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Protein A affinity chromatography has been widely used for initial product capture in recombinant antibody/Fc-fusion purification. However, in general Protein A lacks the capability of separating aggregates (unless the aggregates are too large to enter the pores of resin beads or have their Protein A binding sites buried, in which case the aggregates do not bind). In the current work, we demonstrated that CaptureSelect FcXP affinity medium exhibited strong aggregate separation capability and effectively removed aggregates under pH or conductivity gradient elution in two bispecific antibody (bsAb) cases. For these two cases, aggregate contents were reduced from >16% and >22% (in the feed) to <1% and <5% (in the eluate) for the first and second bsAbs, respectively. While more case studies are required to further demonstrate FcXP's superiority in aggregate removal, findings from the current study suggest that FcXP can potentially be a better alternative than Protein A for product capture in cases where aggregate content is high., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) is an analytical method routinely used for assessing aggregation content in protein samples. As SEC-HPLC separates analytes based on their hydrodynamic radius, it generally lacks the capability of differentiating species that are similar in size. Recently while purifying a bispecific antibody (bsAb), we noticed that SEC-HPLC can provide certain degree of resolution between the target bsAb and a disulfide scrambled form, although these two species were identical in molecular weight. In seeing the unexpected potential of SEC-HPLC at resolving species with similar size, we further tested Zenix SEC-300, a mixed-mode SEC-HPLC column from Sepax, which was reported to be capable of separating protein analytes based on other factors besides size. The Zenix column indeed provided resolution much better than the regular SEC-HPLC column. Upon further optimization, the Zenix column allowed close to baseline separation of the correctly folded and the disulfide scrambled species. The current study, as a complement to the previous reports, further demonstrates that mix-mode SEC-HPLC is capable of separating protein analytes that are close in size but are different in conformation and/or surface characteristics., (Copyright © 2024 Elsevier Inc. All rights reserved.)

For a certain number of mAbs, bispecific antibodies (bsAbs) and Fc-fusion proteins that we worked on, the Protein A capture step experienced low yield (i.e., ∼80%). A previous case study suggested that non-binding aggregate formed in cell culture was the root cause of low Protein A step yield. In the current work, we selected five projects with the low Protein A yield issue to further illustrate this phenomenon. In all cases, existence of non-binding aggregates was confirmed by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis of Protein A load and flow-through. In addition, we demonstrated that aggregates failed to bind to Protein A resin mainly due to their large sizes, which prevented them from entering the resin beads. As the data suggested, SEC-HPLC analysis of Protein A load and flow-through, although not a standard procedure, can provide information that is critical for understanding the unexpected performance of Protein A chromatography in cases like those being presented here. Thus, SEC-HPLC analysis of Protein A load and flow-through is highly recommended for antibodies/Fc-fusions suffering from low Protein A yield., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Protein A affinity chromatography has been widely used for antibody capture and initial purification. In general, the yield of this step is relatively high (i.e., >90%). However, recently while purifying a bispecific antibody (bsAb) in appended IgG format, the Protein A capture step experienced low yield (i.e., ∼80%). It was found that the target bsAb started appearing in flow-through at a relatively low load density, suggesting that a portion of the expressed bsAb has compromised Protein A binding capability. Further studies indicated that the bsAb in flow-through was mainly in aggregate form. In addition, normal Protein A step yield was restored when the column was loaded with bsAb monomer. Thus, all the evidence pointed to the fact that aggregate with compromised binding capacity was the cause of low Protein A step yield in this case., (Copyright © 2023 Elsevier Inc. All rights reserved.)

