14.current Applications of Pharmaceutical Biotechnology
14.current Applications of Pharmaceutical Biotechnology
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
Hugo Almeida
UCIBIO, REQUIMTE, MEDTECH, Laboratory of Pharmaceutical
Technology, Department of Drug Sciences, Faculty of Pharmacy, University
of Porto, Porto, Portugal
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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
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.
2.1 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.
2.4 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.
3.2 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.
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.
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.
6.4 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].
Fig. 4 Different binding isotherms. Proteins and biotechnology products often have a Langmuir
shape with a very high slope at low concentration [24]
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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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
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].
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
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].
Humira® Adalimumab Mannitol, polysorbate 80, water for injection Solution for
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
Lantus® Insulin Zinc chloride, metacresol, glycerol, hydrochloric acid, Solution for
sodium hydroxide, water for injections, polysorbate 20 injection
(when stored in vial)
Neulasta® Pegfilgrastim Sodium acetate, sorbitol, polysorbate 20, water for Solution for
injection injection
Fig. 1 Flow diagram of the high-throughput formulation development concept (Adapted from Martinus et al.
[145])
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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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].
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].
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]
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]
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
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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_105
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
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.
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
aBiosimilarmedicine
bMedicine withdrawn
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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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
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
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].
2.2 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
Nutropin AQ® Children with short stature associated with GH [142, 143]
deficiency, idiopathic short stature, Turner
syndrome, and chronic kidney disease
aMedicine withdrawn
bBiosimilar medicine
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
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].
aBiosimilar medicine
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.
3.2 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].
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
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.
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.
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.
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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© 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_117
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
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].
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.
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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A. C. Silva et al. (eds.), Current Applications of Pharmaceutical Biotechnology, Advances in
Biochemical Engineering/Biotechnology 171
https://doi.org/10.1007/10_2019_118
* 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
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.
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].
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.
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.
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.
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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
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
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
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.
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].
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].
4.3 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
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
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© 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_109
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
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
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.
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].
3.2.2 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.
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
Fig. 6 Vectors used in gene therapy clinical trials. Data consulted in gene therapy clinical trials worldwide
[54]
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].
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].
Fig. 9 General structure of adenoviruses and adenoviral genome. ITR inverted terminal repeats, ψ packaging
signal
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]
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.
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.
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
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].
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].
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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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
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
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]
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
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.
5.2.5 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.
5.2.7 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.
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