Mastering Next-Generation Therapeutic Antibody Impurity Removal
eBook
Last Updated: July 17, 2024
(+ more)
Published: July 16, 2024

Credit: Thermo Fisher Scientific
Monoclonal antibodies (mAbs) are a highly sought-after therapeutic, with hundreds already approved for a wide range of clinical uses. This include novel mAb modalities, such as antibody-drug conjugates and bispecific Abs, developed to tackle difficult therapeutic targets.
However, these new modalities present purification challenges that traditional purification methods, such as ion exchange chromatography, struggle to overcome, resulting in higher costs and lower yields.
This eBook presents recent developments in purification and polishing solutions that can help address the challenges in therapeutic mAb downstream processing.
Download this eBook to explore:
- The advantages of mixed-mode and hydrophobic interaction chromatography
- Purification advice from mAb specialists
- How to address high aggregate challenges and streamline workflows
Mastering next-generation
therapeutic antibody
impurity removalForeword
Monoclonal antibodies (mAbs) are fast becoming one of the
most sought after therapeutics, with hundreds approved
for a wide range of clinical uses, from cancers to infectious
disease, in the last few decades.
In order to address more difficult therapeutic targets, novel
antibody modalities are being developed, such as bispecific
mAbs, antibody fragments and antibody-drug conjugates.
However, these new modalities present purification
challenges that traditional methods of purification such as
polishing with ion exchange chromatography struggle to
overcome. This results in higher costs and lower yields at
manufacturing scale.
This eBook explores the recent developments in purification
and polishing solutions that can help address the
challenges in downstream processing.
1 thermofisher.com/antibody-derived-therapeuticsForeword 1
The challenges of therapeutic
monoclonal antibodies 3
Purification strategies for monoclonal antibody production 7
Purification of complex mab modalities: ask the specialists 8
Monoclonal antibody aggregate polish and viral clearance using
hydrophobic interaction chromatography 12
Webinar advert–an innovative approach to addressing high aggregate
challenges in engineered monoclonal antibodies 19
Poster–an innovative approach to addressing high aggregate
challenges in engineered monoclonal antibodies 20
Streamline Manufacturing of Antibody-Based Therapeutics with Novel
Purification Approaches 21
Accelerating antibody drug development affinity chromatography
purification 24
Boost your mab purification workflow with a new mixed-mode resin 25
Resources 26
Contents
2Bioreactor
Protein A
capture Capture
Clarification
Polishing
Low pH VI
Cation
exchange
Anion
exchange
Virus
filtration
Waste products Centrifuge
Cells and cell
debris
HCP, DNA,
fragments
Aggregates,
leached
protein A
HCP, DNA
1 Depth
filtration
2 Depth
filtration
2 Depth
filtration
Figure 1. mAb production process flow diagram. Adapted from
Gillespie et al., 2014.
3 thermofisher.com/antibody-derived-therapeutics
Introduction
Since the development of the hybridoma technology in 1975,
monoclonal antibodies (mAbs) have become increasingly
important in both medical research and disease treatment.¹ The
first clinically available mAb, muronomab CD3, was approved
in 1986 to block cytotoxic T cell function and prevent rejection
in transplanted patients.² Since then, hundreds of mAbs have
been approved for clinical use for the treatment of a wide
range of diseases, from cancers and autoimmune disorders to
infectious diseases such as HIV.3,4
Despite the success of mAb therapeutics, developing novel
antibodies is challenging, as many of the best-understood
targets have already been exploited. Remaining targets for
potential novel mAb therapeutics pose challenges such as
poorly accessible sites of action, low immunogenicity and
significant differences in targets between human and the
species used for preclinical testing.⁵ In order to overcome these
roadblocks, novel mAb-based therapies are being developed.
These include bispecific mAbs capable of binding two different
antigens, antibody–drug conjugates (ADCs), isotype-switched
mAbs and antibody fragments.⁶
As biotherapeutics, mAbs must adhere to strict regulatory
requirements to help ensure the safety and efficacy of the final
product.7,8 Due to their production in cell-based expression
systems, the manufacture of mAbs can result in process- and
product-related impurities. These impurities significantly affect
the quality and safety of the final product. Therefore, mAbs
must go through extensive purification steps before release.
However, as the structural complexity of novel mAbs increases,
the traditional purification methods have become less effective,
resulting in high costs and low yields. Recent developments
in affinity purification reagents and polish solutions promise
to simplify purification of complex mAbs and improve the
efficiency of commercial production of novel mAb therapeutics.
Purifying mAbs for clinical use
In order to meet regulatory guidelines, purified mAbs must have
low levels of both process- and product-related impurities,
while maintaining a reasonable and cost-effective yield.
Process-related impurities include host cell proteins (HCPs),
host cell DNA, leached reagents from other purification steps,
process buffers and residual detergents used for viruses.
The challenges of therapeutic
monoclonal antibodies
Product-related impurities include high and low molecular
weight product aggregates, misformed products, viruses,
endotoxins from bacterial expression systems and posttranslational modifications.⁹
Following mAbs harvesting from cell culture by centrifugation
and filtration, the typical mAb purification process begins with
protein A chromatography (Figure 1). Protein A is covalently
immobilized onto porous resins. As the samples pass through
the column, protein A binds to the Fc region of antibodies at
the consensus binding site (CBS), capturing the mAbs and
resulting in relatively high purity and recovery in a single step.⁹
One of the advantages of protein A chromatography is its
sensitivity to low pH. This enables an acidic elution of bound
antibodies, and a subsequent low pH hold to inactivate any
contaminating viruses (in line with regulatory guidelines for viral
safety).10,11
The efficiency of protein A chromatography means that
downstream polishing steps only need to remove a small
amount of the remaining impurities. The most common of
these polishing steps are based on ion exchange, namely
anion exchange (AEX) chromatography and cation exchange4 thermofisher.com/antibody-derived-therapeutics
(CEX) chromatography, depending on the impurity to be removed.
The net charge of mAbs varies, depending on the relative pH
of the antibody’s environment. Changing the environment
therefore allows the manipulation of mAb charge and enables
the removal of a wide range of differently charged impurities.12
CEX chromatography binds positively charged antibodies and
impurities, which are then separated based on charge difference,
eluting at different ionic strengths. AEX chromatography binds
negatively charged impurities, while positively charged antibodies
are allowed to flow through. It is used for the removal of host cell
DNA, HCPs, bacterial endotoxins and furhter viral clearance.13,14
The final polishing steps in purification to help ensure the product
meets regulatory specifications, using methods such as ion
exchange, or hydrophobic interaction chromatography (HIC).15
The Protein A chromatography workflow is highly efficient
and selective for traditional mAbs, as it removes the majority
of impurities in a single robust step. Both AEX and CEX
chromatography have high loading capacities and can
accommodate both bind-elute and flow-through modes, for
efficient purification at manufacturing scale.
The challenges of traditional
polishing
Despite the successes of current purification and polishing
processes, these methods still show limitations for both traditional
and more complex antibody technologies. Thus, there is a
significant demand for better manufacturing and purification
strategies. Inherent challenges in mAb production begin even
before the purification stages. mAbs are limited to production
in a live-cell expression system, which can be difficult to scale
to a commercial manufacturing level and can produce variable
yields. Historically, mammalian expression systems were used for
full-length antibodies, though recent advances have also allowed
the use of Escherichia coli.16 Emerging technologies using in vitro,
cell-free systems could reduce some of the challenges associated
with live-cell systems (e.g., reducing the amount of cellular debris
and cell-related impurities). However, these methods are not yet
ready for industrial-scale manufacture.17
Extraction of any high-purity product from a complex mixture
such as a cellular expression system is a challenge, but the
development of emerging mAb technologies has complicated
this process further. The diversity of novel mAb structures has
led to increased process- and product-related impurities, for
which traditional methods are not suitable. For example, bispecific
antibodies may express heavy chains (HCs) and light chains (LCs)
from two different parental mAbs. Should these HCs and LCs
mispair to form a homodimer during production, they represent
an additional impurity that must be removed from the final
product, via additional purification steps or specific resins.18 The
complex structure of bispecific mAbs also leads to increased
levels of aggregation and the generation of antibody fragments,
which can prove difficult to remove by conventional means.18
Other novel modalities, such as antibody fusion proteins, bring
their own unique challenges to purification. In this type of
therapeutic, the long half-lives of antibodies are leveraged to
enhance the effectiveness of shorter-lived therapeutic proteins
such as cytokines. However, these too show increased levels
of misformed and mismatched products and aggregates.
In addition, instability in acidic conditions can give rise to
further aggregation and cleavage during the low pH steps of
conventional purification methods.18
For both standard and novel mAbs modalities, traditional polishing
methods can be further optimized for more efficient purification
at almost every stage of the process. In some cases, the type
of chromatography itself can inhibit efficient purification. Despite
its importance for viral clearance, the low pH hold step results
in aggregate formation, exacerbated by the process of protein
A chromatography itself.19 Protein A can leech into the eluted
fraction during the chromatography, both as fragments and fullsize proteins. This results in additional impurities to be removed
from the final product. Furthermore, the protein A fragments can
be even more difficult to remove than the whole proteins by ion
exchange chromatography.20 HIC is often used as an orthogonal
polishing step during mAb purification due to its high aggregate
clearance capability. However, the high concentrations of
kosmotropic salts used during the process require extra washing,
dilution and ultrafiltration steps to remove the salts from the final
product, reducing overall efficiency.21
In addition to limitations in the current processes, the impurities
to be removed can also cause issues. HCPs are a critical impurity
that must be cleared to ensure the safety of the final product.
Although the majority of HCPs can be cleared across multiple
polishing steps, the remaining HCPs can be highly difficult to
remove using traditional means.22 Residual HCPs can decrease
product stability and cause unwanted immune reactions in the
recipients.22 Finally, purification bottlenecks can occur at several
steps in the purification process.thermofisher.com/antibody-derived-therapeutics 5
Optimizing downstream
processing for improved
purification
The challenges of traditional purification and polishing methods
can be overcome through careful optimization and selection of
the most appropriate polishing platforms, ensuring complete
removal of impurities. Ion exchange chromatography is a
common post-protein A step in most polishing workflows to
remove high molecular weight contaminants.9 Ion exchange
chromatography can be employed in both bind-and-elute and
flow-through modes, with the optimal method dependent on
the isoelectric point of the mAb to be purified. A version of AEX
chromatography known as weak partitioning chromatography
(WPC) can remove weakly binding impurities, increasing
product yield.⁹
CEX chromatography has been shown to effectively remove
HCPs, and maximum removal can be assured by selecting
an optimal stationary phase with the chemical and physical
properties best for the impurities and mAb in question.23 To
reduce purification bottlenecks and increase throughput, highcapacity resins can be selected for CEX which may allow the
omission of a rate-limiting protein A capture step.24 Washing,
dilution and filtration steps that increase the time cost of HIC
may be reduced through the use of a salt-free method, using
an extremely hydrophobic resin in the flow-through step without
kosmotropic salts.21
As the technology of novel mAb modalities advances, it is only
fitting that the technology of mAb purification also advances
in step. If optimal yield and efficiency are to be achieved, and
regulatory guidelines for purity are to be met, then traditional
purification methods and resins are no longer the best, or only,
choice. With the wide range of resins now available for all types
of chromatography, use of historical and outdated resins is
no longer necessary. Resin screening studies can be used to
easily select the resin with the highest binding capacity, best
selectivity and best resolution for the antibody in question. For
example, the ideal resin for bispecific antibody purification and
removal of mispaired dimers would consist of a small particle
size combined with large pore diameters for perfusive flow and
mass transfer.18
Further advances in efficiency and purification outcomes
have been achieved by the combination of ion exchange
chromatography with other methods of purification such as
HIC. Known as mixed-mode chromatography, the stationary
phases involved consist of both hydrophobic and charged
functional groups. High levels of aggregates and highmolecular-weight impurities can be removed in a single
step. This eliminates the need for multiple ion exchange
chromatography polishing steps, improving cost and timeefficiency, and helping to ensure a highly pure product, even for
complex mAb modalities.25
Conclusion
Chromatography plays an essential role in therapeutic mAb
manufacture and purification. Until recently, lags in the
development of updated resins and methods have decreased
the efficiency and effectiveness of traditional purification
pathways in the face of novel mAb technologies. However, new
resins and methods, such as mixed mode chromatography,
promise to address the challenges posed by more conventional
methods, enabling more thorough removal of impurities in fewer
steps. Selection of the best resin and platform for superior
mAb purification is crucial, but it doesn’t need to be complex or
confusing. The following articles will outline the tools available
for high-quality mAb purification and polishing, and the novel
technologies that can significantly enhance the efficiency and
economy of mAb manufacture.