WuXiBody is a bispecific antibody (bsAb) platform developed by WuXi Biologics. Its key feature is the replacement of one parental antibody's CH1/CL region with the T-cell receptor (TCR) constant domain, which prevents mispairing between non-cognate heavy chain and light chain. In addition, heavy chain heterodimerization in asymmetric WuXiBody molecule is promoted by the knobs-into-holes (KiH) technology. Despite the great success of KiH strategy in improving heterodimer formation, homodimers (especially the hole-hole homodimer) can still be generated at low levels. In general, detection and monitoring of homodimers during KiH bsAb purification are challenging as homodimers share similar physicochemical properties with the target heterodimeric bsAb. Nevertheless, the unique design of WuXiBody allows homodimers to be effectively detected and monitored by multiple methods. In the current work, with an asymmetric WuXiBody case study, we demonstrated that hole-hole homodimer can be effectively monitored by six chromatography methods including hydrophobic interaction chromatography (HIC), reverse phase (RP), cation exchange (CEX), KappaSelect, CaptureSelect CH1-XL and Protein L., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Knobs-into-holes (KiH) technology has been widely used in asymmetric bispecific antibody (bsAb) construction to promote heavy chain heterodimerization. However, despite the great improvement of heterodimer formation by this strategy, homodimers (especially the holehole homodimer) can still be generated at low levels. Consequently, hole-hole homodimer is a common byproduct associated with the production of KiH bsAbs. In addition, previous studies showed that hole-hole homodimer exists as two different isoforms. As the major difference between these two isoforms lies in the Fc region, we speculated that Protein A media, which bind IgG Fc region with high affinity, and CaptureSelect FcXP, a CH3 domain-specific affinity resin, may provide certain resolution between these two conformational isoforms., Objective: The objective of this study was to study the capability of Protein A and CaptureSelect FcXP affinity resins in differentiating hole-hole homodimer isoforms., Methods: The hole-hole homodimer was produced in CHO cells by expressing the hole half-antibody. The homodimer, along with the half-antibody was initially captured by Protein A chromatography and was then further purified by size-exclusion chromatography (SEC), which separated the homodimer from the unpaired half-antibody. The purified hole-hole homodimer was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analytical hydrophobic interaction chromatography (HIC). The purified hole-hole homodimer was separately processed by columns packed with Protein A and CaptureSelect FcXP resins. The purified hole-hole homodimer was also analyzed by Protein A-high-performance liquid chromatography (HPLC)., Results: SDS-PAGE analysis and analytical HIC study confirmed that hole-hole homodimer exists as two conformational isoforms. When the hole-hole homodimer was processed by Protein A and CaptureSelect FcXP chromatography, the elution profiles contained two peaks, indicating that both affinity resins possess the capability of differentiating hole-hole homodimer isoforms., Conclusion: Our data suggest that Protein A and CaptureSelect FcXP affinity resins both possess the capability of differentiating hole-hole homodimer isoforms and, therefore, can be used for monitoring isoform conversion under various conditions., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

