phases and
delays in information gathering that can endanger the whole bioprocess
[180]. By measuring directly inside the manufacturing unit, these sensors
provide real-time data and do not compromise the sterility barrier. However,
another possibility includes online measurement, which requires sample
displacement and return to the unit through a bypass mechanism but also
maintains vessel sterility [181].
For techniques that have not been developed for in situ integration, off-
line and at-line monitoring are possible alternatives. These require
destructive sampling accoupled with external sample preparation and
analysis. The difference between both is related with the distance of the
assay equipment, with at-line having the advantage of close proximity to
the respective manufacturing unit. Consequently, any process control with
these monitoring strategies will be performed in retrospective with delayed
information [181].
Currently, cell therapy manufacturing possesses process parameters
with different kinds of monitoring methods [181]. Nevertheless,
advancement of PAT aspires for universal in situ monitoring (Fig. 5).

Fig. 5 Comparison of different monitoring paradigms. Conventional and established systems are
outdated and based mostly on off-line methods. Innovative approaches have envisioned a new
perspective that aspires to reach universal online monitoring toward a quality-assured cell-based
product
Conventional CPP have monitoring techniques that have existed for
several decades. The lack of modernization associated with industry
resistance in applying PAT advancements has crippled much needed
innovation in cell therapy manufacturing. Instead of having monitoring
development accompany efforts in making standard bioprocessing units,
cell-centered manufacturers have paradoxically opposed it [182]. This is
clearly evident for traditional univariable parameters, whose monitoring is
based on outdated techniques.
Historically, pH and dissolved oxygen (DO) tracking is performed using
electrochemical sensors [183, 184]. Due to the requirement of a reference
electrode, pH glass electrodes tend to be bulky, and their glass envelope
displays some fragility. Likewise, DO electrodes are also limited, since
consumption of local oxygen requires constant flow around the sensor and
low membrane stability restricts shelf-life [182]. Furthermore, mostly fixed
geometry configurations have impaired adaptation of traditional
electrochemical electrodes into novel bioreactor systems. Nevertheless,
manufacturers have extensive knowledge regarding these sensors, and their
considerable reliability has made their adoption widespread.
Fortunately, advancements in optical fibers have made it possible to
follow pH and DO without the need of electrochemical probes. Optical
sensors are able to quantify these parameters through the presence of
indicator or fluorescent dyes [181]. Continuous optical monitoring of pH in
a perfusion bioreactor for a baby hamster kidney (BHK-21) cell culture was
achieved using phenol red as an indicator [185]. Additionally, instead of
having bulk media constituents as indicators, dye adsorption on a solid
matrix combined with patch technology has originated viable products and
systems for cell culture during manufacture (e.g., Optical pH and DO
sensors by PreSens Precision Sensing GmbH or Ocean Optics). Optical
patch sensors were used to monitor pH and DO in a high-throughput system
for simultaneous operation of 12 bioreactors [186]. Versatility, easy
implementation, and miniaturization potential of patch sensors have also
been exploited in disposable culture technology. Single-use bioreactors,
such as BIOSTAT STR® produced by Sartorius, have integrated patch
sensors for both DO and pH. This system has been used for scalability
studies on AT MSC expansion using microcarriers in a stirred bioreactor
[153]. Although optical sensors exhibit great adaptability and allow for
continuous external monitoring, their dyes are vulnerable to
photobleaching. Nevertheless, a comparison between electrochemical and
optical pH and DO sensors highlighted considerable correlation between
parameter measurements, easing concerns regarding lesser robustness of
optical sensors [187].
Cell-related CPP demand pioneering approaches and instruments that
are capable of following complex and multivariate data. For certain
parameters it would be impossible to individually follow each specific
component in a parallel manner. Culture medium formulation and cell
transcriptomics are some variables that require a widespread perspective.
Spectroscopy is an attractive technique to address multiparametric needs
since a broad range of wavelengths can be covered [181]. Additionally,
electromagnetic radiation interacts with any type of matter, which makes
spectroscopy also applicable to biological parameters [180]. Partition of this
wide-reaching field originates multiple techniques, with several being
adapted as PAT tools for monitoring.
Ultraviolet (UV)/visible spectroscopy focuses on consequences of
sample or analyte excitation through a UV or visible light source.
Information for cell-based bioprocessing monitoring can be retrieved by
observing two different radiation phenomena, namely, scattering and
absorption. Measurement of light absorption through a specific path length
is the basis for optical density (OD) and absorbance techniques [180].
Although restricted to suspension cultures, medium turbidity can be
correlated with cell concentration. Furthermore, significant absorption
targets for this range of the spectrum include aromatic molecules, such as
fluorophores, chromophores, and aromatic amino acids. The latter can be
extremely useful for protein quantification. Quality of cytokines used in
medium supplementation can be certified using this technique.
Instead of absorption, light scattering is another event that can provide
parameter information. Biomass from aggregate-based or suspension cell
cultures can be followed by the amount of scattering of an incoming light
source. Growth of Chinese hamster ovary (CHO) cells was successfully
followed with a backward light scattering platform [188]. While cell
proliferation can be monitored, potential of UV/visible spectroscopy for cell
therapy manufacturing is limited, since high aromatic compound selectivity
cannot be fully exploited in a cell-centered bioprocess.
Moving to higher wavelengths in the spectral window leads to infrared
(IR) spectroscopy. Lower-energy radiation associated with IR spectroscopy
affects the vibrational states of molecules. Fortunately, each molecule after
excitation emits its own unique radiation fingerprint [180]. Spectral
monitoring of culture media allows for multiple quantification of culture
medium components and metabolic by-products in a noninvasive manner.
Unfortunately, water molecules present can also interfere with data
acquisition. Deconstruction of these measured spectra is necessary to
distinguish between individual analytes [189]. Thus, there is a significant
dependence on chemometric algorithms to unravel incoming data. An in
situ near-infrared spectroscopy probe was validated in determining analyte
concentrations in a CHO-K1 cell culture [190]. Fourier transform infrared
(FTIR) spectrometers are a third generation of instruments to reach the field
of IR spectroscopy. With a higher signal-to-noise ratio, this technique has
also been successfully used to follow MSC osteogenic differentiation and
the metabolic profile of MSC expanded on microcarriers in a XF culture
system [191, 192].
Molecular vibrational interactions are also responsible for originating
Raman spectroscopy. This technique is based on sporadic inelastic
scattering of incident light. In contrast to IR spectroscopy, it is less affected
by water interference, and therefore substantial focus is being given to
Raman spectroscopy [180]. This method combines possibility for versatile
in situ probes, continuous and noninvasive real-time monitoring, sensitivity
for most culture components, and a high signal-to-noise ratio. Cellular
events aside from proliferation are also potential targets for Raman
spectroscopy, with differentiation of adipose-derived MSC in multiple
lineages being followed with an in situ probe [193].
Radiation-based monitoring strategies have an extensive application
potential and have unquestionably expanded monitoring capabilities.
Further spectroscopy techniques that have been explored as PAT tools
include fluorescence spectroscopy and dielectric spectroscopy [194]. In situ
probes based on the latter (e.g., iBiomass by Fogale Biotech and Incyte by
Hamilton) have been developed for cell density measurements and are
compatible with microcarrier-based cultures [195, 196].
With an ever-growing listing of CPP, the demand for techniques or
instruments that incorporate multiple culture parameters in an efficient
manner is growing. YSI 2950D by YSI is a metabolite analyzer that has
been used to simultaneously measure up to six different culture parameters
using proprietary immobilized enzyme electrodes [142, 197]. The leading
instrument BioProfile Flex2 by Nova Biomedical has challenged the
boundaries of parameter parallelization with monitoring capacity for 16
different parameters, ranging from glucose concentration to cell viability
[198]. However, these devices do not fulfill in-line monitoring ambitions. In
trying to solve this issue, a prototype capsule (PATsule) currently in
development has heightened hopes for a real-time multiparametric in-line
device [182].
The development of these techniques hopes to give PAT significant
observational power, helping assure cell therapy manufacturing needs. The
need for PAT advancement emphasized beforehand has culminated with a
momentum in solving this issue. Recently, Raman spectroscopy was
employed as a PAT tool in an autologous immunotherapy model for cell
therapy bioprocessing [199].

4 Downstream Processing
For traditional biopharmaceuticals, cellular contribution ends after upstream
processing, where cells are taken advantage of as miniature factories to
produce a desired product. Downstream units from cell-centered
bioprocesses have an unprecedented challenge in trying to design
purification methods that give special care to cell sensitivity without
changing cell identity and potency [84]. Fortunately, cell separation is a
common practice in research, thus manufacturing units can try to scale
existing technology or develop entirely novel techniques.
After upstream manipulation, cells must be harvested from their
respective vessels to proceed to downstream processing. While cells grown
in single-cell suspensions can be easily recovered, adherent cells require
surface detaching techniques. Enzymatic methods disrupt cell-surface
interactions by causing proteolytic cleavage of integrins, which are proteins
responsible for cell adhesion and contribute to cell signaling by transducing
ECM stimuli [200]. While damaging integrins can affect cell phenotype or
function, enzymatic detachment of cells is common in cell-related processes
[201–204], with possibilities varying between Trypsin-EDTA, TrypLE™,
and Accutase™. In order to avoid disrupting cell phenotype, approaches
focused on reversible adherence to microcarriers have been pursued.
Considering that many cell therapies will be administered intravenously,
the presence of particulates or intact microcarriers in the final cell product
represents a major safety risk [205]. In this context, different technologies
have emerged that may address this concern. For instance, Advanced
Corning® Dissolvable Microcarriers (Corning Life Sciences) have been
developed to facilitate adherent cell harvesting. These carriers are
comprised of polygalacturonic acid chains cross-linked by calcium ions,
which can be dissolved with exposure to ethylenediaminetetraacetic acid
(EDTA) and pectinase. Human iPSC expanded in a Vertical-Wheel™
bioreactor were successfully extracted from dissolvable microcarriers [206].
The use of dissolvable microcarriers for expansion of iPSC led to a
detachment efficiency of 92 ± 4% compared with 45 ± 3% when using
plastic microcarriers with common detachment techniques.
Microcarrier functionalization is an alternative strategy to achieve
reversible microcarrier adherence. Poly(N-isopropylacrylamide) is a
thermal-responsive polymer which dramatically alters its conformation
based on temperature fluctuations [207]. Different microcarrier
functionalization methods using this polymer with cell culture validation of
various cell types have been reviewed [207]. Recently, the same concept
was explored using carriers with larger pores (i.e., macrocarriers) for human
dermal fibroblast and MSC culture. Considering the difficulties in
harvesting adherent cells from macroporous carriers, this technology may
give these carriers new potential for implementation in cell therapy
processes [208].
Considering their different size and density, cell-carrier separation has
been explored by filtration or centrifugation techniques. These selected
approaches must be compatible with manufacturing processes of
considerate scale, while respecting cell sensitivity. Dead-end filtration
represents the scalable version for small-scale mesh filters, with
commercial versions (Opticap® series by Merck Millipore) being applied
for efficient separation of MSC from respective microcarriers after cell
expansion and detachment [209]. A screening of several filter mesh sizes
between 30 and 100 μm was performed after detachment of cells from the
microcarriers. Interestingly, 30 μm pore sizes originated a significantly
reduced cell recovery (approximately 67%) when compared with the
performance of meshes with 80 and 100 μm pore sizes (>90%) [209]. In
order to avoid fouling and membrane clogging associated with normal flow
filtration, tangential flow filtration (TFF) might be an improved alternative
for such separation. Hollow fibers (developed by Repligen or GE
Healthcare) allow for separation with less pressure due to feed flow
occurring tangential to the membrane.
Being reliant on pressure for separation, filtration methods can cause
cellular stress. Therefore, centrifugation techniques are a suitable
alternative for such separations. Aforementioned counterflow centrifugation
or fluidized bed centrifugation minimize shear stress during cell separation
and can also be implemented for downstream purposes [210]. However, the
more demanding volume scale might limit application of previously
described instruments. kSep systems developed by Sartorius explore the
same centrifugation principles, having been designed to be able to process
larger volumes and also allow for continuous cell isolation [211].
With both cell types in suspension, cell purification stages may be
required after cell manipulation. These techniques were extensively covered
in Sect. 2.2, and their application is also ever-present during downstream
phases of the manufacturing process. Depending on the production method,
cell-based therapies may demand several cell purification units throughout
the pipeline.
At this point, both adherent and suspension cells converge in their
respective downstream pipeline, requiring concentration and washing units
before entering the formulation and filling stage. Fortunately, reducing
volume possesses coincident methods with cell-carrier separation.
Previously mentioned TFF and counterflow centrifugation are both viable
options for concentration. TFF implementation for MSC concentration after
microcarrier detachment and clarification was studied with a systematic
breakdown of hollow fiber characteristics [209] (i.e., material and pore size)
and filtration operation modes [212]. TFF under continuous and
discontinuous operation resulted in cell recovery rates higher than 80% with
a cell viability of 95%.
Although downstream stages appear to be dominated by filtration and
centrifugation processes, disruptive separation mechanisms are being
explored for cell therapy manufacturing applications. FloDesign Sonics has
harnessed acoustic waves to influence cell movement [213]. Using
acoustophoresis, cells are restrained in produced acoustic waves, which
allows for washing and volume reduction without negatively affecting cells.
Ekko represents a continuous, closed, and scalable platform that has been
commercially developed by FloDesign Sonics for cell therapy manufacture.
Downstream flowcharts for cell therapy manufacturing vary between
adherent and suspension cultures. However, established filtration and
centrifugation techniques are mostly transversal throughout different stages.
Still, the limited amount of approaches for downstream processing is a
critical issue for cell therapy manufacturing.

5 Formulation and Storage


When purified cells are compliant with quality control objectives, their
formulation and filling is required for commercialization. Transport of cell-
based products is much more demanding than conventional
biopharmaceuticals. For minimally manipulated therapies, such as
transplants, cells are delivered fresh in cold storage. Autologous products
can follow similar formulation and filling principles, since their shelf-life is
typically low. However, allogeneic cell therapies that serve a one-fit-all
business model depend heavily on cryopreservation methods. The storage
of cells at extremely low temperatures (e.g., ranges between −135 and
−190°C, using liquid/vapor phase nitrogen tanks) drastically minimizes
metabolic activity, allowing the preservation of cell viability over prolonged
storage. However, cryopreserved products are a logistic burden as
specialized infrastructure and equipment are necessary for handling and
transport [84].
Moreover, cryopreservation processes are prone to inflict cell damage
through multiple mechanisms [214]. These include abrupt temperature
changes and unwanted thermal fluctuations. Therefore, appropriate cooling
rates and temperature maintenance are important parameters for a
successful cryopreservation. Cell injury can be associated with extracellular
and intracellular ice formation. In order to prevent ice formation,
cryoprotectants are usually added to cryopreserved suspensions. However,
cryoprotectants can also be toxic to cells. Finally, cells are also subject to
thawing injury, as a result of intracellular recrystallization.
The median viability of PB-derived HSPC cells can decrease from 98–
99% (at the time of harvest) to 71–76% after thawing these cells from a
liquid/vapor phase nitrogen freezer [215, 216]. UCM MSC, isolated
through enzymatic digestion, presented a mean viability after thawing
ranging from 75 to 81%, depending on the cryopreservation period, while
viability prior to freezing was 83% [217]. When isolated through explant
culture, UCM MSC presented a mean viability after thawing ranging from
71 to 83%, while viability of fresh cells was 93%.
In order to minimize cryopreservation-induced cell damage and enhance
product reproducibility, automated and quantitative tools can be used for
cryopreservation and thawing [218]. Programmable controlled rate freezers
(CRFs) employing liquid nitrogen as a refrigerant offer greater control and
customizability of cooling rates. New freezing systems that do not require
liquid nitrogen have recently emerged such as the Asymptote VIA Quad,
Duo, and Research freezers [218]. These are suitable for GMP cleanroom
facilities where the use of liquid nitrogen tanks leads to risks in terms of
contamination and air quality.
Automated dry-thawing devices can eliminate the risks associated with
manual thawing in a water bath, such as water-borne contaminants and
operator variability [218]. Examples of this technology include the
Asymptote VIA Thaw SC2 and CB1000, Biocision ThawSTAR, Medcision
ThawCB, and Sarstedt Sahara.
To some extent, it might be desirable to avoid cryopreservation at all,
either to prevent cryo-induced cell damage or to circumvent laborious and
expensive freeze and thawing procedures. Storage at 2–8°C can be
sufficient, especially in situations that only require short-term storage.
Specialized hypothermic storage media such as HypoThermosol (BioLife
Solutions) allow an increased product stability at hypothermic temperatures
(e.g., 2–8°C), avoiding the need for cryopreservation procedures [219]. In
2012, TiGenix completed the first phase I clinical trial with AT MSC (i.e.,
Alofisel) stored and administered in HypoThermosol (NCT01743222)
[220].
An innovative technique has been developed that could have major
implications on tissue and cell preservation. Osiris Therapeutics has
designed a lyophilization technology (Prestige Lyotechnology) to preserve
living cells and tissue. A proof-of-concept was performed with placental
tissue, which can be used as biological dressings for treatment of burns and
deep wounds [221]. Lyophilized tissue was compared directly with
cryopreserved samples concerning cell viability, ECM components, and
tissue organization, showing comparable results. Lyopreservation of cells
has major consequences, as lyophilized samples can be stored at room
temperature without the need for expensive cryopreservation equipment.
For cell therapy manufacturing, this breakthrough might imply considerable
cost reductions, with deep structural changes to formulation and filling.

6 Cost of Goods
Feasibility is a key concept concerning cell therapy development. Although
possessing great therapeutic potential, cell therapies have inherently
complex manufacturing processes that can impact their commercial
viability. The introduction of cells as a therapeutic product has caused a
paradigm shift in bioproduction. Every manufacture stage has had
difficulties in translating typical manufacturing units to cell-based products.
More sophisticated microenvironments during upstream production are
necessary when producing cell-based therapies, which can include feeder
layers and biomaterial-based scaffolds. During the downstream phase,
sensitivity of living cells to typical separation and purification processes
demands innovative approaches, which usually are costly. Also, end-stage
product transportation cannot yet fully rely on more conventional
lyophilization options, since cells still depend either on fresh or
cryopreserved storage for transport [84].
In turn, production has a high risk of becoming exceptionally expensive,
leading to an unsustainable commercial product. Even after achieving
regulatory approval, recent products have suffered with suspicions against
long-term commercial sustainability. CAR-T therapies, such as Yescarta
and Kymriah, have battled with health insurers to achieve coverage deals in
several countries [222]. In England, its health cost-effectiveness watchdog
(NICE) had initially ruled against acceptance of Yescarta into the national
healthcare system, claiming £300,000 per patient was an excessive strain on
its healthcare budget [223]. However, progress was made when Yescarta-
producing Gilead offered a confidential discount on the listed price, leading
to the approval of Yescarta for treatment of adult patients with diffuse large
B-cell lymphoma [224]. Being potential cures for blood malignancies, their
pricing must recognize a less favorable commercial and manufacturing
scenario associated with one-time treatments. Additionally, their therapeutic
indication has been for cancer patients who have shown resistance to
chemotherapy and have ruled out bone marrow transplants. Thus, expected
demand for such cell therapies is relatively low. Every one of these
constraints is a threat against reliable commercialization of these potentially
life-saving cell therapies.
CAR-T cell therapies are not isolated cases, since approved Alofisel has
also suffered from similar concerns. In early 2019, NICE released its final
appraisal on this cell-based product, advising against its adoption for
treatment of perianal fistulas in adults with non-active or mildly active
luminal Crohn’s disease [225]. Although possessing very promising clinical
trial results with 50% of patients treated with Alofisel during its Phase 3
trial showing fistula remission [226], lack of long-term remission studies
has raised suspicions on the durability of its treatment benefit.
Consequently, the proposed therapeutic value of Alofisel reflected in its
pricing (list price – £13,500/vial with one dose consisting in four vials)
cannot be assured, leading to rejection of coverage by NICE.
In order to assure the sustainability of approved therapies, with
regulatory consent and adoption by healthcare providers, detection of cost
reductions at any stage of a cell therapy bioprocess is crucial [35].
Incorporation of cost of goods (COG) analysis allows for a systemic
search for cost drivers and should be included during any decision related
with the manufacture process [39]. Assessing COG at a preliminary level
facilitates process modifications that would become laborious and critically
expensive at a later stage. In the case of cell therapies, there are some
inherent characteristics that differentiate their production backbone and are
responsible for immediate distinctions in the COG analysis approach.
Interestingly, cell origin has a profound impact on manufacture,
commercialization, and business model choice. Production of allogeneic
therapies is an economy of scale, which becomes increasingly cost-effective
as the demand increases. Aligned with common concerns regarding the
need of high cell doses for most cell therapies, donor to patient treatments
have a desired production profile. Scale-up allows for reductions of
consumable costs and operating labor. However, allogeneic therapies have
yet to fully harness the potential for COG reduction of an economy of scale
[39]. The above-mentioned lack of automation and closed production
pipelines for “off-the-shelf” allogeneic products have been holding these
therapies back. Nevertheless, these concerns have been identified, and
efforts are being made to overcome them. Decisional tools for the
manufacture process based on risk management analysis that incorporate
COG breakdown have been developed for allogeneic cell-based therapies
[227]. Recently, an open-source bioprocess economics tool revealed that the
Vertical-Wheel™ bioreactor system would allow cost savings in the
manufacturing of MSC for cell therapy purposes [104].
Autologous therapies have their own distinct scenario concerning COG.
Being patient specific, each produced lot is restricted to only one patient.
Instead of implementing a scale-up strategy, these therapies require
parallelization as their manufacturing dogma. This has clear consequences
in COG analysis, with single-use modular options becoming more attractive
than traditional larger-scale production vessels [39]. Furthermore,
autologous cell therapies do not follow conventional demand-supply
relationships. In this case, demand is simultaneously supply, since the
patient possesses the cellular component for its own cell therapy. This
significantly reduces any implementation of cost-effectiveness measures
due to demand predictability. Although demand can be tracked, increasing
cell therapy stock is not possible for autologous therapies, as they are not an
“off-the-shelf” product. In terms of manufacturing facility policy,
autologous therapies require prioritized point-of-care. Since cells from the
patient are extracted, altered/expanded, and reinfused, proximity to the
patient is critical. Accordingly, the concept of decentralized facilities for
COG reduction in autologous approaches has been explored [228].
Tailored COG analysis models are necessary to accurately reduce costs
in autologous and allogeneic therapies. However, there have been attempts
to increase cell therapy versatility by changing its cell source requirement.
By removing the endogenous T-cell receptor (TCR) from allogeneic T-cells,
advances were made toward the creation of a universal CAR-T product
[229]. Consequently, an allogeneic COG model was able to be developed
for such a cell therapy [230]. Altering the nature of tissue procurement may
be a COG solution when trying to convert an unsustainable cell therapy into
a commercially available product. Although cell source is able to deeply
condition cost drivers due to different production models, other
manufacturing parameters also have impact on commercial sustainability.
Having recognized the importance of COG analysis in cell therapy
manufacturing, the ISCT conducted a survey for its members to quantify
and discriminate costs in their production pipelines [39]. Acquisition of
material and consumables had the highest mean response, being responsible
for 31% of the total costs. Labor-related costs followed, occupying 20% of
the cost burden. Processing and facility costs shared a similar slice, having
been attributed 16% and 17%, respectively. Quality control and distribution
were at the rear, representing 11% and 5% of total manufacturing costs.
Although this breakdown cannot be applied as a universal template for cell
therapies, it helps in comprehending their COG scenario.
Production of an MSC-based therapy was used as a case study in order
to explore the full potential of cost optimization through COG analysis. As
mentioned beforehand, allogeneic therapies benefit from an economy of
scale and an “off-the-shelf” business model. By predicting increases in
annual demand and batch sizes, COG layout recommended adoption of
specific expansion strategies [227]. While multiplate bioreactors and
multilayered flasks competed at annual demands between 1010–1011 cells
and at a batch size of 109 cells, single-use bioreactors in combination with
microcarriers dominated higher targets in annual demand and batch size.
Although planar technologies are normally chosen as the initial expansion
platform, this simulation demonstrated that single-use bioreactors have
comparable costs even at the lower values of demand and batch size. This
observation was an improvement of a previous study that only considered
adoption of bioreactors when confronted with infeasible scenarios of planar
technology [231]. Additionally, considerable differences were pointed out
concerning the labor associated with the explored platforms. For the first
50 days of an annual production of 100 batches consisting of 5 × 1010 cells
each, multilayered flasks required 14 full-time equivalents with a labor load
surpassing 80 h in a single day [227]. In the same manufacturing
conditions, single-use bioreactors needed only two full-time equivalents
with daily labor loads constantly under 10 h. Both selection of expansion
platform and labor requirements were shown to be highly influenced by a
COG breakdown as cost drivers.
This methodology enhances decision-making, by simulating possible
scenarios and COG reaction to manufacture alterations. COG optimization
should be pursued throughout the whole bioprocess, even as development
advances.

7 Quality by Design
Having cells taking the central role in a therapy has broaden therapeutic
angles to tackle innumerous diseases. However, ensuring high-quality
products with consistency is more challenging for such cell-based therapies.
The complexity of the cellular component associated with lack of complete
comprehension of its machineries demands even more stringent quality
measures during manufacture.
After initial proof-of-concept research, bioprocess development must
include product quality guidelines to guarantee cellular therapeutic
attributes, avoid manufacturing failure and alleviate regulatory concerns
regarding safety. Quality assurance in the biopharmaceutical field was
introduced by the US Food and Drug Administration (FDA) to tackle
alarming waste rates, nonexistence of predictable models, and insufficient
production control [232]. cGMP was delineated to renew the
pharmaceutical sector to address this issue. Quality management of
biopharmaceuticals changed with the creation of the Quality by Design
(QbD) model [233]. Due to its increasing impact, the FDA and EMA
launched a joint pilot program with parallel application assessments in
order to harmonize and integrate QbD guidelines [234]. A holistic view of
the bioprocess allied with extensive scientific knowledge of each
production component and a systematic and iterative method of
improvement serve as the basis of QbD.
Cell therapies have followed traditional biopharmaceutical development
mistakes regarding manufacturing development. Inability to apply QbD and
COG analysis has increased manufacturing failures and unsustainable
bioprocesses. Translation of prominent clinical trial results to a scalable and
cost-effective bioprocess that has led to several letdowns with development
suspension and product withdrawal [235, 236].
A logical sequence of steps is delineated by QbD in order to advise
manufacturers regarding intelligent production pipeline development for
cell therapies (Fig. 6). Initial guidelines concern the end product, with the
identification of therapeutic objectives. These should describe with great
detail cutoff goals for concepts such as identity, potency, and purity [237].
High degree of clarity and detail in defining end product properties will
facilitate identification of critical production processes and problem
localization and solving. By creating a quality target product profile
(QTPP), the framework for QbD is established.
Fig. 6 Global description of the QbD process. An initial QTPP requires defining end product
objectives that satisfy therapeutic needs. Translation of these objectives to their cellular features
uncovers process CQA. In turn, process variables that are responsible for influencing CQA are
identified as CPP. Controlled variation of these parameters originates a normal operating space,
which limits process operability. Continuous process monitoring and control facilitates improvement
implementation, creating a cycle of process optimization. QbD quality by design, QTPP quality
target product profile, CQA critical quality attribute, CPP critical process parameters
With the target profile set, the entire process needs to be broken down to
identify critical variables that have a direct effect on the QTPP. Raw
material attributes (RMA) need to be controlled for quality with selected
checkpoints so that variability and lack of quality cannot compromise the
bioprocess from the start [233]. After controlling process inputs, continuous
monitoring with PAT throughout the entire process should exist to assure
correct transformation or manipulation of raw materials into the final
product.
Considering that following every possible variable is unrealistic, critical
quality attributes (CQA) should be selected. For cell-based therapies, these
attributes correspond to cellular features. Since living cells are multivariate
systems, isolating inputs (e.g., signaling factors) and controlling outputs
(e.g., cell expansion) are not trivial. Knowledge on cell networks is
increasing, but a complete map of cellular machinery is still far from reach.
Thus, identification of CQA, which are crucial for QbD, can be difficult.
Throughout the bioprocess, each unit has an impact on cells and is
accountable for altering their characteristics to some extent. Controlling
CQA will depend on identifying which CPP are responsible for changing
those same features. Control strategies should build on CPP discovery,
which serve as directives for selection of monitoring techniques [233]. The
holistic nature of QbD demands whole process overview, with parallel
interventions as the production pipeline moves forward.
After definition of individual CQA and respective CPP, studying their
interactions should follow. By uncovering and exploring networks, process
knowledge increases greatly. In turn, reaction to process variability can be
achieved quickly and pragmatically by tweaking CPP. Rapid process
correction is particularly relevant for cell therapy manufacturing. Even with
RMA tightly controlled, cells have inherent complexity that leads to
aberrant and unpredictable behavior. Therefore, discerning connections
between CQA and CPP will increase process mapping and improve
decision-making [238].
Interactive effects can be revealed by analyzing a defined design space.
The range of variability of CQA caused by fine-tuning of CPP or RMA will
define boundaries for a normal operating range in the design space.
Working inside the normal operating range admits changes in CPP without
compromising end product quality. CQA-CPP connection can be very
laborious and difficult to obtain, with a substantial need for varied
experimental data. In order to circumvent excess labor and costly
optimizations, design of experiments and systems biology are effective
tools that originate the same results using up only a fraction of expected
time and resources [237, 239].
Design of experiments is dedicated to evidencing relationships between
input and response variables in an optimized manner. Extensive
multivariable exploration inside a selected design window is performed
using the least amount of experimental conditions. Factorial design is
responsible for generating necessary experimental combinations. Obtained
experimental data allows delineation of a response surface for every CPP.
These surfaces are described by behavior functions, which model the
above-mentioned CQA-CPP relationships [240]. Besides enhancing process
knowledge, behavior functions enable CPP optimization resulting in CQA
improvement. Implementation of design of experiments has caused process
improvements from pluripotent stem cell cultivation to ex vivo HSPC
expansion [241–243].
Systems biology tools can also contribute to discerning CQA-CPP
interactions. Omics-based techniques, such as gene expression and protein
production, can contribute to QTTP review, updating end product identity,
and potency. Also, metabolic pathways can derive similar information
without directly compromising cells. In this context, metabolic by-products
were used to construct a fed-batch platform for HSPC expansion in order to
avoid inhibitory feedback signaling [244]. Intrinsically, large amounts of
generated data can also yield reduced dimension mechanistic models,
similar to behavior functions. Overall, the impact of any CPP modifications
on CQA can be more easily detected.
As QbD requires the combination of extensive fundamental knowledge
with engineering principles, applying it to cell therapies is a daunting task.
Biological variability is inherent, making standardization and quality
control a struggle for any process that includes cells. However, QbD
guidelines serve as a backbone for correct cell therapy manufacturing
development. Its implementation is critical to any future bioprocess,
assuring quality and robustness throughout each unit. Additionally,
continuous improvement associated with QbD brings process evolution to
the production pipeline, making it dynamically resistant to unsustainability
[237].
8 The New Cell-Free Therapeutic Paradigm
Recent advances in cell biology have led to a better understanding of cell
communication processes. Cells are able to interact with their surroundings,
influencing other cells and tissues through paracrine and endocrine
signaling. Multiple studies have been developed using cellular secretome
for therapeutic purposes. For instance, MSC-conditioned medium improved
hepatocellular regeneration in a rat model of acute liver injury [245]. On the
other hand, conditioned medium from genetically modified MSC was able
to limit infarct size and improve ventricular function in mice infarcted
hearts [246]. In addition, the secretome obtained from bioreactor cultures of
human BM MSC was able to induce neurogenesis and increase neuronal
cell differentiation in vivo [247].
The secretome consists of every macromolecule a cell transports to the
extracellular space at a given point in time. The secretome contains mainly
proteins such as growth factors, hormones, cytokines, chemokines, and
interferons, as well as extracellular vesicles [248, 249]. In particular,
extracellular vesicles (EVs) have been given great attention recently by the
scientific community, due to their potential both as diagnostic and
therapeutic tools. EVs are lipid membrane enclosed structures actively
secreted by most cell types. These vesicles have emerged as relevant
mediators of intercellular communication, through the transfer of a cargo of
proteins and RNA (i.e., microRNA and mRNA), which prompt alterations
on recipient cells [250–252].
Depending on their biogenesis, EVs are broadly categorized as
exosomes, microvesicles, or apoptotic bodies. Of notice, exosomes (50–
150 nm) are generated via the endosomal pathway [253, 254],
microvesicles (50–1,000 nm) by outward budding of the plasma membrane
[253, 254], and apoptotic bodies (up to 5 μm) released as blebs of cells
undergoing apoptosis [255]. EVs are associated with multiple physiological
processes, such as immune surveillance [250] and tissue repair [256, 257]
but also in the pathology underlying several diseases, such as cancer [258–
260] and neurodegenerative diseases [261].
Given the importance of EVs in cell communication, the scientific
community soon realized their potential to target diseased cells. EV
membranes resemble the cell membrane, allowing high biocompatibility to
target cells for therapeutic purposes. Moreover, EVs show specific targeting
activity [262] and small size, ideal to cross biological barriers, such as the
blood-brain barrier [263].
The therapeutic use of EVs is twofold. On the one hand, EVs were
found to mediate some of the therapeutic effects from their original cells
[264, 265]. Therefore, these could be potentially used in substitution of
their cell of origin, as a cell-free therapy triggering equivalent therapeutic
effect. On the other hand, EVs can be used as drug delivery vehicles,
through loading of EVs with therapeutic cargo [266].
Engineered MSC-derived EVs were able to successfully target and
regenerate ischemic myocardium in mice [267]. Dendritic cell-derived EVs
were able to deliver siRNA to the brain in mice, demonstrating their
potential use as targeted therapy for neurodegenerative diseases [268].
Multiple studies have successfully developed EVs as drug delivery vehicles
for cancer therapy [269–272]. As an example, intravenously injected
exosomes from immature dendritic cells delivered doxorubicin specifically
to tumor tissues in mice, leading to inhibition of tumor growth without
overt toxicity [269].
Despite the promising potential of EVs for therapeutic applications,
robust manufacturing processes that would increase the consistency and
scalability of EV production are still lacking, both in EV production
(upstream processing) and further isolation (downstream processing) [273,
274]. Furthermore, higher efficiency drug loading approaches and strategies
to increase EV cell-specific targeting need to be developed [274].
In conclusion, cell-derived products such as EVs are emerging as novel
therapeutic opportunities. These cell-free therapies present significant
advantages, avoiding the complexity and safety issues in utilizing cells
themselves as therapeutic systems in a clinical context [266, 275].
Nonetheless, this field is still in a very early development stage and requires
substantial research before being successfully translated into clinics.