References
1. Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of
predefined specificity. Nature. 1975;256(5517):495-497. doi:10.1038/256495a0
2. Todd PA, Brogden RN. Muromonab CD3. A review of its
pharmacology and therapeutic potential. Drugs. 1989;37(6):871-899.
doi:10.2165/00003495-198937060-00004
3. Manis JP. Overview of the therapeutic monoclonal antibodies. UpToDate. Wolters
Kluwer. https://www.uptodate.com/contents/overview-of-therapeuticmonoclonal-antibodies/print. Updated April 10, 2024. Accessed June 11,
2024.
4. Lu R-M, Hwang Y-C, Liu I-J, et al. Development of therapeutic antibodies
for the treatment of diseases. J Biomed Sci. 2020;27(1). doi:10.1186/
s12929-019-0592-z
5. Carter PJ, Lazar GA. Next Generation Antibody Drugs: Pursuit of the “highhanging fruit.” Nat Rev Drug Discov. 2017;17(3):197-223. doi:10.1038/
nrd.2017.227
6. Lyu X, Zhao Q, Hui J, et al. The global landscape of approved antibody therapies.
Antibody Therap. 2022;5(4):233-257. doi:10.1093/abt/tbac021
7. Guideline on development, production, characterisation and specification for
monoclonal antibodies and related products. European Medicines Agency.
2016. https://www.ema.europa.eu/en/documents/scientific-guideline/
guideline-development-production-characterisation-and-specificationmonoclonal-antibodies-and-related-products-revision-1_en.pdf Published
July 21, 2016. Accessed June 11, 2024.
8. Guidance for industry for the submission of chemistry, manufacturing, and controls
information for a therapeutic recombinant DNA-derived product or a monoclonal
antibody product for in vivo use. United States Food and Drug Administration.
https://www.fda.gov/media/77528/download. Published August 1996.
Accessed June 11, 2024.
9. Liu HF, Ma J, Winter C, Bayer R. Recovery and purification process development
for monoclonal antibody production. mAbs. 2010;2(5):480-499. doi:10.4161/
mabs.2.5.12645
10. Jin W, Xing Z, Song Y, et al. Protein aggregation and mitigation strategy in low ph
viral inactivation for monoclonal antibody purification. mAbs. 2019;11(8):1479-
1491. doi:10.1080/19420862.2019.16584936 thermofisher.com/antibody-derived-therapeutics
11. ICH Q5A(R2) Guideline on viral safety evaluation of biotechnology
products derived from cell lines of human or animal origin.
European Medicines Agency. https://www.ema.europa.eu/en/
ich-q5ar2-guideline-viral-safety-evaluation-biotechnology-productsderived-cell-lines-human-or-animal-origin-scientific-guideline. Published
December 14, 2023. Accessed June 11, 2024
12. Schneider Z. Importance of isoelectric point (pI) of antibodies. The Antibody
Society. https://www.antibodysociety.org/new-articles/importanceisoelectric-point-pi-antibodies. Published June 28, 2017. Accessed June 11,
2024.
13. Chahar DS, Ravindran S, Pisal SS. Monoclonal antibody purification and its
progression to commercial scale. Biologicals. 2020;63:1-13. doi:10.1016/j.
biologicals.2019.09.007
14. Curtis S, Lee K, Blank GS, Brorson K, Xu Y. Generic/matrix evaluation of SV40
clearance by anion exchange chromatography in flow‐through mode. Biotechnol
Bioeng. 2003;84(2):179-186. doi:10.1002/bit.10746
15. Arora I. Chromatographic Methods for the purification of monoclonal antibodies
and their alternatives: A Review. Int J Emerg Techn Adv Eng. 2013;3(10):475-481.
16. Rashid MH. Full-length recombinant antibodies from Escherichia coli: production,
characterization, effector function (FC) engineering, and clinical evaluation. mAbs.
2022;14(1). doi:10.1080/19420862.2022.2111748
17. Sifniotis V, Cruz E, Eroglu B, Kayser V. Current advancements in addressing
key challenges of therapeutic antibody design, manufacture, and formulation.
Antibodies. 2019;8(2):36. doi:10.3390/antib8020036
18. Chen SW, Zhang W. Current trends and challenges in the downstream purification
of bispecific antibodies. Antib Ther. 2021;4(2):73-88. doi:10.1093/abt/tbab007
19. Mazzer AR, Perraud X, Halley J, O’Hara J, Bracewell DG. Protein a
chromatography increases monoclonal antibody aggregation rate during
subsequent low ph virus inactivation hold. J Chromatogr A. 2015;1415:83-90.
doi:10.1016/j.chroma.2015.08.068
20. Carter-Franklin JN, Victa C, McDonald P, Fahrner R. Fragments of protein a eluted
during protein a affinity chromatography. J of Chromatogr A. 2007;1163(1-2):105-
111. doi:10.1016/j.chroma.2007.06.012
21. Ghose S, Tao Y, Conley L, Cecchini D. Purification of monoclonal antibodies
by hydrophobic interaction chromatography under no-salt conditions. mAbs.
2013;5(5):795-800. doi:10.4161/mabs.25552
22. Ito T, Lutz H, Tan L, et al. Host cell proteins in monoclonal antibody processing:
Control, detection, and removal. Biotechnol Progr. 2024;e3448. doi:10.1002/
btpr.3448
23. Bruch T, Graalfs H, Jacob L, Frech C. Influence of surface modification on protein
retention in ion-exchange chromatography. J Chromatogr A. 2009;1216(6):919-
926. doi:10.1016/j.chroma.2008.12.008
24. Tao Y, Ibraheem A, Conley L, Cecchini D, Ghose S. Evaluation of high‐capacity
cation exchange chromatography for direct capture of monoclonal antibodies from
high‐titer cell culture processes. Biotechnol Bioeng. 2014;111(7):1354-1364.
doi:10.1002/bit.25192
25. Mixed-mode chromatography. ThermoFisher Scientific. https://www.
thermofisher.com/uk/en/home/life-science/bioproduction/poroschromatography-resin/bioprocess-resins/mixed-mode-chromatography.
html. Accessed June 11, 2024.Monoclonal antibodies (mAbs) are becoming an increasingly
prominent therapeutic agent which can be engineered to treat a wide range of diseases and
disorders, from cancers to autoimmune
diseases.1 Consequently, many developers have multiple mAbs in their pipeline,
increasing the demand for rapid,
efficient purification processes.
Purification of mAbs is a complex,
multistep process. After initial
harvesting and filtration steps,
mAbs undergo capture, affinity
purification and polishing steps to
remove impurities.
However, the complexity of engineered
mAbs can hinder the purification
process, creating mis-formed
products, antibody fragments
and aggregates. Failure of the
process requires modifications
to both the capture step and
the polish strategy.
This infographic will explore
the modifications needed for an
optimized platform process, focusing
on impurity removal and the Thermo
Fisher Scientific solutions available.
As the complexity and sophistication of mAb therapies increases, structural features such as symmetric,
asymmetric and fragment-based bispecifics are becoming more common, resulting in new purification
challenges. The traditional polishing process can have limited success for these mAbs, resulting in
incomplete impurity removal, and risks non-adherence to regulatory guidelines.
Alternative processes, such as hydrophobic interaction chromatography (HIC) and mixed-mode
chromatography (MMC) are emerging as more efficient platforms for more modern, engineered mAbs.
PURIFICATION
STRATEGIES
FOR
MONOCLONAL
ANTIBODY
PRODUCTION
Typically, an affinity step and two polish steps are needed to remove a range of impurities:
A typical purification process consists of three steps: affinity chromatography, often using Protein A, followed by
cation exchange (CEX) then anion exchange (AEX).
Productrelated
impurities (e.g.,
aggregates,
mis-formed
product, dimers,
post-translational
modifications)
Viruses
Host cell
proteins
(HCPs)
DNA
Endotoxins
(from bacterial
expression
systems)
Affinity
Antibody isolation
HCP reduction
Thermo ScientificTM
CaptureSelectTM
Affinity Resins
HIC
• HCP reduction
• Leached ligand removal
• Aggregate, fragments
• Product related isoforms
AEX as needed
• Final HCP removal
• DNA removal
• Virus removal
AEX
• Final HCP removal
• DNA removal
• Virus removal
Thermo ScientificTM
POROSTM Caprylate
Mixed-Mode Cation
Exchange Resins
Thermo ScientificTM
POROSTM AEX Resins
• POROS XQ
• POROS 50 HQ
• POROS 50 D
• POROS 50 PI
Thermo ScientificTM
POROSTM AEX Resins:
•POROS XQ
• POROS 50 HQ
• POROS 50 D
• POROS 50 PI
Thermo ScientificTM
PorosTM CEX Resins:
• POROS XS
• POROS HS
CEX as needed
• Final HCP removal
• DNA removal
• Virus removal
Thermo ScientificTM
POROSTMHIC Resins:
• POROS ETHYL
• POROS BENZYL
• POROS BENZYL
ULTRA
Mixed Mode-CEX
• HCP reduction
• Leached ligand removal
• Aggregate, fragments
• Product related isoforms
References:
1. Quinteros DA, Bermúdez JM, Ravetti S, Cid A, Allemandi DA, Palma SD. Therapeutic use of monoclonal antibodies: General
aspects and challenges for drug delivery. Nanostruct Drug Deliv. Published online 2017:807-833. doi:10.1016/b978-0-323-
46143-6.00025-7
Thermo ScientificTM
CaptureSelectTM
Affinity Resins
High purity and
yield in a single step
Affinity
• Antibody isolation
• HCP reduction
Thermo ScientificTM
POROSTM CEX
Resins:
Cation Exchange
Removal of:
Complex mAb
platform process
Challenging mAb
platform process
Thermo ScientificTM
POROSTM AEX
Resins:
Anion Exchange
For removal of:
• Final HCP
• DNA
• Viruses
The POROS Caprylate Mixed-Mode Cation Exchange Resins have a superior pore structure for rapid mass transport and
unique selectivity. This can help increase productivity, purity and yield in flow-through modes.
Wide operating range
80–90% mAb monomer
recovery with minimal
optimization
Cost-effective
due to reduced
chromatography
steps and reagents
Linear pressureflow curve results
in excellent and
predictable scalability
High aggregate
selectivity, effective up
to 20% aggregation
Improve your polish process for engineered mAbs with
Thermo Scientific™ POROS™ Caprylate Mixed-Mode
Cation Exchange Resin
MMC
Typically combines ion exchange and
hydrophobic interaction principles for improved
selectivity and resolution of impurities.
Able to purify diverse populations
of proteins.
Removes high levels of aggregates and a
wide range of impurities.
Suitable for bind-elute and flow-through
chromatography.
HIC
Removes proteins based on interactions
between hydrophobic ligands on the resin with
hydrophobic motifs on proteins.