In a previous work we demonstrated that CHO protease caused fragmentation of an expressed bispecific antibody (bsAb) and this detrimental host cell protein (HCP) can be effectively removed through an optimized Protein A wash step. In addition, preliminary evidence suggested that the responsible protease belongs to the threonine or cysteine protease family. In the current study, this protease was further identified as cathepsin B. First, we identified several CHO proteases in the further fractionated Protein A wash using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and this allowed us to select four candidate proteases. Next, by examining the cleavage pattern of each individual protease and comparing it with that observed during purification, cathepsin B was identified as the protease responsible for the observed bsAb fragmentation., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Half-antibody is a frequent byproduct associated with the recombinant production of many asymmetric bispecific antibodies (bsAbs). Although this byproduct can be largely removed by post-capture polishing steps, it is ideal to have it partially cleared at the capture step to achieve a more robust downstream process. Previously we showed that Protein A affinity chromatography possesses the capability to separate half-antibody. In this study, we assessed the half-antibody separation capability of four less commonly used subdomain-specific affinity resins. The data suggest that these resins exhibit different capabilities for separating half-antibody from the corresponding bsAb. In specific, whereas Protein A affinity resin can always provide partial separation under typical conditions, the separation efficiency of three subdomain-specific affinity resins (i.e., Capto L, CaptureSelect CH1-XL and CaptureSelect FcXP) heavily relies on the property of the two parental monospecific antibodies from which the bsAb is derived, which may range from complete separation to no separation at all. This novel information provides more options for half-antibody clearance at the capture step., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In general, purification of bispecific antibody (bsAb) is more challenging than that of monospecific antibody due to the increased complexity in byproduct profile. Like in the case of monospecific antibody purification, immunoglobulin-binding protein-based affinity chromatography is an indispensable tool for bsAb purification. For example, Protein A affinity chromatography has been widely used to capture Fc-containing bsAbs whereas other affinity media such as Protein L and KappaSelect, which bind kappa light chain, are used to capture bsAbs that do not contain a Protein A-binding site. In fact, affinity chromatography also possesses the capability of removing certain product-related impurities in bsAb purification when it is conducted with suitable medium and under appropriate conditions. Fully exploring the potential of affinity chromatography in bsAb purification to achieve both product capture and byproduct removal is highly desirable, as this can greatly alleviate the purification burden on subsequent polishing steps and hence improves the overall robustness of the downstream process. This article briefly reviews the byproduct clearance potential of several commonly used affinity media under relevant bsAb purification scenarios., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Assembly of IgG-like asymmetric bispecific antibodies (bsAbs) requires heavy chain heterodimerization and cognate heavy-light chain pairings. Multiple strategies have been developed to solve these chain association issues. While these strategies greatly promote correct chain pairing, they normally cannot prevent low amount of chain mispaired byproducts from being generated. Besides, byproducts can also be generated as a result of discordant chain expression. The existence of various byproducts poses considerable challenges to downstream processing during the production of recombinant IgG-like bsAbs. In many cases, yield is greatly compromised for purity improvement. This mini review introduces eight IgG-like bsAb platforms, which share a common feature: they all contain built-in purification-facilitating elements in addition to chain pairing control designs. These platforms, by simultaneously providing solutions to the two issues associated with bsAb production (i.e., correct chain pairing and efficient purification), improve both efficiency and robustness of bsAb production., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A certain impurity may bind weaker or tighter to a particular type of chromatography column (e.g., ion exchange, hydrophobic interaction or mixed-mode) than the target protein, and this forms the basis for separation. For impurities that bind weaker, they can be removed by an appropriate pre-elution wash. However, we previously showed that wash-enabled impurity clearance is usually sensitive to loading density, causing poor robustness of the process. In this work, with a bispecific antibody case study, we compared the relative robustness of anion exchange and the corresponding mixed-mode chromatography, which mediates both anion exchange and hydrophobic interactions, on removing a weakly-bound byproduct by wash. It was learned that under a fixed appropriate wash condition, the latter achieves consistent byproduct clearance and good yield over a much wider range of loading density than the former. As wide loading density range is highly desirable for large-scale manufacturing, the above finding suggests that for a chromatography step that employs stepwise elution and relies on pre-elution wash for removing weakly-bound impurities, mixed-mode chromatography could be a better choice than the corresponding monomode ion exchange chromatography., (Copyright © 2021 Elsevier Inc. All rights reserved.)

WuXiBody is a bispecific antibody (bsAb) platform developed by WuXi Biologics. Its key feature is the replacement of one parental antibody's CH1/CL region with the T cell receptor (TCR) constant domain, a design that promotes cognate heavy chain (HC)-light chain (LC) pairing. BsAbs based on WuXiBody can adopt either asymmetric or symmetric format. For purifying a WuXiBody-based symmetric bsAb, we identified a LC-missing species as a major byproduct. While for bsAbs based on other platforms removal of such byproduct can pose considerable challenge to the downstream team, in this case WuXiBody's unique design makes separation relatively straightforward. We previously showed that Capto MMC ImpRes mixed-mode chromatography under bind-elute mode can effectively remove this LC-missing species. However, the dynamic binding capacity (DBC) of Capto MMC ImpRes is relatively low under the selected condition, making the process less desirable for large-scale manufacturing. In this study, we demonstrated that when Capto MMC ImpRes chromatography is conducted under weak partitioning mode, high throughput, good yield, and effective byproduct removal are simultaneously achieved., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Diamond MMC Mustang is a relatively new mixed-mode resin, which mediates both cation exchange and hydrophobic interactions. In this work, we evaluated this resin using Cytiva's Capto MMC ImpRes, a well-established mixed-mode resin with similar properties, as a benchmark. The data suggest that in comparison with Capto MMC ImpRes, Diamond MMC Mustang exhibits comparable binding capacity and resolution. In addition, the resin under evaluation shows good lot-to-lot consistency. The information provided in this study allows users to have additional options when selecting mixed-mode resin for intermediate purification or final polishing, which is favourable especially at the present time when the supply chains of many manufacturers are negatively impacted by the coronavirus pandemic., (Copyright © 2021 Elsevier Inc. All rights reserved.)

