Mastering Next-Generation Therapeutic Antibody Impurity Removal

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 a‹nity 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 a“er 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 buŽer. Novel antibody modalities have driven development of new options for purification such as CaptureSelect technology. CaptureSelect ligands oŽer 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 diŽerent 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 oŽer 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 e
cient 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