Salt buffer conditions promote binding and
stabilize mAb structure.
Removes high levels of aggregates and a wide
range of impurities.
Suitable for bind-elute and flow-through
chromatography.
Removal of impurities
A traditional mAb downstream platform process
Typical process steps
Optimizing polish strategies for complex mAbs
Complex mAb process steps
The advantages of Mixed Mode-CEX resins
© 2024 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. This information is not intended to
encourage use of these products in any manner that might infringe the intellectual property rights of others.
Pharmaceutical Grade Reagent. For Manufacturing and Laboratory Use Only.
Affinity chromatography
Captures proteins based on reversible
protein/ligand interactions, where the ligand
is coupled to a chromatography resin.
Cation exchange
Captures positively charged mAbs and
impurities. Captured mAbs are then eluted by
increasing conductivity or increasing buffer pH.
Anion exchange
Removes negatively charged product and
process-related impurities. It can be used in
both flow-through and bind-elute modes.
Discover more
• HCP
• Leached ligand
• Aggregates and
fragments
• Product-related
isoforms
• POROS XS
• POROS 50 HS
• POROS XQ
• POROS 50 HQ
• POROS 50 D
• POROS 50 PI8 thermofisher.com/antibody-derived-therapeutics
Jett Appel
Field Application Scientist,
Thermo Fisher Scientific
Jett Appel has been a Purification Field Applications Scientist at Thermo Fisher Scientific
since 2021, supporting purification development and scale-up for monoclonal antibodies,
viral vectors, nucleic acids, and recombinant proteins. Prior to joining Thermo Fisher,
Jett worked at Avid Bioservices, a contract development manufacturing organization
(CDMO) based in Orange County, CA. During his 5 years of working at Avid, Jett first
worked in process development and eventually transitioned to a senior engineer role in
the Manufacturing Sciences & Technology (MSAT) team, where he supported purification
development and technology transfers for monoclonal IgGs, IgMs, bispecific antibodies, scFvs, and recombinant enzymes up to
2000 L bioreactor scale. Jett received a bachelor’s degree in Chemical and Biomolecular Engineering from UCLA in 2016. While
at UCLA, he was involved in epigenetics research at the Steve Jacobsen Lab, which included studying the pathways involved in
RNA-directed DNA methylation in Arabidopsis thaliana.
Nicholas Bardol
Manager, Manufacturing Services,
Thermo Fisher Scientific
Nicholas has been working in the pharmaceutical industry for the past 13 years as a
process development scientist focusing on biologics purification and small molecule
conjugation. His work at CDMOs has supported over a dozen novel molecules
through GMP Manufacturing and FDA filings. He joined the Saint Louis, MO, Thermo
Fisher Scientific site supporting Pharma Services location in 2018 as a member of the
downstream process development group.
Kelly Flook
Senior Manager, Product Management,
Purification Products, Thermo Fisher Scientific
Kelly Flook is a dynamic product manager specializing in downstream purification
of biotherapeutics and new product development. With over 15 years’ experience in
product development Kelly has an excellent track record of bringing the right product
to the market. She started her career as a scientist specializing in the development of
polymer based analytical columns for HPLC before moving into purification R&D and then
product management. Kelly went to school in the UK, receiving a bachelor’s degree in
Analytical Chemistry from University of Northumbria, Newcastle, and a PhD in Polymer Chemistry from University of Durham.
Purification of complex mab modalities:
ask the specialists
As the complexity of novel mAb modalities increases, traditional purification and polishing processes may
no longer be effective. Outdated resins and archaic polishing methods can decrease the efficiency and
effectiveness of mAb purification, while new mAb structures can also bring new, difficult to remove impurities.
We asked three in-house specialists at Thermo Fisher Scientific for their advice on how to overcome the
challenges of purifiying complex mAbs, and how to optimize your polishing strategies for maximum efficiencythermofisher.com/antibody-derived-therapeutics 9
Q: What are the main challenges faced in the
purification of complex mAbs compared to
traditional monoclonal antibodies?
Jett Appel (JA): It’s hard to pinpoint the main challenges
because it depends a lot on antibody structure – and antibody
structures are diverse, creating diverse challenges. Often, when
we talk about complex mAbs, we are talking about bispecific or
multispecific antibodies, antibodies where the Fc domain – or
even antibody fragments, such as Fabs or scFvs – are modified.
So, for example, you may not be able to utilize traditional affinity
steps for purification, or it could result in product impurities
that are harder to separate. So, good yield or purity tends to be
obtained differently from these different complex mAbs.
Nicholas Bardol (NB): An increasing number of drug
candidates are bispecific, which are very difficult to separate,
because bispecific expression systems produce a lot of parent
mAb fragments and paratopes as impurities. Most fragments
not only share a lot of the primary characteristics we would
usually exploit in traditional purification strategies, but they can
also be more biologically active. So if they outcompete the drug
candidate for the epitope they’re targeting, then the efficacy of
the drug candidate really diminishes.
Kelly Flook (KF): Because structures are growing more
and more complex, heterogeneity and aggregation become
increasingly challenging. However, it’s not just complex mAbs
that are suffering from increased aggregation levels. As the
industry moves towards more cost-efficient mAb production,
higher cell densities and processes to increase titer can impose
additional challenges downstream with respect to process- and
product-related impurities.
Q: How do the structural characteristics of
complex mAbs impact the purification process
and pose unique challenges?
JA: It depends on the exact structure of the complex mAb.
Bispecific or multispecific antibodies often have different
heavy chains or light chains and this can result in mispairing.
Consequently, these antibodies are very similar in physicochemical
properties and can be hard to separate or may get co-purified with
traditional Protein A affinity steps.
Other formats of complex mAbs – for example, antibodies that
have been multimerized – are much larger than traditional mAbs.
This can result in steric hindrance or lower diffusivities. That’s a
unique challenge because it may lead to lower resin capacities and
therefore larger columns, which could ultimately lower productivity
as scale increases.
NB: In the case of antibody–drug conjugates, they’ve been
modified specifically to take on a drug linker, and that can pose
many challenges – we see issues with free thiol or unbound drug
linker. In the case of bispecifics, it’s hard to use charge-based
separation because the sequence of impurities will overlap a lot with
the sequence of the target antibody.
Just as importantly, we may not be able to use our typical polishing
strategies of anion and cation exchange. Instead, we have to
rely more on things like multimodal or hydrophobic interaction
chromatography (HIC) as a different tool to target another
characteristic of those impurities and see if we can remove them
with a different mechanism than one the industry typically relies on.
Q: What are some of the common impurities or
contaminants encountered during the purification
of complex mAbs? And how are they typically
addressed?
JA: Some common impurities overlap with traditional mAbs –
e.g., host cell proteins, residual DNA and aggregates. Often in
these complex mAbs, we tend to see higher levels of aggregates
because as these structures are engineered the impurities are
either produced more in the cell culture itself or can be generated
under traditional mAb purification parameters, such as exposure
to low pH conditions. The complex mAbs can be less stable and
form impurities inherently within the process.
Other common impurities can be mispaired homodimers, for
example. They’re typically addressed through a traditional ion
exchange to separate the target product from these impurities.
But, as Nick was alluding to earlier, this can often be a
challenge. So, you may need to implement HIC or multimodal
chromatography as well.
NB: I already alluded to impurities that we see with unbound drug
linker or bispecific isoforms, but the biggest one that we deal with
is host cell protein (HCP). In some of the newer mAbs, there is a
lot of change in the pI of the target antibody, and it can overlap
with the HCP pI. Traditionally, antibodies have high pI relative to
the HCPs, making charge-based separation very straightforward:
you bind up your antibody, and flow through all the HCP. But,
suppose the target is sitting right in the middle. Then you either
need multiple steps to capture and resolve everything on either
side of the pI or you have to look at another option like multimodal
chromatography or HIC again, just to get rid of those impurities
that have historically been relatively simple to remove.
KF: Many manufacturers have developed platform purification
workflows for their traditional mAbs, but these don’t always
work for their more complex molecules. This is when you
need to turn to HIC or multimodal chromatography. For
affinity purification, although Protein A continues to be the
workhorse, it’s not always the best option, depending on the
binding domains of the specific antibody. This is where our
CaptureSelect affinity resin product line comes in.10 thermofisher.com/antibody-derived-therapeutics
Q: Could you discuss any specific strategies
or technologies employed to overcome the
challenges associated with complex mAb
purification?
JA: We’ve seen customers implementing unique affinity
technologies. Typically, we’ve utilized traditional protein A
affinity chromatography but we can see that, depending
on how these complex mAbs are engineered, other affinity
ligands that target different domains of the antibody could
be advantageous. For bispecifics, we’ve seen engineered
antibodies where one arm doesn’t have a CH1 domain. So,
for example, you could utilize an affinity resin that targets
CH1. That homodimer won’t bind to the resin and will just flow
through instead of getting co-purified with protein A.
Another interesting strategy that customers use is to leverage
the avidity of the target molecule or the impurity to the resin
and utilize pH gradients. This is used, depending on the
antibody format, when there is more than one specific domain,
or if the target molecule or the impurity has fewer domains
than the other. Where traditional operating conditions for
polishing steps work for standard mAbs, they may not work
for complex mAbs. These may then need a unique set of
conditions; for example, operating cation exchange under a
more alkaline condition may help improve resolution. As Nick
mentioned earlier, other technologies – such as multimodal
chromatography or HIC – can be a useful tool if the standard
methods do not work.
KF: Our ion exchange resins have high capacity and robust salt
tolerance to be able to handle higher titers in bind-and-elute
mode. Using a flowthrough process, your capacity becomes
capacity for the impurity, which is typically <5% at this point,
meaning you can significantly improve productivity and reduce
resin consumption.
NB: I want to highlight a couple of things both upstream and
downstream of purification. The industry has been chasing high
titers in their clone selection for as long as biologics have been
around, but choosing the clone with the highest titer may not be
the best strategy. Instead, choosing the cell line with the lowest
levels of a complex impurity might be the best mechanism. So
be very forward when thinking about clone selection.
Another thing is analytical strategies. A lot of these new
impurities come with bispecifics and isoforms that share the
same charge; using all of the traditional analytical techniques,
you might not get total resolution. They may co-elute and you
won’t know the contaminant is present until the end stages,
or you may chase something that isn’t there. So, looking at
orthogonal assays for size or isoform pattern and charge
heterogeneity can be hugely beneficial. Some less commonly
used techniques can be really powerful here; using 2D-LC and
native mass spec to see those hidden isoforms can save a lot
of time when you’re trying to develop your purification strategy.
Q: Are there any particular purification
techniques or methods that have shown
promising results in effectively purifying
complex monoclonal antibodies?
JA: Utilizing innovative affinity technologies that target different
domains can be useful to leverage the unique structures of these
complex mAbs. That’s one technique that has shown promise.
Innovations looking at different optimization conditions for
polishing steps and unique polishing resins can be a useful
strategy as well. A lot of these complex mAbs may not be
as stable and can form higher-level aggregates, therefore
the ability to operate a wider set of conditions and separate
species using resins that might be more selective for different
product species can be useful.
NB: Affinity capture is the big space where we’re seeing major
changes and alternative mechanisms for purifying complex
mAbs. For bispecifics, they have both a lambda and kappa
light chain, which is something that you can target – you
can capture one side, then capture the other side. That’s a
perfect mechanism to remove all of the fragments of the parent
antibodies that would otherwise need convoluted polishing
strategies to remove.
KF: We recently launched the new POROS Caprylate, our
first mixed-mode cation exchange resin. As many may know,
caprylic acid is used to pull down high levels of aggregates
upstream. Attaching this to the bead allows aggregation to be
effectively removed downstream, reducing aggregation from
20% to less than 2% without the need to then remove the
caprylic acid. An added benefit is that this resin was designed
specifically to be used in flow-through mode to allow high
recovery over a wider range of loads compared to other resins
positioned for aggregate removal in flow-through mode.