9 Concluding Remarks and Future Perspectives


Exciting developments in the cell therapy field over the last decades has led
to an increasing number of clinical trials testing multiple cell products for
therapeutic purposes. This has been followed by an increasing (although
still small) number of products receiving marketing authorization by
regulatory agencies. Even though there was a substantial progress in the
field, manufacturing of cell-based therapies still presents multiple
challenges that need to be addressed in order to assure the development of
safe, efficacious, and cost-effective cell therapies.
Multiple human tissues have been used to derive cells for therapy,
showing different therapeutic capacity depending on the source that cells
were isolated from. Identifying the most appropriate cell source for each
application would allow increased therapeutic efficacy. Moreover, most
cells isolated from human sources are extremely heterogeneous. Identifying
and expanding specific functional cell subpopulations could allow the
development of more efficacious and reproducible products for a specific
application.
FBS and other animal-derived products comprise the majority of cell
culture media supplements used for cell manufacturing. These supplements
are ill-defined, increase the risk of contamination with virus and prions, and
lead to poor reproducibility, limiting their application in the clinical setting.
The development and application of S/XF culture media formulations
aiming at a chemically defined medium are essential for the clinical
translation of cell-based products.
Bioreactor technology has allowed the establishment of scalable
manufacturing processes for cell expansion. However, these dynamic
systems also present challenges for cell culture, namely, shear stress, which
may impact product features. New agitation designs such as the Vertical-
Wheel™ and wave bioreactors have been developed to provide a more
homogenous and gentler particle suspension at lower agitation rates. These
bioreactors also present the opportunity to implement single-use
technologies, more suitable for the establishment of GMP-compliant
processes. Furthermore, bioreactors could be designed for a specific
application and further optimized relying on simulation techniques.
Computer-monitored culture systems, capable of controlling feeding
regimes and maintaining concentrations of physicochemical parameters
(e.g., O2 and pH) and certain molecules (e.g., growth factors and cytokines)
within an optimal range, would offer a great improvement in the robustness
of the manufacturing process.
Currently, isolation and expansion protocols are performed in open
systems, involving a two-stage protocol, which lead to increased risk of
contamination, thus compromising the safety and standardization of the
final cell product. Ideally, cell isolation and expansion could be performed
in a single-stage, closed system. This can be achieved using the Quantum®
bioreactor and other strategies such as the one developed by
Papadimitropoulos and colleagues to culture freshly isolated BM nucleated
cells within 3D porous scaffolds in a perfusion-based bioreactor system
[276].
The implementation of automated and closed systems would provide a
major contribution to achieve a GMP-compliant, clinical-grade cell
manufacturing. CliniMACS Prodigy, developed by Miltenyi Biotec, has
successfully shown how manufacturing automation and closed systems can
be applied to cell therapies. This platform has integrated an entire
manufacturing process in an automated manner. Upstream stages (cell
fractionation, isolation, and cultivation) were combined with downstream
units (cell purification, formulation, and filling) to enable a fully closed
clinical scale cell therapy platform [277].
Advancements in cell therapy manufacturing and their individual
process units are fruitless if the overall production pipeline is not cost-
effective. Commercial sustainability will ultimately be responsible for
dictating the future of a certain cell therapy. Continuous COG analysis
represents the most pertinent tool for identifying critical cost drivers, which
should be flagged and made priorities for improvement.
Early process development by design can avoid considerable and costly
time constraints with manufacture innovation being focused for ensuring
product quality. Continuous monitoring improves process knowledge, and
respective control assures maximized pipeline stability, promoting
reproducibility of cell therapy production. Furthermore, QbD cooperation
with COG analysis can assist in parameter targeting for optimization,
contributing toward cyclical process improvements.
Although cell-based therapies represent a new level in bioprocessing
complexity in every manufacturing stage, these also show unprecedented
levels of therapeutic potential, already radically changing the landscape of
medical care.

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A. C. Silva et al. (eds.), Current Applications of Pharmaceutical Biotechnology, Advances in Biochemical
Engineering/Biotechnology 171
https://doi.org/10.1007/10_2019_108

Bioprinting Technologies in Tissue Engineering


Bengi Yilmaz1, 2, Aydin Tahmasebifar1, 2 and Erkan Türker Baran1, 2
(1) Institute of Health Sciences, Department of Tissue Engineering, University of
Health Sciences, Istanbul, Turkey
(2) Institute of Health Sciences, Department of Biomaterials, University of Health
Sciences, Istanbul, Turkey

Erkan Türker Baran


Email: [email protected]

Abstract
Bioprinting technology is a strong tool in producing living functional tissues and
organs from cells, biomaterial-based bioinks, and growth factors in computer-
controlled platform. The aim of this chapter is to present recent progresses in
bioprinting of nerve, skin, cardiac, bone, cartilage, skeletal muscle, and other soft
tissues and highlight the challenges in these applications. Various composite bioinks
with bioactive ceramic-based scaffolds having patient-specific design and controlled
micro-architectures were used at clinical and preclinical applications successfully
for regeneration of bone. In nerve tissue engineering, bioprinting of alginate- and
gelatin-based gel bioinks by extrusion presented a controllable 3D microstructures
and showed satisfactory cytocompatibility and axonal regeneration. Bioprinting of
cardiac progenitors in biopolymers resulted in limited success, while the use of
bioinks from extracellular matrix induced satisfactory results in cardiac
regeneration. Osteochondral scaffold bioprinting is challenging due to the complex
hierarchical structure and limited chondral regeneration. Therefore, current
approaches focused on osteochondral scaffold with vascular network and mimicking
hierarchical structures. The applications of bioprinting in other types of tissues were
also studied, and results showed significant potentials in regeneration of tissues such
as cornea, liver, and urinary bladder.
Graphical Abstract
Keywords Bioprinting – Bone – Cardiac – Cartilage – Nerve – Skin – Tissue
engineering

1 Introduction
Bioprinting is a variant of 3D printing and refers to the three-dimensional (3D)
manufacturing of biological constructs through the layer-by-layer deposition of inks
with living cells. It can also be defined as the use of material science and production
techniques to build tissues and organs with viable cells and biomolecules in specific
organization to perform a particular biological function [1]. The first international
conference on bioprinting was held at the University of Manchester, UK, in
September 2004, and the definition of bioprinting was first proposed in this
conference as: “the use of material transfer processes for patterning and assembling
biologically relevant materials – molecules, cells, tissues, and biodegradable
biomaterials – with a prescribed organization to accomplish one or more biological
functions” [2]. Since the ultimate goal of bioprinting is to produce living functional
tissues and organs to be transplanted, Mironov et al. [3] proposed another term
“organ printing” which is defined as: “computer aided 3D tissue engineering of
living organs based on the simultaneous deposition of cells and hydrogels with the
principles of self-assembly.”
Bioprinting, or 3D bioprinting, was defined in “Workshop on Bioprinting,
Biopatterning and Bioassembly” at the University of Manchester, UK, in 2004, as:
“The use of material transfer processes for patterning and assembling biologically
relevant materials—molecules, cells, tissues, and biodegradable biomaterials—with
a prescribed organization to accomplish one or more biological functions” [4]. 3D
bioprinting for the production of biological structures typically involves layer-by-
layer deposition of bioink to form 3D tissue or organ structures with an input from a
computer-aided design (CAD) program [5]. 3D bioprinting has been gathering
enormous attention from the scientific community of regenerative medicine due to
its ability to manufacture organs from native cells. Figure 1 demonstrates the
increasing scientific interest to bioprinting and shows the related scientific
categories according to Web of Science database. Although the bio-additive
production of a transplantable entire organ has not been achieved yet, this
technology is advancing, and it is thought that it will soon resolve the crisis of organ
shortages [6].
Fig. 1 (a) Number of records for “bioprinting” in academic publications by year; (b) scientific categories
related to bioprinting. Source: Web of Science, December 2018

According to the Organ Procurement and Transplantation Network of US


Department of Health and Human Services, 114,417 people are actively waiting for
a lifesaving organ transplant in the USA, and only 30,415 organ transplants were
performed in 2018; see Fig. 2 [7]. Regenerative medicine gives hope to fill the gap
between the number of organ transplants and organ need through the engineering of
functional tissues or organs to improve or modify abnormal and necrotic tissues and
organs [8]. Furthermore, 3D printed human tissues can be a better model for
preclinical testing of potential drugs than in vivo testing on animal models in order
to predict the clinical outcome in humans [9].

Fig. 2 According to Organ Procurement and Transplantation Network: (a) number of waiting list candidates by
organ type as of December 2018 in the USA; (b) source of transplants performed in January to October 2018 in
the USA [7]

There are three basic variables that should be considered in order to be able to
produce living and functional tissue structures successfully by using the bioprinting
method. The first is the cellular component which is the living part of the structure
and containing at least one or multiple cell types. The second one is the scaffolding
or supportive biomaterials comprising mostly proteins and polymers that can
provide support and protection to cells during and after the manufacturing process.
These biomaterials can mimic the physical environment and the biochemical signal
cells as in the body and serve as a bioink bridge between cells and hardware. Third
is the actual bioprinting device in which the manufacturing process is performed.
Most importantly, the biomaterial used is a necessary bridge between the bioink,
cells, and hardware [9].
In a historical point of view, Wyn Kelly Swainson owned a patent for a method
and an apparatus for the first time in 1971 to produce 3D structures in a medium
which is selectively sensitive to different parameters of electromagnetic radiation
[10]. Later, in 1984, Charles W. Hull patented an apparatus for the production of
three-dimensional objects by stereolithography [11], which was considered as the
world’s first 3D printer [12].
The use of 3D printing in healthcare applications was started with customized
and patient-specific surgical guides and implants in orthopedics [13]. In 2002,
Envisiontec GmbH began to sell a bioprinter named Bioplotter which was able to
produce scaffold structures from various biomaterials for tissue engineering [14]. In
2010, a research center Chemical Institute of Sarria (IQS – Instituto Químico de
Sarrià) announced two hydroxyapatite (HA) formulations for use in 3D printers
[14]. In 2011, Objet introduced a biocompatible colorless photopolymer material,
MED610, for medical and dental markets [15]. In 2012, Organovo Holdings and
Autodesk announced a collaboration to create the first commercial 3D bioprinting
software [14]. Organovo’s NovoGen MMX Bioprinter was the first commercial
bioprinter tested for 3D-printed tissues [16, 17].

2 3D Bioprinting
Custom-made and patient-specific scaffolds/tissues/organs can be printed according
to the 3D models constructed from the data acquired by medical imaging
techniques, such as 3D computed tomography (CT) and magnetic resonance
imaging (MRI), or generated digitally by using many CAD software. Figure 3
shows the flow diagram for the manufacturing of bioprinted tissues that can be used
either directly in animal models or patients or as in vitro constructs that can be used
for drug testing and disease modeling.
Fig. 3 A typical production flowchart for bioprinting

3D bioprinting is a comprehensive process that requires adjustment of various


design parameters, such as imaging, modeling, printer choice, selection of bioink,
culture conditions, and development of 3D construct [8]. The manufacturing process
can be categorized in three different steps: pre-bioprinting, bioprinting, and post-
bioprinting [18]. The pre-bioprinting (modeling) step involves acquisition of 3D
images by using techniques like 3D scanner, CT, and MRI, designing a digital 3D
model suitable for printing from these imaging modalities or by using CAD-CAM
and mathematical modeling techniques, and finally the selection of bioink,
biomaterial, and cells. In the bioprinting step, 3D-rendered model is sliced into
customizable horizontal portions that are imported into the bioprinter system. One
or more bioinks, which are composed of cells, bioactive molecules, and
biomaterials, are placed in the printer cartridges and printed layer by layer in aseptic
conditions. The post-printing, which is biomimetic remodeling of tissue with the
help of mechanical and chemical signals, is also an important step for obtaining
mechanical integrity and biological functionality.
The main technologies used for 3D bioprinting with biological materials are
inkjet, micro-extrusion, and laser-assisted printing [19]. Table 1 summarizes the
different features of these technologies.
Table 1 Comparison of bioprinter types [19]

Inkjet Extrusion Laser assisted


Material 3.5–12 mPa/s 30 mPa/s to >6 × 107 mPa/s 1–300 mPa/s
viscosities
Gelation Chemical, photo-cross- Chemical, photo-cross-linking, sheer Chemical, photo-
methods linking thinning, temperature cross-linking
Preparation time Low Low to medium Medium to high
Print speed Fast (1–10,000 droplets Slow (10–50 μm/s) Medium-fast (200–
per s) 1,600 mm/s)
Resolution or <1 pL to >300 pL 5 μm to millimeters wide Microscale resolution
droplet size droplets, 50 μm wide
Cell viability >85% 40–80% >95%
Cell densities Low, <106 cells/mL High, cell spheroids Medium,
108 cells/mL
Printer cost Low Medium High

2.1 Inkjet Bioprinting


2D inkjet printing is a noncontact technique that accepts a digital signal representing
an image and deposits tiny ink drops to form the image onto a substrate, which is
mostly paper. It is possible to fabricate 3D tissues by using a technology very
similar to that of a common inkjet printer. In fact, the first attempts of bioprinting
started with working on the hardware and software necessary to convert commercial
tabletop inkjet printers into cell printers [20], and the first patent for printing viable
cells with inkjet printer came in 2006 by Thomas Boland and coworkers at Clemson
University [21]. One of the early researchers Anthony Atala (the director of the
Wake Forest Institute for Regenerative Medicine in Winston-Salem, NC, USA) also
began on a desktop inkjet printer that was modified to print 3D structures [22].
Droplet-based inkjet bioprinting works in a similar manner to the inkjet
technology as described above. The most common types of inkjet printing devices
that are adapted to bioprinting have thermal and piezoelectric mechanisms to
generate the droplets in drop-on-demand principle (see Fig. 4). The devices with a
thermal printhead have an electric heating unit that vaporizes the binder material to
form a vapor bubble. This vapor bubble expands due to pressure and exits as a
droplet from the printhead. Although the temperature rises to 200–300°C while
forming a bubble, the printhead temperature increases only about 10°C, because the
process only takes a few microseconds (about 2 μs) [8]. In the devices using a
printhead with a piezoelectric actuator, the voltage pulse in the printhead causes a
rapid shape change in the piezo-material, and this generates a pressure pulse in the
binding fluid, resulting in a droplet. These printers ensure fast and precise
deposition of the binder liquid.

Fig. 4 Inkjet, extrusion, and laser-assisted bioprinters

2.2 Extrusion Bioprinting


Fused deposition modeling (FDM) or fused filament fabrication (FFF) was
developed in the early 1990s, and this extrusion-based 3D printing strategy is the
most common and inexpensive type of additive manufacturing technology [8, 23].
FDM technique is based on extrusion of molten thermoplastic polymer filaments or
small granules from a small nozzle, which then hardens to form a solid construct
(see Fig. 5) [24]. FDM offers many advantages, such as easy material switching,
low maintenance costs, compact size, and flexibility in working temperatures;
however, the narrow range of printable materials limits its applications [25].
Fig. 5 A schematic diagram showing fused deposition modeling

Extrusion-based (dispensing or direct writing) bioprinting originates from FDM


printing technology. These devices use pneumatic-, mechanical-, or
electromagnetic-driven needle-syringe-type systems to deposit cells and
biomaterials [8]. The cell-laden bioinks, which are mostly composed of hydrogels
and some bioactive components, are dispensed by the printhead to form the desired
3D structures. In order to produce 3D structures with high accuracy, bioinks must be
designed with shear-thinning or fast-solidifying properties. It is possible to obtain
multiwalled and core-shell fibers from different bioinks by using multi-
compartment type of nozzles and to adapt microfluidic strategy to extrusion
bioprinting [26].

2.3 Laser-Assisted Bioprinting


In 1999, Odde and Renn [27] reported the possible tissue engineering applications
of a laser-guided direct-writing system which has the ability to organize cells
spatially into well-defined 3D arrays. Laser-assisted bioprinting is also a droplet-
based system which is also known as laser-induced forward transfer (LIFT). This
system consists of a pulsed laser source; a focusing system; a ribbon, which
contains a layer of biological material to be printed and a glass covered with a laser-
energy-absorbing layer (such as gold or titanium); and a substrate that collects the
printed material [19, 28]; see Fig. 4. The metal film is vaporized by a laser pulse,
and this produces a jet of liquid solution of organic materials (cells and molecules)
which is then deposited onto the facing substrate [29]. Although laser-assisted
bioprinting is less common than other bioprinting techniques, it has many
advantages, such as having high spatial resolution and capability of printing a
variety of biological materials and high cell viability.

2.4 Stereolithography (SLA)


Stereolithography was developed as a solid free-form manufacturing technique in
the early 1980s [30]. Today, most 3D printers and recent bioprinters accept input
files in the STL format. The acronym “STL” stands for STereoLithography, since it
was the first 3D printing process, and the STL file format emerged as a native file
format from a CAD software [31]. This STL file is sliced to generate a G-code,
which provides instructions for the stereolithography apparatus (SLA).
The main components of the system are a reservoir filled with a photocurable
polymer solution or resin, a x-y axis controlled laser, and a fabrication stage with z-
axis control [9]. A SLA is depicted in Fig. 6. Stereolithography is a laser-assisted
additive manufacturing technology that utilizes an ultraviolet (UV) laser to
photopolymerize the surface of a bath of photosensitive polymer. The stage is
gradually lowered, allowing layers to be polymerized on top of each other, thereby
forming 3D structures in a bottom-up manner. The unscanned areas stay in the
liquid phase.

Fig. 6 A schematic diagram showing stereolithography apparatus

The use of SLA in bioprinting was reported by the study of Dhariwala and
coworkers in 2004 for the first time [32]. They filled a vat with cells and a
photocurable hydrogel, which consists of poly(ethylene oxide) and poly(ethylene
glycol dimethacrylate) in a ratio of 3:2 and a photoinitiator (Irgacure). The vat was
placed on a table where the movement could be controlled in the vertical axis. UV
light cured designated places in the vat according to the CAD file. For each layer,
the process was repeated to produce 3D structures. High cell viability was achieved
with a laser of UV light at 365 nm. Today, there are various applications of SLA
from bioprinting to other rapid prototyping purposes.

2.5 Microvalve-Based Bioprinting


Microvalve-based bioprinting is a kind of drop-on-demand systems which is mostly
preferred for hydrogel dispensation [33]. This technology was developed by
Demirci and Montesano in 2007. The main focus of their custom-made microvalve-
based bioprinting is cell printing [34]. A conventional microvalve bioprinting
system is composed of robotic platform and electromechanical printheads which are
connected to a gas regulator [34, 35]. The gas regulator provides positive pressure
on the system as it is in conventional systems. The solenoid coil and the plunger
movement controls the microvalve opening time as low as 0.1 ms [34, 35]. The
valve can be controlled by mechanical, electrical, and magnetic forces. Thus, the
positive pressure and valve opening time controls allocating of bioink during
printing [35].
The pressure, which is required for dispensing of material from the nozzle, leads
to significant shear stress on cells. In addition, the parameters such as nozzle
diameter and the viscosity of bioink have considerable effect on shear stress [36].
Although the moderate shear stress plays a crucial role in cell signaling and protein
expression and improves stem cell differentiation, the disproportionate shear stress
may rapture cell membrane [36]. The narrow range of printable viscosity is the main
limitation of microvalve-based bioprinting systems (1–70 Mpa) [37].

3 4D Bioprinting
4D printing technology was invented by the Self-Assembly Laboratory at the
Massachusetts Institute of Technology (MIT) in collaboration with Stratasys Ltd.
[38]. The main difference from 3D printing is the production of multi-material
prints with shape-changing ability over time or the use of customized materials
which can be changed from one shape to another after being taken from the print
bed. 3D bioprinting technology relies on the assumption that the printed cells can
rapidly form tissues and start to synthesize the extracellular matrices, which can
provide geometrical shape and mechanical support. 4D bioprinting, where time is
the fourth dimension, the printed materials or living cellular constructs continue to
evolve over time after being printed [39] (see Fig. 7). The stimuli for these
constructs to evolve can be one or more of specific triggers, such as temperature,
pH, humidity, electricity, magnetic field, light, and acoustics [40]. Integrating new
smart materials, which can respond to these stimuli and transform with time, into
3D printing is the basis of 4D printing concept. One such smart feature is the shear-
thinning property of bioinks that allows reversible changes in the shear viscosity of
printable inks to assist 4D printing [41].

Fig. 7 Schematic depiction of printing of 3D and 4D objects

Since bioprinting requires the use of viable cells, 4D printable smart materials
should be biocompatible, and rheological properties should be adequate in order to
maintain high cell viability at optimum printability [42]. 4D printed cell-laden
structures face many challenges as it is in 3D printing technique: (1) cell damage
after printing, (2) deterioration of cell proliferation, and (3) deposition of low or
excess cell population [43].
The number of sensitive materials, which can be printed by using the 4D
printing technique, is very limited, and most are capable of responding to only one
type of stimulus. Furthermore, the deformation ability of the 4D bioprinted
structures formed with these materials is also limited to simple deformations such as
folding/unfolding or self-organization. The precise control of the spatiotemporal
evolution of the printed structure is restricted because these deformations can occur
only in macroscale [39]. The 4D printing technique also needs to overcome the
major difficulties in mimicking the complex dynamic deformation of natural tissues
such as the blood pumping of the heart and the peristaltic movement of the
esophagus or intestine [44].

4 Tissue Engineering Applications of Bioprinting


4.1 Bone Tissue
Bone tissue provides mechanical strength, creates a structural framework, and
allows the body to move. It also serves as a mineral storage and plays a vital role in
homeostasis and regulation of blood pH [45]. Bone is a connective tissue with a
complex hierarchical structure. It is composed mainly of a mineral phase, which
constitutes between 60 and 70 wt% of its total mass, and water between 5 and
10 wt%, while the remaining part is an organic matrix of collagen and other proteins
[46]. Collagen and agglomerated HA mineral crystals intertwine to form several
hundred nanometers of mineralized fibrils [47].
Bone is the second most widely transplanted tissue around the world, and more
than four million operations are performed each year by using natural or synthetic
bone grafts to treat bone defects [48]. The main solutions to facilitate bone repair
are still the autograft and allograft techniques, even though autografting is
expensive, invasive, and subject to infections and hematoma, frequently affecting
both donor sites and surgical sites, and allografts may cause an immune reaction
followed by a rejection [49]. The role of biomaterials and tissue-engineered
constructs has become more important in the last several decades. Among the most
challenging and one of the most studied tissues to reconstruct is bone tissue. The
design of bone tissue engineering scaffold should comprise a three-dimensional
structure with interconnected pore network for cell growth and transport of
nutrients/waste. The scaffold should certainly be biocompatible, and its surface
chemistry should be suitable for cell attachment, proliferation, and differentiation.
Finally, the absorbable scaffold should degrade at a rate that matches the tissue
regeneration while keeping its mechanical properties very close to that of the host
tissue [50].
Several techniques previously used in bone tissue engineering to obtain an ideal
cell-laden scaffold did not yield significant successes due to many limiting factors,
majorly vascularity [51]. Another substantial challenge is the critical size of the
construct while meeting the clinical requirements of structural support,
osteoinductive property, and controllable biodegradability [52].
The main focus of bioprinting for bone tissue engineering is the advancement in
printing methods and the development of compatible ink materials. Various
materials have been developed for use in 3D printing to create new alternatives to
conventional bone grafts. The material groups, such as polymers, ceramics, and
hydrogels, cannot fully mimic the properties of bone when used alone. For example,
in order to increase the bioactivity, hydrogels are generally combined with
osteoconductive elements such as calcium phosphates, tricalcium phosphate (TCP),
biphasic calcium phosphate (BCP), hydroxyapatite (HAp), silicate, or silica and
bioactive glass nanoparticles. Also soluble molecules such as bone morphogenetic
protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) are used to
induce angiogenesis and osteogenesis [51]. The simultaneous integration of these
temperature-sensitive biological agents, living cells, and the bioink materials to
create a 3D artificial bone implant or a complex bone tissue construct with
controlled cell distribution, geometry, and biodegradability, good mechanical
properties, and high osteoinductivity and osteoconductivity is today’s most
important challenge in 3D bioprinting. Table 2 reviews the studies investigating
these possible bioink formulations for specific fabrication methods and their
osteogenic, osteochondral, or vasculogenic/angiogenic potential.
Table 2 Bioprinted cells and bioinks for bone tissue engineering

Bioinksa, b Cells Fabrication 3D printer Main outcomes about


method osteogenic potentialb
Alginate hydrogel (3% w/v in Mouse calvaria Syringe Fab@Home • Chitosan was superior
PBS); alginate-HAp pre-osteoblast extrusion Model 3, to alginate
(20 mg/mL) + CaCl2 (2% v/v as cell line Seraph • The deposition of
cross-linker); chitosan hydrogel (MC3T3-E1) Robotics, USA 48 mg Ca ion per 1 g of
(2% w/v in acetic acid); and chitosan – HAp hydrogel
chitosan-HAp (20 mg/mL) [53] was detected on 21st day
of culture
VEGF-conjugated (5% w/v) Human Pneumatic- NovoGen • The encapsulated
GelMA with low methacryloyl umbilical vein based MMX hMSCs formed a mature
substitution (in core) and VEGF- endothelial cells syringe Bioprinter, bone niche after 21 days
conjugated (10% w/v) GelMA (HUVECs), extrusion Organovo, USA of culture under the
with high methacryloyl human medium perfused
substitution and loaded with mesenchymal condition
silicate nanoplatelets (around) stem cells • Medium perfusion
[54] (hMSCs) resulted in significant
increases in gene
expression levels of
osteogenic differentiation
markers with respect to
nonperfused construct
Alginate (50 mg/mL)-gelatin Human primary Pneumatic 3D-Bioplotter, • Ca2+-polyP complex
(50 mg/mL) hydrogel + CaCl2 osteogenic syringe EnvisionTEC, caused the cells to
(0.4% w/v as cross-linking sarcoma (SaOS- extrusion Germany proliferate faster with an
solution) cell-laden bioink and 2) cells average generation time
agarose hydrogel (0.3% in of approximately 47–
growth medium) with Ca2+- 55 h during the 6 days
polyP complex (100 μM) as incubation period
overlay layer [55] • After 7 days of
culturing, the cells
exposed to the
osteogenic medium and
Ca2+-polyP complex
exhibited significantly
higher mineralization
with respect to the cells
that were not exposed to
Ca2+-polyP complex but
treated with the
osteogenic medium
Bioinksa, b Cells Fabrication 3D printer Main outcomes about
method osteogenic potentialb
GMs of two different Primary human Volumetric TR300 tabletop • The changes in
methacrylation degree (7 wt% adipose-derived dispensing robot, rheological
and 5 wt%)-HAM (1 wt%)-HAp stem cells (extrusion) Unitechnologies characteristics are
(5 wt%) in PBS + LAP (hASCs) SA, Switzerland attributed to extensive
(0.135 wt% of polymer mass as matrix production in the
photoinitiator) [56] hydrogels by the
encapsulated cells
• The expression of bone
matrix components, such
as collagen I, fibronectin
ALP, and osteopontin,
was shown to increase
with the presence of
HAp but even more with
the osteogenic media
supplements after
28 days of culture
PEGDA (20% w/v)-Laponite Primary rat Two- BioScaffolder • 7% nanoclay was
XLG nanoclay (3, 5, 7, 10% osteoblasts channel 3.1, GeSiM, selected as the optimal
w/v) + Irgacure 1173 (3 wt% (ROBs) pneumatic Germany concentration to add to
relative to the mass of PEGDA extrusion the 20% PEGDA
cross-linker as photoinitiator); aqueous solutions
HA (20% w/v) in PBS [57] • The viability of the
printed cells was higher
than 95%
• The release of Mg2+
and Si4+ bioactive ions
from the PEG-clay
scaffolds stimulated the
osteogenic differentiation
of ROBs
• ROB-laden PEG-clay
constructs exhibited
excellent osteogenic
capability both in vitro
and in vivo
Bioinksa, b Cells Fabrication 3D printer Main outcomes about
method osteogenic potentialb
Alginate (3 w/v % in PBS)-MC Immortalized Pneumatic BioScaffolder • Calcium phosphate
powder (9 w/v %) [58] human extrusion 3.1, GeSiM, cement and bioink
mesenchymal Germany biphasic constructs were
stem cell line printed
expressing • Initial and local
human decrease in cell viability
telomerase was detected at the
reverse interface of calcium
transcriptase phosphate cements, and
(hTERT-MSC) cells started to migrate
from the alginate-MC
bioink to the calcium
phosphate cement
strands between day 7
and day 21
Type B gelatin (30%), alginate Wharton’s jelly Pneumatic BioScaffolder • The HUVEC-laden
(5%), and Ca-free DMEM [59] mesenchymal syringe and 3.1, GeSiM, bioink and molten
stem cells melt Germany PDACS/PCL were
(WJMSCs), extrusion dispensed alternately in
human each layer of structure,
umbilical vein and WJMSCs were
endothelial cells ejected by a piezoelectric
(HUVECs) nozzle on PDACS/PCL
layers
• Expressed ion levels of
both angiogenic markers
(vWF and Ang-1) of
HUVECs increased at
day 7
• ALP, BSP, and OC
protein expression levels
of WJMSCs were highest
on the PDASC/PCL with
WJMSC and alginate-
gelatin hydrogels with
HUVEC group for
14 days
Matrigel (9 mg/mL in PBS) [60] Human adipose Pneumatic Custom- • ASCs suspended in
stem cells syringe modified Prusa Matrigel bioink were
(ASCs) extrusion I3 A Pro with printed with
two additional PCL/bioactive borate
syringes, glass composite in
Geeetech, chloroform
China • The live/dead assay
showed 58 ± 11% viable
ASCs on the scaffold
after 1 week of
incubation
Bioinksa, b Cells Fabrication 3D printer Main outcomes about
method osteogenic potentialb
Alginate-PVA-HAp (2.5% w/v)- Mouse calvaria Ball screw- System 30 3D • The constructs were
collagen (5.0–6.5% pre-osteoblast driven printer with a unable to promote cell
w/v) + CaCl2 (0.5 and 1.0% as cell line extrusion modified EMO- proliferation over
cross-linker) [61] (MC3T3-E1) 25 extruder, 10 days
HyRel 3D, USA • Further in vitro
differentiation and in
vivo studies are needed

aDifferentcell-laden bioink formulations are separated with semicolons (;)


bPBS phosphate-buffered saline, HAp hydroxyapatite, VEGF vascular endothelial
growth factor, GelMA/GM gelatin methacryloyl/gelatin methacrylate, polyP
poly(phosphate), PVA poly(vinyl alcohol), HAM methacrylated hyaluronic acid,
LAP lithium phenyl-2,4,6-trimethylbenzoylphosphinate, ALP alkaline phosphatase,
PEGDA poly(ethylene glycol diacrylate), Laponite XLG
([Mg5.34Li0.66Si8O20(OH)4]Na0.66, HA hyaluronic acid sodium salt, MC
methylcellulose, DMEM Dulbecco’s modified eagle medium, PDACS
polydopamine-modified calcium silicate, BSP bone sialoprotein, OC osteocalcin, β-
TCP β-tricalcium phosphate

Compared to 3D printing of constructs, which are added in the post-printing,


cell-laden 3D bioprinting, as mentioned above, involves more complex factors, such
as narrow set of printing materials, the strategy of gelling, cell viability, and some
technical issues [62]. On the other hand, cell-free inks, or, in more accurate words,
3D printed biomaterials, offer a wide range of possible scaffolding materials and
more unconstrained operating parameters, such as high temperature. However,
controlling the cell distribution and seeding density on prefabricated 3D scaffolds is
a very hard constraint, and the results are generally poor formations of extracellular
matrix (ECM) microenvironment.
The advantage of using various 3D printable materials to construct cell-free
scaffolds also makes this approach attractive to bone tissue engineering
applications. There are various polymers that were 3D printed with CaPs or other
biomaterials to form bone tissue engineering scaffolds. For example, poly(ε-
caprolactone) (PCL) and (1) Ca2+-poly(phosphate) microparticles (at a ratio of 2:1
w/w) [63] and (2) silanated silica particles (5, 10, 20 wt%) [64] were printed by
pneumatic melt extrusion. Poly(lactic acid) (10 wt%) was previously printed with β-
TCP (5 wt%) with four different hydrogels by syringe extrusion method [65].
Natural polymers, such as chitosan, are also of interest of bone tissue engineering
area, for example, nano-sized HAp-chitosan-silica (15/50/35 molar ratio,
respectively) and chitosan-silica (50/35 molar ratio, respectively) were previously
printed by using pneumatic syringe extrusion [66]. Several forms of coating were
also applied on 3D printed bone scaffolds. HAp/β-TCP (at a mass ratio of 60/40)
(67% w/w) and 20 wt% Pluronic F-127 solution (33% w/w) was previously used as
3D printed biomaterial, and RGD (Arg-Gly-Asp)-phage nanofibers (1014 pfu/mL)
and chitosan (1 mg/mL) were used as coating material [67].

