Five Key Considerations for Optimizing Resin Selection for Efficient mAb Purification

5 key considerations for optimizing resin selection for efficient mab purification Article Introduction Monoclonal antibodies (mAbs) have become a major class of therapeutics and are now approved for clinical use in various fields, including cancer, autoimmune disorders and infectious diseases such as Ebola and COVID-19.1 In order to adhere to regulatory guidelines, mAbs must meet certain purity standards, making purification a critical step in the bioprocessing workflow. As the complexity of novel antibody modalities has increased (e.g., antibody–drug conjugates, bispecific antibodies, etc.), traditional purification techniques have struggled to meet required purity levels while also maintaining high yields and cost-effective processes. Complex engineered antibodies can be prone to aggregation and fragmentation and may have increased sensitivity to pH. In addition, they may also have altered or absent protein A binding sites, complicating purification further. Recent advances in purification methods have resulted in the emergence of novel affinity and polish purification platforms that can help to overcome many of these challenges. Something as simple as selecting the optimal resin for purification can help achieve high purity and efficient yields. This guide will discuss some of the key considerations for mAb purification, helping you to optimize your purification and polishing processes while still balancing cost efficiency. 1. Addressing challenges in complex purification Although leaps are being made in polishing and purification processes, inherent challenges in the purification of complex mAbs still arise at every stage of the mAb manufacturing process. Cellular expression systems lead to high levels of process-related impurities, such as residual host cell proteins (HCPs) and host cell DNA. In addition, the inherent heterogeneity of mAbs leads to product-related impurities such as aggregates, misformed products and post-translational modifications. These impurities must be removed before product release to meet stringent regulatory and safety guidelines and prevent adverse effects in recipients. This is a complex process, especially for novel antibody modalities. Instead of relying on traditional purification and polishing methods, look to recent developments and emerging platforms in mAb processing for solutions to complex challenges. Hydrophobic interaction chromatography (HIC) (which removes proteins based on hydrophobic interactions between resins and target proteins), and mixed-mode chromatography (MMC), which typically combines ion exchange and HIC properties for improved selectivity, are two such alternative processes, already offering buffers to a range of challenges.2,3 For example, Chinese hamster ovary (CHO) cells are one of the most common cell lines for mAb production. However, these cells produce several notoriously difficult-to-remove HCPs, including clusterin, which can co-elute with the target mAb.4 MMC has been shown to be effective in removing or significantly reducing the most challenging and high-risk HCPs to acceptable levels.5 2. Evaluating affinity chromatography options for mAb capture Affinity chromatography is widely accepted as one of the most efficient and effective techniques for the initial stages of mAb purification, due to its specificity, ease of operation, high yields and high throughput.6 While other chromatography methods rely on separation by differences in size or ionic charge, affinity chromatography relies on specific binding interactions between an immobilized ligand and the mAb to be purified (Figure 1). Due to the specificity of affinity chromatography, it is important to select the right resin for the mAb in question. One of the most common resins for affinity purification is protein A, which binds primarily to the Fc region between the CH2 and CH3 domains of most IgG subtypes.7,8 Although protein A is considered a trusted resin of choice for the purification of traditionally structured mAbs, it can struggle with novel modalities such as bispecific antibodies (bsAb), due to formats that produce Fc-containing mispaired product variants, formats that have modified Fc regions that limit or eliminate binding to protein A, or fragment-based antibody formats. In addition, the acidic elution used during protein A chromatography can lead to aggregation of some modalities, making impurity removal more difficult. In these cases, alternative resins such as protein L are available. Though still requiring a low pH, protein L binding is specific for the variable light chains and can bind most antibody classes and fragment fragment antigen-binding region (Fab) fragments.6,9 Other resins based on camelid-derived single-domain antibody fragments have been developed specifically for bsAbs and antibody fragments. These come in a range of different binding sites and have high affinity, specificity and capacity, allowing for a streamlined workflow and high-purity products.10 They can also enable mild elution, preventing pHinduced aggregation. In addition to careful resin selection, recovery rates can also be maximized by optimizing the purification process and its steps. If bind/elute steps are providing suboptimal monomer recovery, changing the workflow to a flow-through (FT) operation using a HIC resin has been shown to improve recovery.11 HIC resins are particularly well suited for manufacturing-scale workflows, due to their high linear binding capacity over a wider range of flow rates. 