Q: How do the scalability and throughput of
complex mAb purification processes compare
to traditional mAbs? And what considerations
need to be taken into account?
JA: It depends on the antibody and the specific challenges
present. Some of these complex mAbs can have lower
capacities, so you may need to utilize resins at lower loading
densities or, in order to achieve appropriate resolution, operate
at lower flow rates. Ultimately, this can lead to larger columns
as you scale up, increased buffer consumption and longer
processing times, which can reduce efficiency.
Therefore, it’s important to optimize early on and utilize different
strategies – e.g., using conditions that allow you to operate at
faster processing times and higher capacities to reduce that
burden as you scale up more complex mAbs.
NB: We’ve seen a need to prioritize resolution, and we
were seeing lower loading on columns through the entirethermofisher.com/antibody-derived-therapeutics 11
downstream process as we tried to get higher resolution. This
can mean more unit operations in manufacturing and taller
polishing columns that are more difficult to work with, as well
as big increases of buffer volumes and longer residence times.
So, the variations may become a little more complicated to
transfer and scale up. To alleviate these issues, many programs
are using very specific pH ranges or non-traditional buffers
(e.g., Good’s buffers such as MES or bicine) that we haven’t
seen used on a manufacturing scale historically. Pressuretolerant resins can also be very beneficial – you can get the
tall bed heights and the high resolution that you need. New
inline dilution strategies to make the buffer volumes more
manageable are also hugely beneficial when you’re scaling up.
Q: What role does process optimization play in
improving the efficiency and yield of complex
mAb purification? And what are some key
factors to consider during optimization?
JA: Process optimization plays a critical role because your goal
is to maximize purity and recovery and still maintain the quality
of the product. But you also want to ensure that it’s scalable,
robust and overall low-cost once you have a fully scaledup process. Monitoring the process is important to better
understand how it operates – ensuring that you’re not operating
at the edge of failure and identifying the parameters that could
impact your process
NB: Complex mAbs tend to have lower bioreactor titers overall
because they are more difficult to grow, so even a couple of
percent gain can be meaningful. Relying more heavily on DOEs
and quality by design frameworks can help labs to get the
most information possible with the least material, so that those
optimization strategies can be executed with just one batch.
Q: What are the potential future advancements
or innovations in complex mAb purification that
could address existing challenges and further
improve the process?
JA: Firstly, the continuing advancements of different affinity
technologies. Different affinity ligands target different domains
– so designing new ligands that are specific for different
domains or have different characteristics that can be leveraged
for separating difficult-to-resolve impurities in these complex
mAbs is important.
Another interesting advancement is the use of chromatography
modeling software. Mathematical models can reduce the
number of DOE experiments required and help us have a
better understanding of the process. We’re even starting to see
customers utilize AI or machine learning techniques for their
large datasets of different antibody sequences and structures.
AI can look at these datasets and use pattern identification to
predict the behavior of different chromatographic resins.
NB: The other thing that we’re seeing customers move towards
is process analytical technologies. Not all of these mAbs can
be tightly controlled just by looking at a UV signal. Having
inline or online detection or mass-based detection – instead
of just looking for chromophores that may not exist or may
completely overlap with your antibody – has real potential to
solve challenging separation issues that we may not be able to
resolve at scale.
KF: Achieving high purity is all about having the right resin for
your molecule. Chromatographic efficiency can be achieved
by reducing the particle size, but that comes with the expense
of flow rate or bead height due to the pressure limitations of
hardware. Modulating or designing the right chemistry or ligand
on the surface to be selective between target and impurities
is a more effective way to increase purity and yield. But as
molecules become more complex, this means more tools are
needed across different variations from the traditional mAb.
mAb developers can engineer in things like a C-tag to enable
selective removal or a specific sequence that interacts with
specific ion exchange or HIC groups.12 thermofisher.com/antibody-derived-therapeutics
Monoclonal antibody aggregate polish
and viral clearance using hydrophobic
interaction chromatography
cthermofisher.com/antibody-derived-therapeutics 13
T h p m clin leeardyaieipcci daelnuaietnmicriepmmvaopcoltnouriotntciaolnonent,lapa ma dlsarsktnuin itncibod hgthsaiesgiepon ni se(rcMscooalAonnbgatsyliz ), headve
immunology, neurology, and infectious diseases. To
date, approximately 100 MAbs have been designated
as drugs, and the rate of approvals is increasing
rapidly. MAbs currently are a $100 billion industry,
and their demand is likely to be high and continue to
dominate the biologics market for years to come.
As a result of such increasing demand, the past
three decades have seen signicant improvements to
the productivity of large-scale MAb
biomanufacturing. Resin manufacturers invested
greatly in developing better polishing
chromatography solutions to clear residual
impurities, provide additional virus clearance, and
ultimately meet critical quality attributes (CQAs)
required for antibody drug products. For MAb
processes, at least one anion-exchange (AEX) polish
step typically is required. Depending on a specic
residual impurity prole and process challenges
being addressed, an AEX step can be preceded or
followed by a second polish step, typically using
either cation-exchange (CEX) or hydrophobicinteraction chromatography (HIC).
The ultimate goal of downstream purication is
to reliably and predictably produce a safe drug
product suitable for therapeutic use in humans. To
this end, biomanufacturing process- and productrelated impurities such as host cell proteins (HCPs)
from upstream cell culture, residual DNA, leached
protein A from the anity step, process leachables
and extractables, adventitious and endogenous
viruses, endotoxins, antibody aggregates, and other
antibody variants all must be removed to acceptable
levels in conformance with regulatory guidelines.
Among all the impurities mentioned, aggregate
removal can be especially challenging if levels are
high. The FDA generally recommends that MAb
aggregates be reduced to <1% for later phase clinical
campaigns. For reference, a typical MAb process will
start from 1–5% aggregates aer protein A capture,
but this percentage can be >10% for certain
challenging MAbs. To address the need for aggregate
clearance in MAb downstream processing, Thermo
Monoclonal antibody aggregate
polish and viral clearance using
hydrophobic-interaction
chromatography
John J. Li, Moira Lynch, and David Cetlin
MAb A Clinical Process: Mixed-mode in bind–elute mode,
monomer recovery 90%, 25 g/L resin loading, 6-minute
residence time
MAb B Clinical Process: Cation-exchange in bind–elute
mode, monomer recovery 65–70%, 40–45 g/L resin
loading, 6-minute residence time
Figure 1: MAb A and MAb B were run on the UltiMate™
3000 HPLC system using MAbPac™ SEC-1 LC column,
with run conditions of isocratic elution using 50 mM
sodium phosphate pH 6.8, 250 mM NaCl. Aggregate
and monomer peaks are highlighted. Both MAb A and
MAb B contain high aggregate levels ~7–12%. The
clinical processes developed for MAb A and MAb B
aggregate polish are highlighted in the box below.
Absorbance (mAu)
Time (min)
500
400
300
200
100
0 0
.0 5.0 10.0 15.0
MAb A
MAb B
Monomer
~10% Aggregate
H2L3
Dimer
HMW14 thermofisher.com/antibody-derived-therapeutics
SPONSORED NOVEMBER–DECEMBER 2019 17(11–12)Si BioProcess International 3
Fisher Scientific offers a wide range of polish
purification tools based on POROS through-pore resin
technology. POROS HQ and XQ are AEX resins and
POROS HS and XS are CEX resins with different
characteristics and selectivity that can be leveraged
to remove low-to-moderate levels of MAb aggregates
(1, 2). Herein, we discuss a family of POROS HIC
resins with novel ethyl and benzyl chemistries for
the successful polish of two challenging drug
products: MAbs A and B, both with high levels of
aggregates of >10% (Figure 1). In addition to
aggregate clearance, viral clearance strategy on
POROS HIC for MAb A and MAb B processes is
discussed extensively, including a novel prediction
technique that uses parvovirus surrogate mock virus
particles (MVPs) from MockV Solutions as well as
live viral clearance data using xenotropic murine
leukemia virus (XmuLV) and minute virus of mice
(MVM).
POROS HIC CASE STUDY: TWO CLINICAL
MABS REQUIRING AGGRESSIVE STRATEGY
FOR AGGREGATE CLEARANCE
MAb A and MAb B are two clinical-stage MAbs with
difficult downstream processes. Significant
aggregates remained after protein A capture and AEX
flowthrough (FT) polish, requiring an additional
polishing step. For this second polish step, MAb A
uses a mixed-mode bind–elute (BE) process, with low
resin load density, slow flow, and high yield,
whereas MAb B uses a cation-exchange BE process,
with high load density, slow flow, and low yield
(Figure 1).
We asked the biologics company whether we could
design and develop more productive processes for
both MAb A and MAb B using POROS HIC resins and
compared our approaches with existing, optimized
clinical processes. For MAb A, we designed and
verified two strategies: one using POROS Benzyl resin
in BE mode and the other using POROS Benzyl Ultra
resin in FT mode. Our processes were four times and
12 times more productive than the original mixedmode process, respectively (Table 1). For full process
development details, please refer to our previous
publications (3). For MAb B, we optimized a POROS
Benzyl Ultra BE process that improved yield
significantly and shortened residence time compared
with the existing clinical CEX process (Table 2).
MAb A Impurity Clearance and Viral Clearance
Study: In a separate study, we performed highthroughput screening for MAb A and demonstrated
Table 1: Overall comparison of the clinical mixed-mode
process for MAb A compared with the two POROS HIC
processes; process performance metrics have been
improved, resulting in ~4× and ~12× higher productivity.
MAb A
Process
Mixed-Mode
BE (Clinical)
POROS Benzyl
BE Resin
POROS
Benzyl Ultra
FT
Load monomer
purity (%)
90 89 89
Load density
(g/L resin)
25 32 120
Monomer
purity pool (%)
99 99 >99
Monomer
recovery (%)
90 >99 98
HCP (ppm) NA 120–12 ppm 100–35 ppm
Residence time
(min)
6 2 1.2
Pool volume
(50–50 mAu)
5 CV 4 CV NA
Productivity
(g/L/h)
7 27 89
Figure 2: POROS Benzyl Ultra FT viral clearance (MAb A); complete XmuLV and minimal MVM clearance
Log Clearance
40-mg/mL FT 80-mg/mL FT
Mock Pool
120-mg/mL FT
Mock Pool
Water Aggregate
Strip
Hold Control
XmuLV (log clearance)
MVM (log clearance)
6 5 4 3 2 1 0
Column: 0.5 × 5 cm, CV = 1 mL Load buffer: MAb A at 2.5 mg/mL (Tris pH 7, 2 mS/cm) Flow rate: 0.5 mL/min
Residence time: 2 min Load density: Up to 120 mg/mL resinthermofisher.com/antibody-derived-therapeutics 15
4 BioProcess International 17(11–12)si NOVEMBER–DECEMBER 2019 SPONSORED
enhanced selectivity of POROS Benzyl and POROS
Benzyl Ultra resins for clearing MAb A aggregates
using sodium citrate (3). Compared with results from
the clinical mixed-mode process, POROS Benzyl resin
in BE mode demonstrated higher load capacity and
lower residence time (from six to two minutes) with
10% higher monomer recovery, while maintaining
aggregate and HCP clearance performance (Table 1).
Overall productivity of the process increased about
fourfold while achieving product CQAs.
We then optimized an even higher productivity
process using POROS Benzyl Ultra resin in FT mode
with similar impurity clearance ability (Table 1). A
<2-minute residence time combined with a resin load
density >100 g/L resulted in a 12-fold increase in
productivity. Moreover, using a HIC FT process has
the added benefit of a robust AEX-HIC straightthrough process design with no additional
conductivity or pH adjustments and no intermediate
hold up required between AEX and HIC (data not
shown).