4.2 Osteochondral and Cartilage


Osteochondral tissue is composed of hyaline cartilage and subchondral bone and is
found at synovial fluid joints [68]. The main responsibility of hyaline cartilage is to
decrease surface friction and act as a shock absorber on the joint during daily
activity, while the subchondral bone tolerates load and supports hyaline cartilage
[68]. Osteochondral tissue can be degenerated through tumor, aging, or disease that
can result in poor life quality. The vascular structure of articular cartilage has
limited self-reparability, so the presence of defect in articular cartilage can progress
to the underlying subchondral bone creating osteochondral defects.
Osteochondral tissue has a hierarchical and organizational structure which is a
big challenge in designing of scaffold for osteochondral defects [69]. Articular
cartilage is composed of four different zones which, respectively, are the calcified,
deep, middle, and superficial zones [70]. These zones have different extracellular
matrix composition, collagen orientation, and chondrocyte phenotype [70]. The
orientation of collagen fibers is changed in these zones as follows: (a) parallel
alignment to the articular surface (superficial zone), (b) random alignment in the
middle zone, and (c) perpendicular form to the articular surface (deep zone); the
calcified zone has collagen X [70]. The orientation of collagen fibers has a key role
in mechanical properties of articular cartilage.
The surgical treatments are usually required in osteochondral defects [68]. There
are clinical treatments such as debridement, microfracture, autologous chondrocyte
implantation, matrix-induced autologous chondrocyte implantation, and
osteochondral autografting and allografting, but these are effective in early stages
and only postpone tissue degeneration. All of these are complicated and costly
treatments and did not result in proper biomechanical restoration [71].
There are different tissue-engineered cartilage products which are under clinical
trials to overcome limitations of current treatments. Table 3 reviews the list of
cartilage products [71].
Table 3 The list of commercial tissue-engineered cartilage scaffolds

Product name Company Biomaterial Cell


BioCart™II Histogenics, Waltham, MA Fibrinogen/hyaluronic Chondrocytes
acid

BioSeed®-C BioTissue Technologies Polyglactin 910/poly- Fibrin and expanded


GmbH, Freiburg, Germany p-dioxanone fleece autologous chondrocytes
Product name Company Biomaterial Cell

Cartipatch® Tissue Bank of France, Agarose-alginate Chondrocytes


Lyon, France hydrogel

Chondrosphere® Co.don AG, Teltow, Spheroids of Chondrocytes


Germany neocartilage

Hyalograft® C Anika therapeutics, Hyaluronic acid Chondrocytes


Bedford, MA
Matrix-induced autologous MACI, Vericel, Cambridge, Collagen II/III Chondrocytes
chondrocyte implantation MA

NeoCart® Histogenics, Waltham, MA Bovine collagen I Chondrocytes

NOVOCART® 3D TETEC, Melsungen, Biphasic collagen I Chondrocytes


Germany

Recently, there are significant research in tissue engineering approach for


osteochondral defect treatment and cartilage scaffold design [68]. The architecture
of scaffold plays an important role in vascularization, mechanical properties, cell
proliferation, and infiltration. The 3D printing technology is widely used for
printing of osteochondral and cartilage scaffolds due to its ability to create complex
structures and control pore size (Table 4).
Table 4 Bioprinted biomaterials and cells for osteochondral and cartilage

Biomaterial Cells Fabrication 3D printer Main outcome


method
15% methacrylated gelatin Bone marrow Pneumatic Customized, The in vivo
(GelMA) hydrogel for cartilage on mesenchymal dispensing pneumatic results showed
top layer, a combination of 20% stem cells dispensing that GelMa/nHA-
GelMA and 3% system based tri-layered
nanohydroxyapatite (nHA) (20/3% scaffold is useful
GelMA/nHA) hydrogel for in repairing of
interfacial layer, a 30/3% hyaline cartilage
GelMA/nHA hydrogel for and subchondral
subchondral bone at bottom layer bone
[68]
L2C4S4 apatite (1.8 g) + sodium Rabbit Three-axis 3D scaffold The L2C4S4
alginate (0.1 g) + Pluronic F-127 chondrocytes, positioning system printer extracts promote
(1.8 g) [72] rabbit bone osteogenic
marrow stem cells differentiation
and bioactivity
HA + polycaprolactone (in different – Extrusion printing 3D- The porosity is
ratios) [73] Bioplotter; more effective
EnvisionTEC, than addition of
Gladbeck, HA in terms of
Germany mechanical strain
Biomaterial Cells Fabrication 3D printer Main outcome
method
PLGA + 1% ß-TCP 3D printed as a Mesenchymal Pneumatic 3D biological The advantages of
subchondral part and articular stem cell from the printing printer this system
cartilage part was prepared from femoral marrow include high cell
fresh bovine limb by freeze-drying cavity utility efficiency,
[74] easy production,
and precise tissue
repair, which
could provide a
promising
alternative to
current
approaches to
repair joints
defects
PLLA filament used for 3D Murine fibroblasts Fused deposition – Gelatin
printing + 6% gelatin membrane L929 (ATCC, modeling concentration has
prepared by USA) significant effect
electrospinning + Osteogenon drug on mechanical
[75] strength and also
1.5% of
Osteogenon
promote
mechanical
properties of
scaffold
PCL + 20% GelMa + 1:1 Mesenchymal Microextrusion 3D discovery, The reinforced
GelMa/cell suspension [76] stem and inkjet printing RegenHU, polymeric
cell + chondrocyte Switzerland framework by
printing of
spheroids and the
collagen natural
structure was
mimicked
LCS (Li2Ca2Si2O7) powders, Rabbit Pneumatic – The in vitro
sodium alginate + LCS powder chondrocytes printing studies proved
(5:100) added to 20% F-127 that release of Li
solution [77] and Si augmented
osteogenic
differentiation of
rabbit
chondrocytes
Biomaterial Cells Fabrication 3D printer Main outcome
method
HAMA-GelMa + photoinitiator as Allogeneic Core-shell BioPen The pilot study
a shell bioink adipose-derived extrusion printing (custom- proved that real-
HAMA-GelMa + cells as a core mesenchymal made) time 3D printing
bioink [78] stem cells promotes
cartilage
regeneration
The shell
thickness should
be enough to
protect cells from
UV light
Nanocellulose + alginate [79] IPSC Microextrusion 3D discovery, The
printing RegenHU, nanocellulose+
Switzerland alginate bioink is
suitable for
cartilage tissue;
also cell density
in bioink plays
important role in
cartilage
regeneration
Gelatin methacrylate (GelMa) [80] – Pneumatic 3D- The architecture
printing Bioplotter; of scaffold has an
EnvisionTEC, important role in
Germany mechanical
properties
Nanocellulose/alginate Human nasal – – The new
bioink + cells (10:1) [81] chondrocytes cell/bioink
mixing system
(passive mixing)
was used and
proved that this
system increase
cell viability in
comparison with
other
conventional
mixing
10% GelMA + different Human bone Stereolithography- Custom-made The
concentrations of polyethylene marrow based 3D printing stereolithography-
glycol mesenchymal based 3D printing
diacrylate + photoinitiator + TGFβ1 stem cell system promotes
[82] cell viability and
protects growth
factors after
printing

4.3 Nerve
In the treatment of peripheral nerve and spine injuries, neural conduits have been
proposed as an effective neural regeneration matrix that support and guide neural
cells using tissue engineering approaches. Neural tissue harbors neurons and glial
cells (microglia, astrocytes, oligodendrocytes), while the vascular component
consists of endothelial cells, pericytes, and vascular smooth muscle cells [83].
Bioprinting offers a relatively recent approach for engineering of controllable 3D
scaffolds for neural tissues with diverse cell types and complex microscale features
[28]. The major points for the design of nerve guide conduits are (1) mechanical
support while aligning the proximal and distal nerve ends and prevent nerve
compression, (2) permeability to nutrients and waste products through the conduit
wall, (3) low immunogenicity, and (4) biodegradability to eliminate the need for
secondary surgery [84].
Among the printing techniques used in neural conduit construction, extrusion
printing has been used frequently as gels can be deposited conveniently with high
speed. However, laser-based bioprinting, such as stereolithography and inkjet
printing, allows better print resolution, which is a significant advantage in
mimicking anisotropic architecture of nerve tissue. The selection of bioink has a
tremendous effect on neural regeneration as neural cells are relatively more
sensitive to their extracellular milieu; hence neural tissue applications should mimic
the natural ECM of the target tissue [85]. Table 5 summarizes recent studies that
reported application of bioinks in neural conduit bioprinting.
Table 5 The bioinks and bioprinting methods used in neural conduit construction

Bioink Fabrication method Neural regeneration outcome


Alginate [86] An indirect bioprinting Decreased PRSCs viability with culture time. The scaffold
process of alginate with with low alginate concentration showed mechanical
primary rat Schwann unstability during culture
cells (PRSCs)
Alginate conjugated 3D gel extrusion of Cell viability was over 90% after 1 week for RGD and
with RGD or alginate peptides with YIGSR-gel composites, while lower viability was observed
YIGSR peptide [87] Schwann cells without peptides
Composite Extrusion of gel with Cells were able to sustain viability and proliferate in the
hydrogels of Schwann cell using 3D- scaffolds
alginate, fibrin, Bioplotter
hyaluronic acid,
and/or RGD peptide
[88]
Composite alginate- Rat Schwann cell in The printed hydrogel constructs contained more cells and
gelatin hydrogel composite gel was support long-term cell proliferation (after day 9)
[89] printed with an
extrusion-based
bioprinter
Bioink Fabrication method Neural regeneration outcome
Fibrin-factor XIII- 3D gel extrusion with a Schwann cells encapsulated within these scaffolds during
hyaluronate bioplotter fabrication were viable and proliferated in culture. Extrusion
hydrogel [90] induced longitudinal alignment of fine fibrin fibers which in
turn enabled the alignment of encapsulated Schwann cells and
dorsal root ganglion neurites along the 3D printed strands
(a) Gelatin Extrusion-based multi- (a) The printed cells did not proliferate, and axon propagation
methacrylate material 3D bioprinting was not observed in the matrix
(GelMA) and with iPSC-derived (b) When printed in 50% Matrigel concentration as the cell-
gelatin mixed with neural progenitor cells laden bioink, the overall cell viability over 4 days in culture
fibrin (GEL/FIB) for bioengineering of was >75% for both iPSC-derived sNPCs and oligodendrocyte
hydrogels spinal cord progenitor cells (OPCs)
(b) Commercial
extracellular matrix
(Matrigel) [91]
Water-based 3D dispersion extrusion Murine neural stem cells (NSC) which was extruded in
biodegradable with fused deposition hydrogels would proliferate and differentiate. NSC-laden
polyurethane manufacturing hydrogels could reestablish the function of impaired nervous
dispersions which equipment system in the zebrafish embryo
may later form gel
at mild conditions
[92]
GelMA, PEGDA Engineered nerve Complete sciatic nerve transections of mouse models
[93] guidance conduit using demonstrated directional guidance of regenerating sciatic
digital light processing nerves via branching into the microchannels and extended
(DLP)-based rapid toward the distal end of the injury site
continuous 3D-printing
platform

Alginate, a seaweed polysaccharide, can form stable hydrogels upon ionic cross-
link with bivalent ions like Ca+2 and Mg+2. In a recent study, Naghieh et al.
constructed alginate scaffolds at low concentration to enable effective neural
network formation by using indirect bioprinting technique [86]. After a sacrificial
gelatin framework was printed by extrusion-based device, a low-concentration
alginate incorporating primary rat Schwann cells (PRSCs) was casted into the
framework. Later, the gelatin framework was removed, and low-concentration
alginate scaffold was exposed. However, although low alginate provided favorable
conditions for cells, it was not mechanically stable, and cell viability decreased with
time.
Recent studies showed that the cell compatibility of alginate bioink would be
increased by using cell adhesion factors. In one of such studies, Sarker et al. tested
the potential of RGD or YIGSR peptide in increasing cytocompatibility of alginate
in bioprinting application [87]. An extrusion-based system was used to bioprint
RGD, YIGSR, or both peptide-conjugated sodium alginate with Schwann cells into
3D scaffolds. After 9 days of culture, the printed hydrogel constructs contained
more cells and supported long-term cell proliferation compared to 2D culture
conditions (petri dish) with respect to control alginate construct. Furthermore, the
conjugation of both RGD and YIGSR peptides in the same alginate molecules
increased the proliferation of neural cells in bioprinted constructs significantly. To
increase alginate biocompatibility, fibrin, hyaluronic acid, and RGD peptide were
mixed [88]. A 3D plotter was used to construct a grid-type scaffold by extruding the
blended biopolymer solution mixed with Schwann cells, which was later stabilized
with a cross-linking solution containing CaCl2 and thrombin that cross-link alginate
and fibroin, respectively. As indicated by in vitro techniques, cells were able to
sustain viability and proliferation in the scaffolds effectively. Similarly, the
composite technique was used by Li et al. to increase the cell compatibility of
alginate by using gelatin [89]. With the use of a bioplotter, Schwann cells were co-
extruded in alginate-gelatin into a grid-type scaffold structure. In vitro culture of the
printed hydrogel scaffolds showed that more cells were present in the scaffolds and
the composite gel support long-term cell proliferation compared to 2D culture (petri
dish) conditions.
Hyaluronate, a highly hydrophilic polysaccharide, is also known to not support
cells like alginate. England et al. suggested the use of fibrin-factor XIII in
hyaluronate gel precursor for the extrusion of Schwann cells [90]. A bioplotter
extruded hyaluronan-fibrin with Schwann cells into a 3D structure by stacking of
longitudinal strands. Schwann cells encapsulated within these scaffolds during
fabrication were seen viable and proliferated in culture. Interestingly, extrusion-
induced longitudinal alignment of fine fibrin fibers in gel enabled the alignment of
encapsulated Schwann cells and dorsal root ganglion neurites along the 3D printed
strands.
GelMA gels, on the other hand, were proven to be not effective in keeping the
viability and proliferation of induced pluripotent stem cell-derived neural progenitor
cells, which are known to depend on extracellular matrix [91]. Either GelMA or
gelatin blended with fibrin gels was bioprinted via extrusion-based plotter for
construction of scaffold, intended for spinal cord regeneration. In vitro tests showed
that the printed cells did not proliferate, and axon propagation was not observed in
the matrix. Therefore, in the same study, Matrigel, which is composed of growth
factors and proteins that mimic the basement membrane and extracellular matrix
from mouse sarcoma cells, was used in bioprinting of neural progenitors [91]. The
results showed that after extrusion plotting of cell-laden bioink, which was prepared
at 50% Matrigel concentration, the overall cell viability over 4 days culture was
>75% for both iPSC-derived sNPCs and oligodendrocyte progenitor cells (OPCs).
Water-based biodegradable polyurethane dispersions were used in bioprinting of
murine neural stem cells (NSCs) which may later form gel at mild conditions [92].
NSCs were embedded into the polyurethane nanoparticle dispersions before
gelation and extruded by using fused deposition manufacturing equipment. In vitro
results reported that NSCs could proliferate and differentiate and hence showed the
mild effect of bioprinting of cells with PU bioink. In addition, NSC-laden hydrogels
that were injected into the zebrafish embryo neural injury model could reestablish
the function of impaired nervous system.
Hydrogels based on collagen and gelatin have been proposed as an efficient
bioink for neural regeneration. Due to insolubility of collagen at neutral pH, gelatin,
which is a shorter-chain, denatured collagen product, is preferred candidate with
high water solubility at physiologic pH. Gelatin itself has a random coil structure in
solution and forms a helix below 35°C where helix-chain aggregation causes
gelation [94]. Especially, the acrylated gelatin, gelatin methacryloyl (GelMA), has
found particular interest as viable cells could be encapsulated via
photopolymerization during bioprinting process. Tao et al. attempted bioprinting of
GelMA with poly(ethylene glycol)-poly(3-caprolactone) (MPEG-PCH)
nanoparticles which encapsulated Hippo pathway inhibitor that can block MST1/2
kinase activity [95]. The bioprinted nerve conduit-drug construct induced higher
rate of recovery of sciatic injuries with respect to morphology, histopathology, and
functions in in vivo rat sciatic nerve model. In a similar approach, dopamine (DA)
was conjugated to GelMA molecules and used in bioprinting of a scaffold for
culturing of neural stem cells (NSCs) [96]. A custom-made stereolithography was
used in bioprinting of GelMA-DA, and UV laser beam was effective in gelation at
25 μJ intensity. The observation of a significant neural network on the 3D printed
GelMA-DA scaffolds after 12 days of culture indicated a stimulatory effect of
conjugated dopamine on neural progenitor cell when compared to unconjugated
GelMA scaffolds. In another study, GelMA was blended with poly(ethylene glycol
diacrylate) (PEGDA) for the engineering of nerve conduit [93]. A digital light
processing (DLP) apparatus, which could generate the desired patterns, was adapted
to the laser beam processing in a rapid continuous 3D-printing platform for
engineering of nerve guidance conduit. The implantation of the conduits with
microchannels and sleeve design into mouse models of complete sciatic nerve
transections demonstrated directional guidance of regenerating sciatic nerves via
branching into the microchannels and extension toward the distal end of the injury
site.

4.4 Skin
The skin being the outermost organ is susceptible to injuries, infections, and
environmental effects. Skin tissue is composed of the upper epidermis and inner
dermal layers with their respective cells of keratinocytes and fibroblasts. Tissue
engineering of skin grafts is a lifesaving approach for patients having major injuries,
burns, and skin diseases. The membrane preparation techniques, such as
electrospinning, freeze-drying, and solvent casting, have long been used for skin
graft constructions conveniently. Bioprinting of the skin attracted ever-growing
interest due to its sophisticated and controlled production properties which would be
rather difficult with conventional skin graft production methods.
Vijayavenkataraman et al. pointed out commercial initiatives and collaborations
among cosmetic companies (L’Oréal USA, NovoGen, Procter & Gamble) and
bioprinter producers (Organovo, Rokit) mainly aimed at fulfilling the huge demand
in production of skin products to test cosmetic products [97]. The potentials of 3D
bioprinting technique for skin tissue engineering have been investigated recently by
several researchers (see Table 6). Bioprinting facilitates the specific deposition of
multiple types of skin cells simultaneously [105]. In addition, 3D bioprinting
enables the precise localization of multiple cell types and appendages within a
construct [106]. However, there are challenges that limit skin bioprinting, such as
the technical difficulties associated with nozzle blockage and shearing stresses on
the cells [107]. Patient-specific construction of scaffolds is one of the most
important advantages of 3D printing technology. There is one recent study that
investigated segmenting chronic wounds and transmitting the coordinates to a
bioprinter robot to facilitate the treatment of chronic wounds [98]. As a result,
semiautomatic segmentation of wound images and image processing methods
improved the control of bioprinting process through more accurate coordinates.
Table 6 Bioprinted skin scaffolds with various bioinks and cell sources

Bioink Fabrication method Skin regeneration outcome


Sodium alginate [98] Extrusion by 3D plotting Bioprinted patches were produced
according to patient’s wound size and
contour
Type I collagen [99] Pneumatic extrusion by a The bioprinted skin structure showed the
3D plotter dermal and epidermal layers as well as the
terminal differentiation of the KC that
formed the stratum corneum. MC-
containing epidermal layer showed freckle-
like pigmentations at the dermal-epidermal
junction
Collagen [100] The 3D bioprinted Skin constructs had a higher degree of
constructs were fabricated resemblance to native skin tissue in terms
using a bioplotter with of the presence of well-developed stratified
multiple microvalve-based epidermal layers and the presence of a
printheads in two separate continuous layer of basement membrane
steps: proteins as compared to the manually cast
1. Bioprinting of collagen samples
fibroblast matrices
2. Keratinocytes and
melanocytes were directly
printed onto the bioprinted
collagen fibroblast matrices
Bioink Fabrication method Skin regeneration outcome
Bovine fibrinogen-thrombin [101] 3D cell spraying with a Bioprinted layered human dermal
bioplotter fibroblasts and epidermal keratinocytes in a
hydrogel showed rapid wound closure,
reduced contraction, and accelerated
reepithelialization in nude mice model
Adipose-derived ECM mixed with The simultaneous inkjet and Perfusable channels could supply nutrients
bovine fibrinogen mixture for extrusion 3D printing throughout the dECM-based construct.
hypodermal compartment. Skin- Printed HUVEC could cover the surface of
derived ECM (dECM) and channel, forming endothelium
fibrinogen mixture for dermal
compartment. Gelatin for vascular
channels [102]
Cells embedded in collagen gel or Cell lines of fibroblasts and Printed fibroblasts and keratinocytes
a mixture of blood plasma and keratinocytes embedded in proliferated and were vital. Extensive
alginate [103] collagen were printed in 3D formation of intercellular adherens
as a simple example for skin junctions between keratinocytes and their
tissue. On the principle of minor formation between fibroblasts, which
laser-induced forward proves the tissue formation, were observed
transfer
Amniotic fluid-derived stem Pneumatic-driven and Bioprinted constructs of collagen/fibrin gel
(AFS) cells and mesenchymal extrusion-based 3D with AFS or MSC cells had faster wound
stem cells (MSCs) were separately hydrogel solutions, with or closure rate than the gels without cells in
suspended in the without cells vivo. High vessel formation per unit area
fibrinogen/collagen solution [104] was detected for both kinds of cells as
compared to constructs without cells in
vivo

For increased vascularization and efficient epidermal growth, a perfusable,


vascularized full-thickness skin equivalent composed of epidermis, dermis, and
hypodermis was investigated by Kim et al. [102]. It was observed that the printed
HUVECs were covered on the surface of vascular channel, forming endothelium
resembling vascular structures. The results showed that keratin 10 and filaggrin
were expressed at the early and late stages of differentiation of the epidermis.
Within the dermal compartment, laminin and dermal ECMs were expressed at the
boundary between the dermis and epidermis similar to natural process.
In a different approach, Koch et al. deposited 20 layers of fibroblasts (mouse
NIH-3T3) and 20 layers of keratinocytes (human HaCaT) embedded in collagen gel
onto a sheet of MatriDerm® (decellularized dermal matrix) by laser-assisted
bioprinting for constructing dermis and epidermis layers, respectively [103]. The
printed constructs could show the presence of intercellular junctions, such as
cadherins and connexin 43 (Cx43), in the epidermis after 10 days of cultivation in
culture.
In addition, bioprinting of pigmented skin was tested to obtain a better
resemblance to native skin [100]. Primary human keratinocytes, melanocytes, and
fibroblasts were used in bioprinting process, while a two-step drop-on-demand
bioprinting strategy was used to deposit the cell droplets to form the melanin units
of epidermal layer. As a result, 3D bioprinted skin constructs had a better
resemblance to native skin as the printed tissue had well-developed stratified
epidermal layers and a continuous layer of basement membrane proteins as
compared to the manually cast samples.
Skin regeneration potential of amniotic fluid-derived stem (AFS) cells and bone
marrow-derived mesenchymal stem cells (MSCs) was compared by suspending
cells in fibrin-collagen gel and printing over the wound site [104]. A bioprinter with
pneumatic-driven nozzles was used to deposit layers of fibrin-collagen and
thrombin gel solutions containing AFS cells or MSCs deposited over full-thickness
wounds, which were created in nude mice models. In vivo tests showed that the
constructs of collagen/fibrin gel with AFS cells accelerated closure of full-thickness
wounds faster than the construct without cells, and they were as effective as the gels
with MSC. In addition, the vessel formation per unit area, which is a significant
indication of regeneration, was significantly higher with both kinds of cells than
only-gel printed grafts as the histology staining revealed.
As a conclusion, the bioprinting technology has enabled controlled fabrication
platform for construction of artificial skin, which potential has yet to be realized in
skin tissue engineering [108]. However, the resolution, vascularity, optimal cell and
scaffold combinations, and cost of bioprinted skin are some issues that should be
overcome to harness the new technology [109]. Histological complexity of the skin,
such as neural and immuno-components and hair follicles, should also be
considered in bioprinting of the skin to obtain fully functional native skin [108,
110].