MabSelect PrismA is an affinity resin whose ligand is derived from the B-domain of Protein A. Compared to its predecessor resins (i.e., MabSelect SuRe LX and MabSelect SuRe), the PrismA ligand has enhanced affinity for the VH3 chain. Consistently, when being used for the purification of an asymmetric IgG-like bispecific antibody (bsAb), MabSelect PrismA resin shows certain resolution between the intact product and a single-arm byproduct, which contain the same Fc region but different numbers of VH3 domain. Fibro PrismA is a newly launched product which has the same PrismA Protein A ligand as MabSelect PrismA but uses cellulose fiber instead of resin as its matrix. It was learned that Fibro PrismA, in comparison to PrismA resin, exhibits further improved resolution, allowing efficient clearance of the single-arm byproduct as well as good recovery of the target bsAb. This finding suggests that Fibro PrismA provides a potential solution for separating antibody species containing the same Fc region but different numbers of VH3 domain, which can otherwise be difficult to separate., (Copyright © 2021 Elsevier Inc. All rights reserved.)

WuXiBody is a novel bispecific antibody (bsAb) platform developed by WuXi Biologics. BsAbs based on WuXiBody can adopt either asymmetric or symmetric format. In general, recombinant production of asymmetric bsAbs has a more complex impurity profile than that of symmetric ones, as assembly of the former usually involves an increased number of distinct chains. Consequently, purification of asymmetric bsAb typically poses greater challenges to the downstream team. Recently while purifying a WuXiBody-based bsAb in asymmetric format, we learned that MMC ImpRes mixed-mode chromatography under linear pH or salt gradient elution can effectively remove two out of the three low-molecular-weight byproducts post Protein A chromatography but not the third one, which was eventually removed under pH-salt dual gradient elution. This finding suggests that MMC ImpRes chromatography performed under pH-salt dual gradient elution provides better resolution than that performed under pH/salt mono gradient elution., (Copyright © 2021 Elsevier Inc. All rights reserved.)

For recombinant antibody purification, removal of product-related impurities usually relies on the two polishing steps post Protein A chromatography. A certain impurity may bind weaker or tighter to a particular type of column than the target antibody, and this forms the basis for separation. For impurities that bind weaker, they can be removed by pre-elution wash under appropriate conditions. For impurities that bind stronger, they can be separated by using a suitable condition that selectively elutes the product. In this study, with a bispecific antibody case, we compared the relative robustness of byproduct removal by wash and by elution using two different types of chromatography. The data suggest that elution-enabled byproduct clearance is more robust than wash-enabled clearance, and the former approach provides consistent impurity clearance over a relatively wide range of loading density., (Copyright © 2020 Elsevier Inc. All rights reserved.)

WuXiBody is a novel bispecific antibody (bsAb) platform developed by WuXi Biologics. Its key feature is the replacement of one parental antibody's CH1/CL constant region with the T cell receptor (TCR) constant domain, a design aimed at promoting cognate heavy chain (HC)-light chain (LC) pairing. BsAbs based on WuXiBody can adopt either asymmetric or symmetric format. LC-missing species, a byproduct frequently associated with bsAb containing sequence engineered Fab arm, is also identified during WuXiBody production. Nevertheless, WuXiBody's unique design greatly facilitates removal of this type of impurity, which can otherwise be difficult to clear. In this work, with two concrete cases (WuXiBodies with asymmetric and symmetric designs, respectively), we showed that Capto MMC ImpRes mixed-mode chromatography can effectively remove LC-missing byproducts as well as aggregates, demonstrating that this resin is a powerful tool for WuXiBody purification., (Copyright © 2020 Elsevier Inc. All rights reserved.)