3. Strategies for effective polishing steps Following mAb affinity capture, product purity of non-complex antibodies may be as high as 95%. For more complex, engineered mabs, purity levels of 80% or below are not uncommon. To achieve higher purity, the next steps in the downstream mAb purification process are intermediate and final polishing. This typically consists of a cation exchange chromatography step (CEX), followed by anion exchange chromatography (AEX) and HIC. For relatively clean feeds, a single polishing step may be sufficient, provided the product purity and safety requirements are met. Selecting an appropriate resin combination along with optimized process conditions enables orthogonal separations that maximize yields, reduce process and product-related impurities, and improve process robustness. Resins should align with the purification step and the charge properties of the target molecule or impurities to be removed. In AEX, the chromatography matrix is positively charged, so negatively charged molecules can be captured. For example, impurities such as viruses, HCPs, DNA, endotoxins and aggregates can be captured and removed from the final product. In comparison, CEX captures positively charged mAbs, which can then be eluted by increasing the conductivity or buffer pH. For complex mAbs, different polishing strategies may be needed. IEX using standard conditions may result in inadequate impurity removal, risking non-adherence to regulatory guidelines. HIC and MMC offer unique selectivity and can often be more effective platforms for novel mAb modalities. HIC resins can be utilized in either bind and elute or flow-through mode, depending on the hydrophobicity of the resin, the salt concentration and the salt type used (e.g., kosmotropic salts like ammonium sulfate). This allows for highly customizable options for targeting specific impurities, such as product variants in bind and elute mode or removal of aggregates and HCPs in flow-through mode. HIC Figure 1. Protein A chromatography. Sample Washing buffer Elution buffer Purified antibodies Unwanted components Target antibodies Affinity resin Purification column Sample loading Washing Elution 2 thermofisher.com/resin-selection-tool resins can support high resolution, even under low conductivity conditions often encountered post protein A, making HIC an excellent option for manufacturing scalability. HIC resins are often utilized for the purification of engineered mAbs, including antibody fragments and antibody–drug conjugates. MMC combines the benefits of IEX and HIC for even greater selectivity and purification due to a unique stationary phase that contains both hydrophobic and charged functional groups (Figure 2). MMC is ideal for a diverse range of proteins and particularly challenging mAbs with high aggregate levels and complex impurity profiles, as it can reduce 10% aggregation to ~1% in a single polish step.3 4. Enhancing recovery rates through optimal resin selection A generic purification workflow may be able to meet regulatory guidelines, but if it isn’t achieving maximal recovery rates, it could be costing you product and efficiency. Additionally, a process that works well at a pilot or development scale may not run efficiently at a manufacturing scale. It’s important to optimize your workflow in a manner that maximizes recovery rates while still maintaining high purity and efficiency. To achieve this, resin screening studies can be used to select the resins with the highest binding capacity, best selectivity and best resolution for the antibody in question. Recovery can be affected by resin characteristics such as binding capacity, elution efficiency and stability, therefore these are important aspects to consider when selecting the best resin. Protein A affinity resins are more commonly used in bioprocessing, especially for traditional humanized mAbs However, some engineered antibodies can have altered, blocked or absent protein A binding sites, causing issues for traditional protein A-based purification. In these cases, specialized matrices can be used, designed to target different binding sites while still maintaining high dynamic binding capacity. For example, for Fab purification, ligands that bind the constant heavy (CH1) domain of IgG subclasses, or the kappa and lambda Fab regions, can be used. For Fc-fusion proteins and chimeric antibodies, ligands that bind only the constant heavy domain (CH3) are available. Resin selection is also key in polishing steps. While IEX is often considered as a first choice for mAb polishing, HIC resins are an effective alternative to address high levels of aggregation or in antibody–drug conjugates (ADCs), where further purification is needed to refine the drug–antibody ratio (DAR). HIC resins can operate using various salt types, over a range of salt concentrations, and come in a range of hydrophobicities. They offer improved resolution and can help achieve enhanced clearance of aggregates with higher productivity when other processes fail.12 You can also consider mixed-mode (MMC) resins. These resins combine properties of IEX and HIC, and they can remove even high levels of aggregates in a single flowthrough step, thereby reducing the number of polishing steps and improving efficiency.13 5. Balancing cost and performance in resin selection The core goal of purification is to isolate a high-purity product in a reliable and cost-efficient manner. In addition to ensuring optimal performance, economic aspects – such as initial costs, lifespan and regeneration capabilities – must also be considered when selecting a resin. Reducing the number of steps in the downstream purification process can improve cost-efficiency, as it reduces processing time and is faster to optimize. A streamlined process can also reduce the amounts of reagents needed and the