In addition to the impurity clearance study, we
tested the ability of the optimized MAb A POROS HIC
BE and FT processes to clear retrovirus XmuLV and
parvovirus MVM. In the POROS Benzyl Ultra FT
process, we observed complete clearance of XmuLV
(>4 log) and minimal clearance of MVM (<1 log) up to
120 g/Lr load at 2-minute residence time (Figure 2).
XmuLV is more hydrophobic than MVM and thus
bound tightly to the resin in FT mode. MVM is less
hydrophobic and did not bind. It is interesting that a
water strip was insufficient to remove bound XmuLV
from POROS Benzyl Ultra resin.
In the POROS Benzyl BE process, MAb A load was
bound at high salt levels, and we performed specific
monomer elution with 260 mM sodium citrate. We
observed complete clearance of XmuLV (>4 log) and
partial clearance of MVM (1.5 log) in the elution
fraction. We then performed sequential washes of
decreasing salt concentrations to 130 mM sodium
citrate, then buffer alone where bound aggregates
eluted, followed by a water strip and finally a 1M
arginine strip. It is interesting to note that water and
arginine but not buffer alone were sufficient to
remove the highly hydrophobic XmuLV from POROS
Benzyl resin. The differential stripping behavior for
XmuLV is consistent with the relatively less
hydrophobic character of POROS Benzyl resin
compared with POROS Benzyl Ultra resin.
Table 2: Comparing the clinical cation-exchange bind–
elute process for MAb B with the POROS Benzyl bind–
elute process; POROS Benzyl resin achieved complete
clearance of a difficult H2L3 aggregate species while
improving yield by 20% and decreasing residence time
threefold.
MAb B
Process
CationExchange BE
(Clinical)
POROS Benzyl
Ultra BE Resin
POROS Ethyl
BE Resin
Load monomer
purity (%)
90 90 NA
Load density
(g/L resin)
40 40 18
Monomer
purity pool (%)
>98 >98 NA
H2L3 (%) <1 <1 NA
Monomer
recovery (%)
>65 >85 NA
Residence time
(min)
6 2 NA
Pool volume
(50–50 mAu)
4 CV 4 CV NA
Figure 3: MAb A POROS Benzyl bind–elute process viral clearance study; complete XmuLV and modest MVM
clearance
Log Clearance
Tris pH 7.0
260 mM
Sodium Citrate
(Monomer Elution)
XmuLV (log clearance)
MVM (log clearance)
Column: 0.8 × 10 cm, CV = 5 mL
Load buffer: MAb A at 3 mg/mL (Tris pH 7, 575 mM Na Citrate)
Flow rate: 2.5 mL/min Residence time: 2 min
Load density: 32 mg/mL resin
Tris pH 7.0
130 mM
Sodium Citrate
(Exploratory Wash)
Tris pH 7.0
0 mM
Sodium Citrate
(AggregateSolution)
Water Strip 1 M Arginine
pH 8.5 Strip
Hold Control
4.5
4
3.5
3
2.5
2
1.5
1016 thermofisher.com/antibody-derived-therapeutics
SPONSORED NOVEMBER–DECEMBER 2019 17(11–12)Si BioProcess International 5
MAb B Impurity Clearance and Viral Clearance
Study: MAb B also was difficult to polish from an
aggregation standpoint, with >5% aggregation
consistently post AEX-FT and a highly unique
cysteine-mediated H2L3 aggregate species (one light
chain more than the monomer) that did not behave
similarly to dimer or higher molecular-weight
aggregate species. To clear H2L3 and dimer species
completely, we developed MAb B’s clinical process
as a CEX-BE step that tolerated a 30–40% yield loss.
Using POROS Benzyl Ultra resin in BE mode, we
optimized a process for MAb B that decreased
residence time from six to two minutes, while
yielding a 20% increase in monomer recovery
(Table 2).
In preliminary studies, we also observed complete
clearance of MAb B H2L3 and other aggregate species
in FT mode on POROS Benzyl Ultra resin, though
further salt type and concentration optimization was
required (results not shown). Finally, we tested
stability of MAb B in high-salt buffers and did not
observe de novo aggregate formation over a sevenday time course at 4 °C and 25 °C (results not
shown).
We tested the ability of the POROS Benzyl Ultra BE
process for MAb B to clear retrovirus XmuLV and
parvovirus MVM (Figure 4). MAb B was loaded with
700 mM citrate, and specific monomer elution was
performed using buffer with no salt added. Note that
both XMuLV and MVM bound POROS Benzyl Ultra
resin under those load conditions, but buffer alone
removed only MVM — not XMuLV. For the MAb B
monomer elution fraction, we again observed
complete XMuLV clearance and minimal MVM
Figure 5: MAb B POROS Ethyl bind–elute process viral clearance study; complete XmuLV and modest MVM
clearance
Log Clearance
4 3 2 1 0
FT + 3 CV
700 mM
Sodium Citrate
Tris pH 7.0
500 mM
Sodium Citrate
(Elution)
Tris pH 7.0
360 mM
Sodium Citrate
Tris pH 7.0
0 mM
Sodium Citrate
Water Hold Control
Column: 0.8 × 10 cm, CV = 5 mL Load buffer: MAb B (Tris pH 7, 700 mM sodium citrate)
Flow rate: 2.5 mL/min Residence time: 2 min
XmuLV (log clearance)
MVM (log clearance)
Figure 4: MAb B POROS Benzyl Ultra bind–elute process viral clearance study; complete XmuLV and minimal MVM
clearance
FT + 3 CV 700 mM wash
Log Clearance
5 4 3 2 1 0
MAb G Elutes Tris, pH 7.0
0 mM Sodium Citrate
Water
Hold Control
XmuLV (log clearance)
MVM (log clearance)
Column: 0.8 × 10 cm, CV = 5 mL Load buffer: MAb B (Tris pH 7, 700 mM sodium citrate) Flow rate: 2.5 mL/min Residence time: 2 min17 thermofisher.com/antibody-derived-therapeutics
6 BioProcess International 17(11–12)si NOVEMBER–DECEMBER 2019 SPONSORED
clearance. Water strip did not remove XmuLV from
POROS Benzyl Ultra resin, a finding consistent with
results obtained for the MAb A FT process on the
same resin.
To test virus-resin hydrophobic interaction, we
performed MAb B BE experiments using POROS Ethyl
resin without further co-optimizing for H2L3 or
aggregate clearance. At 700 mM sodium citrate load
phase, we saw both strong XmuLV and moderate
MVM binding to POROS Ethyl resin. Using sequential
salt drops of 500 mM and 360 mM, then buffer
alone, both XmuLV and MVM were stripped from the
resin using the minimal buffer wash with no salt
added. The relative ease of elution for even the
highly hydrophobic XmuLV virus is consistent with
POROS Ethyl resin being the least hydrophobic
member of the POROS HIC resin family.
VIRAL CLEARANCE PREDICTION AND STRATEGY
USING MVPS BY MOCKV SOLUTIONS
In the HIC virus clearance studies discussed above, we
observed minimal to modest clearance of MVM in all
POROS HIC processes conducted in BE and FT modes.
To better understand MVM hydrophobicity, binding,
and clearance for the three POROS HIC resins, we used
MockV’s MVM MVP in high-throughput screening
(HTS) format. Binding was tested under increasing
concentrations of sodium citrate (Figure 6).
Clearance of the MVP on all three HIC resins was
<0.5 log, even under the highest salt concentrations
(600 mM sodium citrate for MAb A). Overall, our
observations point to the low hydrophobicity of MVP
and (by extension) MVM — a finding supported by
Johnson et al. (8). Our finding also is consistent with
our live viral clearance observations above in which
minimal binding of MVM was detected for POROS
Benzyl Ultra resin in FT mode polish of MAb A.
Figure 6: MVP mimics MVM, with transmission electron
microscopy (A). High-throughput screening of MVP
binding to POROS Ethyl, Benzyl, and Benzyl Ultra resins
under increasing sodium citrate concentrations (B) for a
load spike of 1 × 109 MVP/mL MVP did not bind to any of
the HIC resins under all conditions tested.
100 nm 100 nm
Spike E9 MVP/mL
~0.5 log
clearance
600 nM
citrate
MVP/mL (×108)
10
8 6 4 2 0
[Sodium Citrate] (M)
0.0 0.2 0.4 0.6 0.8
MVP biochemically and physically resemble MVM
Assay = immuno-qPCR readout MVP are BSL-1 safe (empty)
MVP does not significantly bind to POROS HIC resins,
consistent with lack of MVM binding to POROS HIC resins
A B
MVM MOCK VIRUS PARTICLE (MVP) KIT
Commercial kits for analyzing the removal of impurities
such as host cell proteins and residual host DNA are used
routinely to ensure a quality-by-design (QbD) approach to
biologics manufacturing. The information amassed from
these types of kits feeds decision-making processes
throughout the evolution of a downstream purification
process — from early stage process development to latestage commercial manufacturing. However, today’s
analytical toolbox lacks an easy-to-implement kit that can
provide information about process capability for viral
clearance. That is mainly because viral clearance studies
require propagation of live mammalian viruses. The cost,
time, logistics, and environmental challenges demand
specialized laboratories, personnel, and expertise for such
studies, leading many drug developers to outsource them
or postpone that work until late in process validation or just
before filing a biologics license application (BLA).
Viral surrogates such as bacteriophages have been used
for over a decade in virus filtration applications because
they do a satisfactory job at mimicking the size and
physical properties of mammalian viruses (4). However,
they fail to mimic the overall physiochemical and surface
properties of the live mammalian viruses used in viral
clearance spiking studies. Moreover, off-the-shelf
quantification assays and consistent sources of
bacteriophage spiking material are not readily available in
standardized kit formats.
MockV Solutions commercialized the MVM mock virus
particle (MVP) kit, which uses an immuno-qPCR assay to
quantify a noninfectious MVM-surrogate viral particle.
These MVPs have similar morphology and physiochemical
characteristics to MVM (4). The ability of MVM MVPs to
predict MVM clearance has been studied for virus filters (5)
and ion-exchange chromatography (6). POROS AEX resins
have shown superior salt tolerance in FT mode retention of
MVP for a viral vaccine polish application (7).18 thermofisher.com/antibody-derived-therapeutics
SPONSORED NOVEMBER–DECEMBER 2019 17(11–12)Si BioProcess International 7
Similarly, for the POROS Benzyl BE operation,
negligible MVM binding was observed during MAb A
loading at 600 mM citrate. Taking advantage of this
result, the addition of a high-salt chase in the BE
process could improve the MVM clearance achieved
in the elution pool. We also performed parallel
column studies using MVP and the optimized POROS
HIC processes for MAb A (Figure 7). We showed that
for both Benzyl Ultra FT and Benzyl BE processes,
MVP clearance mirrored MVM clearance. Taken
together, our results highlight the utility of MockV’s
MVM MVP kit as a process development and
prediction tool for live MVM clearance.
DISCUSSION
HIC is an excellent tool for the downstream
purification of biotherapeutics. Thermo Fisher
Scientfic has designed a family of POROS HIC resins to
confer high selectivity for impurity removal. They are
highly efficient for aggregate polish and offer
orthogonal HCP and viral clearance capabilities. Here
we showed highly productive POROS HIC polishing
processes in both BE and FT modes that outcompeted
legacy clinical processes designed for two highly
challenging case studies (MAbs A and B).
We demonstrated complete XmuLV and partial
MVM viral clearance for both MAb A and MAb B
processes. Finally, we used MockV Solutions MVM
MVP viral surrogate to better understand MVM virus
and HIC interactions. Because these MVPs
physiochemically resemble live MVM used for
spiking studies, we also presented data highlighting
the utility of MVP for predicting MVM clearance both
in high-throughput screening and column studies
using optimized POROS HIC polish processes. As
predicted, the clearance of MockV MVPs closely
paralleled MVM clearance in all POROS HIC studies.