4.5 Cardiac and Skeletal Muscle


Irreversible loss of cardiomyocytes due to ischemic heart attack leads to lethal heart
diseases and high mortality rates. Therefore, the regeneration of damaged cardiac
muscle is the only way to restore heart functions. Cellular cardiomyoplasty is the
implantation of in vitro cultured cardiomyocytes into the damaged myocardium, to
facilitate regeneration [111]. However, when injected to the heart directly, low cell
retention and viability of cardiomyocytes are encountered. Tissue engineering of
heart muscle by using bioprinting is carrying significant potential as the scaffold
matrix architecture would be customized while cardiac cells are placed in situ. In
this part of the chapter, the skeletal tissue engineering by bioprinting approach is
also discussed. Table 7 summarizes recent applications of bioprinting in cardiac and
skeletal tissue engineering.
Table 7 Bioprinted muscle scaffolds with various bioinks and cell sources

Bioink Fabrication method Muscle regeneration outcome


Bioink Fabrication method Muscle regeneration outcome
Silicone rubber 3D bioprinter with pneumatic PCL scaffolds showed more efficacy as there was a
and syringe extrusion was used in the higher percentage of adult primary human
poly(caprolactone) deposition of silicone rubber or cardiomyocyte (pHCM)) attachment and more cell
(PCL) [111] PCL in a grid structure migration, compared to the silicone rubber. Electrical
stimulation increased cell density on the PCL scaffold
Poly(ester Laser-induced forward transfer Increased vessel formation and found significant
urethane urea) (LIFT) cell (human mesenchymal functional improvement of infarcted hearts following
(PEUU) cardiac stem cells) printing technique transplantation of a LIFT tissue-engineered cardiac
patch immersed in patch in immune-deficient rat model
Matrigel was used
to collect
patterned cells
[112]
Carbon nanotubes A UV-integrated pneumatic 3D- Human coronary artery endothelial cells in MeCol gel
(CNT)- Bioplotter system was employed presented significant cellular proliferation, migration,
incorporated to create hybrid cell-laden and differentiation (lumen-like formation) over
alginate and MeCol. Alginate was printed as 10 days of incubation in vitro
methacrylated the implant framework to contain
collagen (MeCol) cell-laden MeCol within its
[113] spaces in each printed layer
Heart tissue- Human cardiac progenitor cells MixC/M (with VEFGs and MSCs) and patternC/M
derived (hCPCs) or endothelial cells were (generated patches by patterning of CPCs and MSCs
decellularized extruded in a disk-shape with VEGFs) greatly promoted vascularization
extracellular structures (8 mm in diameter and compared with the CPC patch after 4 weeks
matrix (hdECM) 0.5 mm in thickness) by a implantation; the patterned patch exhibited enhanced
bioink [114] bioplotter cardiac functions, reduced cardiac hypertrophy and
fibrosis, increased migration from patch to the infarct
area, and neo-muscle and capillary formation in rat
myocardial infarction model
Gelatin Human embryonic stem cell- A contractable cardiac tissue was obtained. hESC-
methacrylate derived cardiomyocytes (hESC- CMs printed in isotropic slabs beat synchronously;
(GelMA) [115] CMs) were mixed with GelMA however they contract without directional preference,
printed into a scaffold by using whereas hESC-CMs encapsulated in the parallel line
micro-continuous optical printing pattern contract in the direction of patterning
(μCOP)
No bioink was Human-induced pluripotent stem Patches exhibited ventricular-like action potential
used [116] cell-derived cardiomyocytes waveforms and uniform electrical conduction
(hiPSC-CMs), fibroblasts (FB), throughout the patch. In vivo implantations of
and endothelial cells (EC) were assembled cardiac patch showed vascularization and
aggregated to create mixed cell engraftment of 3D bioprinted cardiac patches
spheroids. The 3D bioprinter
picks up individual cardiospheres
using vacuum suction and loads
them onto a needle array
Bioink Fabrication method Muscle regeneration outcome
Fibrin-based Primary cardiomyocytes Dense, uniformly aligned, and electromechanically
(containing gelatin suspended in a fibrin-based coupled cardiac cells were observed after 3 weeks of
and hyaluronic bioink and cell-laden hydrogel culture
acid) bioink [117] were sequentially printed with a
sacrificial hydrogel and a
supporting polymeric frame by
using 3D pneumatic printing
The decellularized Human skeletal muscle cells Improved cell viability, myotube formation, and de
skeletal muscle (hSKMs)-encapsulated bioink novo myofiber regeneration in rat models of
extracellular solution was 3D extruded into 3D volumetric muscle loss (VML). Improved de novo
matrix (dECM) scaffold by a plotter. muscle fiber formation and vascularization were
and vascular Prevascularized muscle constructs observed in rats
dECM (vdECM) were fabricated through coaxial
bioinks [118] nozzle printing with dECM and
vdECM bioinks
PEG-fibrinogen Microfluidic printing head with An enhanced myogenic differentiation with the
and alginate [119] coaxial needle was used to formation of parallel aligned, long-range, tightly
extrude muscle precursor cells packed, and completely striated myotubes
(C2C12) into 3D constructs
composed of aligned hydrogel
fibers

Adams et al. studied bioprinting of silicone rubber and poly(caprolactone)


(PCL), which are known as printable and biocompatible materials, into scaffolds for
cardiac patch construction [111]. A grid-type scaffold was 3D printed with
pneumatic syringe extrusion from silicone rubber or PCL bioink. In vitro tests
showed that PCL scaffolds had more efficacy as there was a higher percentage of
adult primary human cardiomyocyte (pHCM) attachment and more cell migration,
compared to the silicone rubber. In addition, an increase in cell density of cardiac
cells was observed on the PCL scaffold after electrical stimulation.
Laser-induced forward transfer (LIFT) cell printing technique performs
controlled transfer of inorganic and biological materials in conjunction with
proteins, peptides, DNA, RNA, and cells in three-dimensional (3D) patterns with
survival rates of printed cells at nearly 100% [112]. Poly(ester urethane urea)
(PEUU) cardiac patch immersed in Matrigel was used to collect patterned cells from
laser beam focused on the donor slide. Human mesenchymal stem cells (hMSCs)
and endothelial cells (EC) were patterned in such a way that the rectangular islands
of hMSC were surrounded by the EC lanes, mimicking vascular tissue model on a
PEUU cardiac patch that was immersed in Matrigel. The therapeutic potential of the
patch in cardiac regeneration was tested in immune-deficient Rowett nude rat left
anterior descending ligation model with respect to cell survival, infarct wall
thickness, angiogenesis, cardiac remodeling, and functional improvement. Increased
vessel formation and significant functional improvement of infarcted hearts were
observed in in vivo results.
Carbon nanotubes (CNT) were incorporated into alginate and methacrylated
collagen (MeCol) bioink for building cardiac patch with electrical, mechanical
attributes [113]. A UV-integrated pneumatic 3D-Bioplotter system was used to
construct human coronary artery endothelial cells (HCAECs) encapsulated in
MeCol. An indirect bioprinting technique was used for MeCol bioink. Alginate was
printed as the implant framework in order to hold cell-laden MeCol within its
spaces in each printed layer. As a result, HCAECs in MeCol gel presented
significant cellular proliferation, migration, and differentiation (lumen-like
formation) over 10 days of incubation in in vitro cell culture.
Jang et al. tested heart tissue-derived decellularized extracellular matrix
(hdECM) bioink to encapsulate and print human cardiac progenitor cells (hCPCs)
[114]. Human cardiac progenitor cells (hCPCs) or endothelial cells were extruded in
a disk-shape structures (8 mm in diameter and 0.5 mm in thickness) by using a
bioplotter. Three patch types were constructed as: (1) only CPCs (CPC), (2)
randomly mixed of both CPCs and human turbinate tissue-derived mesenchymal
stem cells (MSCs) with vascular endothelial growth factors (VEGFs) (mixC/M), or
(3) generated patches by patterning of CPCs and MSCs with VEGFs alternatively
(patternC/M). Neovascularization and tissue formation potential of cell-laden
constructs were studied in Balb/c nude mice model, while hdECM patch without
cells was used at the epicardium of the rat myocardial infarct (MI) model to study
the functional benefits of hdECM. The tests of vascularization and tissue formation
of stem cell patch in vivo showed that mixC/M and patternC/M greatly promoted
vascularization compared with the CPC patch after 4 weeks implantation. The
mixC/M and patternC/M patches both seemed to induce a potent angiogenic
response in the host tissues: blood vessels developed in the implanted patches and
appeared to be functional vessels because red blood cells could be observed in the
lumens. The cardiac functions in rat myocardial infarction model revealed that the
patterned patch could exhibit enhanced cardiac functions, reduced cardiac
hypertrophy and fibrosis, increased migration from patch to the infarct area, and
neo-muscle and capillary formation.
In another study, gelatin methacrylate (GelMA) bioink was used in bioprinting
of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) [115]. With
the aim of cardiac patch construction, hESC-CMs were mixed GelMA and
bioprinted by using Micro-continuous optical printing (μCOP) platform. μCOP
system consists of programmable digital masks to control millions of individual
mirrors to pattern light onto a liquid volume. The digital patterns are casted on
photopolymerizable polymer on a high-precision stage, which is computer
controlled, to create 3D scaffolds. hESC-CMs were mixed with GelMA and
polymerized into micropatterned (parallel lines) cardiac patch. After 3–7 days of
cell culture, a contractable cardiac tissue was obtained. hESC-CMs printed in
isotropic slabs beat synchronously; however they contract without directional
preference, whereas hESC-CMs encapsulated in the parallel lines pattern contract in
the direction of patterning.
In a different approach, a vacuum-assisted bioprinter device, which picks up
individual cardiospheres using vacuum suction and loads them onto a needle array,
could be used to construct a cardiac patch without using any bioink material [116].
Cardiospheres that were produced by cell aggregation of human-induced pluripotent
stem cell-derived cardiomyocytes (hiPSC-CMs), fibroblast (FB), and endothelial
cells (EC) were assembled into a cardiac patch by pickup function of dispenser
needles of the bioprinter. Electrophysiological results indicated that patches
exhibited ventricular-like action potential waveforms and uniform electrical
conduction throughout the patch.
Wang et al. tested fibrin-based (containing gelatin and hyaluronic acid) bioink
for primary cardiomyocytes (infant rat hearts) by co-printing with sacrificial gel and
supportive frames [117]. Three dispensing modules for cell-laden hydrogel,
sacrificial hydrogel, and PCL were operated separately. Primary cardiomyocytes
suspended in a fibrin-based bioink and cell-laden hydrogel were bioprinted along
with supporting frame (PCL) and a sacrificial hydrogel by using 3D pneumatic
printing. Bioprinted cardiac tissue constructs had a spontaneous synchronous
contraction in culture medium. Progressive cardiac tissue development was
confirmed by immunostaining for actinin and a calcium influx spreading across the
entire tissue after 1 week of culture. After 3 weeks, cardiac tissues were formed
with uniformly aligned, dense, and electromechanically coupled cardiac cells.
In skeletal tissue engineering, Choi et al. used decellularized skeletal muscle
extracellular matrix (dECM) and vascular dECM (vdECM) as bioinks for printing
vascularized human skeletal muscle cells [118]. hSKMs were mixed in dECM while
endothels (HUVECs) were mixed in vdECM bioinks for coaxial bioprinting of them
into thick constructs by using extrusion-based 3D plotting system. The 3D muscle
constructs showed enhanced cell viability, myotube formation, and de novo
myofiber regeneration for rat models of volumetric muscle loss (VML). In vivo
results showed that coaxial nozzle printing mimicked the hierarchical architecture of
vascularized muscles, and allogenic human cells in the constructs improved de novo
muscle fiber formation, vascularization, and innervation, as well as 85% of
functional recovery could be observed in VML injury. In another muscle bioprinting
trial, Constantini et al. used PEG-fibrinogen- and alginate-based bioinks by
extrusion-based construction of aligned hydrogel fibers encapsulating muscle
precursor cells (C2C12) [119]. With a unique approach, the printing head had a
microfluidic device with a coaxial needle that can be used to extrude C2C12 cells
into 3D constructs while producing aligned hydrogel fibers. By this method an
enhanced myogenic differentiation with the formation of parallel aligned, long-
range, tightly packed, and completely striated myotube structures could be
achieved.
4.6 Other Soft Tissues
The 3D printing technique gives the chance to control the size and features at
micro-/nanoscale for various types of soft tissues. The printing of complex tissues,
such as liver and cornea, is possible, but many issues exist until today. In printing of
multicellular tissues, cells and matrix materials should be printed together to mimic
tissue micro-architecture [120, 121]. Therefore, many tissue scaffolds have been
designed to mimic native tissue with complex 3D morphology. Table 8 shows some
important researches that investigated those challenging soft tissues such as liver,
cornea, and urinary bladder.
Table 8 Biomaterials and cells used for bioprinting of selected soft tissues

Tissue Biomaterials Cells Fabrication 3D printer Main outcome


method
Liver [122] GelMa + PEG-bis- Human Cellbricks – It was proved
chloroacetate + hepatic stellate printing- liver organoid
photoinitiator + cells cells, HepaRG stereolithography can be printed by
cells using
stereolithography
technique and
hydrogel as a
bioink
Liver [123] 2% w/v alginate, 3% w/v Human Microextrusion Inkredible, The desirable
gelatin, 0; 0.25; 0.5; 1, or bipotent printer Cellink, bioprinting was
2 mg/mL w/v hECM, hepatic Gothenburg, achieved in
0.03 M CaSO4, and progenitor Sweden alginate-gelatin
cells, human with 0.5 and
7 × 106 mature HepaRG epithelial lung 1 mg/mL hECM
cells/mL carcinoma bioink
cells
Urinary Bone marrow-derived cell Bone marrow- – Regenova, The urinary
bladder [124] spheroids derived cells Cyfuse bladder can be
Biomedical printed by using
K.K., bone marrow
Tokyo, stem cells, and
Japan vascularization is
seen after
transplantation to
rabbit
Cornea [125] Sodium Human Microextrusion Inkredible, The bioinks with
alginate + methacrylated corneal printer Cellink, low viscosity are
collagen stromal cells Gothenburg, suitable for 3D
Sweden printing of
cornea
Tissue Biomaterials Cells Fabrication 3D printer Main outcome
method
Hepatic 3% alginate + cells Mouse Pneumatic 3D The collagen is
structures embryonic printing bioprinting not printable
[126] fibroblasts system, alone, and
(MEFs) Korea alginate was
Institute of added to modify
Machinery its pH and
and printing
Materials, temperature
South Korea
Cornea [127] Gelatin + lyophilized Human Extrusion INVIVO, 3D bioprinted
bovine AM corneal printing Rokit, HCEC-laden
endothelial Seoul, AM grafts being
cells (HCEC) Korea well engrafted
and
demonstrated
significantly
improved
corneal thickness
and edema
compared to the
control
conditions
Human Human intestinal Human Extrusion Organovo, Fully human 3D
intestinal myofibroblast (IMF) primary printing 3D intestinal tissue
tissues [128] interstitium, human intestinal NovoGen model composed
intestinal epithelial cells epithelial Bioprinter, of primary cells
(HIEC) cells, San Diego, with increased
myofibroblast USA complexity and
function
compared with
standard in vitro
models can be
designed
Photoreceptor- Human retinal pigmented Human retinal Microvalve- RegenHU The microvalve-
retinal tissue epithelial cell line, human pigmented based bioprinting bioprinter, based bioprinting
[129] retinoblastoma cell line epithelial cell Switzerland is efficient and
line, human accurate to build
retinoblastoma the in vitro tissue
cell line models with the
potential to
mimic natural
tissue
Cornea [130] Collagen I, Limbal Laser-assisted – The laser-
ethylenediaminetetraacetic epithelial stem bioprinting assisted
acid, human female AB cells, human bioprinting
blood plasma, human adipose- systems can be
recombinant laminin-521, derived stem used in cell
hyaluronic acid sodium cells printing by
salt optimizing laser
source power
5 Conclusion
The significant factors which should be taken into account in designing of
biological substitutes are pore size, mechanical integrity, architecture, and mass
transportation. Thus, the success of biological substitute requires a deep knowledge
about contribution of mechanical properties, biological factors, and material
composition during regeneration period. The 3D print technology gives opportunity
to accurately control the architecture of printing substitute which gives the ability of
mimicking natural tissues. In addition, the bioink composition has a tremendous
effect on success of bioprinting. The bioinks that mimic extracellular matrix of
target tissue are a promising approach in bioprinting. Therefore, formulations of
tissue-specific bioink for each type of tissue will become an effective strategy. 3D
printing of complex and large-scale constructs with high spatial resolution is a
major issue that needs to be investigated. Further inventions on these issues will
unblock challenges in bioprinting of functional and complex tissues with high
mechanical integrity.

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Gene Therapy
Ana del Pozo-Rodríguez1, Alicia Rodríguez-Gascón1, Julen Rodríguez-Castejón1,
Mónica Vicente-Pascual1, Itziar Gómez-Aguado1, Luigi S. Battaglia2 and
María Ángeles Solinís1
(1) Pharmacokinetic, Nanotechnology and Gene Therapy Group
(PharmaNanoGene), Faculty of Pharmacy, Centro de investigación Lascaray
ikergunea, University of the Basque Country UPV/EHU, Vitoria-Gasteiz,
Spain
(2) Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di
Torino, Turin, Italy

María Ángeles Solinís


Email: [email protected]

Abstract
Gene therapy medicinal products (GTMPs) are one of the most promising
biopharmaceuticals, which are beginning to show encouraging results. The broad
clinical research activity has been addressed mainly to cancer, primarily to those
cancers that do not respond well to conventional treatment. GTMPs to treat rare
disorders caused by single-gene mutations have also made important
advancements toward market availability, with eye and hematopoietic system
diseases as the main applications.
Nucleic acid-marketed products are based on both in vivo and ex vivo
strategies. Apart from DNA-based therapies, antisense oligonucleotides, small
interfering RNA, and, recently, T-cell-based therapies have been also marketed.
Moreover, the gene-editing tool CRISPR is boosting the development of new gene
therapy-based medicines, and it is expected to have a substantial impact on the
gene therapy biopharmaceutical market in the near future.
However, despite the important advancements of gene therapy, many
challenges have still to be overcome, which are discussed in this book chapter.
Issues such as efficacy and safety of the gene delivery systems and manufacturing
capacity of biotechnological companies to produce viral vectors are usually
considered, but problems related to cost and patient affordability must be also
faced to ensure the success of this emerging therapy.

Graphical Abstract

Keywords Delivery vectors – Ex vivo – Gene therapy medicinal product – In


vivo – Manufacturing – Quality control

1 Introduction
Biopharmaceuticals, pharmaceuticals produced in biotechnological processes by
molecular biology methods, have become one of the most effective clinical
treatments for a wide variety of diseases. The biopharmaceutical market includes
gene therapy products, based on the use of nucleic acids as active pharmaceutical
ingredients for the modulation of the gene expression. Gene therapy based on the
administration of DNA and messenger RNA (mRNA) acts by means of
therapeutic protein expression, whereas the use of small interfering RNA
(siRNA), microRNA, oligonucleotides, or aptamers provides posttranslational
gene silencing. An emerging area in this field is genome editing, which corrects
the disease by replacing a sequence of a defective gene by a healthy copy in order
to restore the “wild-type” DNA. Most of those nucleic acids are produced as
biopharmaceuticals, although some of them, such as antisense oligonucleotides,
are made by chemistry means.
Despite that gene therapy entered clinical trials in the early 1990s, the first
nucleic acid-based product registered in the European Union was Glybera in 2012,
for lipoprotein lipase deficiency. Currently, only 15 gene therapy medicinal
products have received approval worldwide; nevertheless, since 1989 almost
2,700 gene therapy-based clinical trials have been completed, are ongoing, or have
been approved for a broad range of applications. Therefore, it is expected that
nucleic acid-based products will have a substantial impact on the
biopharmaceutical market in the near future.

2 Concept of Gene Therapy


Gene therapy is a novel approach to treat, cure, or ultimately prevent disease by
changing the expression of a person’s gene [1]. According to the European
Medicines Agency (EMA), a gene therapy medicinal product generally consists of
a vector or delivery formulation/system containing a genetic construct engineered
to express a specific transgene (“therapeutic sequence”) for the regulation, repair,
replacement, addition, or deletion of a genetic sequence [2]. By far the most
common vector systems used for gene therapy to date have been viral vectors and
plasmid DNA vectors. Gene therapy works by repairing, deactivating, or replacing
dysfunctional genes that cause disease with the aim of (re)establishing normal
function.
Gene therapy has long been regarded a promising treatment for many diseases,
including those inherited through a genetic disorder (such as hemophilia, human
severe combined immunodeficiency, spinal muscular atrophy, or cystic fibrosis) or
acquired (such as cancers of different kinds).
Depending on the cell type to be modified, gene therapy can be classified into
two categories: somatic and germline gene therapy [3]. Somatic gene therapy
targets to body somatic cells such as bone marrow or blood cells. This type of
gene therapy cannot be passed on to descendants. In germline gene therapy, egg
and sperm cells (germ cells) are the objective of therapy, and the inserted gene
passes on to future generations. The idea of germline gene therapy is controversial
due to ethical concerns, and it is not permitted in many countries. In spite of that,
at the end of 2018, a research team in China announced the birth of two babies
whose genomes had been edited, and there may be a third one [4]. These practices
have been widely condemned as irresponsible and as failing to conform with
international norms [5].
Two fundamental strategies have evolved to restore or modify target cell
function: ex vivo or in vivo gene delivery [6]. In ex vivo therapy, cells from the
patient or a donor are harvested, and the therapeutic gene is then transduced in a
cell therapy manufacturing setting. The modified cells are later reinfused into the
patient. In vivo gene therapy consists on functional modification of targets by
direct transgene injection into the patient. Figure 1 features a scheme with the ex
vivo and in vivo approaches to gene therapy.
Fig. 1 Ex vivo and in vivo approaches to gene therapy

Thanks to the advances of genetics and bioengineering, gene therapy has


become possible, although at present, this is predominantly an experimental area.
However, in the last 5 years, enormous advances have occurred, with the approval
of a few drug products by the Food and Drug Administration (FDA) and the EMA
and others that are expected to be in the near future.

3 Nucleic Acids for Gene Therapy


Historically many gene therapy approaches have been based on expression of a
transgene encoding a functional protein (i.e., the transgene product). However,
newer tools including directly acting nucleic acid sequences such as microRNA,
interference RNA (RNAi) via short hairpin RNAs (shRNA) or short interference
RNA (siRNA), molecular scissor and gene-editing approaches such as zinc finger
nucleases (ZFNs), transcription activator-like effector nucleases (TALEN), and
clustered regularly interspaced short palindromic repeats (CRISP)-associated
nuclease Cas9 (CRISPR/Cas9) are being extensively applied in research and for
developing new medicinal products. The development of this emerging field is
still maturing, although there are many nucleic acid-based drugs in clinical trials,
and some of them have been already approved. There are also other promising
candidates to be used in a variety of genetic and nongenetic disorders such a
monogenic, infectious, cardiovascular, inflammatory, neurodegenerative, and a
wide variety of cancers.
Nucleic acids are negatively charged and high molecular weight molecules,
with physical-chemical properties very different to that of conventional drugs.
They present limited stability in the biological medium and must access to an
intracellular compartment (the cytoplasm or the nucleus); these two characteristics
contribute to the difficulties to develop a medicinal product.
Depending on the application, the objective of the therapy can be gene
augmentation, gene silencing, or gene editing [7].

3.1 Gene Augmentation


The objective of gene augmentation is to restore normal cellular function by
delivering a functional copy of a gene (DNA) or messenger RNA (mRNA).

3.1.1 DNA
Typically, the gene of interest is inserted into a plasmid or expression cassette,
which is a high molecular weight, double-stranded DNA construct containing
transgenes, which encode specific proteins [8]. Plasmids also contain a promoter
and a terminator signal to drive and end gene transcription, respectively [9].
Transfection with DNA leads to much higher protein and persistent expression
than those obtained with mRNA. However, with mRNA, once it reaches the
cytoplasm, translation starts instantly, without the need to enter the nucleus to be
functional [10]; on the contrary, DNA has to reach the nucleus of the target cell,
being this process one of the most limiting steps for transfection.
Apart from plasmid DNA, minicircle DNA (mcDNA) are emerging due to
their safety and persistent transgene expression in both quiescent and actively
dividing cells [11]. mcDNA are episomal, covalently closed circular gene
expression systems, generally biosynthesized in recombinant bacteria, that consist
in minimalistic backbones with potential to meet the clinical requirements for safe
and long-lasting expression [12]. An alternative approach to sustain prolonged
gene expression is the inclusion of scaffold matrix attachment regions (S/MAR)
moieties in mcDNA constructs. Among others, mcDNA has been proposed as a
potential therapeutic strategy for cancer [13].

3.1.2 Messenger RNA


Messenger RNA (mRNA) is the template for the synthesis of proteins. The use of
synthetic mRNA to produce a desired protein in cells is a very promising
technology to apply in clinic. Contrary to plasmid DNA, mRNA-based
therapeutics are still in their infancy, in spite of important advantages. mRNA
must only be delivered to the cytoplasm where cellular translation machinery is
located. From a therapeutic perspective, protein expression arising from mRNA is
more transient than that from DNA. From a safety point of view, mRNA does not
integrate into the genome and so poses no risk of insertional mutagenesis [14].
Moreover, mRNA gene therapy circumvents the need for selecting a specific
promoter, and thus the transfection process is relatively efficient and simple [15,
16]. Another advantage of mRNA refers to the production process, raw material
synthesis, and the quality product, which are more easily standardized than that
for DNA, that leads to higher reproducibility [17].
Foreign RNA possesses inherent immune-activating adjuvant properties, and
this effect has been studied for the intracellular delivery of mRNAs coding for
specific antigens, with potential application in cancer immunotherapy [18],
prophylactic vaccines [19], and allergy tolerization [20].
Since the protein expression profile of mRNA and DNA is completely
different, the codelivery of these two nucleic acids has been proposed to take
advantage of both [6].

3.2 Gene Silencing


3.2.1 Antisense Oligonucleotides
Single-stranded antisense oligonucleotides (ASO) are short sequences of modified
DNA or RNA that can be used as therapeutic tools through (1) activation of
RNase H to achieve specific knockdown of the target transcript or (2) modulation
of pre-mRNA splicing to enable the restoration of a (partially) functional protein
or alternatively a protein with reduced toxicity [21]. The ASO’s unprecedented
specificity for transcripts makes them unique as a therapeutic entity as it allows
very specific targeting and provides the opportunity to, for example, correct
genetic defects for rare genetic diseases with a current unmet medical need,
modulate splice defects in autoimmune or neurodegenerative diseases, or target
transcripts expressed by tumors or viruses.
The utilization of synthetic ASOs is increasing in areas ranging from clinical
diagnostics to novel pharmaceutical therapeutics, and the efficacy and safety of
ASOs are being investigated for the treatment of various genetic disorders where
no treatment is currently available. Recently, several first-in-class ASO drugs have
been approved by the FDA or EMA, including mipomersen for the treatment of
familial hypercholesterolemia, eteplirsen for the treatment of Duchenne muscular
dystrophy, and nusinersen for the treatment of spinal muscular atrophy.

3.2.2 Aptamers
Aptamers are short single-stranded RNA or DNA oligonucleotides, normally 15–
80 nucleotides, with the capacity to fold in stable three-dimensional structures.
These molecules present very high affinity with nucleic acids through structural
recognition and bind to them through electrostatic interactions, hydrogen bonding,
van der Waals forces, base stacking, or a combination of them [22].
Aptamers recognize and bind targets of interest just like antibodies and have
important advantages over conventional antibodies: (1) easy to synthesize by
automated methods; (2) easy to modify to improve the stability, binding strength,
and specificity to the target nucleic acid; (3) structure very flexible; and (4)
display low to no immunogenicity when administered in preclinical doses 1,000-
fold greater than doses used in animal and human therapeutic applications [23].
Due to the molecular recognition of their targets, aptamers have a variety of
diagnostic and therapeutic applications, such as biosensors and target inhibitors.
Due to simple preparation, easy modification, and stability, aptamers have been
used in diverse areas within molecular biology, biotechnology, and biomedicine
[24]. However, up to now, the introduction of aptamers into the market has not
been very successful, and only one aptamer-based product have been approved for
clinical use, Macugen® (pegaptanib), for the treatment of age-related macular
degeneration.

3.2.3 RNA Interference


RNA interference (RNAi) is a posttranscriptional mechanism of gene silencing
through chromatin remodeling, inhibition of protein translation, or direct mRNA
degradation. RNAi is a naturally occurring process of gene regulation present in
plants and mammalian cells, and it can be used to downregulate disease-causing
genes. Typically, there are three different types of commonly used RNAi
molecules [25]:
– MicroRNA (miRNA)
– Short interfering RNA (siRNA)
– Short hairpin RNA (shRNA), also called expressed RNAi activators
miRNA
miRNA and their role in regulating normal physiological processes were
discovered in the last decade, as well as their involvement in pathological
disorders such as cancer [26]. They are noncoding RNA molecules of 18–25
nucleotide in length that regulate at posttranscriptional level the expression of
genes by binding to the 3′-UTR of target genes [27]. A miRNA can regulate
different mRNAs, because they are not specific to a single mRNA [28]. miRNA is
transcribed from DNA as primary miRNA (pri-miRNA), which is later processed
into a precursor miRNA (pre-miRNA) by two proteins: Pasha and Drosha. The
pre-miRNA is transported to the cytoplasm, where it is processed by Dicer to
obtain the miRNA, which is incorporated into the RNA-induced silencing
complex (RISC), where a helicase unwinds the miRNA. The resulting antisense
stand guides the RISC to its complementary mRNA, which is cleaved (Fig. 2).
Fig. 2 Schematic representation of the mechanism of action of miRNA. Pri-miRNA is transcribed from DNA
and is later processed into pre-miRNA. In the cytoplasm, the pre-miRNA is processed to give the miRNA,
which incorporates into the RNA-induced silencing complex (RISC), where a helicase unwinds the miRNA.
Finally, the resulting antisense strand binds to its complementary mRNA and cleaves it

miRNAs have emerged as key players in a wide array of biological processes,


and changes in their expression and/or function have been associated with plethora
of human diseases, such as myocardial infarction and stroke [29], Parkinson’s
disease [30], or cancer. The application of miRNA in cancer therapy is based on
the finding that miRNA expression is deregulated in cancer tissues and also due to
the ability of miRNA to target multiple genes and alter cancer phenotypes [31]. In
fact, in neoplastic diseases, miRNA can be downregulated when they function as
tumor suppressors or overexpressed when they function as oncogenes [32].
siRNA
siRNAs are short double-stranded RNA segments with 21–23 nucleotides and are
complementary to the mRNA sequence of the protein whose transcription is to be
blocked. siRNA molecules are incorporated into the RISC complex, which bind to
the mRNA of interest and stimulate degradation of mRNA or the suppression of
the translation process [22]. Figure 3 shows the mechanism of siRNA.
Fig. 3 Schematic representation of the mechanism of action of siRNA. The molecules of siRNA are
incorporated into the RNA-induced silencing complex (RISC), where a helicase unwinds it. Finally, the
resulting antisense strand binds to its complementary mRNA and cleaves it

The main advantage of synthesized siRNA is that these molecules do not need
to reach the nucleus to induce the therapeutic effect. As a drawback, stability must
be improved in order to optimize the efficacy [22].
Because of their small size and low potential to elicit adaptive immune
responses, several antihuman immunodeficiency virus (HIV) RNAs have
advanced to clinical trials. A potential advantage of anti-HIV-1 siRNAs over
current therapies is that their sequences could be tailored to target a patient’s
particular viral strains and provide a personalized approach to therapy [33]. A
major challenge for the development of anti-HIV-1 siRNAs is that lymphocytes,
which represent the major cell type for HIV-1 replication, are widely distributed in
the body and extremely difficult to penetrate with existing siRNA delivery
technologies [34]. Other viral infections with limited treatment options and more
easily accessible target that could be treated with siRNA include hepatitis B virus,
Ebola, and respiratory syncytial virus [29]. Other indications of siRNA under
clinical investigation are hepatocellular carcinoma, hepatic fibrosis, dry eye
syndrome, melanoma, and pancreatic ductal adenocarcinoma.
At present there is a siRNA-based therapy (patisiran) recently approved by the
FDA and the EMA for the treatment of hereditary transthyretin-mediated
amyloidosis, a rapidly progressive, heterogeneous disease caused by the
accumulation of misfolded transthyretin protein as amyloid fibrils at multiple sites
and characterized by peripheral sensorimotor neuropathy, autonomic neuropathy,
and/or cardiomyopathy [35]. Another product, inclisiran, is an experimental
therapeutic agent for the treatment of hypercholesterolemia, which is being tested
in late-stage clinical trials.
Short Hairpin RNA
Short hairpin RNA (shRNA), also called expressed RNAi activator, is a plasmid-
coded RNA that needs to be transcribed in the nucleus to downregulate the
expression of a desired gene. It can be transcribed through either RNA polymerase
II or III. The first transcript generates a hairpin-like stem-loop structure and is
then processed in the nucleus by a complex containing the RNase II enzyme
Drosha. The individual pre-shRNAs generated are finally transported to the
cytoplasm by exportin 5. Once in the cytoplasm, the complex Dicer processes the
loop of the hairpin to form a double-stranded siRNA [36]. Figure 4 features a
scheme with the mechanism of action of shRNA. Since shRNA is constantly
synthesized in the target cells, more durable gene silencing is achieved in
comparison to other forms of RNAi [37]. shRNAs represent an important tool in
the assessment of gene function in mammals and are largely used as a research
tool. Although shRNA has been assayed to develop new therapies for retinal
diseases [38], viral infections [22, 33], or cancer [39], no therapeutic product
based on shRNA has been approved.
Fig. 4 Schematic representation of the mechanism of action of shRNA. The molecules of shRNA are
transcribed in the nucleus and are exported to the cytoplasm, where the complex Dicer processes them to form
a double-stranded siRNA. The siRNA is later incorporated into the RNA-induced silencing complex (RISC),
where a helicase unwinds it. Finally, the resulting antisense strand binds to its complementary mRNA and
cleaves it

3.3 Gene Editing


Gene-editing technology has recently emerged as a new treatment modality for a
variety of diseases, including hereditary, infectious, and neoplastic diseases. This
technology is based on programmable nucleases, which consist of a nuclease that
can be reprogrammed to cleave a precise target sequence [40, 41]. Consequently,
they induce a double-strand break (DSB) at a specific and desired location.
There are four types of gene-editing nucleases: meganucleases, ZFN, TALEN,
and CRISPR/Cas9 [36]. Meganucleases, also called homing endonucleases,
stimulate the cellular recombination and repair of DNA to fix the break by simply
copying the gene encoding them (the homing endonuclease gene) and flanking
DNA into the broken chromosome [42]. ZFN contains zinc finger proteins, the
most common class of DNA-binding proteins across all of biology, and FokI
nuclease as the DNA-binding and cleavage domains. ZFNs have now been widely
used for genome editing in many species and cell types for basic science,
biotechnology, and medical applications, such as targeted disruption of the CCR5
gene for HIV-1 therapy [43]. One important disadvantage of ZFN is that it is an
expensive and time-consuming technology. TALEN use the same FokI-derived
nuclease domain as ZFN, but differ in that they employ distinctive DNA-binding
arrays: TALE effector repeat arrays [44]. TALEN technology for the recognition
of a wider range or target gene sequences requires complicated engineering that is
a matter of concern. The CRISPR/Cas9 system is based on a prokaryotic antiviral
mechanism in which the bacteria insert a partial gene sequence from an infection
source, such as bacteriophage, into their own genomes to defend against repeat
infection [45]. This system includes a RNA guide to bind to a complementary
sequence in a target gene, which is recognized and cut by the Cas9 [46]. Once the
target gene is cleaved by the programmed nuclease, the reparation of the DSB can
be done by two different mechanisms: nonhomologous end joining (NHEJ) and
homology-dependent repair (HDR) [43]. In the NHEJ, the target region is
eliminated by joining the DSB, and it can be used to silence or correct a
pathogenic gene. On the contrary, with the HDR modality, a homologous
sequence can be introduced into the DSB, enabling donor DNA to be inserted to
either correct an existing gene or add a new one. Figure 5 shows the different
modalities of DSB reparation.
Fig. 5 Schematic representation of the different modalities of gene repair with nucleases

Contrary to gene augmentation and suppression, therapeutics based on gene


editing can lead to a permanent effect at the genome, and therefore, this recent
technology represents a key development of gene therapy [37].
In the last years, there has been tremendous progress in gene editing, mainly
with CRISPR/Cas9, thanks to the simplicity of the manufacturing procedures in
comparison to the earlier tools, meganucleases, ZFN, and TALEN. However, it is
important to remark that in comparison to standard gene transfer approaches,
genome editing, particularly that based on CRISPR/Cas9, is in its translational and
clinical infancy. In spite of that, several clinical trials with gene-editing
technologies have been completed or are undergoing [47]. For instance,
engineering ZFN have been tested in clinical trials to disrupt CCR5 (C-C motif
chemokine receptor type 5) expressed in human T cells and hematopoietic stem
cells to provide to them resistance to human immunodeficiency virus (HIV)
infection [48]. Other clinical trials with ZFN have been approved for delivering
the factor IX gene for hemophilia, the α-iduronidase gene for
mucopolysaccharidosis I, and the iuronidate-2-sulfatase gene for
mucopolysaccharidosis II. The first-in-human use of TALEN gene-edited T cells
in two infants with refractory relapsed B-cell acute lymphoblastic leukemia led to
a successful induction of molecular remission ahead of allogeneic stem cell
transplantation [49].
The rapid technological advances in genome editing have allowed
manipulating germ cells, gametes, zygotes, or embryos. In a recent study [50], the
CRISPR/Cas9 technology repaired in an embryo DSBs induced at the mutant
paternal allele, by predominantly using the homologous wild-type maternal allele
instead of a synthetic DNA template. The authors were able to avoid mosaicism in
cleaving embryos and achieve a high yield of homozygous embryos carrying the
wild-type MYBPC3 gene (whose mutation causes hypertrophic cardiomyopathy),
without evidence of off-target mutation. In spite of the potential use for the
correction of heritable mutations in human embryos, much remains to be
considered before clinical applications, including the reproducibility of the
technique with other heterozygous mutations.
Human application of these technologies still presents a substantial challenge.
Collaboration between regulatory bodies and the scientists developing these life-
changing treatments is very important for gene editing to progress to the clinic,
with special emphasis to the critical assessment of risk vs benefit [41]. Among all
the potential applications of gene editing, it is impossible to predict which
approaches will ultimately be successful, both in the clinic and on the market, but
it is expected that the application of gene-editing technologies is poised to change
the practice of medicine dramatically in the years to come [51]. The long-term
follow-up of patients who will participate in genome-editing clinical trials will
likely provide invaluable insight into the in vivo activity and specificity of
programmable nucleases.

