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Articles Article Archives Removal of Endotoxin from Protein in Pharmaceutical Processes
Removal of Endotoxin from Protein in Pharmaceutical
Processes
Posted: July 29, 2016 Tweet Share 0 Share
Tim Sandle, PhD
Bio Products Laboratory Ltd
Introduction
Bacterial endotoxin is the lipopolysaccharide component of
the cell wall of Gram-negative bacteria, together with other Follow APR
cellular material that combines to form an endotoxin complex.
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Endotoxin is pyrogenic and it presents a risk to patients who
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are administered intravenous and intramuscular preparations.1
Thus bacterial endotoxins pose a risk to many pharmaceutical Your Email Address
processes and, where not controlled, to the finished products.
There are different methods for endotoxin removal. These
include depyrogenation,2 such as dry-heat processes applied Connect with Us
to glassware, and rinsing,3 as might be applied to closures.
These areas receive reasonable coverage within the
pharmaceutical sector. Areas that are actively discussed within
biotechnology fields but which receive less attention in the
wider pharmaceutical context are steps to remove endotoxin
bound to protein. This article considers some of the Related Categories
biotechnological applications for endotoxin removal.
Endotoxin Detection and Testing Equipment »
Pharmaceutical Processing Risks
Limulus Amebocyte Lysate (LAL) Endotoxin
Detection Assays »
In the pharmaceutical industry endotoxin testing of parenteral
drugs is important because lipopolysaccharide is ubiquitous in
Endotoxin Detection and Testing »
waterborne bacterial species and water is the main ingredient
in many parenteral products; water is also used for cleaning. As
Pharmaceutical Microbiology »
an example of the risk, a single Escherichia coli cell contains
approximately 2 million lipopolysaccharide molecules. These
molecules consist of a hydrophobic lipid A moiety, a complex
array of sugar residues and negatively charged phosphate groups. Endotoxins are heat stable, making them resistant
to most conventional sterilization processes and thus necessitating separate tests for viable cells (bioburden) and
endotoxin.
Pharmaceutical processes and equipment are at risk from endotoxin. Hazards stem from human handling, dust,
packaging, contaminated rinse water and microbial growth can all contaminate components with endotoxin.4 The
emergence of multiple drug resistant bacteria has led to patients receiving larger doses of a number of drugs at once,
each contributing to the potential endotoxin load on the patient. As medical technology improves there is also a larger
number of at-risk patients such as the immunosuppressed, premature babies and the elderly, with increased sensitivity
to pyrogens.
Risks not only arise as a result of contamination; they can also occur as a result of the process. With biotechnology, for
example, Gram-negative bacteria, such as E. coli¸ are often used to produce recombinant DNA products like peptides
and proteins. These products are always contaminated with endotoxins and removal steps are required to eliminate
the endotoxin from the product.5
Endotoxin Removal
There are two difficulties that relate to endotoxin removal
from products. The first is that the process applied must
not alter the product during the step of endotoxin
clearance. The second is with the relatively low endotoxin
concentration in presence of the product and the
difficulty in removing bound endotoxin. The binding of
endotoxin can become enhanced through protein
concentration processing steps.
The basis of many techniques for endotoxin removal is the
structure of endotoxin complexes themselves: an
endotoxin molecule possesses hydrophobic, hydrophilic, and charged regions. These features shape a range of
possible interactions with other molecules.6 A secondary means of endotoxin removal uses the weight of the
endotoxin complex.
Two-Phase partitioning
The use of aqueous two phase systems is becoming popular with certain processes, as an alternative to organic-water
solvent extraction systems, partly because the method can generate milder conditions that do not harm or denature
unstable/labile biomolecules. With endotoxin removal, the hydrophobic nature of endotoxin means that two-phase
partitioning serves as an effective endotoxin removal method. This involves the optimization of the conditions leading
to the partitioning of the target protein into one phase while the endotoxin is separated from the product by being
partitioned into another phase.7 Here two-phase systems can be manipulated by altering factors like polymer mass,
pH, ionic strength, and concentration of the phase component or, alternatively, through the addition of affinity
ligands.