In conclusion, POROS HIC is a highly productive and
platform-ready purification step that confer
orthogonal selectivity for aggregate and impurity
removal as well as viral clearance potential.
ACKNOWLEDGMENTS
MockV Solutions acknowledges Dale Dembrow, head of
CMC at MockV Solutions, as well as Dan Gowetski and
Krishna Gula at NIH. Thermo Fisher acknowledges
Immunogen for graciously providing the MAb A and MAb
B samples.
REFERENCES
1 Welsh J, et al. A Practical Strategy for Using
Miniature Chromatography Columns in a Standardized
High-Throughput Workflow for Purification Development of
Monoclonal Antibodies. Biotechnol Prog. 30(3) 2014: 626–
635; doi:10.1002/btpr.1905.
2 Kang K, et al. Development of an Acidic/Neutral
Antibody Flow-Through Polishing Step Using Salt Tolerant
Anion Exchange Chromatography. Pharm. Bioprocess. 3(8)
2015: 477–487.
3 Li JJ, et al. Improving Aggregate Removal to Enhance
Clinical Manufacturing of MAbs. BioPharm Int. 28
September 2018.
4 Aranha-Creado H, Brandwein H. Application of
Bacteriophages as Surrogates for Mammalian Viruses: A
Case for Use in Filter Validation Based on Precedents and
Current Practices in Medical and Environmental Virology.
PDA J. Pharm. Sci. Technol. 53(2) 1999: 75–82.
5 Cetlin D, et al. Use of a Noninfectious Surrogate to
Predict Minute Virus of Mice Removal During
Nanofiltration. Biotechnol. Progress 34(5) 2018: 1213–1220.
6 Herbig K, et al. Modeling Virus Clearance: Use of a
Noninfectious Surrogate of Mouse Minute Virus As a Tool
for Evaluating an Anion-Exchange Chromatography
Method. BioProcess Int. 17(5) 2019: 34–41.
7 Clearing the Way for Viral Clearance. BioProcess Int.
Ask the Expert Webinar; https://bioprocessintl.com/
sponsored-content/clearing-the-way-for-viral-clearance.
8 Johnson S, et al. A Step-Wise Framework to Design a
Chromatography-Based Hydrophobicity Assay for Viral
Particles. J. Chromatog. B 2017; doi:10.1016/j.
jchromb.2017.08.002. c
Corresponding author John Li, PhD, (john.li3@thermofisher.com) is
staff scientist, bioproduction business of Thermo Fisher Scientific.
Moira Lynch (moira.lynch@thermofisher.com) is a senior manager of
innovation, bioproduction business of Thermo Fisher Scientific, and
David Cetlin (dcetlin@mockvsolutions.com) is founder and CEO of
MockV Solutions.
This article is based on an “Ask the Expert” webinar for BioProcess
International. Access the entire presentation at https://bioprocessintl.
com/sponsored-content/clearing-the-way-for-viral-clearance.
Figure 7: MVM and MVP clearance for MAb A BE and
FT processes on POROS HIC resins; MVM was spiked
as a single virus, and clearance was measured by
infectivity assay. MVP was spiked as a single agent and
tracked by immuno-qPCR using the MockV analytical
kit. MockV MVP clearance at mirrored MVM live virus
clearance for all processes tested.
Log Clearance
MVM (BSL-2) MVP (BSL-1)
2.0 Benzyl BE Benzyl Ultra FT
1.5
1.0
0.5
0
1.6
1.0 1.0
0.5
MVP clearance is comparable to MVM clearance
in both MAb A FT and bind–elute polish processes.
*Data originally cited and published from Bioprocess International19 thermofisher.com/antibody-derived-therapeutics
An innovative approach to addressing
high aggregate challenges in
engineered monoclonal antibodies
As the demand for therapeutic antibodies increases, new monoclonal antibodies (mAbs) and mAb derivatives are continuously
being developed.
Ion exchange chromatography is typically used during the purification of standard mAbs. However, engineering nextgeneration antibodies can result in high levels of aggregates, which cannot be efficiently cleared using traditional purification
methods.
Watch this webinar to discover new mixed-mode ion exchange chromatography solutions, designed to
effectively remove aggregates and maximise mAb recovery.
Watch Now
*webinar originally published and cited on Bioprocess InternationalYing Chen, Al de Leon, Kelly Flook, Thermo Fisher Scientific, Bedford, MA 01730 USA
An innovative approach to addressing high aggregate challenges
In engineered monoclonal antibodies
Learn more at thermofisher.com/purification-contact
Abstract
With advances in engineered antibody designs, treatment performance improves, but higher
aggregate levels are often produced in the cell culture creating new purification challenges.
Current solutions for aggregate removal include bind and elute strategies with cation
exchange or hydrophobic interaction chromatography resins which, whilst effective, often
result in poor process economics and low recoveries. Alternatively, caprylic acid has been
successfully used as a flocculant for antibody aggregates but requires a filtration step
resulting in a more labor intensive and complicated process. The work in this poster
describes the performance of a resin-based approach using immobilized Caprylic acid. It
effectively removes high levels of aggregates as well as host cell protein residues and
leached ligand from protein A affinity resin.
Introduction
With the need of designing therapeutics with higher efficacy, more engineered monoclonal
antibody derivatives are actively pursued for the next generation of mAb-based drugs. With
the more complex structures, like symmetric, asymmetric or fragment-based bispecifics, the
downstream process developer is challenged by mis-paired products, undesired fragments
and higher levels of aggregates. Alternative new mAb designs are equally challenging. The
use of caprylic acid as a flocculant for aggregate removal and high molecular weight species
has been earlier suggested by Brodsky et al.[1] in 2012. The precipitation step though
requires to introduce additional filtration and sedimentation steps.
By chemically attaching caprylic acid (octanoic acid) to large pore POROS™ divinylbenzene
polymeric beads, a chromatography resin with excellent aggregate removal capabilities was
developed. The work described here tests the final design of the resin on loading, aggregate
elimination and also best operational conditions (for our simulated mAb high aggregate
test solution).
Trademarks/licensing
© 2023 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of
Thermo Fisher Scientific and its subsidiaries unless otherwise specified. This information is
not intended to encourage use of these products in any manner that might infringe the
intellectual property rights of others.
Materials and methods
Sample preparation
A IgG1 type mAb was produced in-house and purified using Thermo Scientific™
MabCaptureA™ affinity resin. In order to mimic high aggregate levels, the mAb was then
stressed through multiple exposures to high and low pH adjustments, until the aggregate
level reached approximately 10%. [2]
Hydrophobic Weak
Cation
Exchange
Figure 1: POROS™ and Caprylic Acid form a mixed-mode, hydrophobic weak cation
exchange resin – Thermo Scientific™ POROS™ Caprylate Mixed-Mode Cation
Exchange Chromatography Resin
Purified mAb was then applied to 1mL POROS Caprylate Mixed-Mode resin packed into
OmniFit glass column (6.6mmID x 30 mmL).
HPLC-SEC was performed with a Thermo Scientific MabPac™ SEC-1 on Thermo
Scientific UltiMate™ 3000. Buffer: 50mM Sodium Phosphate, 300 mM NaCl, pH 6.5; flow
rate: 0.2 mL/min; detection: UV at 280nm.
HCP and Protein A ligand leach was performed with Cygnus CHO Host Cell Protein
ELISA-kit and Repligen Protein A ELISA-Kit, respectively.
Creating Modelled Sample for Resin Test
POROS Caprylate resin –
Aggregates, Impurity Clearance (HCP, LPrA)
Monoclonal Antibody (Herceptin )
Affinity Capture Select Resin
(MabCapture A )
Low pH hold, Depth Filtration
Viral inactivation
Aggregation production
Generated ~10% HMW aggregates
Clarified cell culture fluid
Protein A resin selectively interact
with Fc region of antibody
Virus inactivation
Aggregate induction by pH cycling,
monitored by HPLC analysis using
Thermo Scientific MAbPac™ SEC-1
Evaluated over a broad range of pH
and conductivities to maximize
impurity removal.
Figure 2: Schematic of sample generation, aggregate induction and resin
performance test.
HPLC – SEC used for aggregate level determination on mock-up feed solution
Retention time, mins
50 0
100
150
200
250
Absorbance, 280nm mAU
1
2
3
4
0.00 2.50 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00
0
40
6.5 18
5.00
Figure 3: SEC chromatograph of mAb feed prior to purification using POROS
Caprylate (blue) and after (black). Inset is an expanded section of high molecular
weight species.
Results – DoE study
Finding optimal conditions
A Design of Experiment (DoE) study was used to evaluate the optimum mobile phase pH
and conductivity to achieve monomer yield > 80% and reduction of aggregate levels to < 2%.
The design space: pH range 4.5 – 6.0, [NaCl] from 0 – 500mM. Load density was kept
constant at 100mg / mL resin.
The DoE study centered around
conditions found favorable in previous
flow-through experiments and qualitative,
wellplate based HTS screening. The pH
range was chosen from 4.5 to 6.0, the
NaCl concentration from 0 to 500 mM.
Figure 4: Design Space, [NaCl] and pH
vs. monomer and aggregate response
The 2-dimensional representation of the design space below show relatively large design
conditions for high yield and purity expectations..
Even with lower conductivity conditions, POROS
Caprylate Mixed-Mode resin is able to reduce
aggregate levels down to 1–2%
This option is favorable for a directly following
low salt anion exchange process step in the
overall polishing process. As the AEX polishing
is also often run in flow-through mode, the
suggested savings pull through then at that
step as well (lower buffer consumption, lower
COGS, smaller column sizes, fast high yield
break through).
Figure 5: Design space for monomer
vs. aggregate percentage
POROS Caprylate Mixed-Mode Cation Exchange resin is also effective in removing
other high molecular weight species (HMWS), like host cell proteins (HCP) or leached
Protein A resin ligand.
Results—Load density study
Conditions used for load density study
Feed: Buffer & Residence Time:
Max loading: 325 g/L resin Sodium Acetate pH 5.25
Monomer Purity: 89.4% 275mM NaCl (28.62 mS/cm)
% Aggregate: 10.6% Residence Time: 3 min
Figure 6: Monomer recovery (dark blue) vs aggregate accumulation (orange), with
aggregate levels marked for 1%, 2% and 3%
9 8 7 6 5 4 3 2 1 0
10
0
20
40
60
80
100
0 50 100 150 200 250 300 350
% Aggregate
Cumulative Monomer Recovery (%)
Loading Density (g/L resin)
Result show very favorable monomer yield for the given aggregate impurity levels.