4 Delivery Systems Used in Gene Therapy


One of the main challenges of gene therapy is the development of safe and
effective administration systems that are able to overcome the main limitations of
nucleic acids when they are administered in the body [52]. Therefore, a
fundamental aspect for the success of gene therapy is the availability of delivery
systems capable of protecting the genetic material from degradation, facilitating
its internalization in target cells, and releasing them intracellularly. The ideal
delivery system depends on the target cells, the kind of nucleic acid to be
delivered, and the duration of expression [53]. The delivery systems are classified
into two large groups: viral and non-viral vectors.
Viral vectors are prepared from genetically modified viruses so that they are
not able to replicate in the target cells, but they express the therapeutic gene they
transport. Viral vectors allow high transfection efficiencies; however, they present
important safety limitations due to the oncogenic and immunogenic potential (due
to viral proteins). Another problem associated with viral vectors is the inability to
transport large nucleic acids.
Non-viral vectors are safer, simpler, cheaper, and more reproducible systems.
In addition, they do not present limitations regarding the size of the genetic
material they can incorporate. Nevertheless, a disadvantage of non-viral systems is
that their transfection efficiency is lower compared to viral vectors, although in
recent years new non-viral systems have been developed with materials that
exhibit higher transfection efficiencies. In fact, the number of clinical trials with
products based on non-viral vectors (Fig. 6) has increased in the last decade, and
those based on lipid nanocarriers (lipofection) are used in 4.1% of all trials [54].

Fig. 6 Vectors used in gene therapy clinical trials. Data consulted in gene therapy clinical trials worldwide
[54]

4.1 Viral Vectors


Viruses used as delivery systems of genetic material include adenoviruses, adeno-
associated viruses (AAV), retroviruses, and lentiviruses among the most evaluated
in clinical trials. Other viruses, such as those derived from herpesvirus or
poxvirus, have also been studied as possible viral vectors.
The selection of the most suitable viral system in each case depends on
different factors: the organ or the target cell, the ability to integrate the genetic
material carried by the vector in the genome of the host cell, the duration of
expression gene over time (short-term or long-term response), or the size of
therapeutic nucleic acid. Table 1 shows the main characteristics of the most
frequently studied viral vectors, to be taken into account for their application in
gene therapy.
Table 1 Features of the most studied viral vectors in gene therapy

Retroviruses Lentiviruses Adenoviruses AAV


Viral genome RNA RNA DNA DNA
Retroviruses Lentiviruses Adenoviruses AAV
Target cells Dividing Dividing and Dividing and Dividing and
cells nondividing cells nondividing cells nondividing cells
Integration in the host Yes Yes No Yes
genome
Response Long-term Long-term Short-term Long-term
Size of the genetic material 8 kb 8 kb 7.5–30 kb 4.5 kb
to be carried

AAV adeno-associated viruses

4.1.1 Retroviral Vectors


Retroviruses are RNA viruses that contain two strands of RNA enveloped by an
icosahedral capsid of peptide nature (Fig. 7). The capsid is surrounded by a
phospholipid envelope, in which different types of glycoproteins act as ligands for
specific receptors on cell surfaces and therefore determine the tropism of the virus.

Fig. 7 General structure of retroviruses and retroviral genome. LTR long terminal repeats, pbs first binding
site, ppt poly-purine sequence, ψ packaging signal

The genome of retroviruses (Fig. 7) contains three types of genes: gag genes
that encode capsid proteins, pol genes that encode the enzymes necessary for the
replicative cycle of the virus (protease, integrase, reverse transcriptase), and env
genes that encode the envelope glycoproteins. This genome also contains a
packaging signal, ψ, thanks to which the RNA molecules bind to the capsid
proteins and are effectively packaged, and the long terminal repeats (LTR) at each
end of the viral genome. The left LTR contains a region for the start of
transcription (U3 promoter) and a first binding site (pbs) for the start of reverse
transcription. The right LTR contains a poly-purine sequence (ppt) for replication
of the second strand. For application in gene therapy, the retroviral vectors are
generated by the substitution of the gag, pol, and env sequences of the viral
genome by the therapeutic gene. As a consequence, the therapeutic sequence in
this case cannot be greater than 7–8 kb.
The replicative cycle of a retrovirus begins with entry into the cell, which is
mediated by receptors [55]. Once inside the cell, the viral reverse transcriptase
enzyme produces a DNA molecule from the viral RNA. Subsequently, the DNA is
integrated into the genome of the host cell, and the transcription yields different
RNAs, which are exported to the cellular cytoplasm, where they are translated into
structural proteins of the virus, and directs synthesis of new virion nucleocapsids.
The nucleocapsids leave the cell and keep enveloped by a plasma membrane-
derived outer coat.
It is important to point out that the DNA obtained by reverse transcription
from the RNA is not able to cross the nuclear membrane of the cell to be treated.
Therefore, retroviral vectors only transfect efficiently dividing cells, because the
DNA takes advantage of the disruption of the nuclear envelope during the mitosis,
to reach the interior of the nucleus [56].
On the other hand, the integrase enzyme allows the integration of the genetic
material in the genome of the host cell. Thanks to this, it is possible to obtain a
long-lasting expression of the therapeutic sequence; however, the insertion into
the genome of the target cell is also one of the major problems of viral gene
therapy, since if it takes place in an unwanted region of the genome of the
transfected cells, there is a risk of mutagenesis and oncogenesis. In fact, in clinical
trials with these vectors, several patients developed leukemia and dysplasia of the
bone marrow due to insertional mutagenesis [57, 58]. These adverse effects were
partially reduced by the design of so-called self-inactivating vectors in which the
genome sequences of the virus identified as responsible for mutagenesis, 3′LTR,
are deleted. This idea for self-inactivating vectors had been already described in
the literature by Yu et al. [59]. Another limitation of retroviral vectors is that they
are recognized and inactivated by the complement system, which means that they
are mainly used in ex vivo gene therapy.
Despite the mentioned limitations, and due to its high transfection efficiency,
since 1989, the year in which the first clinical trial with gene therapy was
launched, 514 clinical trials with retroviral vectors have been started (17.5% of the
total of clinical trials with gene therapy) [54].
One of the most commonly used retroviruses in gene therapy is the murine
leukemia virus (MLV), which has shown efficacy in different types of
immunodeficiency. In fact, recently the European Medicines Agency (EMA) has
approved a drug based on ex vivo gene therapy with this retroviral vector for the
treatment of patients with severe combined immunodeficiency due to adenosine
deaminase (ADA-SCID) deficiency that cannot be treated with bone marrow
transplant (Strimvelis; Orchard Therapeutics) [60].

4.1.2 Lentiviral Vectors


Lentiviruses also belong to the Retroviridae family. However, they have certain
differential genes with respect to the rest of retroviruses that facilitate the entry of
genetic material into the cell nucleus, so lentiviral vectors can also transfect
nondividing cells. One of the most known and studied viruses of this subfamily is
the human immunodeficiency virus (HIV).
The general structure of lentiviruses is the same as that described for
retroviruses: two RNA strands included in a protein capsid, surrounded by a
phospholipid envelope that includes different types of glycoproteins.
The genome of the lentiviruses (Fig. 8) shares with the retroviruses the gag,
pol, and env genes, as well as the LTR, pbs, and ppt sequences. However, it
presents other specific genes: tat genes (transcription regulators), rev genes
(regulators of the expression of viral proteins), vif genes (necessary for the
infection of different cell types), vpr genes (participate in the entrance to the
nucleus), vpu genes (involved in the release of viral particles from infected cells),
and nef genes (increase the infectivity of the virus).

Fig. 8 General structure of lentiviral genome. LTR long terminal repeats, pbs first binding site, ppt poly-
purine sequence, ψ packaging signal

The cycle of life is similar to that described for retroviral vectors, with the
difference that lentiviruses present genes encoding nuclear localization signals that
favor the entry of DNA (synthesized by the reverse transcription process) into the
nucleus.
In order to use lentiviruses as gene delivery systems, the tat gene is removed,
and the gag and pol genes are encoded on a different plasmid from that of the rev
or env genes. The final vector results from three separate plasmids containing the
necessary viral sequences for packaging [61]. In addition, the 3′LTR sequence of
the viral genome may be deleted to generate self-inactivating (SIN) lentiviral
vectors [62], as previously mentioned in the case of retroviral vectors.
Due to the tropism of lentiviruses and their ability to transfect cells that are not
in division, the main application of lentiviral vectors is directed to introduce
genetic material in vivo in cells of the central nervous system [63], for example,
for the treatment of Parkinson’s disease [64] or in cells of the retina [65] (suitable
for the treatment of retinitis pigmentosa). Furthermore, lentiviral vectors
efficiently transfect ex vivo cells of the hematopoietic system, which are difficult
to transfect with other vectors. In fact, ex vivo gene therapy with lentiviral vectors
to genetically modify CD34+ cells has been evaluated in more than 100 clinical
trials in recent years for the treatment of monogenic diseases (i.e., β-thalassemia
[66], X-linked adrenoleukodystrophy [67], metachromatic leukodystrophy [68], or
Wiskott-Aldrich syndrome [69]), of different types of cancer [70], and of
infectious diseases such as HIV infection [71].

4.1.3 Adenoviral Vectors


Adenoviruses encompass more than 50 different virus serotypes and are
responsible for 5–10% of respiratory infections in children and adults. In gene
therapy, serotypes 2 and 5 are the most used. Adenoviruses are non-enveloped
viruses that consist of a double strand of DNA within an icosahedral capsid (Fig.
9).

Fig. 9 General structure of adenoviruses and adenoviral genome. ITR inverted terminal repeats, ψ packaging
signal

The genome of an adenovirus (Fig. 9) has a size of approximately 35 kb. At


the ends are the inverted terminal repeats (ITR), and close to the left ITR, the
packing signal, ψ, is arranged. The numerous genes it contains differ in the early
(E) and late (L) regions. The latter, responsible for the coding of structural
proteins, are transcribed after replication of the viral genome, and the E regions
are transcribed before synthesizing the viral DNA, since they give rise to
regulatory proteins. These proteins alter the expression of host cell proteins that
are necessary for DNA synthesis, intervene in viral replication, and also prevent
the death of infected cells by blocking the apoptosis or avoiding recognition by the
immune system.
Adenoviruses are endocytosized into the cell after binding to the CAR receptor
(coxsackievirus and adenovirus receptor). The nucleocapsids are released into the
cytoplasm, and with the help of cellular microtubules, they reach the nuclear
membrane, and the genetic material is introduced to the nucleus of the cell
through the nuclear pores. Replication then begins, and once all the components of
the virus have been synthesized, they are assembled and released from the cell by
cell lysis induced by the virus itself. During this process the adenoviral genome
does not integrate into the genome of the host cell. This is one of the advantages
of adenoviruses which makes them safer compared to other viral systems,
although this also means that the viral life cycle is not adapted for long-term
transgene expression.
To be used as vectors for gene therapy, E regions are deleted from the genome,
and various levels of attenuation can be achieved by removal of different numbers
of genes: only one E1B gene (first-generation vectors), the majority of early genes
(second-generation vectors), and even full deletion of all genetic information of an
adenovirus (so-called gutless vectors) [72]. The large size of the genome and the
possibility to delete a major part of it provide high coding capacity for these
vectors: 1–2 kb can be inserted in early generation vectors and up to 30 kb in
gutless vectors. However, they need another helper virus that replicates normally
and expresses all the proteins needed to assemble the adenoviral vector [73].
Despite its advantages, the total elimination of impurities from helper viruses is
complicated and limits its clinical application. In fact, at high doses adenoviruses
are toxic [74], and one of their main limitations is that they are very immunogenic
[75], which decreases their effectiveness.
In spite of their limitations, the advantages of adenoviral vectors have meant
that they have been evaluated in more than 500 clinical trials [76], most of them
aimed at the treatment of cancer.

4.1.4 Adeno-Associated Viral (AAV) Vectors


Adeno-associated viruses (AAV) are small non-enveloped viruses with single-
stranded DNA (Fig. 10). The genome has a size of about 4.7 kb and is composed
of three regions, called rep, cap, and aap, flanked by the corresponding ITR. The
rep region encodes nonstructural proteins involved in viral replication, packaging,
and integration into the host genome, genes in the cap region encode the structural
proteins of the capsid, and aap gene encodes the assembly-activating protein [77].
AAV vectors for clinical application are generated by replacing the rep, cap, and
aap genes with the gene of interest.

Fig. 10 General structure of AAV and AAV genome. ITR inverted terminal repeats
The entry of AAV into the cell takes place by endocytosis after binding to the
receptor-mediated cellular surface. Once the nucleocapsid escapes from the
endosome and is transported to the nucleus, the viral genome is able to cross the
nuclear membrane. In the nucleus a second strand of DNA, necessary for the
replication of the virus, will be synthesized. In the presence of a helper virus
(coinfection by an adenovirus or herpesvirus), the double-helix DNA generated
can be integrated into the genome of the host cell, and replication of the virus will
take place. In the absence of a helper virus, the AAV genome usually remains
latent in the form of an episome. Replication of the viral genome and subsequent
packaging results in the generation of viral particles that will escape from the host
cell by lysis thereof [78].
These vectors are quite safe, can transfect cells with or without capacity of
division, and provide long-term gene expression, up to 6 years. Another advantage
of AAV is the possibility of selecting the most suitable serotype depending on the
target tissue [79]. Table 2 shows the most suitable AAV serotypes for different
tissues.
Table 2 AAV serotypes suitable for specific target tissues [79]

Serotype Target tissue


AAV1 Nervous system, skeletal muscle
AAV2 Nervous system, kidney, photoreceptors, retinal pigment epithelium
AAV4 Nervous system, retinal pigment epithelium
AAV5 Nervous system, lungs, photoreceptors, retinal pigment epithelium
AAV6 Skeletal muscle, lungs
AAV7 Skeletal muscle
AAV8 Nervous system, photoreceptors, retinal pigment epithelial, liver, skeletal muscle, heart, pancreas
AAV9 Nervous system, heart, liver, skeletal muscle, lungs

AAV adeno-associated viruses

The main disadvantage of AAV is the limited size of the genetic material they
can transport, which must not exceed 4.5 kb. However, viral vectors based on
AAV have been evaluated in more than 180 clinical trials, most of them aimed at
the treatment of monogenic diseases. In fact, a medicinal product called Luxturna
and based on AAV2 have reached the market. Luxturna (voretigene neparvovec) is
a gene transfer vector that employs an AAV2 as a delivery vehicle for the human
retinal pigment epithelium 65 kDa protein (hRPE65) cDNA to the retina [80].
This medicine is indicated for the treatment of adult and pediatric patients with
vision loss due to inherited retinal dystrophy caused by confirmed biallelic RPE65
mutations and who have sufficient viable retinal cells to express the protein and
respond to the treatment.

4.1.5 Manufacturing of Viral Vectors


Viral vector production for clinical application requires viral propagation in
suitable animal cell lines, viral recovery, concentration purification, and
formulation [81]. To meet commercial and regulatory requirements, each process
must be scalable and reproducible and must yield high virus titers. For large-scale
manufacturing, suspension-adapted cell lines cultured in bioreactors are more
appropriate than adherent cells systems, conventionally used at laboratory scale
[82]. Depending on the viral vector and the cell clone used, the most suitable
bioreactor must be chosen [83].
The production of viral vectors may be carried out by transiently transfecting
the producing cells with vector and helper or packaging plasmids or by generating
stable producer cell lines. In the first case, culture cells are co-transfected with
multiple plasmids, one containing the expression cassette for the transgene and
other plasmids encoding regulatory and structural viral proteins. The main
limitations of transient transfected cells are the amount of transfected cells
achieved and the variability in transfection [84]. In addition, transient transfection
results in contaminations of the final product due to excess plasmids [85] and
residual transfection reagent. In the second case, cells are genetically modified, so
that they contain, inserted in the cellular genome, the genes that encode the
structural proteins necessary for the formation of viral particles containing the
transgene of interest [86]. Processes using stable producer cell lines are easier to
scale-up and result in less contaminated vectors, although some drawbacks have to
be also considered: the gene products necessary to produce vectors are toxic to
cells, each vector-produced cell line requires specific certifications as master cell
bank, and, upon cell expansion, high-titer vectors are not always ensured [84].
One of the limitations of viral vector manufacturing is to purify a sufficient
amount of viral particles even to start a clinical trial. The downstream processing
or purification of viral vectors aims to eliminate contaminants, whether process or
product-related. Process-related impurities derive from starting materials (residual
DNA and residual host cell protein from each cell bank) or raw materials (culture
reagents, purification reagents and equipment materials, helper viruses, and helper
virus nucleic acid used in production), whereas product-related impurities include
vectors with deleted, rearranged, hybrid, or mutated sequences. The ultimate goal
of the downstream processing is to obtain a product with high purity, potency, and
quality that can meet the guidelines of the FDA [87] and EMA [2] regulatory
agencies. With this aim, different concentration methods have been developed:
centrifugation, tangential flow filtration, ultrafiltration, polyethylene glycol
precipitation, two-phase extraction, membrane filtration, liquid chromatography,
or adsorption chromatography [88].

4.1.6 Quality Control of Viral Vectors


Another aspect to take into account are the quality controls that these vectors must
overcome to ensure that each batch manufactured meets the specifications of
purity, potency, safety, and identity and there are consistency and comparability
between batches. This is a challenge given the high complexity of the viral
systems due to the large number of protein subunits that make up the viral capsid
and the composition of the lipid membrane present in enveloped viruses. In
addition, it is necessary to develop specific analytical methods for each type of
virus and even for each serotype. These methods can be divided into those that are
similar to others already validated for recombinant proteins and vaccines and
those that are specific to each vector. The former include, for example, the
analysis of impurities related to the production process, such as packaging cell
proteins. Specific assays of viral vectors include the analysis of the activity of the
resulting product of the therapeutic genetic material, as a measure of potency. It is
also necessary to determine the impurities due to the presence of residual genetic
material encapsulated in the vector. In any case, it must be considered that many
of these methods developed to analyze the specific quality controls of viral vectors
are not yet validated according to the standards established to license and market
these products.

4.2 Non-viral Vectors


Non-viral systems can be defined as those physical or chemical methods that help
in the process of transfer of exogenous genetic material to the cell, facilitating the
entry and intracellular bioavailability thereof. Non-viral systems try to imitate the
capacities of viruses as gene transfer vehicles, providing greater security from the
point of view of biological risk and pathogenicity. However, reproducing with a
non-viral system what a virus performs naturally is a great challenge that has led
to the development of different strategies. The selection and design of the most
appropriate non-viral system are conditioned by its efficacy and safety, by the
tissue or target cell, and by the type of therapeutic genetic material [53].

4.2.1 Physical Methods


Physical methods are based on the application of physical forces to temporarily
alter the permeability of the cell membrane, allowing the genetic material to cross
the cytoplasmic membrane and reach the interior of the cell. It is important that
there is a balance between the efficiency of cellular internalization and the damage
exerted on the cell. These methods have been frequently evaluated as systems of
administration of naked genetic material, without the need to formulate it or
include it in a viral or non-viral vector, which gives them great simplicity.
Nevertheless, physical methods are mainly evaluated and used in preclinical
studies. Table 3 summarizes the main characteristics of physical delivery methods
used in gene therapy [89].
Table 3 Types of physical delivery systems in gene therapy and main features

Method Advantages Limitations


Needle injection Simple Low efficiency
Direct needle injection on a specific tissue Safe Local inflammation
Hydrofection or hydrodynamic injection High efficacy in the Hemodynamic
Intravascular injection of high volumes of a solution liver changes
containing the nucleic acids
Microinjection High efficiency Cell by cell
Direct injection into host cell by microneedles administration
Time-consuming
Need of specialist
Biolistic injection or gene gun Simple and fast Low efficiency
Administration of metal microparticles at high velocity Reproducibility Low tissue
penetration
Cell damage
High cost
Electroporation Noninvasive Risk of tissue
Application of electric pulses that open pores on cell Simple damage
membrane High efficiency Surgery necessary to
target internal organs
Low cost
Widely employed
Sonoporation Noninvasive Low reproducibility
Application of ultrasounds (combined with microbubbles or Safe Tissue damage
nanocarriers) to permeabilize temporally cell membrane Targeting to specific
tissues
Magnetofection Noninvasive Effective only on
Application of external magnetic fields combined with Effective in primary surface areas
magnetic particles cells (difficult to Mainly applied in
transfect) vitro
Optofection Nucleic acids release Tissue damage
Application of laser pulses combined with nucleic acid from endosomes Inflammation
complexes or nanoparticles Restricted to single
cells or small areas

4.2.2 Chemical Carriers


Chemical vectors are based on the use of different types of compounds capable of
encapsulating or binding, electrostatically or covalently, the genetic material.
In order to develop a suitable non-viral delivery system, the selected vector
must have the capacity to enter the cell and to overcome different barriers
maintaining the stability of the nucleic acid throughout the entire transfection
process. Once within the cell, it must guarantee the proper intracellular
distribution of genetic material. Therefore, the main steps that these delivery
systems have to overcome to reach the cytoplasm (in the case of RNAs) or the
nucleus (in the case of DNAs) are the following: interaction with cell membrane,
entry into the cytoplasm, intracellular distribution, and entry into the nucleus (Fig.
11). To date, different strategies for overcoming these limitations have been
proposed, and the evolution of non-viral vector transfection has been significantly
improved in recent years.

Fig. 11 Main stages during transfection process: (1) interaction with cell membrane, (2) entry into the
cytoplasm, (3) intracellular distribution, and (4) entry into the nucleus. NPC nuclear pore complex
The first step in the genetic transfer process is the interaction between vectors
and cell membranes. Cationic vectors interact electrostatically with the negative
charge of the cell membrane surface, and the internalization process is started.
Additionally, in order to enhance the interaction with specific cells, different
ligands can be added to the vectors to improve binding to surface receptors [90,
91].
Once the vector has bound to the cell surface, it must penetrate into the
cytoplasm. The internalization or entry into the cell can take place through two
mechanisms: fusion with cell membrane or endocytosis. These two entry routes
are not exclusive, and depending on the type of cell and vector, one or the other
may predominate. However, in most cases vectors penetrate into the cells mainly
through endocytic pathways. Endocytosis begins with the formation of a vesicle
from the invagination of the plasma membrane, called an endosome. These
endosomes fuse with lysosomes by creating endolysosomes, and their hydrolytic
enzymes can degrade genetic material. Therefore, to achieve efficient gene
transfer, it is necessary for the nucleic acid material to be protected from
degradation by lysosomes to ensure release of nucleic acids into the cytoplasm. In
fact, the endosomal escape represents an important barrier to achieve efficient
transfection in the case of non-viral gene therapy [92].
In the case of DNA, once it is cytoplasm, it must be able to enter to the
nucleus. However, the nuclear membrane is a selective barrier for
macromolecules, such as DNA. The transport through this membrane is a highly
regulated process, facilitated by a series of water channels of about 10 nm, called
nuclear pore complexes (NPCs). The genetic material transported by non-viral
vectors penetrates the nucleus through two main routes: NPCs or during cellular
mitosis, when the nuclear membrane is temporally disrupted. The passage through
the NPCs is carried out by means of an energy-dependent process that generally
involves the recognition of specific nuclear localization signals (NLS) [93]. The
NLS consist of one or more short sequences of amino acids with positive charges
containing arginines and lysines [94]. The formulation of DNA with compounds
containing NLS is a strategy commonly used in non-viral gene therapy.
Chemical delivery systems or non-viral vectors are broadly categorized into
inorganic, polymeric, lipidic, or peptidic particles. In many cases, the combination
of some of different kinds of chemical compounds is used in order to improve
their profile of efficiency, cellular specificity, and safety, giving rise to hybrid
systems [53].
Inorganic Particles
Inorganic particles are nanostructured systems with different sizes, shapes, and
porosity, designed to protect the genetic material from degradation and to escape
from the reticuloendothelial system after its systemic administration. They can be
composed of different elements, being used in gene therapy calcium phosphate
[95], silica [96], gold [97], or magnetic compounds such as iron oxide [98].
These inorganic particles are of interest since they are easy to produce and
ligands can be added to their surface that facilitate the union to the genetic
material through electrostatic interactions. Cationic components are usually
incorporated to the surface of the particle. An example of this type of system
consists of combining iron oxide particles with PEI, which favors the
condensation of nucleic acids, with polyethylene glycol (PEG), which favors the
colloidal stability of the particles, and with cell penetration peptides, which favor
cellular internalization [99]. In the case of gold particles, nucleic acids are
previously thiolated to covalently bound to the delivery system [100].
Other types of inorganic materials used to develop inorganic particles that are
showing encouraging results in vitro and in vivo in animal models are graphene
[101] or fullerene [102]. However, it is still necessary to study in greater depth the
long-term safety and the influence of functionalization, size, and shape in
transfection efficiency to facilitate the clinical application of these newer
compounds.
Polymeric Particles
The main component of these vectors is a cationic polymer that binds and
condenses the genetic material, giving rise to the so-called polyplexes [103].
Cationic polymers bind by electrostatic interactions the negatively charged genetic
material, so that the nucleic acid is adsorbed to the surface of the nanoparticulate
system or is encapsulated in its interior. In addition, these systems allow the
incorporation of different ligands that improve the transfection efficiency in the
target tissue. In general, polymeric vectors are more stable than lipid vectors, and
even in some cases, the progressive degradation of the polymer allows controlling
the rate of release of the genetic material once it is inside the cell.
The polymers used in the preparation of non-viral vectors can be subdivided
into synthetic and natural polymers (also called biopolymers).
The synthetic polymers most used in gene therapy are polyethyleneimine
(PEI), the dendrimers [104], the polyesters (i.e., poly(lactic-co-glycolic) or PLGA)
[105], or polymethacrylates [106]. Among them, PEI has been evaluated in
various clinical trials for the treatment by local gene therapy of different types of
cancer [107], but the high toxicity of this polymer has limited its application.
In the group of the biopolymers applied in gene therapy are polysaccharides,
such as chitosan, cyclodextrins, alginate, pullulan, or dextran. Some of these
polysaccharides are used by themselves as delivery systems, but most of them are
generally used in combination with other non-viral vectors to improve their
efficacy, safety, or biodistribution [90, 108, 109].
Lipidic Particles
Lipid-based systems are the most studied non-viral vectors at the clinical level. Up
to 119 clinical trials have been documented, most of them in phases I and II, in
which lipid vectors have been used as delivery systems for the genetic material. In
most cases, vectors have been designed for the treatment of different types of
cancer but also for the treatment of cardiovascular diseases, hepatitis C virus
infection, or monogenic diseases such as cystic fibrosis [54]. Recently, a lipid-
based siRNA delivery system called patisiran (Alnylam® Pharmaceuticals) has
reached the market, as treatment of hereditary transthyretin-induced amyloidosis
[35].
The main components of the lipid-based vectors are cationic lipids, formed by
hydrophobic alkyl chains, linked through an intermediate binding structure to a
polar group. The most used cationic lipids are 1,2-di-O-octadecenyl-3-
trimethylammonium propane (DOTMA), 1,2-dioleoyloxy-3-trimethylammonium
propane (DOTAP), 1,2-dimyristyloxypropyl-3-dimethyldhydroxyethylammonium
bromide (DMRIE), or 3ß-[N-(N′, N′-dimethylaminoethane)-carbamoyl]-
cholesterol (DC-cholesterol), although derivatives of these lipids are also being
studied in order to improve their efficacy and safety [110]. Thanks to their cationic
nature, these lipids are able to condense and protect the genetic material, as well
as to bind to the negative charges of the cell membranes. The main limitations of
non-viral vectors based on cationic lipids are the low efficacy in vivo due to the
fact that they are not stable and that they undergo rapid clearance, as well as the
possibility of generating inflammatory or anti-inflammatory responses.
Cationic lipids can be used by themselves to form complexes, known as
lipoplexes, by mixing them directly with the negatively charged genetic material,
but they are normally used to prepare colloidal systems that are then bound to the
genetic material to obtain the lipoplexes. The preparation of these colloidal
systems can involve other lipid components, which may improve the transfection
efficiency of cationic lipids, such as the phospholipid 1,2-dioleoyl-sn-glycero-3-
phosphoethanolamine (DOPE), which has fusogenic function and facilitates
endosomal escape, or polyethylene glycol (PEG), which forms a steric coating that
makes vectors more stable in vivo. The colloidal lipid systems used in gene
therapy are liposomes, nanoemulsions, and solid lipid nanoparticles (SLNs) [92].
Nanoemulsions consist of a dispersion of an oil phase stabilized in an aqueous
phase by means of a third component that acts as a surfactant, so that droplets of
about 200 nm are formed. From the technological point of view, nanoemulsions
are simple to manufacture, and they are very stable during storage. In spite of this,
the application of cationic nanoemulsions in gene therapy is still quite limited
[111].
SLNs are spherical particles in the range of nanometers, formed by a core
composed of a solid lipid at room temperature surrounded by a layer of
surfactants. In the case of SLNs designed to be applied in gene therapy, cationic
lipids exert part of the surfactant effect and, in turn, confer positive charge to the
surface of the particles. SLNs have shown efficacy as systems for administering
different types of genetic material at preclinical level in vitro and in vivo, after
their systemic or local administration, showing promising results especially in
ocular pathologies [112–115], as well as in infectious diseases [37], lysosomal
storage disorders [116], and various types of cancer [92].
Liposomes are spherical vesicles composed of one or more lipid bilayers
surrounding an aqueous core, which show a size ranging from 20 nm to a few
microns. Cationic liposomes are effective transfection systems in very varied
types of cells in vitro and also in vivo after their local or systemic administration.
In fact, in most clinical trials using lipid vectors, these are cationic liposomes.
Peptidic Particles
Some peptides are capable of condensing nucleic acids by themselves resulting in
the formation of nanoparticulate systems. These include cationic peptides
composed of short sequences of positively charged amino acids such as histidine,
arginine, or lysine; in fact, poly-L-lysine is one of the peptide vectors with the
highest transfection efficiency [117]. Proteins of natural origin, such as collagen or
albumin [118], are also used as peptide vectors. In addition, it is very common to
use peptides as ligands to functionalize some of the previously described
polyplexes and lipoplexes. In this sense, peptides may be used to target non-viral
vectors to a specific tissue (i.e., transferrin to target tumor tissue or hepatocytes
[119] or RGD (arginine-glycine-aspartic acid) sequences to target specific tissues
[120]). Another application of peptide as ligands is to improve their effectiveness
by helping the genetic material to overcome some of the barriers of the
transfection process: cell penetrating peptides [121] to improve cell entry,
fusogenic peptides [122] to increase endosomal escape, or NLS [94] to entry into
the nucleus.
Manufacturing and Quality Control of Non-viral Vectors
The production of nanoparticles for clinical gene therapy presents important
hurdles, which are still hampering the translation from laboratory to patients. Non-
viral vectors are complex formulations that must be customized depending on the
nucleic acid to be delivered, the variety of target diseases, and the administration
route [107]. Due to their complexity, nanoparticulate systems show unique
Chemistry, Manufacturing and Controls (CMC) challenges [123]. In fact, suitable
methods for large-scale production of simple nanosystems, such as liposomes,
have been developed [124]. However, when formulation becomes more complex,
for example, with the addition of surface modification or ligands, the number of
steps in the production process and the cost of the final product increase, and
quality control is also more difficult [125].
Regarding quality attributes, parameters that must be especially considered
because of their impact on biological yield are size, shape, surface charge,
presence of ligands to provide effective targeting, surface modification with PEG,
impurities associated to starting materials and the production process, and stability
during manufacturing and long-term storage and upon administration [126].
Batch-to-batch variability of non-viral vectors can potentially led to changes in all
these parameters. Therefore, small changes in manufacturing process variables
(such as temperature, pH, time, agitation speed, quality of starting materials, etc.)
can significantly affect the quality, efficacy, and safety of the final vector [127]. It
is important to stablish procedures to assess nanotherapeutics not only at final
steps but also at intermediate ones. Moreover, the application of concepts of
quality by design (QbD) based on quality guidelines introduced by the
International Conference on Harmonisation (ICH Q8, Q9, and Q10 [128]) has
been proposed to address questions related to manufacturing processes and CMC
complexities [123, 127]. The aim of QbD fundamentally aims at building quality
and safety from the first design steps of the product [129]. This methodology
intends for establishing a multidimensional design that defines process input
requirements and operational ranges necessary to ensure that the product meets
critical quality attributes. Designers of new nanotherapeutics will gain an
understanding of these concepts and the role their preliminary data plays in
preparing and positioning a potential nanoparticulate system for a gene therapy
product development.