Ultra ltration
Since endotoxin molecules tend to form micelles or vesicles in aqueous solution these can be removed from a solution
by filtration. Ultrafiltration (the process works by excluding endotoxin by molecular weight using an ultra-fine filter
which blocks molecules of 10,000 Daltons or greater (the molar mass of an endotoxin monomer varies from 10,000 to
20,000 Da.)8 This process is often coupled with 0.1μm filter for bioburden control. While effective for water, methods
used for decontamination of water, such as ultrafiltration, have little effect on endotoxin levels in protein solutions.9
Chromatography
For many applications, a negative chromatographic technique is the preferred method for endotoxin clearance.10
Affinity chromatographic methods (such as affinity ligands including DEAE sepharose, poly-L-lysine and polymyxin-B)
act to bind endotoxin through biding affinity. In contrast ion-exchange chromatography uses a positive charge to
attract the negatively charged endotoxin and then allowing its elution. Both processes are affected by the pH range;
temperature; flow rate and amount of electrolytes in the solution.11 Modifications include large bead hydrogel-based
methods, including the relatively recent technique of inside-out ligand attachment.12 In addition size-exclusion
chromatography can be considered, although this depends upon the size of the proteins.13 Also considered are
endotoxin-selective adsorber matrices designed for endotoxin removal through endotoxinprotein dissociation.14
Complications can arise, however, through the tendency of endotoxin to form micellular (cellular aggregate)
structures. Thus the success of affinity and ion-exchange chromatography in separating endotoxin from proteins is
affected by the properties of the target protein. With size exclusion resins, for example, the relatively large size of the
micellular form of endotoxin can cause the molecule to function like a larger biological molecule. Furthermore, with
anion-exchange chromatography, the negative charge of endotoxin can lead to interactions with anion exchange
resins, resulting in the co-purification of endotoxins with the other biological molecules. A further downside is that
negatively charged proteins pose the problem of product loss.
Electrophoresis
Although not widely used for endotoxin removal, some researchers have reported success with the application of slab-
polyacrylamide or sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
These methods could be used to separate of lipopolysaccharide from protein.15
Detergents
The dissociation of endotoxin from protein solutions can through the use of non-ionic surfactants in a washing step.16
However, this can present a problem for later purification since the detergent requires removal. The additional step can
lead to some loss of product yield.
Other Puri cation Steps
Less commonly used purification steps for endotoxin removal include ultracentrifugation (devices acceleration as high
as 1 000 000 g) and enzymatic digestion of protein.17 These are not suitable for many processes.
Summary
This article has, within the space provided only surveyed some of the methods of endotoxin removal. Not every
method described will be applicable and, depending upon the product of concern, alternative approaches such as
bespoke endotoxinselective adsorber matrices will be needed for the prevention of endotoxin contamination.18
Remaining with the more common methods described in this article, for approaching endotoxin removal some users
elect to work through the following steps:19
1. The use endotoxin removal resin, which is especially applicable at small scale. If this is unsuccessful then,
2. Use of a cation exchanger to bind endotoxin at a low pH and wash extensively. If endotoxin remain after elution,
then try:
3. Binding to anion exchanger at an alkali pH. If endotoxin remains in the eluate, then:
4. Reduce protein and dissolve in a buffer. Proceed with a cation exchanger and extensive washing. If this is
unsuccessful:
5. Use a buffer containing 0.5% Triton X-100 and try repeated extractions.
Consideration of endotoxin removal steps should form part of a contamination control risk assessment. The types of
removal steps selected will be process dependent. Where hazards are identified and risks cannot be completely
eliminated through endotoxin removal, the critical stages of the process should be subjected to endotoxin testing.
References
1. Caroff, M., Kariban, D., Cavaillon, J. et al. (2002). Structural and functional analyses of bacterial
lipopolysaccharides. Microbes and Infection 4: 915-926
2. Hecker, W., Witthauser, D. & Staerk, A. (1994). Validation of dry heat inactivation of bacteria endotoxins. PDA
Journal of Pharmaceutical Science and Technology 48 (4): 197-204
3. Sandle, T. (2004). Three aspects of LAL testing: Glucans, depyrogenation and water system qualification. PharMIG
News 16: 3-12
4. Williams, K.L. (2001). Endotoxins: Pyrogens, LAL testing and Depyrogenation 2nd Edition. Drugs and the
Pharmaceutical Sciences Volume 111. Marcel Dekker Inc., New York, USA. Chapters 1, 2, 7 & 8
5. Hirayama C, Sakata M. (2002) Chromatographic removal of endotoxin from protein solutions by polymer
particles. J Chrom B 781:419-432.