% Aggregate Loading density (g/L resin) Monomer recovery (%)
1% 85.6 80.4
2% 181.9 96.3
3% 256.8 99.2
Table 1: Loading density and monomer recovery at assigned aggregate
impurity levels
Results—Reduction of other HWMS
Parameter Unit
Loading density
experiment R&D
Batch A
Loading density
Experiment R&D
Batch B
Production
Validation Batch
MMCEX-001
Total load [mg] 160 175 100
Buffer
conditions
25mM sodium acetate
275mM NaCl,
pH 5.25
25mM sodium
acetate, 75mM NaCl,
pH 5.30
25mM sodium
acetate, 12mM NaCl,
pH 4.5
Host cell
protein in load [ppm] 555 450 648
Host cell
protein after
column
[ppm] 24 14 36
Leached
protein A in
load
[ppm] 60.3 67.5 78.5
Leached
protein A after
column
[ppm] 3.1 4.7 1.3
Text System 1mL CV Omnifit column 6.6mm ID x 300mmL residence time 3
minutes
Table 3: HCP & Leached Protein A ligand reduction, 3 different
experiments/conditions
Conclusions
Simulated high aggregate levels in our mAb test solution has shown that POROS Caprylate
Mixed-Mode resin operated in flow-through mode, is very promising for
Effective removal of high (10%) aggregate levels in mAbs using flow-through mode
Delivering high monomer yields (> 80%) with low aggregate impurity levels (< 2%)
Improved mAb purification process designs, were flow through can be used for the cation
exchange step and the anion exchange-based final polishing step
The economics of a such intensified process design can be highly advantageously for
existing and new modalities
References
1. Brodsky Y, Zhang C, Yigzaw Y, Vedantham G. CaprylicVan, Biotechnol Bioeng. 2012 Oct
2. Stress-Induced Antibody Aggregates, Ajish SR Potty and Alex Xenopoulos, p44 ff,
BioProcess International 11(3) March 2013, BioProcess
Intended use statement
POROS resins: Pharmaceutical Grade Reagent. For Manufacturing and Laboratory Use
Only.
Materials and methods (continued) Results—DoE study (continued)
d21 thermofisher.com/antibody-derived-therapeutics
U sp ethinuneargibofiaclveaestrnial doitenlyvnocelo ufamappbntueteirrbo resoitdfnoieresduce
chromatography steps in their
downstream processes. It can increase
product yield, reduce the time required
for bioprocess development, and
ultimately reduce time to market and
the overall cost of goods (CoG).
Because of its ability to bind to the Fc
region of monoclonal antibodies
(MAbs), protein A has been used as the
platform for anity capture of IgGbased therapeutics for decades.
In addition to standard IgG MAbs,
many antibody formats are in
development: e.g., bispecifics, fusion
proteins based on the crystallizable
fraction (Fc) of antibodies, proteins
made up of just the antigen-binding
(Fab) fragments of antibodies, and
antibody–drug conjugates (ADCs).
Such engineered antibodies can pose a
challenge to protein A anity capture if
they have altered or absent protein A
binding sites. These novel formats also
can be dicult to purify because some
tend to aggregate, form dimers, and
can come with elevated levels of high–
and low–molecular-weight (HMW, LMW)
species. Light-chain dimers can present
alongside some antibody fragments,
and overexpressed light chains can
complicate their downstream
processing further. In addition, some
engineered antibodies are sensitive to
low-pH/acidic conditions, which can
limit a developer’s choice of elution
buer.
Novel antibody modalities have driven
development of new options for
purification such as CaptureSelect
technology. CaptureSelect ligands oer
a unique anity purification solution
based on camelid-derived single
domain (VHH) antibody fragments. The
small, 14-kD anity ligands provide a
platform solution to many
biopharmaceutical purification
challenges and have been proven to
deliver increased yields and purities for
proteins of interest in many
applications. VHH anity ligands are
produced by recombinant yeast culture
in a process that is free of animal-origin
materials. The molecules are highly
stable and screened for high specificity,
binding capacity, and desired elution
conditions for a target therapeutic of
interest. Thermo Fisher Scientific has
developed a number of unique
CaptureSelect anity matrices to target
dierent binding sites on an array of
human therapeutic proteins (Figure 1).
CaptureSelect CH1-XL ligands bind
to the constant heavy domain (CH1) of
all human IgG subclasses and purify
100% of kappa and lambda Fabs,
providing an excellent platform for Fab
purification. Because of its high
selectivity, the CH1-XL ligand
precludes copurification of free light
chains and light-chain dimers. The
resin has a high dynamic binding
capacity (DBC) of ~19 mg/mL for a
polyclonal Fab with ecient elution at
relatively mild pH. Fab purity of 98%
has been achieved using
CaptureSelect CH1-XL resin, with a
nearly 90% yield.
CaptureSelect KappaXP resin was
designed for purification of Fab
fragments and bispecific antibodies.
The ligand provides 100% kappa
subtype coverage for all
immunoglobulins that contain a kappa
light chain. This resin has a high DBC
of 20–30 g/L for kappa Fabs and has a
DBC of 30-45 g/L IgG. Ecient elution
can be achieved at relatively mild
acidity (up to pH 6).
CaptureSelect LambdaXP ligands
oer 100% lambda-subtype coverage
and can be used to purify IgGs that
contain a lambda light chain, including
bispecifics and Fab fragments. The
resin has a high DBC of >35 g/L for
IgGs and can be eluted at pH 3.5–4.0.
CaptureSelect FcXP ligands bind
only the constant heavy domain CH3.
These were developed for Fc-fusion
proteins, chimeric antibodies, and
molecular formats in which the protein
SUPPLIER SIDE
Streamline manufacturing of
antibody-based therapeutics with
novel purication approaches
Laurens Sierkstra
VH
VL
CL
CH1
CH2
CH3
VH
VL
CL
CH1
CH2
CH3
Example antibody subdomain structure22 thermofisher.com/antibody-derived-therapeutics
Sponsored November 2023 21(5)e BioProcess International 3
A binding site is either removed or
blocked. The resin also has a high
DBC — >40 g/L with 10% breakthrough
(BT) and 5-minute residence time —
and elutes efficiently at pH 4.0–4.5.
Case Study in
Streamlining Purification
This case study exemplifies rapid
implementation of CaptureSelect FcXL
resin, a predecessor of the FcXP resin
mentioned above, into a commercial
next-generation therapeutic
manufacturing process. A purification
process had been established for a
novel-format antibody based on an
affinity capture step followed by three
polishing steps. The process had a low
DBC because the antibody did not
bind well to protein A, and lower-pH
elution limited product stability. Other
problems included elevated fragment
levels and an environmentally
unfriendly regeneration solution. The
developer wanted to remove one
process step to enable a better fit
within a particular manufacturing
facility.
Several capture options were
screened for load density, yield, and
reduction of host-cell protein (HCP),
HMW, and LMW impurities.
CaptureSelect FcXL resin scored the
best on most attributes, including the
sum of LMW (Table 1). Following
implementation this resin for affinity
capture, the following results were
achieved:
• reduction of a four-step
chromatography process to three
steps, removing the need for one of
the polish steps
• consistent yields of ~80% in one
pilot and three good manufacturing
practice (GMP) runs
• increased binding capacity to
improve facility fit
• an improved impurity profile
• increased pool stability due to
elution at mild pH
• excellent scalability
• use of a more environmentally
friendly regeneration solution.
Case Study in
Improved Recovery
Using bind–elute polishing steps to
remove impurities such as HMW
components and dimers can be
cumbersome. In this case study, a
customer with a three-step purification
process step sought to improve product
recovery and process efficiency. The
original process used protein A capture
followed by anion-exchange polish in
flow-through (FT) mode and a mixedmode bind–elute to remove a high
percentage of aggregates (12%). Cation
exchange had been evaluated for
aggregate removal but was not efficient
for the MAb in this case. The existing
process provided only 90% monomer
recovery, with loading at 25 g/L and a
6-minute residence time.
The goal of our collaboration was to
replace the bind–elute mixed-mode
step with a FT operation to improve
recovery, aggregate clearance, and
efficiency. Mixed-mode
chromatography was replaced with
Poros Benzyl Ultra hydrophobicinteraction resin. Three attributes of
such resins make them extremely wellsuited for manufacturing scale: Product
resolution is maintained as linear flow
rate increases. High linear binding
capacity is possible over a large range
of flow rates. And scalability is linear
and predictable.
The final process cleared
aggregates to <1%, with 90% monomer
recovery after 25-g/L resin loading
Table 1: Comparing affinity-capture options for a novel-format antibody that had
suboptimal binding to protein A
(Existing)
Affinity A Affinity B Affinity C Nonaffinity
CaptureSelect
FcXL
Load density Medium Low Medium Very High High
HCP reduction High High Very High Low Medium
Yield High High Low Low High
HMW Medium Medium Medium Medium Medium
Main peak Medium Medium Medium High High
LMW High High High Medium Low
Figure 1: CaptureSelect ligands offer a range of highly specific affinity-capture options.
CaptureSelect
CH1-XL
CaptureSelect
KappaXP
LambdaXP
CaptureSelect
FcXP
Fab purification platform
Binding the CH1 domain
No binding of free light chains
Developed for Fab fragments and bispecifics
Binding CL-kappa domain
High dynamic binding capacity
Mild elution conditions
Developed for Fc-fusion proteins and IgGs that bind
to protein A poorly
Binding the CH3 domain (Fc)
Mild elution conditions
Fab F(ab')2
Fab
Bispecific IgG
containing κ or λ CL
IgG Fc-fusion
protein23 thermofisher.com/antibody-derived-therapeutics
4 BioProcess International 21(5)e November 2023 Sponsored
and a 6-minute residence time (Figure
2b). The FT process was coupled
directly to the upstream AEX process
with no need for buffer exchange,
which saved both time and money.
These results demonstrate the power
of HIC as an alternative strategy in
MAb-aggregate polishing.
Removing HCPs with
Affinity Polishing
Product- and expression-related HCPs
can be difficult to remove even by
multiple polishing options. HCPs that
coelute with proteins of interest are
particularly difficult to remove. If they
are not addressed sufficiently, a
product’s advancement from one
clinical phase to the next can be
delayed. Clusterin, cathepsin D,
galectin-3 binding protein, and
G-protein coupled receptor 56 are
among the many HCPs that can be
difficult to remove. To address such
challenges, Thermo Fisher Scientific
developed an approach called “affinity
for polish” that applies CaptureSelect
affinity ligands on POROS beads to
enable fast and efficient HCP removal
in FT applications. POROS
CaptureSelect ClusterinClear beads
were used to remove >95% of clusterin
as well as >65% of other top HCPs in
one customer evaluation.
New Solutions for New
Antibody Formats
Purifying the next generation of
antibody therapeutics requires a
diverse set of high-performance
chromatography options. The tools
described herein can deliver important
benefits including high purity and yield
in a single capture step, a reduction of
the number of necessary steps in a
downstream process, and seamless
upscaling for biomanufacturing as
products move through clinical phases
toward commercialization.
Laurens Sierkstra is senior director
of research and development for
bioproduction purification and analytics
at Thermo Fisher Scientific in Leiden,
Netherlands; laurens.sierkstra@
thermofisher.com.
POROS resin is a pharmaceutical grade
reagent for manufacturing and
laboratory use only. CaptureSelect
ligands and affinity matrix are for
research use or further manufacturing,
not for diagnostic use or direct
administration in humans or animals.
Figure 2: Results of the new process — final verification
Load Purified Antibody
HMW Dimer Monomer LMW
Eective reduction of dimer and HMW aggregates
in low-conductivity solutions, with high recovery
A successful MAb polish step for highly ecient aggregate
removal and near-complete monomer recovery
Mixed-Mode
MAb-A Process BE HIC FT
Load density (g/L resin) 25 80
Monomer purity FT (%) 99 >99
Monomer recovery FT (%) 90 98
Host-cell proteins (ppm) <LLoQ <LLoQ
Residence time (minutes) 6.0 1.2
0.1% 0.1%
85.5%
9.5% 0.5%
5.0% 0.1%
99.3%
Final Verification
*Data originally cited and published from Bioprocess InternationalLearn more at thermofisher.com/antibody 24 -therapeutics thermofisher.com/antibody-derived-therapeutics
Bioprocessing
INTRODUCTION
Pim Hermans, Frank Detmers, Kevin Sleijpen, Simon Adema, Hendrik Adams, Elina Klijs, Anja Overweel,
Paul Janszen, Jessica de Rooij & Laurens Sierkstra
Thermo Fisher Scientific, Leiden, the Netherlands
Accelerating antibody drug development with
subdomain-specific affinity chromatography
TRADEMARKS/LICENSING
Caution: For Research Use or further manufacturing, not for diagnostic use or direct administration in humans or animals. © 2021 Thermo Fisher Scientific Inc. All
rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. This information is not intended to
encourage use of these products in any manner that might infringe the intellectual property rights of others. EvolveD is a registered trademark of Delta Precision Ltd.