5 Applications of Gene Therapy


The first major clinical advance in the state of the field of gene therapy was in
1990, when the adenosine deaminase (ADA) gene was administered to a 4-year-
old girl to treat the severe combined immunodeficiency (SCID) she suffered
[130]. This clinical trial fostered the launching of additional studies, one of them
in the year 2000 for patients with the X-linked form of SCID [131], which was an
important landmark for gene therapy. On the one hand, it provided for the first
time a demonstration of therapeutic effect of gene transfer for the treatment of a
genetic disease. On the other hand, two of the patients developed T-cell leukemia
as a result of insertional oncogenesis related to the retroviral vector used [57],
which dampened the perspectives on gene therapy. Nonetheless, other potential
hazards derived from the use of viral vectors to administer the genetic material
were already known. In 1999, a systemic inflammatory response to the dosage of
adenoviral vector administered into the right hepatic artery for the treatment of
ornithine transcarbamylase (OTC) deficiency caused the death of Jesse Gelsinger,
an 18-year-old male with partial OTC deficiency who participated in a pilot
(safety) study of gene therapy [74].
Gene therapy has survived its previous failures, and it has emerged, thanks to
the improvements of viral and non-viral vectors, the management of immune
reactions, and the use of new mechanisms of action. Actually, a large number of
clinical trials, almost 2,700 undertaken in 38 different countries, have been
approved globally since 1989, and as can be seen in Fig. 12, most of them
addressed cancer [54].

Fig. 12 Indications addressed by gene therapy clinical trials [54]

The extensive research activity in this field has not led to a significant number
of gene therapy-based approvals. Since 2003, when Gencidine®, indicated for the
treatment of head and neck squamous cell carcinoma, received approval in China
as the first gene therapy medicinal product (GTMP) marketed worldwide [132],
only 15 new products have been approved. However, GTMPs are becoming an
emerging and expanding class of innovative medicinal products that can offer a
more specific and causal/targeted treatment of many rare diseases, including rare
cancers [133]. Gene therapy may be initially approved for patients who are
lacking other therapeutic options, including conditions that in absence of treatment
can cause disability or early death and conditions that require intensive and
onerous maintenance therapy in form of enzyme or protein replacement. For these
patients, gene therapy could offer long-term stabilization or improvement of their
health, with the ultimate objective of obtaining a cure [134]. Table 4 shows
nucleic acid-based products, including antisense oligonucleotides (ASOs) and
gene-engineered cells, commercialized until present [135–137].
Table 4 Nucleic acid-based products approved

Product Year/agency Company Indication/administration route Strategy/vector


(first
approval)

Vitravene® 1998/FDA Isis CMV retinitis in AIDS In vivo – ASO


(fomivirsen) Pharmaceuticals patients/intravitreal injection

Gencidine® 2003/SDFA SiBiono Head and neck In vivo – gene


GeneTech carcinoma/intratumoral augmentation/AD5

Macugen® 2004/FDA EyeTech Wet form of AMD/intravitreal In vivo – aptamer


(pegaptanib) injection

Oncorine® 2005/SDFA Sunway Biotech Nasopharyngeal cancer/local In vivo – oncolytic


virus/AD5

Glybera® 2012/EMA UniQure Lipoprotein lipase In vivo – gene


(alipogene deficiency/intramuscular augmentation/AAV1
tiparvovec)

Kynamro® 2013/FDA Kastle Familial In vivo – ASO


(mipomersen) Therapeutics hypercholesterolemia/subcutaneous
injection

Imlygic® 2015/FDA BioVex Melanoma/local In vivo – oncolitic


(Talimogene virus/HSV-1
laherparepvec)

Spinraza® 2016/FDA Biogen Spinal muscular atrophy/intrathecal In vivo – ASO


(nusinersen)

Exondys 51® 2016/FDA Sarepta Duchenne muscular In vivo – ASO


(eteplirsen) Therapeutics dystrophy/intravenous infusion

Zalmoxis® 2016/EMA MolMed Haploidentical HSC Ex vivo – allogeneic


(nalotimagene transplant/intravenous infusion T cells genetically
carmaleucel) modified

Strimvelis™ 2016/EMA Orchard ADA-SCID/intravenous infusion Ex vivo –


Therapeutics autologous
CD34+ genetically
modified

Luxturna® 2017/FDA Spark Retinal dystrophy In vivo – gene


(voretigene Therapeutics (RPE65)/subretinal augmentation/AAV2
neparvovec)

Kymriah® 2017/FDA Novartis ALL and DLBCL/intravenous Ex vivo –


(tisagenlecleucel) infusion autologous
CAR T cell

Yescarta® 2018/FDA Kite Pharma DLBCL and PMBCL/intravenous Ex vivo –


(axicabtagene infusion autologous
ciloleucel) CAR T cell
Product Year/agency Company Indication/administration route Strategy/vector
(first
approval)

Tegsedi® 2018/EMA Akcea hATTR/subcutaneous injection In vivo – ASO


(inotersen) Therapeutics

Onpattro® 2018/FDA Alnylam hATTR/intravenous infusion In vivo –


(patisiran) Pharmaceuticals siRNA/lipid
nanoparticles

Zolgensma® 2019/FDA AveXis, Inc. Spinal muscular In vivo – gene


(onasemnogene atrophy/intravenous infusion augmentation/AAV9
abeparvovec-
xioi)

FDA Food and Drug Administration, CMV cytomegalovirus, AIDS acquired


immunodeficiency syndrome, ASO antisense oligonucleotides, SFDA China State
Food and Drug Administration, AD5 adenovirus serotype 5, AMD age-related
macular degeneration, EMA European Medicines Agency, AAV1 adeno-associated
virus serotype 1, HSV-1 herpes simplex virus type 1, HSCT hematopoietic stem
cell transplant, ADA-SCID severe combined immunodeficiency due to adenosine
deaminase deficiency, AAV2 adeno-associated virus serotype 1, ALL B-cell acute
lymphoblastic leukemia, DLBCL diffuse large B-cell lymphoma, PMBCL primary
mediastinal large B-cell lymphoma, CAR chimeric antigen receptor, hATTR
hereditary transthyretin amyloidosis, siRNA small interfering ribonucleic acid

Apart from the five gene therapy products approved (Gencidine®, Oncorine®,
Glybera®, Imlygic®, and Luxturna®), products based on ASOs, small interfering
RNAs (siRNA), or aptamers have been also authorized, which have yet to exert a
profound influence on the biopharma product landscape.
Kymriah®, Yescarta®, Zalmoxis®, and Strimvelis™ may be categorized as
both cell and gene therapies. In all these cases, genetic modification is undertaken
ex vivo using a viral vector to achieve transduction, followed by infusion of the
genetically modified cells into the patient. Kymriah®, Yescarta®, and Strimvelis™
use autologous cells, whereas Zalmoxis® uses allogeneic cells as a starting point.
Strimvelis™ is a hematopoietic stem cell therapy, and the other three are T-cell
therapies, being Kymriah® and Yescarta® the first chimeric antigen receptor
(CAR) T-cell-based products. All four products have orphan status or target niche
conditions and either are under additional monitoring or require further post-
authorization safety studies.
Out of the total of GTMPs approvals, Vitravene® and Glybera® were
withdrawn from market in 2002 and 2017, respectively. Moreover, the
authorizations for use in the EU of Kynamro® and Exondys 51® were refused in
2012 and 2018, respectively.
A major challenge that faces most of the GTMPs is high development and
production cost, which has led to pricing and reimbursement issues. A
representative example is Glybera®, an adeno-associated virus serotype 1-based
gene therapy for intramuscular administration in adult patients with familial
lipoprotein lipase deficiency, a rare autosomal recessive disorder. Glybera® was
commercialized in 2012 with a price of $1m per treatment, and it was pulled from
market in 2017 despite being therapeutically successful [138].
As challenging as the generally high price is the developmental timeline of
GTMPs that typically span two or even three decades from concept introduction to
commercialization [138]. For instance, Kymriah®, the first CAR T-cell therapy,
was approved by the FDA in 2017, after almost 30 years since the concept of
redirecting T cells’ potential to kill cancerous cells was introduced [139]. In the
same way, in the case of Strimvelis™, it took nearly 15 years since the onset of
the preclinical in vivo study [140] before its developer Fondazione Telethon
(Italy) received orphan designation status from the European Commission in 2005.
In this sense, there is already a similar precedent with the monoclonal antibodies;
it took more than three decades to get the commercialization of these products to
become the primary drivers of the pharmaceutical market.
Besides the economic factors, other aspects have contributed to the low level
of commercialization of GTMPs, such as the complexity of the technologies,
difficulties in manufacturing processes, and regulatory barriers [138]. GTMPs face
significant additional regulatory challenges when pursuing market approval due to
the risks and concerns gene therapies which must be accounted during the
regulatory process [141]. Moreover, the mismatch between the capacity of
manufacturing vectors and the requirement of these emerging therapies is an
important hurdle that is slowing down gene therapy progress [142].
Nevertheless, considering not only the intensive investigation performed but
also the recent advances in the gene-editing field and T-cell-based therapies,
GTMPs will undoubtedly have a substantial contribution on the biopharmaceutical
market over the years to come. Furthermore, with several product candidates now
undergoing regulatory review, it appears likely that clinicians will have increasing
opportunities to generate their own assessments of gene therapy as a treatment
modality [7].

5.1 Gene Therapy Medicinal Products for Cancer


Cancer diseases that have been targeted by gene therapy are primarily those that
do not respond well to conventional treatment such as metastatic melanoma or
glioblastoma. As mentioned above, the first gene therapy marketed was
Gencidine®, approved in 2003 for the treatment of head and neck squamous cell
carcinoma by the China State Food and Drug Administration (SFDA), although it
is not available in the United States or Europe. Gencidine is a type 5 recombinant
adenovirus, which has the E1 region replaced by a Rous sarcoma virus promoter
linked with the human wild-type p53 gene and a poly (A) tail [143]. The tumor
suppressor p53 and its target genes are essential regulators of cell cycle control
and induction of apoptosis. The p53 signaling cascade modulates cell cycle and
DNA repair to maintain the genetic integrity of cells. If irreparable DNA damages
occur, p53 activates cellular apoptotic pathways to eliminate genetically damaged
cells [144]. Gencidine®, administered by intratumoral injection, induces the
expression of the tumor suppressor protein p53 causing growth arrest and
apoptosis in tumor cells. However, the antitumor effects depend on the expression
level of transduced p53 and on the integrity in p53-mediated cascades in the target
tumors [145].
Another approach to address the treatment of cancer is the use of oncolytic
viruses (OV) that selectively replicate in tumor cells without harming normal
cells. Recombinant virus technology has allowed the development of conditionally
replicating viruses, being Oncorine® (H101) the first OV marketed, which
received approval in China in 2005 for treatment of nasopharyngeal cancer after
intratumoral administration [146]. Oncorine® is a type 5 adenovirus with E1B-
55KD and partial E3 deletion that cannot replicate in normal cells where p53 is
active; therefore, it can selectively infect and kill tumor cells via the targeting of
pro-apoptosis. The first OV to gain approval by the FDA and EMA as an
anticancer therapy was talimogene laherparepvec (Imlygic®), approved in 2015
for melanoma treatment. It is a modified type 1 herpes simplex virus (HSV-1)
engineered to express human granulocyte-macrophage colony-stimulating factor
(GM-CSF). The insertion of GM-CSF in place of both loci of the ICP34.5 gene, as
well as by the deletion of the ICP47 gene, increased the selective replication
within tumor cells, enhancing the tumor-specific immune response [147, 148].
The treatment is administered as a series of subcutaneous or intranodal injections
over at least 6 months, and it has an estimated average cost of US$65,000 [134].
Targeting a sufficient number of cells, even when the vector could be injected
into the tumors directly and repeatedly, represented a serious obstacle to achieving
full efficacy. Furthermore, considering that metastasis is the source of mortality
for most cancers, systemic gene therapy is of considerable interest, and nowadays
it is available with the ex vivo infusion of genetically modified hematopoietic T
cells. In this sense, genetically modified immune T cells represent a new class of
therapeutics that has shown encouraging success for the treatment of some types
of cancer. However, specialized manufacturing facilities and personal trained to
conduct customized procedures for such therapies are vital to ensure accessibility
and quality of care [134].
Zalmoxis® (nalotimagene carmaleucel) is an ex vivo GTMP approved by
EMA in 2016 as adjunctive treatment in haploidentical hematopoietic stem cell
transplantation (Haplo-HSCT) of adult patients with high-risk hematological
malignancies. Haplo-HSCT can be associated with prolonged immunodeficiency
posttransplantation, and Zalmoxis® aids immune reconstitution and reduces the
risk of graft-versus-host disease [149]. This GTMP is based on allogenic somatic
T cells genetically modified with a retroviral vector to express the herpes simplex
thymidine kinase (HSV-TK) suicide gene and a truncated form of the human low-
affinity nerve growth factor receptor (ΔLNGFR) genes (for identification of
transduced cells). The expression of the HSV-TK gene allows the selective killing
of T cells that have this suicide gene, upon administration of ganciclovir or
valganciclovir, preventing further development if the patient develops graft-
versus-host disease [150].
The most recent therapies approved by FDA and EMA against cancer are
Kymriah® (tisagenlecleucel) and Yescarta® (axicabtagene ciloleucel), the first
chimeric antigen receptor (CAR) T cell-based products, being both CD19-directed
genetically modified autologous CAR T-cell immunotherapies. CARs consist of
an antigen-binding domain, from either an immunoglobulin molecule or a T-cell
receptor, fused to an intracellular signaling domain, from receptors such as CD28,
OX40, and CD137, that mediates activation and costimulation to enhance T-cell
function and persistence [47]. CARs recognize antigens independently of the
major histocompatibility complex (MHC), which endows the CAR T cell with a
fundamental antitumor advantage, because a major mechanism of immunoevasion
by cancer is loss of MHC-associated antigen presentation by tumor cells. Another
advantage is that CARs target nonprotein surface molecules, like carbohydrates
and glycolipids. One limitation of current CAR T-cell strategies is that they
require extracellular surface targets on the tumor cells [151].
CD19 is at present the most common CAR target; CD19 displays frequent and
high-level expression in B-cell malignancies, it is required for normal B-cell
development in humans, and it is not expressed outside of the B-cell lineage,
which makes CD19 a nearly ideal target. For CAR T-cell therapy manufacture, T
cells are isolated from the blood of the patient, activated, and then genetically
engineered to express the CAR construct. T cells are modified by using a lentiviral
or a retroviral vector for Kymriah® and Yescarta®, respectively. After ex vivo
expansion of the CAR T cells, they are formulated into the final product for direct
infusion [152]. However, CAR T-cell administration has been associated with
serious systemic toxicities that often require intensive care and in some instances
have caused patient deaths. To date, the most prevalent adverse effects following
infusion of CAR T cells result from on-target T-cell activation, including cytokine
release syndrome, macrophage activation syndrome, and tumor lysis syndrome
[7].
Kymriah® and Yescarta® have a US list price of $475,000 and of $373,000,
respectively [134]. Both in the United States and in EU, one of the indications of
Kymriah® is the treatment of pediatric and young adult patients up to 25 years of
age with B-cell acute lymphoblastic leukemia (ALL) that is refractory, in relapse
posttransplant or in second or later relapse. The other indication is the treatment of
adult patients with relapsed or refractory diffuse large B-cell lymphoma
(DLBCL), after two or more lines of systemic therapy. Yescarta® is indicated for
the treatment of adult patients with relapsed or refractory diffuse large B-cell
lymphoma (DLBCL) and primary mediastinal large B-cell lymphoma (PMBCL),
after two or more lines of systemic therapy (EMA).

5.2 Gene Therapy Medicinal Products for Other Applications


The resurgence of gene therapy in recent years for the treatment of genetic
diseases is largely due to successes in trials that utilized both ex vivo strategies
(for X-linked SCID and adrenoleukodystrophy) and in vivo approaches (Leber
congenital amaurosis type 2 and hemophilia B) [7]. Gene therapies to treat rare
disorders caused by single-gene mutations have made the most progress toward
market availability. It has to be considered that in many of these diseases, there are
few treatment options apart from supportive and symptomatic care. Development
of gene therapies has also been influenced by ease of administration in target
tissues, i.e., diseases of the eye and hematopoietic system.
Among the organs targeted by gene therapy, the eye has been at the forefront
of translational gene therapy largely due to appropriate disease targets and its
suitable anatomic features [153]. In fact, fomivirsen (Vitravene®) indicated for the
treatment of cytomegalovirus retinitis (CMV) in patients with AIDS was the first
therapeutic ASO approved by FDA in 1998. It was administered intraocularly, and
its target was the mRNA that encoded the CMV immediate early (IE) 2 protein,
which is required for viral replication. EMA also approved this product in 1999;
however, Novartis stopped marketing the drug in 2002 in Europe and in 2006 in
the United States [154].
Likewise, pegaptanib (Macugen®), indicated for the treatment of age-related
macular degeneration, was the first therapeutic aptamer approved by FDA in
2004. Macugen® is an RNA aptamer for intravitreal administration that consists of
28 nucleotides that binds to 165 isoform of VEGF (vascular endothelial growth
factor). Its anti-angiogenic effect not only stops the excessive growth of blood
vessels but also prevents the formation of defective blood vessels [155].
Moreover, FDA and EMA approved Luxturna® in 2017 and 2018,
respectively, for the treatment of adult and pediatric patients with vision loss due
to inherited retinal dystrophy caused by confirmed biallelic RPE65 mutations and
who have sufficient viable retinal cells. Biallelic mutations in the RPE65 gene,
which encodes the all-trans-retinyl ester isomerase, in this gene, can be described
as Leber congenital amaurosis type 2, retinitis pigmentosa type 20, early-onset
retinal dystrophy, and other clinical labels for severe rod-mediated inherited
retinal dystrophies, which all eventually progress to complete blindness [156].
Luxturna® was designated an orphan medicine by EMA for two forms of the
disease, retinitis pigmentosa in 2015 and Leber’s congenital amaurosis in 2012.
Voretigene neparvovec (Luxturna®) consists of a recombinant adeno-associated
virus serotype 2 vector carrying a functional RPE65 gene. This gene augmentation
therapy is given by bilateral subretinal injection and has a list price of
US$425,000 (per eye treatment).
Ex vivo gene therapy approaches have mainly targeted hematopoietic system,
being Strimvelis™ the first ex vivo GTMP approved for the treatment of an
inherited disorder, adenosine deaminase deficiency-severe combined
immunodeficiency (ADA-SCID). This recessive immune disorder is caused by
mutations in the ADA gene and characterized by the absence of cellular and
humoral immune function and a fatal outcome very early in life. Strimvelis™,
with orphan designation and approved in 2016 by EMA under additional
monitoring, saw the first clinical application on a single patient in March 2017.
This hematopoietic stem cell therapy is indicated for the treatment of patients with
ADA-SCID, for whom no suitable human leukocyte antigen (HLA)-matched
related stem cell donor is available [134]. Patients receive CD34+ cells transduced
with retroviral vector that encodes for the human ADA cDNA sequence. The
genetically modified autologous CD34+ cells act by repopulating the
hematopoietic system with cells that express active levels of the ADA enzyme.
Strimvelis™, with a list price of €594,000, should be administered by intravenous
infusion in a specialized transplant center. These ex vivo therapies require
complex procedures and trained personnel to harvest, transduce, and reinfuse the
hematopoietic target cells, and in the case of Strimvelis™, it is currently available
only at a single center in Milan to where the patient and family are required to go
for treatment [7].
Apart from gene therapy medicines, different nucleic acid-based products have
also been approved for the treatment of diverse genetic diseases, including spinal
muscular atrophy (SMA), Duchenne muscular dystrophy (DMD), familial
hypercholesterolemia, and hereditary transthyretin amyloidosis (hATTR).
SMA is an autosomal recessive neurodegenerative rare disease that, in most
cases, involves homozygous deletion of the survival of motor neuron 1 (SMN1)
gene on chromosome 5q. As a consequence, patients suffer the deficiency of the
survival of motor neuron (SNM) protein which plays a critical role in motor
neuron development. SMN1 is one of two nearly identical genes that encode SMN;
the other is survival of motor neuron 2 (SMN2). Infants with a more severe form
of the disease (type 1 SMA) die before 2 years of age; later onset of the disease in
infants is referred to as SMA2. FDA has approved in 2019 Zolgensma®
(onasemnogene abeparvovec-xioi), a recombinant AAV9-based gene therapy
designed to deliver a copy of the gene encoding the human SMN protein.
Zolgensma® is administered as infusion, and it is indicated for the treatment of
pediatric patients less than 2 years of age with SMA with biallelic mutations in the
SMN1 gene. This product, with a price of $2.1 million, is the most expensive drug
in the world.
Nusinersen (Spinraza®) is an ASO approved in 2016 by FDA and in 2017 by
EMA (orphan medicine in 2012) for treating patients with SMA. Biogen has
priced Spinraza® at $750,000 for the first year’s treatment ($125,000 per
injection) and $350,000 per year subsequently [154].
SMN protein is mainly produced from SMN1, whereas SMN2 produces a small
amount of full-length SMN protein. Typically, a higher number of copies of SMN2
are associated with a less severe phenotype of the pathology. However, since the
amount of protein formed is low, even multiple copies of SMN2 do not fully stop
the disease. The intron 7 in SMN2 contains an intronic splicing silencer (termed
ISS-N1) with binding sites for negative splicing factors (NSFs). Binding of these
NSFs to intron 7 pre-mRNA precludes the recognition of exon 7 during the
splicing process. The ASO nusinersen blocks the ISS-N1 site preventing the
binding of the NSFs. As a result, Spinraza® administered via intrathecal injections
modulates the splicing of the SMN2 mRNA transcript to include exon 7, thereby
increasing the production of full-length SMN functional protein [157].
DMD is a rare X-linked disease characterized by loss-of-function mutations in
the DMD gene coding for dystrophin, which disrupt the reading frame of the
dystrophin mRNA and cause the introduction of premature stop codons, leading to
mRNA degradation and the loss of protein synthesis in striated muscle. It is a fatal
disorder characterized by progressive muscle weakening and wasting, with boys
losing ambulation by 12 years of age or earlier; death often occurs in the 20s,
usually due to respiratory or cardiac complications [154, 158]. Eteplirsen
(Exondys 51®) is a 30-nucleotide phosphorodiamidate morpholino oligomer and
was approved as an ASO drug in 2016 by FDA, although authorization for use in
the EU was refused by EMA in 2018. Eteplirsen promotes dystrophin production
by restoring the translational reading frame of DMD through specific skipping of
exon 51 in defective gene variants. The therapeutic strategy of antisense-mediated
“exon skipping” is developed to force exon exclusion from mature mRNA of
DMD with the purpose of restoring reading frame. Eteplirsen is suitable for 14%
of DMD patients with DMD mutations, it is administered by intravenous injection,
and it has a price of $300,000/patient/year [159].
Familial hypercholesterolemia is an autosomal dominant genetic condition
resulting from mutations of the low-density lipoprotein cholesterol (LDL-C)
receptor, apolipoprotein B (ApoB), or pro-protein convertase subtilisin/kexin 9
(PCSK9) [160]. Mipomersen (Kynamro®) is an orphan medicine approved in
2013 by FDA but with refused authorization for use in the EU by EMA in 2012.
Mipomersen is a single-stranded synthetic DNA ASO targeting ApoB-100,
resulting in suppression of the hepatic production of the ApoB, total cholesterol,
LDL-C, and non-high-density lipoprotein (HDL) cholesterol lipoproteins in rare
genetic disorder patients with homozygous familial hypercholesterolemia.
Kynamro® is available as a solution for injection under the skin. Due to the
serious risk of liver toxicity, mipomersen is labeled a black box warning
hepatotoxicity by FDA [159].
hATTR is a rare, autosomal dominantly inherited, progressively debilitating,
and life-threatening disease. Misfolded transthyretin (TTR) proteins accumulate as
amyloid deposits at multiple sites culminating in intractable peripheral
sensorimotor neuropathy and, in many cases, autonomic neuropathy and/or
cardiomyopathy. Recently, two different nucleic acid-based products have been
approved for the treatment of hATTR, by using two different strategies [35].
Inotersen (Tegsedi®) is an ASO designed to suppress the expression of both wild-
type and mutant forms of TTR. It has recently gained marketing authorization
approval by EMA in 2018 (orphan designation in 2014) under additional
monitoring for the treatment of stage 1 or stage 2 polyneuropathy in adult patients
with hATTR and regulatory approval from the FDA for the treatment of the
polyneuropathy of hATTR. It is available as a solution for injection under the skin
in pre-filled syringes, and the recommended dose is one injection once a week
[161]. Onpattro® (Patisiran) is a siRNA designed to target TTR to reduce the
levels of both wild-type and mutant TTR. Patisiran is formulated in lipid
nanoparticles that direct the siRNA to the liver, the primary site of TTR
production. Patisiran, with the same indications that of inotersen, is available as a
solution for infusion and received regulatory approval in 2018 from the FDA and
EMA (orphan designation in 2011) [35].

6 Challenges of Gene Therapy


As has been commented in this chapter, gene therapy has still many challenges to
be overcome: the science is complex, treatment is technically difficult, and the
regulatory approval process is necessarily different to that for conventional
therapies. Actually, it has been considered as the most complex “drugs” ever
developed [47]. Efficacy, safety, and manufacturing issues are important
challenges that must be faced. There are also difficult questions about cost,
accessibility, and social justice that will need answers once the methods are shown
to be effective and safe.

6.1 Efficacy Issues


The low efficacy, which may lead to treatment failure, is one of the most
important challenges of gene therapy [162]. The development of new delivery
systems with higher transduction rates and higher affinity to a specific cell or
tissue is necessary to increase the number of products that reach clinical
evaluation. Another reason that may explain the low efficacy of a gene therapy
product is the presence of endogenous natural antibodies against viral vectors or
the transgene product. This is of particular importance because it prevents
transduction and limits gene therapy product administration more than once.

6.2 Safety Issues


Potential immunogenicity and oncogenicity are the main challenges concerning
safety. When a gene vector integrates into the host genome, there is potential for
gene disruption and insertional mutagenesis giving with an increase in risk of
tumor arising from the genetically modified cells. This occurs as a consequence of
activation/upregulation of oncogenes or inactivation or downregulation of
tumor/suppressing genes [162]. To overcome the potential oncogenicity of viral
vectors, it is crucial to design new vectors that prevent the activation of oncogenic
genes at integrations sites. The use of non-integrating vectors or highly targeted
genomic integration at the desired chromosomal loci is also helpful.
Immunogenicity reactions may be due to both the viral vector and the
transgene product due to the unpredictability of innate and antigen-dependent
immune responses in humans [162]. One additional problem is that these
responses are difficult to detect in animal models, as such effects arising in
animals, who may have been given human product, are not indicative of whether
such would occur in humans. Some alternatives to prevent immunogenicity
include the administration of immunosuppressive agents prior or after the
administration of the gene therapy product, the modification of the capsid proteins
of the vector, and the elimination of viral genes.