6. Magalhães PO, Lopes AM, Mazzola PG, et al (2007) Methods of endotoxin removal from biological preparations: a
review, J Pharm Pharm Sci.;10(3):388-404
7. Lopes, M., Magalhães, P. O., Mazzola, P. G. et al., (2010) LPS removal from an E. coli fermentation broth using
aqueous two-phase micellar system, Biotechnology Progress, 26 (6): 1644–1653
8. Anspach FB. (2001) Endotoxin removal by affinity sorbents. Journal of Biochemical and Biophysical Methods
49:665-681.
9. Jang H, Kim HS, Moon SC et al. (2009) Effect of protein concentration and detergent on endotoxin reduction by
ultrafiltration. BMB reports 42: 462–466
10. Koizumi N, Morozumi A, Imamura M, Tanaka E, Iwahana H, Sato R. Lipopolysaccharide-binding proteins and their
involvement in the bacterial clearance from the hemolymph of the silkworm Bombyx mori. Eur J Biochem
1997;248:217–24
11. M.-F. Lin, C. Williams, M. V. Murray, and P. A. Ropp, (2005) Removal of lipopolysaccharides from protein-
lipopolysaccharide complexes by nonflammable solvents, Journal of Chromatography B, 816 (1-2): 167–174
12. Saraswat, M., Musante, L., Ravidá, A. et al (2013) Preparative Purification of Recombinant Proteins: Current Status
and Future Trends, BioMed Research International Vol. 2013, Article ID 312709, http://dx.doi.
org/10.1155/2013/312709
13. Diogo, M. M. Queiroz, J. A., and Prazeres, D.M. F. (2005) Chromatography of plasmid DNA, Journal of
Chromatography A, 1069 (1): 3–22
14. Petsch D, Anspach FB. (2000) Endotoxin removal from protein solutions, J Biotechnol.;76(2-3):97-119
15. Jann B, Reske K, Jann K. (1975) Heterogeneity of lipopolysaccharides. Analysis of polysaccharide chain lengths by
sodium dodecylsulfate-polyacrylamide gel electrophore. Eur. J. Biochem 60:239-246.
16. Reichelt P, Schwarz C, Donzeau M. (2006) Single step protocol to purify recombinant proteins with low endotoxin
contents, Protein Expr Purif.;46(2):483-8
17. Adam O, Vercellone A, Paul F, et al (1995) A nondegradative route for the removal of endotoxin from
exopolysaccharides, Anal Biochem.;225(2):321-7
18. Petsch, D. and Anspach, F. B. (2000) Endotoxin removal from protein solutions, Journal of Biotechnology, 76 (2-3):
97-119
19. Protein Chemist - Endotoxin Removal from Proteins at:
http://www.proteinchemist.com/tutorial/endotoxin.html (accessed July 17, 2016)
Dr. Tim Sandle has over twenty-five years experience of microbiological research and biopharmaceutical
processing. This includes experience of designing, validating and operating a range of microbiological tests including
sterility testing, bacterial endotoxin testing, bioburden and microbial enumeration, environmental monitoring,
particle counting and water testing. In addition, Dr. Sandle is experienced in pharmaceutical microbiological risk
assessment and investigation.Dr. Sandle is a tutor with the School of Pharmacy and Pharmaceutical Sciences,
University of Manchester for the university’s pharmaceutical microbiology MSc course. In addition, Dr. Sandle serves
on several national and international committees relating to pharmaceutical microbiology and cleanroom
contamination control (including the ISO cleanroom standards). He is a committee member of the Pharmaceutical
Microbiology Interest Group (Pharmig); serves on the National Blood Service advisory cleaning and disinfection
committee; and is a member of several editorials boards for scientific journals.Dr. Sandle has acted as a consultant,
expert witness and technical advisor to sterile and non-sterile manufacturing facilities, microbiology laboratories,
the medical device industry and hospitals. Dr. Sandle has also undertaken several technical writing and review
projects.Dr. Sandle has written over four hundred book chapters, peer reviewed papers and technical articles relating
to microbiology. In addition, Dr. Sandle has written several books. Dr. Sandle has also delivered papers to over fifty
international conferences and he is an active journalist.
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Victor Grayson • 3 years ago
Very informative.
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