Technology based on single domain antibody fragments [VHH]
High target purity in a single step, independent of feedstock
Unique VHH screening technology to determine final resin properties:
target specificity
mild elution
ligand stability
Scalable & animal origin free technology
Suitable for cGMP manufacturing processes
A unique set of CaptureSelect™ affinity resins has been developed directed against a variety
of antibody subdomains, supporting manufacturers to help facilitate purification of novel
antibody formats.
Fig.1 CaptureSelect™ Antibody Selectivity
Binding regions of CaptureSelect™ resins for affinity purification of antibodies and antibody fragments.
• efficient elution at milder conditions protecting the target molecule and
smaller elution pool volumes
• High dynamic binding capacity at shorter residence times
• High dynamic binding capacity: >40 g/L at 4 min residence time
• Efficient elution at milder conditions (pH 4) making it suitable for Fc fusion
proteins
Fig.3 High dynamic binding capacity using
KappaXP for Trastuzumab (humanized IgG1)
purification. Dynamic binding capacity as function
of residence time determined by frontal analysis.
Column format: 0.8cmDx10cm.
Feed concentration 3G/L
CaptureSelect™ KappaXP & LambdaXP affinity matrices –
CL Kappa and Lambda binding domain resins
• Fab purification platform
• Binds the CH1 domain
• No binding of free light chains
• Purification of Fab fragments and
bi-specifics
• Binds CL-kappa or CL- lambda
domain
• High Dynamic Binding Capacity
• Mild elution
• Binds the CH3 domain
• Solution for non-Protein A binding
IgGS and Fc-fusion proteins
• Mild elution
CaptureSelect™ FcXP affinity matrix – CH3 binding domain resin
Start FT EL ST
150 kDa
100 kDa
50 kDa
25 kDa
Solving purification challenges in the downstream process of
bi-specific molecules
A purification platform for all IgG subclass molecules with an altered
Protein A binding site or pH sensitivity such as Fc-fusion proteins
Fig.5 High dynamic binding capacity using FcXP in
Rituximab and polyclonal IgG purification. Dynamic Binding
Capacity as function of residence time determined by frontal
analysis. Column format: 0.5cmDx20cm. Feed concentration 5
g/L Rituximab and 7 g/L polyclonal IgG.
Fig.6 One-step purification from crude
material with high purity. Overexpressed
light chain dimers are present in the flow
through (FT) but not in the elution fraction
(E). ST = strip pH 2
2B
Fig.2 Ranibuzimab purification from HEK293
cells. Analysis of the fractions after purification
with CaptureSelect CH1-XL resin shows high
yield and purity in a single step.
2A: SDS-PAGE silver staining of the load (L), flow
through (FT) and elution (E) fractions, showing no
presence of light chains in the elution pool.
2B: Gel filtration analysis showing 98% purity of
the Fab fragment in the elution fraction with a
yield of 86% 2A
• No co-purification of free light chains (only correct assembled Fabs)
• Efficient elution at milder pH (4 – 4,5)
PURIFICATION OF ANTIBODY THERAPEUTICS - EXAMPLES
CaptureSelect™ CH1-XL affinity matrix – CH1 binding domain resin
Scalable platform solution for efficient Fab fragment purification
Fig.4 LambdaXP dynamic binding capacity (left) and elution (right) of a bi-specific antibody. Dynamic
binding capacity at 10% breakthrough ~40 mg/mL (4 min residence time). Elution performance using 25mM
sodium acetate at pH 3.6 using a load concentration of 32 mg/mL demonstrates an efficient elution of 3CVs.
CaptureSelect LambdaXP dynamic binding
(bi-specific antibody)
Affinity purification platforms such as Protein A or L are well-established in the manufacturing
process of therapeutic monoclonal antibodies. However, with the development of engineered
modalities such as bi-specific antibodies, fragments and Fc-fusion proteins, challenges in the
downstream process of these molecules arise. Affinity chromatography resins, specifically
developed to bind antibody-subdomain regions, can provide an alternative solution in the
purification process of these new formats. Thereby, advancing the commercial production of
new antibody therapeutics.
Thermo Scientific resin Dynamic Binding Capacity Elution properties
CaptureSelect KappaXP 40 g/L at 2 min residence time Efficient elution at milder conditions (pH 5-
6) with additives
CaptureSelect LambdaXP > 35 g/L at 4 min residence time Efficient elution at pH 3.5-4 – small elution
pool volume
CaptureSelect LambdaXP elution efficiency
(bi-specific antibody)
CaptureSelect FcXP purity
(Rituximab)
CDR1
Camelid Ig VHH
CDR2
CDR3
CaptureSelect technology – unique affinity purification solution
CaptureSelect resin family for therapeutic antibody development
CaptureSelect antibody subdomain-specific affinity resins address the
purification challenges in therapeutic antibody development by providing
unique selectivity, high purity and yields in a one-step purification
process.
CONCLUSIONS
Learn more at thermofisher.com/antibody-therapeutics
Bioprocessing
INTRODUCTION
Pim Hermans, Frank Detmers, Kevin Sleijpen, Simon Adema, Hendrik Adams, Elina Klijs, Anja Overweel,
Paul Janszen, Jessica de Rooij & Laurens Sierkstra
Thermo Fisher Scientific, Leiden, the Netherlands
Accelerating antibody drug development with
subdomain-specific affinity chromatography
TRADEMARKS/LICENSING
Caution: For Research Use or further manufacturing, not for diagnostic use or direct administration in humans or animals. © 2021 Thermo Fisher Scientific Inc. All
rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. This information is not intended to
encourage use of these products in any manner that might infringe the intellectual property rights of others. EvolveD is a registered trademark of Delta Precision Ltd.
Technology based on single domain antibody fragments [VHH]
High target purity in a single step, independent of feedstock
Unique VHH screening technology to determine final resin properties:
target specificity
mild elution
ligand stability
Scalable & animal origin free technology
Suitable for cGMP manufacturing processes
A unique set of CaptureSelect™ affinity resins has been developed directed against a variety
of antibody subdomains, supporting manufacturers to help facilitate purification of novel
antibody formats.
Fig.1 CaptureSelect™ Antibody Selectivity
Binding regions of CaptureSelect™ resins for affinity purification of antibodies and antibody fragments.
• efficient elution at milder conditions protecting the target molecule and
smaller elution pool volumes
• High dynamic binding capacity at shorter residence times
• High dynamic binding capacity: >40 g/L at 4 min residence time
• Efficient elution at milder conditions (pH 4) making it suitable for Fc fusion
proteins
Fig.3 High dynamic binding capacity using
KappaXP for Trastuzumab (humanized IgG1)
purification. Dynamic binding capacity as function
of residence time determined by frontal analysis.
Column format: 0.8cmDx10cm.
Feed concentration 3G/L
CaptureSelect™ KappaXP & LambdaXP affinity matrices –
CL Kappa and Lambda binding domain resins
• Fab purification platform
• Binds the CH1 domain
• No binding of free light chains
• Purification of Fab fragments and
bi-specifics
• Binds CL-kappa or CL- lambda
domain
• High Dynamic Binding Capacity
• Mild elution
• Binds the CH3 domain
• Solution for non-Protein A binding
IgGS and Fc-fusion proteins
• Mild elution
CaptureSelect™ FcXP affinity matrix – CH3 binding domain resin
Start FT EL ST
150 kDa
100 kDa
50 kDa
25 kDa
Solving purification challenges in the downstream process of
bi-specific molecules
A purification platform for all IgG subclass molecules with an altered
Protein A binding site or pH sensitivity such as Fc-fusion proteins
Fig.5 High dynamic binding capacity using FcXP in
Rituximab and polyclonal IgG purification. Dynamic Binding
Capacity as function of residence time determined by frontal
analysis. Column format: 0.5cmDx20cm. Feed concentration 5
g/L Rituximab and 7 g/L polyclonal IgG.
Fig.6 One-step purification from crude
material with high purity. Overexpressed
light chain dimers are present in the flow
through (FT) but not in the elution fraction
(E). ST = strip pH 2
2B
Fig.2 Ranibuzimab purification from HEK293
cells. Analysis of the fractions after purification
with CaptureSelect CH1-XL resin shows high
yield and purity in a single step.
2A: SDS-PAGE silver staining of the load (L), flow
through (FT) and elution (E) fractions, showing no
presence of light chains in the elution pool.
2B: Gel filtration analysis showing 98% purity of
the Fab fragment in the elution fraction with a
yield of 86% 2A
• No co-purification of free light chains (only correct assembled Fabs)
• Efficient elution at milder pH (4 – 4,5)
PURIFICATION OF ANTIBODY THERAPEUTICS - EXAMPLES
CaptureSelect™ CH1-XL affinity matrix – CH1 binding domain resin
Scalable platform solution for efficient Fab fragment purification
Fig.4 LambdaXP dynamic binding capacity (left) and elution (right) of a bi-specific antibody. Dynamic
binding capacity at 10% breakthrough ~40 mg/mL (4 min residence time). Elution performance using 25mM
sodium acetate at pH 3.6 using a load concentration of 32 mg/mL demonstrates an efficient elution of 3CVs.
CaptureSelect LambdaXP dynamic binding
(bi-specific antibody)
Affinity purification platforms such as Protein A or L are well-established in the manufacturing
process of therapeutic monoclonal antibodies. However, with the development of engineered
modalities such as bi-specific antibodies, fragments and Fc-fusion proteins, challenges in the
downstream process of these molecules arise. Affinity chromatography resins, specifically
developed to bind antibody-subdomain regions, can provide an alternative solution in the
purification process of these new formats. Thereby, advancing the commercial production of
new antibody therapeutics.
Thermo Scientific resin Dynamic Binding Capacity Elution properties
CaptureSelect KappaXP 40 g/L at 2 min residence time Efficient elution at milder conditions (pH 5-
6) with additives
CaptureSelect LambdaXP > 35 g/L at 4 min residence time Efficient elution at pH 3.5-4 – small elution
pool volume
CaptureSelect LambdaXP elution efficiency
(bi-specific antibody)
CaptureSelect FcXP purity
(Rituximab)
CDR1
Camelid Ig VHH
CDR2
CDR3
CaptureSelect technology – unique affinity purification solution
CaptureSelect resin family for therapeutic antibody development
CaptureSelect antibody subdomain-specific affinity resins address the
purification challenges in therapeutic antibody development by providing
unique selectivity, high purity and yields in a one-step purification
process.
CONCLUSIONS25 thermofisher.com/mixed-mode-chromatography
Achieve aggregate clearance in mAb purification
Thermo Scientific™ POROS™ Caprylate Mixed-Mode Cation
Exchange Chromatography Resin is a scalable solution
specifically designed to purify monoclonal antibodies (mAbs)
and antibody derivatives in flow-through applications for
process intensification.
Why choose POROS Caprylate Mixed-Mode Cation
Exchange Chromatography Resin?
• Handles high levels of aggregates
• Can reduce 10% aggregation to <2% in a single polish step
• >80–90% mAb monomer recovery with minimal
process optimization
• Reduced cost of goods—bind-elute vs. flow-through (2–5x less)
based on applicable capacities and processing parameters
Boost your mAb
purification
workflow w ith a new
mixed-mode resinResources
Gain insight into an innovative
solution for mixed-mode
chromatography, specifically
designed to remove high level of
aggregates in the downstream
process of therapeutic antibodies.
Read more
Learn about a variety of CaptureSelect
resins for affinity purification to
address today’s challenges in the mAb
therapeutics workflow.
Read more
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