6.3 Drug Development and Manufacture Issues


Because of its unique set of characteristics, the nonclinical development package
of a gene therapy medicinal product is rather more complex than conventional
medicinal products. One important difference with respect to a conventional drug
product is that the approval of a gene therapy product must be based not only on
data of the therapeutic transgene but also of data on the vector/delivery system.
Manufacturing of gene therapy products is an additional complexity factor. In
the near future, there is a challenge for the development of manufacturing
capacities, which must be sufficient to meet the coming demand, specifically the
AAV production. In this sense, several companies of viral vector production are
amplifying their manufacturing facilities to face the future increase demand for
viral production [142].

6.4 Ethical Issues


Currently, at least in the Western countries, clinical use of gene therapy is limited
to somatic cells for the treatment of a specific disease. As it has been explained
above, germline gene therapy leads to hereditary modifications that pass on to
subsequent generations, and therefore, it is the object of a heated discussion [163].
The recent announcement by a Chinese research of the birth of twins whose
genomes were edited by CRISPR/Cas9 during in vitro fertilization has engendered
broad condemnation for the premature clinical deployment of a still experimental
biomedical area [164]. The organizing committee of “the Second International
Summit on Human Genome Editing,” held in Hong Kong in November 2018
under the auspices of the US National Academies of Science and Medicine, the
UK Royal Society, and the Hong Kong Academy of Sciences, reiterated that “the
scientific understanding and technical requirements for clinical practice remain
too uncertain and the risks too great to permit clinical trials of germline editing at
this time” [163].
The potential use of gene therapy for purposes other than diseases treatment is
another important topic to be addressed. For instance, the application to eugenics,
that is, the attempt to change or improve complex human traits related to a broader
number of genes; such as, personality, intelligence, or character [162].

6.5 Affordability
Gene therapy-based medicines have a high cost of development, production,
product storage, and transportation, which lead to very high prices. For instance,
the price of Glybera was around 1,000,000 €; Strimvelis, 600,000 €; and Yescarta
and Kymriah 300,000 and 400,000 €, respectively. Gene therapy medicinal
products are expensive to develop and to manufacture, and sometimes, they are
one-time treatments. These reasons, among others, may justify the high cost [164].
Affordability of novel innovative and high budget impact therapies has become an
important topic in Europe, and so far, each country has come up with individual
approaches to improve affordability [165]. For example, in the case of
Zolgensma®, with an annualized cost of $425,000 per year for 5 years, the
company proposes to payers to create 5-year outcomes-based agreements and
novel pay-over-time options [166].

6.6 Intellectual Property Complexity


The complexity of the intellectual property “territories” that can surround a given
gene therapy development also explains the slow progress of gene therapy [142].
In fact, a new gene therapy product under development may be conditioned by
patents involving not only the therapeutic transgene itself but also its mechanism
of action, the non-viral or viral vector used as the delivery system, and the method
for delivery of the nucleic acid therapy to the patient (e.g., the delivery devices,
surgical techniques, and treatment protocols to be used).
Despite the numerous challenges, in the last years, important unified efforts by
research and clinical scientists in academic, translational, and industry settings
have resulted in tangible outcomes, with several marketing authorizations and
approved commercial products. Initiatives for willingness to participate in clinical
trials and equable access of patient population to somatic gene editing as a
treatment option in clinical care are necessary to increase the opportunities to
successful of gene therapy.

7 Conclusion
Gene therapy, regarded as one of the most promising and most active fields of
medicine, is beginning to show encouraging results. Current therapies are
primarily experimental with only a few gene therapy medicinal products on the
market, although several product candidates are undergoing regulatory review.
The efforts and advances made in this area have led to the development of new
therapeutic strategies to treat several disorders, many of them without currently
available treatments. The remarkable basic, translational, and clinical research
activity in gene therapy has been addressed mainly to cancer, but a significant
number of clinical trials have also targeted a broad variety of diseases including
monogenic, infectious, and cardiovascular diseases.
Nucleic acid-marketed products are based mainly on in vivo strategies.
Initially gene augmentation was the main option, although ex vivo therapies and
new ASOs seem to be major strategies at present. Moreover, the first product
containing a siRNA has been already marketed. Recent strategies also include T-
cell-based therapies, with two products marketed in 2018 for the treatment of
hematological malignancies by immunotherapy, and the gene-editing tool
CRISPR, whose rapid progress is boosting the development of new gene therapy-
based medicines.
Despite the positive forecast that the nucleic acid-based products have on the
biopharmaceutical market, different hurdles are slowing down their progress. The
success of gene therapy may be compromised by two main challenges, the cost
and reimbursement of the treatments, as well as the technological issues to ensure
accessibility and quality of the treatments. The availability of specialized
manufacturing personnel and facilities to conduct customized procedures and the
progress in the manufacturing capacity of efficient and safe vectors to meet the
upcoming demand of gene therapies are essential for the advance of this emerging
therapy in the future.

Acknowledgments
This work was supported by the University of the Basque Country UPV/EHU
(GIU17/32) and by the Spanish Ministry of Economy and Competitiveness
(SAF2014-53092-R) and FEDER funds. I Gómez-Aguado thanks UPV/EHU for
her research grant.

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The Impact of Pharmacogenomics in


Personalized Medicine
Dev Bukhsh Singh1
(1) Department of Biotechnology, Institute of Biosciences and
Biotechnology, Chhatrapati Shahu Ji Maharaj University, Kanpur,
Uttar Pradesh, India

Dev Bukhsh Singh

Abstract
Recent advances in Pharmacogenomics have made it possible to understand
the reasons behind the different response of a drug. Discovery of genetic
variants and its association with the varying response of drug provide the
basis for recommending a drug and its dose to an individual patient. Genetic
makeup-based prescription, design, and implementation of therapy not only
improve the outcome of treatments but also reduce the risk of toxicity and
other adverse effects. A better understanding of individual variations and
their effect on drug response, metabolism excretion, and toxicity will
replace the trial-and-error approach of treatment. Evidence of the clinical
utility of pharmacogenetics testing is only available for a few medications,
and FDA labels only require pharmacogenetics testing for a small number
of drugs. Although there is a great promise, there are not many examples
where Pharmacogenomics impacts clinical utility. Some genetic variants
related to different diseases have been reported, and many have not been
studied yet. The information related to the outcome of treatment with a
particular drug and a genetic variant can be used to release a warning/label
for the use of that drug. There are many limitations in the way of
implementing the goal of personalized medicine. Future advances in the
field of genomics, diagnosis approaches, data analysis, clinical decision-
making, and sustainable business model for personalization of therapy can
speed up the individualization of therapy based on genetic makeup.
Graphical Abstract

Keywords Clinical decision – Genetic makeup – Personalized medicine –


Pharmacogenomics – Therapeutic response

1 Pharmacogenomics
Pharmacogenomics deals with the study of the genetic basis for varying
response of drugs among individuals. It is an emerging and challenging
field of therapy with a limited clinical utility and applicability. There are
several factors such as environmental factors, age, weight, gender, and
metabolism which affect the efficacy of a drug. The genetic makeup of the
patient also decides the response drug of a drug [1]. There are many genetic
variants associated with a disease. All genetic variants are not associated
with the drug responses on treatment. Some genetic variants may be
associated with the risk of developing a disease. The expression level of
genes in different patients may be different. Patient with the higher
expression level of a protein will produce a different response as compared
to a patient with lower or no expression level of the same protein [2]. Many
Pharmacogenomics tests have been developed for the treatment of some
disease, but the clinical applicability of these tests is very limited.
Ethical issues related to the study and application of Pharmacogenomics
should be addressed. There is a need to protect the confidentiality of
patients, and all patients should have equal opportunity to get benefitted by
personalized therapy. Pharmacogenomics can bring a significant
improvement in the issues related to the safety and efficacy of the drug.
Recent omics advances have made it possible to understand the significance
of genetic variations on individual patient’s response to a drug. Omics
technologies have enabled us to answer the many medical questions and
also promoted the personalization of therapies based on genetic makeup
(Fig. 1). The long-term goal of Pharmacogenomics is to help medical
practitioners in the diagnosis and prescription of a drug and its dosage,
based on the patient’s genetic makeup. The major objective of
Pharmacogenomics is to study and catalog all the genetic and epigenetic
variants that cause variation in drug response. Pharmacogenomics-related
data, testing, and drug label are available for only some drugs.
Pharmacogenomics-related study has been conducted for drugs used for the
treatment of cancer, diabetes, depression, immunotherapy, anticonvulsant,
anti-infective, cardiovascular, and psychotropic drugs, as well as for some
other therapies.
Fig. 1 Medical questions and use of omics and other approaches for better therapeutic decisions and
individualization of therapy

Several deaths occur due to the adverse effect of the drug.


Pharmacogenomics tests have reduced the cases of adverse drug reactions
and also ignored the trial-and-error approach of therapy. Thus,
Pharmacogenomics approaches may lower down the cost of therapy for
patients and physicians significantly. Currently, most of drug and dose
prescriptions are based on age, weight, sex, lifestyle, and level of liver and
kidney enzymes. Scientists can identify the genetic variation in a small
subset of population which is associated with varying response to a drug
[3]. The Food and Drug Administration (FDA) includes Pharmacogenomics
information such as dosage guidelines, side effects, and efficacy of 150
drugs for patients with certain genetic makeup.

2 Factors Affecting Response of Drug


2.1 Genetic Polymorphism
Eighty-five percent of human diversity at Short Tandem Repeat (STR) and
Restriction Fragment Length Polymorphism (RFLP) autosomal loci is the
reason behind differences between individuals of the same population,
whereas differences between individuals of the same continent account for
5–10% [4]. Variation in drug response is not the only result of a mutation in
a single gene but also happens by the altered function of related genes.
Variations in genes associated with pharmacokinetics and
pharmacodynamics of drug may result in toxic, altered, or no response.
These ADME-related variations decide the effective concentration of drug
that reaches to drug target and its metabolism [5].
The information of genetic variants associated with cancer or other
disease is very useful in early diagnosis of disease and also provides the
basis for personalized therapy. Next-generation sequencing (NGS) and
genome-wide association studies (GWAS) have made it possible to
understand the genetic mechanisms involved in a disease and also promote
the individualization of therapy based on genetic makeup. The GWAS have
discovered many genetic variants related to different lethal diseases. These
variants can be used for developing a biomarker for diagnosis and
therapeutic categorization of a particular disease. The systematic
cataloguing of genetic variants and related outcome on therapy provide the
information of pathways and related enzymes and also explain the reason
behind the varied response of treatment by different patients [6]. Genetic
variants can provide better and accurate clinical decisions along with the
family history of the disease. Rare genetic variants cannot be easily
identified by GWAS, and they may have a significant effect on the risk of
disease [7]. The 1000 genome projects have identified many genetic
variants at lower frequencies [8]. The Human Genome Project has covered
the sequencing of the entire human genome, including the 99.9% of the
genome where all humans are identical in genetic makeup/composition [9].
The HapMap project has characterized the patterns of a DNA sequence
within the 0.1% genome where a genetic variation exists among
individuals. The HapMap has facilitated the development of some
diagnostic tools and also guides the selection of drug target for the
treatment of some disease. Information’s on polymorphism and related risk
of occurrence of a disease can be used as an alert message for a patient.

2.2 Epigenetic and Other Factors


Epigenetic factors such as age, sex, liver and kidney function, lifestyle,
previous disease, and adverse reaction are also decides the therapeutic
response of a drug [10]. Old age patients have a high risk of adverse
reaction due to the poor rate of physiology and metabolism. Gender-specific
physiological differences such as pregnancy and breastfeeding also affect
the outcome of treatment by a drug [11]. A significant difference between
the level of different hormones between a male and female has been
observed, which may cause a different response to a drug in male and
female. Environmental chemicals, drugs, and natural compounds can alter
the efficacy of the drug by drug-drug interaction or drug-herb interactions
[12]. These factors can induce or inhibit drug-metabolizing enzymes and
drug transporters.
The use of Ginkgo biloba (ginkgo) along with warfarin or aspirin results
in bleeding and raises blood pressure, when used in combination with a
thiazide diuretic [13]. The use of Ginkgo biloba (ginkgo) along with
trazodone may cause coma in patients. Herb labels should also be available
to avoid the use of a drug in combination with the herb. A drug-drug
interaction (DDIs) involves pharmacokinetic or pharmacodynamic
mechanisms which may affect the bioavailability, efficacy, and response of
a drug. The pharmacokinetics and pharmacodynamics of many co-
administered drugs are known, but the roles of co-administered herbs are
not well explored due to a complex mixture of herbal extracts [14]. The
pharmacokinetics and pharmacodynamics of drug-drug and herb-drug
interactions cannot be ignored while prescribing a drug for therapy.

3 Pharmacogenomics Biomarker
Biomarkers include genetic or somatic gene variants, changes in expression
level, functional irregularities, and chromosomal abnormality. Biomarkers
may be used for diagnosis of diseases or assessment of therapeutic efficacy.
Pharmacogenomics biomarker is used for prescribing the drug or its dose
based on the level of biomarker and presence or absence of its variants.
Some biomarker-related treatments of different diseases are available and
can describe clinical response variability, risk of adverse drug reaction, a
genotype-specific dose of a drug, mechanism of the drug, and polymorphic
drug target. FDA-approved drugs for some disease and their biomarkers are
listed in Table 1. It represents the drugs that can only be recommended to
patients who carry a particular variant related to the biomarker. This can be
very useful for prescribing safe and effective therapy based on the
biomarker. Discovery of novel and potential biomarkers related to a disease
can play a very important role in diagnosis, prescription, and
personalization of therapy.
Table 1 List of Pharmacogenomics biomarkers used for the therapy of diseases [15]

Drug Therapeutic Biomarker Referenced Outcome/efficacy


area subgroup
Abacavir Infectious HLA-B HLA-B∗5701 High risk of immune-
diseases (HIV) allele carriers mediated hypersensitivity
reaction and should not
receive abacavir [16]
Afatinib Oncology EGFR EGFR exon 19 These mutants incur
(tyrosine deletion or exon 21 sensitivity to afatinib
kinase substitution treatment [17]
inhibitor) (L858R) positive
Aripiprazole Psychiatry CYP2D6 CYP2D6 poor Half of the usual dose should
metabolizers be administered [18]
Carvedilol Cardiology CYP2D6 CYP2D6 poor High plasma concentrations
metabolizers of carvedilol, dose
monitoring required [18]
Clobazam Neurology CYP2C19 CYP2C19 poor Avoid clobazam or start with
metabolizers a low dose (2.5 mg/day) [19]
Celecoxib Rheumatology CYP2C9 CYP2C9 poor Half/lower dose
metabolizers recommended [20]
Cisplatin Oncology TPMT TPMT intermediate Consider alternate drug or use
or poor lower dose to avoid
metabolizers ototoxicity [21]
Diazepam Psychiatry CYP2C19 CYP2C19 poor Consider lower dose to avoid
metabolizers prolonged sedation and
unconsciousness [22]
Omeprazole Gastroenterology CYP2C19 CYP2C19 poor Lower the dose to avoid
metabolizers drug-drug interaction [23]

4 Pharmacogenomics Guidelines
Genotype-based dosing guideline has been published by clinical
pharmacogenetics implementation consortium (CPIC), Dutch
Pharmacogenetics Working Group (DPWG), or other organizations [24].
CPIC provides the supports for implementation of pharmacogenetics tests
into clinical practice [25]. The DPWG is a multidisciplinary organization
and includes the knowledge of experts such as pharmacists,
pharmacologists, chemists, epidemiologists, and toxicologists [26]. The
DPWG is also working for therapeutic (dose) recommendations based on
pharmacogenetics data and guides the health practitioners and doctors in
drug and dose prescription [27]. PGxOne™ provides the clinical
Pharmacogenomics test and generates a relevant medical and clinical report
which can guide the treatment of patients. PGxOne™ provides the dose-
related guidelines for some drugs. [28] stated that “Justice demands that
benefits of personalized medicine must be available to individuals of all
racial and socioeconomic status” [28]. Peterson-Iyer [29] has also suggested
some recommendation for consideration in the policy of
Pharmacogenomics [29].

5 Personalized Medicine
Personalized medicine is a therapeutic approach which utilizes the genetic
and epigenetic information of an individual. The current trend of clinical
therapy is to provide the medication to a particular patient based on its
characteristics. Such personalization is achieved by the use of omics
approaches which enable us to find the inter-individual variability at
genomic, transcriptomic, proteomic, and metabolomic levels. The food and
drug administration has approved some genomic companies for the
screening of genetic health risks related to Alzheimer’s disease and rare
blood disorder. The current clinical treatment paradigm is the right drug, for
the right patient, at the right time. Personalization of therapy requires data
collection through diagnostic from patients, individualization of therapy
based on analysis, and an appropriate and sustainable business model.
Digital and mobile medical applications have played an important role in
the characteristics of disease at the individual level. Many definitions of
personalized medicine are available. Some definitions refer to medicine at
an individual patient and while some consider its scope to a subpopulation.
But, most examples of personalized medicine are not personalized. For
example, a subgroup of women with early breast cancer and HER2 positive
is suitable for treatment with trastuzumab. Personalized medicine is a subset
of personalized health care. Personalized health care not only includes
genomics but also considers the other information’s which predicts the risk
of disease and patients response to treatment.
Genetic variants associated with pharmacokinetics and
pharmacodynamics of a drug decide the outcome or efficacy of treatment.
For the application of Pharmacogenomics in cancer, the selection of a drug
and target of drug both are important in deciding the response of treatment.
It is important to decide whether a drug is based on the genetics of
individuals or genetics of cancer cell or both. If a drug target to cancer cell,
then the outcome of treatment depends on the genetics of individual as well
also on the genetics of particular tumor cell.
A better understanding of heterogeneity that exists among individuals
and diseases can help in implementing the goal of personalized medicine.
The outcomes of the Human Genome Project have generated a lot of
interest in the personalization of therapy by identifying the individual-level
differences of diseases based on genetic makeup. Therapeutic decisions
based on an individual’s genetic profile and medical tests can be more
accurate and efficacious in terms of response. The risk of disease and
possible outcome of treatment can be estimated using information such as
age, sex, genetic profile, expressions related to disease, medical tests, and
history of the disease [30]. Disease screening tests and low-cost diagnostic
tools can help in detection of fatal disease at an early stage, and these
results can be utilized in making therapeutic decisions to improve the health
status of a patient. Genomic information greatly affects the dose and
response of treatment. Dose efficacy and safety monitoring tests play a
significant role in improving the status and cost-effectiveness of patient
care.
As the result of omics effort, more than 10 million SNPs have been
identified, and extensive studies on SNPs and related diseases have also
been performed to find its role in clinical applications for
Pharmacogenomics and personalized medicine [31]. There is a need to
validate the biomarkers for patient stratification and dose selection. Clinical
relevance, molecular mechanism, clinical evidence, and regulatory and
clinical guidelines related to relevant SNPs can trigger the application of
personalized medicine. Genomic informations are highly valuable in
making a medical decision related to drug and dose. But, the overall
therapeutic response is not only based on genomics test alone. Moreover, a
better therapeutic response can be achieved by combining genomic test
results with knowledge of age, sex, lifestyle, size, stage, nature, and origin
of the disease. The nongenetic factors, such as environmental and clinical
covariates, may provide important phenotypic information which can be
used for better therapeutic decision [32]. In addition to CYP2C9 and
VKORC1, the dose of warfarin therapy also depends on age, sex, body
mass index, diet, and concomitant drug therapy.
5.1 Clinical Guidance for Personalization of Therapy
In previous decades, all patients with the disease were receiving the
treatment with the same drug. But, now therapies have become more
personalized. For example, breast cancer patients that produce estrogen
receptor may be treated with the drug that targets the estrogen receptor. A
significant fraction of patients (subtype) possessing a particular
characteristic of the disease can be identified for treatment with a drug. This
can be one of the ways to achieve the goal of personalized medicine.
Different biomarkers related to a disease can be detected and quantified.
Biomarkers can be used to divide the patients into different subtypes based
on the susceptibility to disease and the outcome of treatment. Recent
advances in technology have made it possible to sequence an entire genome
of an individual and quantify all the proteins, metabolites, and microbiome
in a tissue. Advance data analysis tools and algorithms can be used to mine
the clinically significant biomarkers related to a disease. Regulatory
guidelines related to the use of biomarkers as a diagnostic tool are needed,
and it will help in the discovery and use of biomarkers in medical practice.
Biomarker-based tests and related clinical guidance for the personalized
treatment of some diseases are shown in Table 2. Advanced diagnostic
approaches are allowing doctors to prescribe the drug and dose to patients
based on their molecular profile [44]. There are several advanced diagnostic
tests for different types of cancer which identifies the expression or
mutation in genes related to the disease. These diagnostic tests are a key
parameter for prescribing a drug to a patient. Personalized guidance
regarding prevention of a disease can be given to a healthy person by
analyzing his genome, proteome, metabolome, and microbiome.
Table 2 Some examples of personalized medicine and related clinical guidance

Therapy Test and its description Clinical guidance Reference


Breast cancer BRCA1: deleterious BRCA1 or Surveillance and Redekop and
BRCA2 mutants have a high risk of chemoprevention Mladsi [30]
breast and ovarian cancer therapy
Breast cancer HER2: tumors with HER2 Use of Trastuzumab Redekop and
overexpression recommended Mladsi [30]
Therapy Test and its description Clinical guidance Reference
Epilepsy HLA-B∗1502: HLA-B∗1502- Use of Redekop and
positive patients show skin reactions carbamazepine may Mladsi [30]
on treatment with carbamazepine be dangerous,
choose an alternative
drug
Atrial fibrillation CYP2C9, VKORC1: dose of warfarin Dose of Warfarin Redekop and
therapy partly dependent on CYP2C9 Mladsi [30]
and VKORC1 genotypes
Hepatitis C HCV RNA: measures the level of Required duration of Redekop and
viral RNA after treatment with treatment Mladsi [30]
interferon alfa and ribavirin
Antiplatelet CYP2C19: clopidogrel is a prodrug Use alternative Mousa et al.
medications that requires metabolic activation by antiplatelet agent [33]
CYP2C19 other than
clopidogrel for
patients with
decreased CYP2C19
Immunosuppressive Thiopurine methyltransferase Patients with Relling et al.
therapy (TPMT): azathioprine is a prodrug decreased TPMT [34]
converted to mercaptopurine that function have a
undergoes methylation to inactive higher risk for
metabolites by TPMT toxicity with
azathioprine
Immunosuppressive CYP3A5: tacrolimus undergoes Carriers of CYP3A5 Renders et al.
therapy oxidative metabolism to inactive alleles may require [35]
metabolites by CYP3A4/3A5 higher doses
enzymes
Kidney transplants CYP2C19: metabolism of Use an alternative Guinea et al.
and invasive fungal voriconazole occurs predominantly agent in case of [36]
infection through CYP2C19 CYP2C19 rapid or
poor metabolizer
Hyperlipidemia SLCO1B1 Use an alternative Armitage et al.
SLCO1B1 is responsible for uptake agent or lower dose [37]
of simvastatin from the blood to in case of decreased
hepatocytes (metabolized). SLCO1B1 activity
Simvastatin blood concentrations
increases at reduced SLCO1B1 level
Chronic myeloid BCR and ABL gene are present on Imatinib is a BCR- Druker et al.
leukemia (CML) chromosome number 22 and 9, ABL tyrosine kinase [38]
respectively. The BCR-ABL mutation inhibitor and can be
occurs when pieces of BCR and ABL used for treatment of
break off and switch places. BCR- BCR-ABL-based
ABL confirms the diagnosis of CML CML
Therapy Test and its description Clinical guidance Reference
Lung cancer EML4-ALK fusion gene used for Crizotinib works Kwak et al.
diagnosis of non-small cell lung only in cancer with [39]
cancer (NSCLC). Such types of overactive ALK
cancer can be cured by targeting
anaplastic lymphoma kinase (ALK)
inhibitor
Coronary artery CYP2C19 converts clopidogrel into Use of clopidogrel Simon et al.
disease an active metabolite. Individuals who may be toxic or less [40]
carry two nonfunctional copies of the efficacious in poor
CYP2C19 gene are poor metabolizers metabolizer. Use
of clopidogrel another alternative
agent/drug
Autoimmune CXCL13, aberrant expression of Antibodies targeting Leypoldt et al.
encephalitis CXCL13, is linked to the CXCL13-mediated [41]
development of autoimmune signaling pathway
disorders
Cystic fibrosis G551D, G551D mutation in ATP- Ivacaftor Ramsey et al.
binding pockets abolishes ATP- recommended for [42]
dependent gating. It is one of the the patients with the
common mutation associated with G551D mutation
cystic fibrosis
Thrombosis Factor V Leiden is a specific gene Avoid prothrombotic Vandenbroucke
mutation that results in thrombophilia drugs for factor V et al. [43]
Leiden-positive
patients

Pharmacogenomics tests are responsible for drug metabolism, transport,


and drug target. Genetic variations can increase the risk of toxicity or poor
efficacy. Pharmacogenomics helps us to select the most suitable drug and
dose to achieve a better therapeutic response. Several clinical
recommendations for commonly used anticoagulants, antiplatelets,
transplant medications, and other therapies are available [45]. Clinical
decision-making depends on many factors such as variants in the patient or
population and the quality of evidence, availability of testing and
Pharmacogenomics data, pharmacokinetics and pharmacodynamics of drug,
drug history, and drug-drug interactions. Service providers should also have
a good knowledge of Pharmacogenomics resources to implement an
accurate clinical decision. Pharmacogenomics mainly helps in the
implementation of personalized medicine by predicting the patients who
should receive a lower dose/higher dose or an alternative drug. The
implementation of Pharmacogenomics is limited by challenges in clinical
testing, data analysis, lack of education, and ethical, legal, and social
implications. Despite the barriers to clinical Pharmacogenomics, several
academic, medical, and community centers have initiated
Pharmacogenomics implementation programs. Future advances in precision
medicine and data sharing between health service providers and patients
will help in achieving the goal of personalized medicine.
Imaging techniques can perform many diagnoses with good confidence
and also avoids the patients to go for invasive testing and unnecessary
surgery. Now computed tomography and ultrasonography provide better
results with improved sensitivity and specificity in the diagnosis of many
diseases [46]. Positron-emission tomography can detect metabolically
active cancer that is not easily detectable with traditional imaging. A rich
database of electronic health records can be a guide for clinical decision. In
the future, software can be developed to identify patients with disease risk
factors or to follow screening guidelines and also for drug selection and
administration.

5.2 Impact of Pharmacogenomics on Personalized Medicine


5.2.1 Cancer
The concept of personalizing treatments provides the best possible direction
for the treatment of many cancers. Different therapeutic strategies for the
treatment of cancer are surgery, radiotherapy, chemotherapy,
immunotherapy, hormone therapy, gene therapy, and dietary therapy. We
can prefer therapies from a wide range of options based on the type,
location, and stage of cancer. Current knowledge of cancer classification,
predictive markers, and combination therapies holds the best possible
avenues for cancer treatment in the future. The strategy is the most
important issue in cancer therapy. As a result of pharmacogenomic
advances, different therapeutic strategies for the treatment of various
cancers have been adopted. Gene therapy approach has been used for the
treatment of prostate cancer and as result necrosis observed at metastatic
sites [47]. Personalized medicine is used in cancer therapy for screening
formulation in the epidermal growth factor receptor (EGFER) gene in lung
adenocarcinoma.
Variants Associated with Risk of Cancer
Studies have shown an association between genetic variants of a gene and
the risk of developing a disease. BRCA1, BRCA2, TP53, PTEN, STK11,
CDH1, CHEK2, ATM, BRIP1, PALB2, RAD51C, RAD50, and NBN genes
have been identified for breast cancer. Association of these genes with risk
of disease has been further investigated. Three variants rs80358978
(Gly2508Ser), rs80359065 (Lys2729Asn), and rs11571653 (Met784Val)
have been reported in the BRCA2 gene, and these variants have shown
significant associations with risk of developing breast cancer [48]. Variants
rs8176085, rs799923, rs8176173, and rs8176258 in the BRCA1 gene, one
common variant in the CHEK2 gene (rs9620817), and one common variant
in the PALB2 gene (rs13330119) are also associated with risk of this
disease. Similarly, four genetic variants, Phe139Ser in SORD, Ala350Arg
in KRT6A, Gly387Cys in SVEP1, and Gly144Arg in MRPL38, have shown
significant association for risk of liver cancer [49]. These variants can be
used for early detection of liver cancer and designing of relevant
therapeutics for treatment. These variants only predict the risk of
developing a particular type of cancer and may not be used for deciding the
therapeutic outcome of treatment with a drug.
Nearly 100 genetic variants that signal an increased risk of colon cancer
are available. Several combined genetic variants may be clinically relevant
and can impact personalized screening. For breast cancer, 182 breast cancer
susceptibility SNPs are available which can be used to understand the
heritability of breast cancer [50]. Study of risk loci for breast cancer can be
a guide for prevention, prognosis, and treatment of breast cancer. The
polygenic risk score can be calculated to estimate individual risks for
developing breast cancer. GWAS have identified more than 30 epithelial
ovarian cancer risk loci which may involve genome editing to establish the
cell-specific carcinogenic effects [51]. Studies have also identified
susceptibility regions potentially shared between breast, ovarian, and
prostate cancer. For example, in oral cancer, an association between TNF-α
variant, rs361525, and risk of an oral precancerous lesion has been
observed. An allelic variant of rs361525 SNP, located at a transcriptional
factor-binding site of the promoter region of TNF-α, disturbs the expression
of TNF-α [52]. Studies have shown that genetic alterations in immune
system genes and genes with metastatic potential are associated with oral
precancer and oral squamous cell carcinoma.
Variants Associated with Outcome of Treatment
Afatinib is a TKI inhibitor used for the treatment of patients with non-
small-cell lung cancer (NSCLC) whose tumors are activated by EGFR [53].
It is also effective for NSCLC patients with EGFR-T790M mutation who
are resistance to the TKIs erlotinib and gefitinib. Afatinib has shown very
good response among patients with tumors having HER2 or HER4
mutations [54]. HER2 mutations are rare; afatinib has shown high activity
for NSCLC patients with HER2 mutation. Imatinib is a TKI inhibitor used
for the treatment of CML. It has also been used for other BCR-ABL-, c-
KIT-, and PDGFR-driven cancers. Imatinib is metabolized by CYP2C8 and
CYP3A4 in vitro. CYP2C8 genotype affects the metabolism of imatinib and
its systemic exposure [55]. But, no evidence regarding the metabolism of
imatinib in CYP3A4 or CYP3A5 genotype is available. Many studies have
been performed to find the association between genetic variants and the risk
of developing particular cancer. Similarly, the varied response of the same
drug has been noticed in different genotypes which may be affected by
genes involved in pharmacokinetics and pharmacodynamics. FDA label
regarding the clinical use of different therapeutics is available which may
be taken into consideration while prescribing the drug and dose for the
treatment.
Recent advances have made it possible to understand the mechanisms of
the development and progression of cancer, diagnosis, and monitoring of
cancer, clinical outcome, and risk of adverse reactions. The Cancer
Genomics Analysis (TCGA) and the International Cancer Genomics
Consortium (ICGC) projects have identified several genetic mutations
responsible for various cancers. Liquid biopsy is carried out to obtain
genomic information from cell-free DNA for screening, monitoring, and
relapse/recurrence of cancer [56]. Recent advances have made it possible to
detect and quantify biomarkers with high specificity and sensitivity. KRAS
and BRAS are the most frequent mutation in human cancer, and analysis of
these mutations might be an effective approach to screen cancers [57].
There are many mutations for which effective drugs are not available.
Personalized biomarker selection can improve the response rate of targeted
therapy for many cancers. A small subset of patients with a particular type
of mutation can be targeted for the treatment with a drug. Germline variants
related to ADMET are also important for personalization of therapy.
HLA genotypes may be used to predict the risk of drug-induced severe
skin hypersensitivity and drug-induced liver injury. Different variants of
HLA-B are used as a therapeutic biomarker for some diseases including
cancer. Individuals with HLA-B∗15:02 or HLA-A∗31:01, HLAB∗57:01,
and HLA-B∗58:01 variants have shown skin hypersensitivity for
carbamazepine and abacavir drugs [58]. These side effects can be avoided
by HLA genotyping of patients. Immuno-genomic analysis of cancer cells,
such as quantification of infiltrated CD8+ T cells and their T cell receptor,
can be useful for assessment of the immune response on therapy [59].
Chimeric antigen receptor T cell therapy and TCR-engineered T cell
therapy have shown clinical effectiveness in hematological cancers.