For IgG-like bispecific antibodies (bsAbs) whose construction involves sequence engineering to promote desired heavy chain-light chain pairing, ¾ antibody (antibody lacking one light chain) is a frequent byproduct during their recombinant production. As this byproduct shares high similarity in physicochemical properties with the target bsAb, its removal poses a challenge to downstream purification. Capto L is an affinity resin based on Protein L, which binds to the variable region of kappa light chain without interfering with the antigen binding site. In this work, we demonstrated that Capto L provides a convenient means for separating ¾ antibody from the bsAb product., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Asymmetric IgG-like bispecific antibodies (bsAbs) are normally derived from two parental mAbs with different origin. Despite the implementation of heterodimerization-promoting strategy in the design, homodimerization can still occur at a low level during the recombinant production of these molecules. In general, monitoring and removal of homodimers pose great challenges to analytical and purification teams, respectively, as these byproducts share high similarity in physicochemical properties with the target heterodimeric bsAb. Protein L is a bacterial surface protein that binds to the variable region of kappa light chain without interfering with the antigen binding site. In this work, we first showed that different antibodies bind Protein L-conjugated resin with varied strength, and then based on this observation we further demonstrated that Protein L chromatography can be a useful tool for monitoring/separating homodimers during the purification of asymmetric bsAbs., (Copyright © 2020 Elsevier Inc. All rights reserved.)

WuXiBody is a novel bispecific antibody (bsAb) platform developed by WuXi Biologics. It enables almost any monoclonal antibody (mAb) sequence pair to be assembled into a bispecific construct. BsAbs based on WuXiBody can adopt either asymmetric or symmetric format. WuXiBody's unique design not only ensures desired chain pairing but also facilitates removal of potential product-related impurities. In this work, demonstrated with four WuXiBody-based bsAbs (two asymmetric and two symmetric ones), we showed that Protein A followed by anion exchange (AEX) and mixed-mode chromatography (i.e., Capto MMC ImpRes or Capto adhere ImpRes) can be a potential platform approach for WuXiBody purification., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Half antibody (one half of an antibody which comprises a heavy chain and a light chain) is a common byproduct in bispecific antibody (bsAb) production and in many cases it represents the major product-related impurity. As half antibody contains only one Fc-domain, it binds Protein A resin weaker than the target bsAb, which contains the full Fc-region. Indeed, Protein A chromatography provides certain resolution between half antibody and the intact bsAb under linear pH gradient elution. Nevertheless, separation between these two species is far from complete under this condition. In this study, we demonstrated that adding salt additive to Protein A mobile phase can significantly improve resolution between half antibody and the intact bsAb, allowing most of the half antibody impurity in the load to be removed by this capture step. Having the majority of half antibody byproduct removed at this early stage is a big advantage as it improves the overall robustness of the downstream process., (Copyright © 2020 Elsevier Inc. All rights reserved.)

During recombinant production of asymmetric IgG-like bispecific antibodies (bsAbs), various by-products are often observed due to unbalanced chain expression and incorrect chain pairing. Among them, half antibody and homodimer are found with high frequency. In this work, with a case study we demonstrated that Capto MMC ImpRes mixed-mode chromatography can effectively remove these two by-products as well as antibody aggregates under optimized conditions. This makes MMC ImpRes a powerful tool for bsAb purification., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Despite the use of knobs-into-holes (KiH) strategy to promote heterodimerization in recombinant production of bispecific antibody (bsAb), homodimer (especially the hole-hole homodimer) can still be generated in small amount. This by-product needs to be removed by downstream process. However, as homodimer and the target bsAb are usually very close in size, these two species may not be readily differentiated using size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Thus, method other than SEC-HPLC needs to be developed to monitor removal of this by-product. Here, through a case study we demonstrate that analytical hydrophobic interaction chromatography (HIC) is a powerful tool for quantitatively monitoring removal of hole-hole homodimer in bsAb purification., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Bispecific antibodies (bsAbs) are antibodies that can simultaneously bind two distinct targets or epitopes. Their dual-targeting capacity offers expanded therapeutic potential. Currently there is a strong interest in design and production of bsAbs to achieve improved efficacy through novel mechanisms of action. However, due to the co-expression of up to four distinct polypeptide chains or assembly involving chains with extended length (in the appended IgG format), recombinant production of IgG-like bsAbs is often accompanied with increased level of product-related impurities (byproducts and aggregates) resulting from heavy chain homodimerization, heavy chain-light chain mispairing, unbalanced expression of different chains and intermolecular misassociation. As some of the byproducts display close similarity to the target bsAb, their removal poses significant challenges to the downstream processing. This article reviews methods that can effectively remove product-related impurities in IgG-like bsAb purification., (Copyright © 2018 Elsevier Inc. All rights reserved.)