labor and utilities costs. For example, high-performing techniques such as affinity chromatography for purification and MMC for polishing can achieve higher yield and purity than other methods in a single step, increasing efficiency without compromising recovery. To streamline IEX steps, resins that have large through-pore structures can allow higher capacity, while resins optimized for low to high flow rates can help ease the scale-up process from small-scale to process-scale production. Ensuring protocol validation and compliance can be complex, costly and time-consuming. Therefore, consider resins that can help address these issues; for example, resins with animal-free origins can help ease adherence to regulatory guidelines. Good availability and quality of technical support from the resin vendor can also help with validation and optimization, while choosing a vendor with a reliable supply chain and a proven history of delivering high-quality product can help mitigate the risk of unforeseen circumstances and facilitate optimal product manufacture. O O OH Hydrophobic Weak cation exchange Figure 2. The structure of an MMC resin backbone. 3 thermofisher.com/resin-selection-tool Pharmaceutical Grade Reagent. For Manufacturing and Laboratory Use Only. © 2025 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. WTP10912755 0525 Find your perfect resin match today at thermofisher.com/resin-selection-tool Resin reuse is another effective method of improving costefficiency, though this should be approached carefully so as not to affect performance. Consider the chemical stability of the resin, dynamic binding capacity over several cycles, monitoring of product quality attributes, and other parameters when assessing a column lifecycle. For example, single-domain VHH antibody fragment affinity resins are inherently stable, making them suitable for large-scale processing. Conclusion The ultimate goal of every mAb purification process is to produce a high-quality therapeutic that meets stringent efficacy and patient safety guidelines. However, it is also important to consider workflow productivity, in order to keep therapeutics affordable and manufacturing efficient. Significant leaps in efficiency and performance can be achieved by simple changes, such as informed resin selection. Selecting the right resin for your process can address aggregation challenges, improve cost-efficiency and maximize recovery rates, while still adhering to regulatory guidelines. Optimize your purification workflow today and see the real-world benefits to your mAbs. References 1. Lyu X, Zhao Q, Hui J, et al. The global landscape of approved antibody therapies. Antib Ther. 2022;5(4):233-257. doi:10.1093/abt/tbac021 2. Hydrophobic Interaction Chromatography. Thermo Fisher Scientific - US. https://www. thermofisher.com/uk/en/home/life-science/bioproduction/poros-chromatographyresin/bioprocess-resins/hydrophobic-interaction-chromatography.html. Accessed May 7, 2025. 3. Mixed-Mode Chromatography. Thermo Fisher Scientific - US. https://www. thermofisher.com/uk/en/home/life-science/bioproduction/poros-chromatographyresin/bioprocess-resins/mixed-mode-chromatography.html. Accessed May 7, 2025. 4. Singh SK, Mishra A, Yadav D, Budholiya N, Rathore AS. Understanding the mechanism of copurification of “difficult to remove” host cell proteins in rituximab biosimilar products. Biotechnol Prog. 2020;36(2):e2936. doi:10.1002/btpr.2936 5. Kiyonami R, Melani R, Chen Y, De Leon A, Du M. Applying UHPLC-HRAM MS/MS method to assess host cell protein clearance during the purification process development of therapeutic mAbs. Int J Mol Sci. 2024;25(17):9687. doi:10.3390/ijms25179687 6. Arora S, Saxena V, Ayyar BV. Affinity chromatography: A versatile technique for antibody purification. Methods. 2016;116:84-94. doi:10.1016/j.ymeth.2016.12.010 7. Protein A Chromatography Resins. Thermo Fisher Scientific - US. https://www. thermofisher.com/uk/en/home/life-science/bioproduction/poros-chromatographyresin/bioprocess-resins/protein-a-chromatography-resins.html. Accessed May 7, 2025. 8. Hober S, Nord K, Linhult M. Protein A chromatography for antibody purification. J Chromatogr B. 2007;848(1):40-47. doi:10.1016/j.jchromb.2006.09.030 9. Antibody Purification Using Protein L. Thermo Fisher Scientific - US. https://www. thermofisher.com/uk/en/home/life-science/antibodies/antibody-purification-kitsreagents/antibody-purification-using-protein-l.html. Accessed May 7, 2025. 10.Affinity Chromatography Resins—Antibody Therapeutics. Thermo Fisher Scientific - US. https://www.thermofisher.com/uk/en/home/life-science/bioproduction/poroschromatography-resin/bioprocess-resins/antibody-derived-therapeutics.html. Accessed May 7, 2025. 11. Sierkstra L. Streamline manufacturing of antibody-based therapeutics with novel purification approaches. BioProcess International. https://www.bioprocessintl.com/ sponsored-content/streamline-manufacturing-of-antibody-based-therapeutics-withnovel-purification-approaches. Published November 29, 2023. Accessed May 7, 2025. 12.De Rooij J, Li J, Terova O. Novel hydrophobic interaction chromatography resins for next generation biotherapeutic challenges. Thermo Fisher Scientific. https://www.thermofisher. com/uk/en/home/life-science/bioproduction/poros-chromatography-resin/chromatographylearning-lab/antibody-therapeutics/resources.html. Accessed May 7, 2025. 13.Chen Y, de Leon A, Flook K. An innovative approach to addressing high aggregate challenges in engineered monoclonal antibodies. Thermo Fisher Scientific Bioproduction. https://www.cellandgene.com/doc/an-innovative-approach-toaddressing-high-aggregate-challenges-in-engineered-monoclonal-antibodies-0001. Accessed May 7, 2025.