5.2.2 Neurodegenerative Diseases


Use of novel CSF, blood-based, and neuroimaging biomarkers specific to
particular diseases has played a very important role in the treatment of
many neurodegenerative diseases. The use of novel biomarkers for
neurodegenerative diseases has improved the accuracy of diagnosis,
prognosis, and the efficacy of therapy. Personalized therapy is suggested for
Alzheimer’s and Parkinson’s diseases because these diseases are clinically
heterogeneous and have strong genetic connections. Genetic risk for
Alzheimer’s disease was found related to the APOE4 gene. Homozygous
individuals for APOEe4 allele have a 50% risk of developing Alzheimer’s
disease, while heterozygous individuals for the APOEe3/e4 genotype have
a risk of 20–30% [60]. Mutation in other genes such as APP, PSEN1, and
PSEN2 are also associated with early onset of Alzheimer’s disease. High
levels of neurogranin CSF is associated with progression to Alzheimer’s
disease and results in a synaptic loss. Blood-based biomarkers are very
useful in the area of Alzheimer’s disease progression. Neuroimaging allows
for preclinical identification of individuals genetically susceptible to
Alzheimer’s disease.
Parkinson’s disease is characterized by loss of dopaminergic neurons
and loss of striatal dopamine signaling. GWAS have identified many genes
and loci related to Parkinson’s disease. Mutations in the genes SNCA,
LRRK2, PINK1, DJ-1, and Parkin are associated with Parkinson’s disease
[61]. The activity of caspase enzymes is also associated with Parkinson’s
disease. Increased activity of Cas9 is associated with high cellular toxicity
in Parkinson’s disease and has been recommended as a therapeutic target
for the treatment of Parkinson’s disease [62]. CRISPR-Cas9 may also be
used for gene therapy in Parkinson’s disease. Immunoglobulin G exerts pro-
inflammatory responses in the body and may be used as a unique biomarker
for Parkinson’s disease. Targeting ganglioside GM1 levels in patients of
Parkinson’s disease may also be a personalized therapeutic approach.
Discovery of new biomarkers of Parkinson’s disease might be more useful
for the personalized therapy of Parkinson’s disease.
Multiple sclerosis is an autoimmune disease which affects the function
of the central nervous system. The allele HLA-DRB1 is associated with an
increased risk of multiple sclerosis [63]. GWAS has identified other
mutations which increase the risk of multiple sclerosis such as SNPs in
interleukin-7 receptor an (IL7R) gene. The chemokine C-X-C motif ligand
13 (CXCL13) is used as a dual biomarker for prognosis and therapy
monitoring. A high correlation was found between increased CXCL13
levels and multiple sclerosis [64]. Increased soluble CD163 levels in blood
and CSF have been identified in multiple sclerosis patients, and soluble
CD163 may also be used as a biomarker for multiple sclerosis [65].
Technological advances in Pharmacogenomics will ensure the clinical
application of personalized medicine. Genomics, transcriptomics
proteomics, metabolomics, and neuroimaging have to speed up the
identification of novel biomarkers and genetic connection of diseases.
GWAS, NGS, microarray data analysis, and identification of mRNA,
miRNAs, metabolites, proteins, SNPs, copy-number variations, and other
genetic alternations can be useful to assess the individualized risk levels for
a patient and can also suggest a most effective therapeutic approach.

5.2.3 Thrombosis
Novel approaches are required to understand the genetic basis of
thrombosis which can provide better and personalized therapy for
thrombosis. Thrombosis is a complex disease caused by a combination of
numerous factors such as environmental and genetic factors. Both plasma-
based and genetic assays are important for assessing the risk of disease, but
genetic factors are more informative in assessing platelet risk factors for
thrombosis. Five genetic risk factors for venous thromboembolism are
VTE, deficiencies of antithrombin, protein C, protein S factor V Leiden,
and the G20210A prothrombin gene variant [66]. There is a lack of clinical
data set related to thrombosis which limits the goal of personalized therapy.
Clopidogrel is effective in platelet aggregation, but a subset of patients has
shown no response to clopidogrel therapy. Varying response to warfarin
therapy was noticed due to the effect of the gene variants of CYP2C9 and
VCORC1 [67]. Treatment of thrombosis can be personalized by identifying
genetic factors responsible for the development and recurrence of the
disease. VCORC1 and CYP2C9 genotype-based strategies are very useful
for initiating anticoagulant therapies. For CYP2C9 and VKORC1
genotypes, edoxaban produces better and safe response than warfarin [68].
CRISPR/Cas9 gene therapy and anti-microRNA approaches have also been
used to treat thrombosis. More clinical trials on antithrombotic agents are
required to collect and analyze the impact of genetic, clinical
environmental, and dietary factors on response to therapy. Risk of
thrombosis can be assessed by studying the effect of different genetic
variants related to thrombosis.

5.2.4 Cardiovascular Diseases


Cardiovascular diseases are one the leading cause of death in developed
countries. Patients with systemic lupus erythematosus and rheumatoid
arthritis have increased risk of cardiovascular disease. Two new putative
risk loci associated with increased risk for cardiovascular disease are
identified [69]. An IL19 risk allele, rs17581834(T), is associated with
stroke/myocardial infarction in systemic lupus erythematosus and
rheumatoid arthritis, while another SRP54-AS1 risk allele, rs799454(G), is
associated with stroke/transient ischemic attack in systemic lupus
erythematosus but not with rheumatoid arthritis. Several SNPs related to
coronary artery disease have been identified, but their function is not clear
yet. The risk allele of a common coronary artery disease-associated marker
at the TOMM40/APOE locus was found to be associated with a lower level
of high-sensitivity C-reactive protein [70]. GWAS have shown the
association between coronary artery disease-associated risk variants and
common inflammatory markers. Calprotectin, an inflammatory marker for
of plaque instability, is used as a new biomarker of coronary artery disease.
Total 53 loci with significant effects in both coronary artery diseases
were identified and at least 1 of low-density lipoprotein, high-density
lipoprotein, triglycerides, type 2 diabetes mellitus, C-reactive protein,
systolic blood pressure, and type 1 diabetes mellitus [71]. These genetic loci
implicate novel genetic mechanisms involved in coronary artery disease.
These genetic loci may be used for earlier diagnosis, prevention, and
individualization of coronary artery disease therapy.

5.2.5 Anesthesia
Succinylcholine, mivacurium, and other anesthetics are substrates for
enzyme pseudocholinesterase. There are 70 different variants of
pseudocholinesterase, and most common polymorphism is known as
atypical which impairs the function of pseudocholinesterase [72]. An
individual homozygous for atypical allele remains paralyzed for 2 h, while
individuals who are heterozygous paralyzed for up to an hour. Similarly, the
varying response of pseudocholinesterase inhibition has been reported for
dibucaine. An individual with rare S-variant of pseudocholinesterase
remains paralyzed for up to 8 h [73]. An individual with no or lower level
of pseudocholinesterase may have an increased risk of toxicity. Codeine is a
prodrug that is metabolized by CYP2D6 into the active metabolite,
morphine. The dose of codeine may be recommended based on poor,
intermediate, extensive, or rapid metabolizer which will depend on the
expression of CYP2D6 gene [74]. Genes for the 5HT3B receptor, dopamine
D2 receptor, the ABCB1 transporter, and CYP2D6 are associated with
postoperative nausea and vomiting. Some polymorphisms related to these
genes are associated with a high incidence of postoperative nausea and
vomiting. ADRB1encodes for the β-1 adrenergic receptor, and certain
polymorphism related to this gene has also been reported.

5.2.6 Type 2 Diabetes


Type 2 diabetes is one of the major causes of premature mortality. GWAS
has identified hundreds of variants associated with type 2 diabetes which
can be assessed for their clinical utility [75]. Some genetic variants related
to type 2 diabetes are known that may have clinical significance in the
prevention, personalized treatment of type 2 diabetes. TBC1D4 and
nonsense variant (Arg684ter) have different prevalence in different
population and may decide the risk of type 2 diabetes [76]. In Latinos
population, a low-frequency missense variant (E508K) at HNF1A conveys
a type 2 diabetes. These variants may be useful in the assessment of clinical
utility and personalized therapy of type 2 diabetes [77]. Metformin is used
for the treatment of diabetes treatment, and some cases of metformin
intolerance have been found. Genetic variants at ATM and glucose
transporter gene (SLC2A2) are associated with the glycemic response of
metformin treatment [78].

5.2.7 Depression
Many genetic variants of cytochrome p450 (CYP450) system, relevant to
the metabolism of many antidepressants, have been identified. However, all
these variants are not providing the clinically significant information which
can be utilized in decision-making [79]. Many efforts have been made to
understand the impact of common genetic variation of CYP450 on
treatment in depression. CYP450 2D6 (CYP2D6) is responsible for the
oxidative metabolism of most of the antidepressants [80]. For CYP2D6,
60–85% of white individuals were found fast metabolizers, while some
individuals with two disrupted copies of CYP2D6 were poor metabolizers.
An effect of CYP450 on treatment with venlafaxine has been studied.
Individuals who are less efficient in metabolizing venlafaxine will have
lower blood concentrations of the active metabolite desvenlafaxine [81].
For many diseases, more clinical studies are required to take a better
therapeutic decision regarding the recommendation of a drug and its dose.
Other than CYP450 systems, the drug transport protein P-glycoprotein
(ATP-binding cassette, subfamily B (MDR/TAP), member 1 (ABCB1)) was
also studied due to its role in the efflux of drugs across the BBB [82].

5.2.8 Psychiatry
CYP has more than 90 known genetic variations and more than 60 alleles.
Study of these CYP2D6 alleles may provide better clinical information
related to treatment for psychiatry. CYP2D6 metabolizes many
antipsychotics and antidepressants. The FDA has recommended the HLA-
B∗1502 genotyping in Asians before prescribing carbamazepine to avoid
toxic outcome [83]. A better understanding of the pharmacokinetics and
pharmacodynamics of psychiatric drugs has allowed personalized
prescription in clinical practice. Dosing recommendation of risperidone in
psychiatric patients is based on the presence of CYP3A inducers and/or
CYP inhibitors and CYP2D6 [84]. CYP2D6 has a higher affinity for
risperidone and hydroxylating it to 9-hydroxyrisperidone. Poor metabolizer
can be identified by CYP2D6 genotyping or by measuring the level of
risperidone. Knowledge pharmacokinetic, pharmacodynamic, efficacy,
safety, and adverse drug reaction are required to understand personalized
prescription and its applications in psychiatry. The goal of personalized
medicine can be achieved by applying the précised medical information or
knowledge in therapeutic practices. AmpliChip CYP450 test for analysis of
the CYP2D6 and CYP2C19 genes are available to detect poor and fast
metabolizer [85]. Around 5% of the Caucasian population is CYP2D6 poor
metabolizers for psychiatric drugs which may have some side effects.
Metabolism of one drug depends on other drug taken which can inhibit or
stimulate the metabolism of other. CYP450 isoenzymes (CYP1A2,
CYP2C19, CYP2D6, CYP3A4) are involved in the metabolism of
psychotropic drugs and may cause adverse drug reactions [86]. A
concentration-to-dose ratio for prescribing clozapine and risperidone is
known, and CYP genotyping of patients can guide therapeutic dose
monitoring. CYP1A2, CYP2B6, and CYP3A4 genotyping has a small
utility from a clinical point of view, while CYP2C9 genotyping has no
utility in psychiatry [87].

5.2.9 Hypertension
Variation in individual response on treatment with the same drug motivates
us to look for genetic factors associated with this variation. Studies have
shown the association between the response of blood pressure and specific
gene polymorphism. Clinical data related to genetic polymorphism and
therapeutic response can guide the way of personalized medicine for
hypertension. Inhibitors of angiotensin-converting enzyme, β-blockers,
angiotensin 2 blockers, and calcium channel blocker are well-studied
inhibitors for the treatment of hypertension [88]. Effect of β1AR
Arg389Gly polymorphism on responses of blood pressure to β-blocker
therapy has been studied. On treatment with metoprolol, patients
homozygous for Arg389 had shown a high reduction in diastolic blood
pressure [89]. The β1-adrenergic receptor gene (ADRB1) encodes a
51.3 kDa protein with 477 amino acid residues. The β1-adrenergic receptor
is present in the heart, controlling heart rate. Six SNPs located near the
ADBR1 gene region are available in dbSNP. Out of six SNPs, two
polymorphisms Ser49Gly (rs1801252) and Arg389Gly (rs1801253) have
been studied well [90]. Studies have shown that siblings with Gly389 allele
have a low diastolic blood pressure than those homozygous for Arg389.
Such types of knowledge related to polymorphism and outcome of
treatment can be utilized while recommending the drug and dose to any
hypertensive patient. More investigations should be performed to know the
effect of other polymorphism on the outcome of treatment with a
hypertensive drug. Studies have found that there is a genotype group that
shows a less favorable response to β-blocker therapy. For adrenergic
receptor polymorphism, the effect of drug bucindolol on different genotypes
can be assessed [91]. This may help in finding the genotypes which show
better or poor response on treatment with bucindolol. Knowledge of genetic
factors that decides the response of a drug can enable us to choose the most
suitable drug and dose for each patient based on genetic profile. Advances
in “omics” technologies have made it possible to identify genetic markers
that can be used to know the response of a particular drug and guide the
way for individualized therapy [92]. A relationship between nephrosis
(NPNS1) gene variants and response to angiotensin-receptor antagonist
losartan has been found. Also, two other genes (ALDH1A3 and CLIC5)
have shown their influence on blood pressure response to
hydrochlorothiazide treatment. Many genetic polymorphisms that affect the
response of treatment with hypertensive drugs such as enalapril,
perindopril, lisinopril, metoprolol, atenolol, bisoprolol, losartan, and
hydrochlorothiazide have been identified [93]. Some effective approaches
for the personalized treatment of hypertension are aldosterone
measurement, renin profiling, aldosterone-to-renin ratio, SNP and
haplotype approach, and hemodynamic assessments.

5.3 Ethical Issues Related to Personalized Medicine


In hospitals and universities, numerous tests and kits are available to detect
genetic diseases. But the accuracy of some kits and validity of some
biomarkers related to disease have a low level of confidence. The ethics of
personalized medicine became an issue when a healthy person has been
reported to have a high risk of breast or ovarian cancer. Ethical issues are
also associated with the use and storage of genetic information of an
individual. Ethics related to personalized medicines are also affected by the
health policies of a country [94]. The National Institutes of Health (NIH) is
trying to develop the best way of treatment and also delivering clinical
information to the health sector and patients. Many research organizations
have confined their study and tests related to a specific genetic disorder in
certain groups of people or region. Informed consent of a patient is required
for the use of their body material to protect the privacy of an individual.
There is a need to assure the protection of privacy of genetic data of an
individual from the government or insurance companies.

6 Challenges and Future Opportunities


One of the important challenges in the way of personalized medicine is to
manage the complexity associated classification of disease. A large number
of new genetic alterations are being reported by next-generation
sequencing. Association of some of these genetic alternations with a disease
is well established, but the role of many mutations/alternation was not
evaluated yet. Other alternations might have alone or combined effect on
disease prognosis. Systematic storage, retrieval, and analysis platform are
required to store the clinical data and also for decision-making. Better
biomarkers of the disease can assist with detection and also can guide
treatment. The financial incentives for designing of new diagnostics are
required because a better diagnosis of the disease can improve the rate of
cure. Advances in DNA sequencing have enabled studies of the microbiome
which are present in our system. Microbiome studies have been considered
for disease and may offer opportunities for personalized therapy.
Associations between a genetic variation and related outcome should be
quantified. Implementation of Pharmacogenomics is based on these
associations, and no more emphasis has been given on clinical validity and
utility of the test. Pharmacogenetics tests should be adopted based on their
clinical utility and cost-effectiveness. Pharmacogenomics-related data and
tests are available only for some drugs, but still, this knowledge has not
been potentially translated into clinical practice at a bigger scale. However,
the clinical utility of Pharmacogenomics test is limited due to a gap
between clinical validity and its application to patients through health-care
providers. Pharmacogenomics-based CPIC dosing guidelines are available
for some drugs. Primary care providers have no adequate guidelines on how
and when to Pharmacogenomics markers [95]. There exist a gap between
evidence of association and the clinical utility of Pharmacogenomics tests,
and there is need to overcome this challenge by exploring evidence from a
health-care provider, patient, and policy perspective [96]. Well-defined
steps of health care from clinical validity to clinical utility are required for
successful implementation of Pharmacogenomics tests to patients. Many
publications related to Pharmacogenomics reports only about association,
but other pieces of evidence such as guideline development and coverage
decisions are necessary for better implementation of Pharmacogenomics.
Pharmacokinetics and pharmacodynamics of many drugs and its
mechanism of interactions have been reported well, but its clinical utility is
still very limited. The lack of clinical utility of a drug delays the application
of Pharmacogenomics tests into health care [97].
Pharmacogenomics-based therapies will reduce the trial-and-error
prescription and will also improve drug safety by avoiding adverse drug
reaction. Pharmacogenomics will reduce the time and cost of a clinical trial.
Genetic profiling will also help in recommending the use of a failed drug
for a particular group of another population [98]. Availability of large scale
of data related to the association of genetic variations with the disease
across many countries will enable us to achieve the goal of personalized
medicine. Study of genetic variations also speeds up the process of the
clinical trial process by targeting the individuals for testing belonging to a
specific population. The government and other funding agencies should
give more incentive for adopting the practices related to personalized
medicine. Faster, reliable and cost-effective approaches for sequencing,
screening, and diagnosis of diseases should be developed which will bring
the use of personalized medicine in real practice. In recent years, a large
amount of knowledge-related genome, proteome, and metabolic pathways
have been generated which enable us to use, monitor, and assess the effect
of a drug on a patient’s genotype. The government should take an initiative
to frame policies related to the use of genetic material and individualization
of therapy. Health-care centers, educational institutes, and hospitals may
play important in promoting research and clinical practices of personalized
medicine. It is expected that the cases of serious adverse drug reactions will
reduce in the future with the implementation of Pharmacogenomics
approaches for personalized therapy.
The cost of genetic tests and its availability to rural people are also a big
challenge for many poor and developing countries. Governments will also
have to think over the issues raised as a result of discriminations based on
genotype. The pharmaceutical companies may take more interest in
developing the drug for a genotype belonging to the rich group of the
population rather than a disease or patient confined to a poor subgroup of
the population. Pharmacogenomics has brought a big shift in the trend of
therapies for many diseases; still, many patients are not in a position to take
the benefit of pharmacogenomic knowledge in personalizing the therapy.
Benefits of personalized medicine should be utilized without waiting for
more discoveries related to Pharmacogenomics and biomarker. The
personalized prescription can be given based on genetic, environmental, or
personal factors that can affect the outcome of the treatment. The FDA has
set many recommendations and guidelines to promote the use of
Pharmacogenomics in the personalization of therapy. To ensure the success
of personalized medicine, some strategic recommendations have been
suggested [99]. These recommendations are related to the discovery of
more potential biomarkers, framing regulations for genetic privacy and
confidentiality, regulatory incentives to pharmaceutical industries and
equity of access to personalized medicine, standardization of genetic testing
and documentation, regulation for private genetic testing firms, and better
awareness about personalized medicine within the medical imaging
community.

7 Conclusion
Pharmacogenomics has played a very important role in adopting the goal of
personalized medicine for many diseases. It has greatly impacted the
prescription of a drug and its dose for many diseases, but a small population
of the world can enjoy the benefits of Pharmacogenomics. Personalized
medicine offers the opportunity to identify patients for whom a drug can be
both effective and safe. Efficient decision-making on clinical data and
proper utilization of health-care resources can enable us to achieve the goal
of personalized therapy. Pharmacogenomics also improves the process of
drug development by focusing the companies to test the safety and efficacy
of a drug in an individual or subgroup of a population. Pharmaceutical
companies have to reduce the cost of personalized treatment so that poor
patients can also utilize the benefit of pharmacogenomics. Medical imaging
techniques have played a very important role in the diagnosis, prediction,
and treatment of many diseases and can be very helpful in achieving the
goal of personalized therapy.

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Index
A
Acoustic separation, ultrasound
Acquired immunodeficiency syndrome (AIDS)
Activation-induced cytidine deaminase (AID)
Acute myeloid leukemia (AML)
Acute respiratory distress syndrome
Adalimumab (HUMIRA)
Adeno-associated viruses (AAV)
Adenosine deaminase (ADA)
deficiency-severe combined immunodeficiency (ADA-SCID)
Adjuvants
Administration, therapeutic proteins
Adrenoleukodystrophy
Advanced therapy medicinal products (ATMP)
Agitation
Agonists
Alginate
Alteplase
Alzheimer’s disease
Amniotic fluid-derived stem (AFS) cells
Amyotrophic lateral sclerosis (ALS)
Anesthesia
Antagonists
Anthrax vaccine
Anti-adsorption agents
Anti-aggregation agents
Antibodies
anti-drug (ADAs)
engineering
high-affinity
humanized
libraries
therapeutic
Antibody-drug conjugates (ADCs)
Anticoagulants
Antidepressants
Anti-drug antibodies
Antioxidants
Antipsychotics
Antisense oligonucleotides (ASO)
Antithrombin
Aptamers
Asparaginase
Autism spectrum disorders
Autoantigens
Avastin
Avotermin
Axicabtagene ciloleucel
B
B cell receptors (BCR)
Bexsero
Bioavailability
Biobetters, formulation
Biodel insulin (BIOD-531)
Biologics
Biomarkers
Biopharmaceuticals, packaging
safety
Bioprinting
Bioprocessing
Bioreactors
single-use
Biosimilars
formulation
Blood clotting factors (blood factors)
Blood glucose
Blood products
Bone marrow (BM)
graft rejection
transplant
Bone sialoprotein (BSP)
Bone tissue
Buffers
Bulking agents
Buoyancy-activated cell sorting
C
Calcitonin
Calcium channel blockers
Cancer, breast
gene therapy
pancreatic
prostate
thyroid
Carbon nanotubes (CNT)
Cardiac tissue
Cardiomyocytes
CAR T-cell therapy
Cartilage
printing
CD19
Cell retention, cross-flow filtration
Cell separation
Cell therapy
Centrifugation, cell retention
cell separation
Chikungunya
Chimeric antigen receptors (CARs)
Chromatograhpy
Class switch recombination (CSR)
Clinical decision
Clotting factors
Coagulation factors
Collagenase
Colony-forming unit-fibroblasts (CFU-F)
Colony-stimulating factors (CSFs)
Combination vaccines
Complementary determining regions (CDRs)
Computer-aided design (CAD)
Consistency lots
Continuous processing
Cost of goods (COG)
CRISPR/Cas9
Critical quality attributes (CQAs)
Cross-flow filtration
Culture medium homogenization
Culture monitoring
Cyclodextrins
CYP2D6
Cystic fibrosis (CF)
Cytokines
release syndrome
Cytomegalovirus (CMV)
retinitis
D
Deamidation
Degludec
Delivery devices
Delivery vectors
Dengue, vaccine
DENGVAXIA
Depression
Depth filtration
Design of experiments (DoE)
Design space
Developability assessment
Diabetes
type 1
type 2
Dicoumarol
Digital light processing (DLP)
Diphtheria toxoid
Disease modeling
Disposable systems
Donor lymphocyte infusions (DLI)
Downstream processing
Drotrecogin alfa
Drug-drug interaction (DDIs)
DTcP vaccine
Duchenne muscular dystrophy (DMD)
Dupuytren’s contracture
E
Ebola
vaccine
Ad5-EBOV
rVSV-ZEBOV
Embryonic stem (ES) cells
Endotoxin
Enoxaparin sodium
Enterovirus 71 (EV71)
Enzymes, therapeutic
Epidermal growth factor (EGF)
receptor (EGFER)
Epigenetic factors
Epigenetic memory, hiPS cells
Epitopes
Epoetin
Erythropoietin
Eteplirsen
Expression systems
Extracellular vesicles (EVs)
F
Factor VIII
Familial dysautonomia (Riley-Day syndrome)
Familial hypercholesterolemia
Fermentation
Fibrinolytics
Fibroblast growth factors
Filgrastim
Filtration, cross-flow
Follicle-stimulating hormone (FSH)
Follitropin
Fomivirsen
Fused deposition modeling (FDM)
Fused filament fabrication (FFF)
G
GARDASIL 9
Gelatin methacrylate (GelMA) bioink
GelMA gels
Gene augmentation
Gene delivery, in vivo
Gene editing
Gene repair, nucleases
Gene silencing
Gene therapy
Genetic makeup
Genetic polymorphism
Ginkgo biloba (ginkgo)
Glargine
Glucagon
Glucagon-like peptides
Gluconeogenesis
Glycosylation
Gonadotropins
Good manufacturing practice (GMP)
Graft vs. host disease (GVHD)
Graft vs. leukemia (GVL) effect
Granulocyte colony-stimulating factor (G-CSF)
Granulocyte macrophage colony-stimulating factor (GM-CSF)
Growth factors
Growth hormone (GH)
GX-H9
H
Heart disease
Heart tissue-derived decellularized extracellular matrix (hdECM)
Hematopoietic cell transplantation (HCT)
Hematopoietic growth factors (HGFs)
Hematopoietic stem/progenitor cells (HSPC)
Hemophilia
Heparin
Hepatitis
alcoholic
B
recombinant vaccine
C
Heplisav-B
Hereditary transthyretin amyloidosis (hATTR)
Herpes zoster
High cell density reactors
Hirschsprung’s disease
Hirudin
Homology-dependent repair (HDR)
Human cardiac progenitor cells (hCPCs)
Human chorionic gonadotropin (hCG)
Human coronary artery endothelial cells (HCAECs)
Human immunodeficiency virus (HIV)
RNAs
vaccine
Human leukocyte antigens (HLA)
Human papillomavirus (HPV) vaccine
Human platelet lysate (hPL)
Human pluripotent stem (hPS) cells
Huntington disease (HD)
Hyaluronate/hyaluronic acid
Hyaluronidase
Hydrogels
Hypertension
I
Immune potentiators
Immunogenicity
mechanisms
Immunoglobulins
IN-105 (tregopil)
Induced tissue-specific stem (iTS) cells
Insulin
Insulin-like growth factors (IGFs)
Interferons (INFs)
Interleukins (ILs)
receptor
In vitro compartmentalization
Isotonicity
L
Laron syndrome
Laser-induced forward transfer (LIFT) cell printing
Lassa virus
vaccine
Leber congenital amaurosis type 2
Lenograstim
Leprechaunism
Luteinizing hormone (LH)
LY3209590
LY900014
Lyophilization
Lyoprotectants
M
Malaria
vaccine
Mammalian cell display
Manufacturing
single-use
upstream
Marburg virus
Measles
vaccine
Mecasermin
Meningococcal ACWY conjugate vaccine (MenACYW135)
Meningococcal subgroup B vaccine (MenB)
Mesenchymal stem/stromal cells (MSC)
Metoprolol
Middle East respiratory syndrome (MERS)
Miller-Dieker syndrome
Mipomersen
MOD-4023
Molgramostim
Monoclonal antibodies (mAbs)
Monophosphoryl lipid A (MPL)
MRSA
Multiple sclerosis
Multiplex automated genome engineering (MAGE)
Mutagenesis
Myocardial infarction
N
Nalotimagene carmaleucel
Neisseria meningitidis
Neoantigens
Nerve growth factor (NGF)
Nerve tissue
printing
Neurodegenerative diseases
Neutropenia
Nipah virus
NOD-like receptors (NLR)
Nonhomologous end joining (NHEJ)
Novel excipients
Nucleic acids, gene therapy
Nusinersen
O
OKT3 (muromonab)
Oncolytic viruses (OV)
Organoids
Ornithine transcarbamylase (OTC) deficiency
Orphan products
Osteopontin (OP)
P
Palifermin
Pancreatic progenitors (PPs)
Pancreatitis
Panning
Paracrine cell therapy
Parathyroid hormone (PTH)
Paratope
Parkinson’s disease (PD)
Pattern-recognition receptors (PRR) agonists
PCV vaccine
PDGF-BB
Pediatric applications
Pegaptanib
Pegfilgrastim
Perfusion reactors
Personalized medicine
Pertussis
Peyronie’s disease
Phage display technology
Pharmacoeconomics
Pichia pastoris (Komagataella pastoris )
Placental growth factor (PlGF)
Plastic bioreactors
Platelet-derived growth factor (PDGF)
Pneumococcus conjugated vaccines
Polyclonal antibodies (pAbs)
Preservatives
Process engineering
Product information
Promoters
Protein-protein interactions
Proteins, aggregation
characterization
chemical degradation
expression
formulation
lyophilized
physical degradation
recombinant
structure
Prothrombin
Q
QT syndrome
Quality by design (QbD)
Quality control
Quality risk management
R
Recombinant drugs
Recombinant enzymes
Recombinant proteins
formulation
Recombinant technology
Regenerative medicine
Replacement therapy
Respiratory syncytial virus (RSV) vaccine
Respiratory syndrome virus (RSV) disease
Reteplase
Rett syndrome
Ribosome/mRNA display
RNA-induced silencing complex (RISC)
RNA interference (RNAi)
RNA, mRNA vaccines
microRNA (miRNA)
S
Saccharomyces cerevisiae
Sargramostim
Scalable culture vessels
Schizophrenia
Sedimentation, cell separation
Separation, cells
Severe combined immunodeficiency (SCID)
Shingles
vaccine
Shingrix vaccine
Short hairpin RNA (shRNA, expressed RNAi activators)
Short interfering RNA (siRNA)
Skin
printing
regeneration
Small molecules (SMs)
Solubility enhancers
Somatic hypermutation (SHM)
Somatotropin
Spinal muscular atrophy (SMA)
Stability
Stabilizers
Stem cells, human pluripotent
Stereolithography (SLA)
Streptococcus pneumoniae
Surfactants
Survival of motor neuron (SNM) protein
T
Tangential flow filtration, alternating
Tenecteplase
Teriparatide
Tetanus toxoid
Therapeutic biologics
Therapeutic response
Thrombin
Thrombolytic agents
Thrombopoietin
Thrombosis
Thrombus formation
Thyroid-stimulating hormone (TSH)
Tissue engineering
Toll-like receptors (TLR)
Tonicifiers
Traceless affinity cell selection
Trafermin
Transcription activator-like effector nucleases (TALEN)
Transdermal delivery
Transfection
Transforming growth factors (TGFs)
Transplant rejection
Transthyretin (TTR)
Trastuzumab
Trehalose fatty acid esters
Trumenba vaccine
Tuberculosis (TB), booster vaccine
Tumor necrosis factors (TNFs)
U
Umbilical cord blood (UCB)
V
Vaccines, accelerated approval
characterization
clinical trials
development
immunogenicity
manufacturing
Valproic acid (VPA)
Varicella zoster virus
Vascular endothelial growth factor (VEGF)
V(D)J recombination
Viral vaccine
Viral vectors, manufacturing
Virus-like particles (VLPs)
W
Warfarin
Y
Yeast display technique
Z
Zika virus
vaccine
Zinc finger nucleases (ZFNs)
Zoster vaccine
Zymogen plasminogen

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