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Abstract 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. Highlights • Approaches that enforce heavy chain heterodimerization and cognate heavy/light chain pairing in bsAb assembly are introduced. • Sequence engineering that can facilitate bsAb purification is briefly discussed. • Methods that can effectively remove commonly found product-related impurities in IgG-like bsAb purification are reviewed. • General suggestions on reducing the type and amount of bsAb specific byproducts are proposed. [ABSTRACT FROM AUTHOR]

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. • During KiH bsAb purification, homodimers are difficult to be monitored as they share similar properties with the bsAb. • WuXiBody is a bsAb platform developed by WuXi Biologics. • The unique design of WuXiBody allows homodimers to be effectively detected and monitored by multiple methods. • In a case study, HH homodimer was effectively monitored by HIC, RP, CEX, KappaSelect, CaptureSelect CH1-XL and Protein L. [ABSTRACT FROM AUTHOR]

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. • Immunoglobulin-binding protein based affinity chromatography can play roles other than product capture in bsAb purification. • Several commonly used affinity resins have the potential for removing half antibody, homodimer and aggregates. • Affinity chromatography mediated separation is mainly based on the difference in binding valency among different species. • Effective byproduct removal by affinity resins may require extensive optimization. [ABSTRACT FROM AUTHOR]

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. • Purification of IgG-like bsAb is challenging despite implementation of byproduct-minimizing chain pairing control strategies. • Including purification-facilitating element as a component of the designed bsAb is highly desirable. • Eight bsAb platforms with built-in purification-facilitating elements besides chain pairing control designs are introduced. • These production-efficient bsAb platforms provide guidance and inspiration on design of new platforms. [ABSTRACT FROM AUTHOR]

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. • In general, Protein A affinity chromatography lacks the capability of separating aggregates. • FcXP, a CH3-specific affinity medium, exhibits good aggregate separation potential under linear pH gradient elution. • Linear conductivity gradient, which enables improved resolution, facilitates convenient conversion into stepwise elution. • FcXP can potentially be a better alternative than Protein A for product capture in cases where aggregate content is high. [ABSTRACT FROM AUTHOR]

In recombinant protein purification, differences in isoelectric point (pI)/surface charge and hydrophobicity between the product and byproducts generally form the basis for separation. For bispecific antibodies (bsAbs), in many cases the physicochemical difference between product and byproducts is subtle, making byproduct removal considerably challenging. In a previous report, with a bsAb case study, we showed that partition coefficient (K p) screening for the product and byproducts under various conditions facilitated finding conditions under which effective separation of two difficult-to-remove byproducts was achieved by anion exchange (AEX) chromatography. In the current work, as a follow-up study, we demonstrated that the same approach enabled identification of conditions allowing equally good byproduct removal by mixed-mode chromatography with remarkably improved yield. Results from the current and previous studies proved that separation factor determination based on K p screening for product and byproduct is an effective approach for finding conditions enabling efficient and maximum byproduct removal, especially in challenging cases. • For a given type of resin, partition coefficient (K p) for any species under various conditions can be determined using HTS. • Conditions under which the highest K p -byproduct and K p -product ratio is obtained provide the best chance for separation. • For a bsAb case, the above approach allowed us to find resin and conditions to effectively remove two truncated byproducts. • Partition coefficient screening is an effective approach for guiding byproduct removal in bispecific antibody purification. [ABSTRACT FROM AUTHOR]

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. • During the purification of a bsAb, one byproduct identified was a disulfide scrambled form. • The native and scrambled species were well resolved on native gel, suggesting that they adopted different conformations. • Regular SEC-HPLC provided limited resolution between the native and the scrambled species, which are identical in size. • Mixed-mode SEC-HPLC provided much better resolution between the correctly folded and the disulfide scrambled species. [ABSTRACT FROM AUTHOR]

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. • For impurities bind weaker, they can be removed by a pre-elution wash under appropriate conditions. • This wash step's effect on impurity removal and impact on step yield are usually sensitive to the loading density. • Here we compared the robustness of AEX and the corresponding mixed-mode chromatography on removing a weakly-bound byproduct. • Under stepwise elution with fixed wash, mixed-mode column maintains good performance over a wider range of loading density. [ABSTRACT FROM AUTHOR]

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. • Low Protein A step yield has been observed for certain mAbs, bsAbs and Fc-fusions. • SDS-PAGE analysis of Protein A flow-through suggested abnormal binding in these cases but missed important information. • SEC-HPLC analysis of column load and flow-through revealed that poor Protein A performance was due to non-binding aggregates. • Aggregates failed to bind mainly due to their large sizes, which prevented the aggregates from entering the resin beads. • For projects experiencing low Protein A yield, SEC-HPLC analysis of load and flow-through is highly recommended. [ABSTRACT FROM AUTHOR]

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. • For bsAb whose construction involves protein engineering to avoid HC-LC mispairing, ¾ antibody is a frequent byproduct. • Removing ¾ antibody can be difficult by commonly used chromatography (e.g., Protein A, IEX, CHT, etc.). • Protein L binds to the variable region of the kappa LC without interfering with the antigen binding site. • Capto L, a Protein L-conjugated affinity resin, provides a straightforward and effective means for ¾ antibody removal. [ABSTRACT FROM AUTHOR]

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. • Homodimers are commonly found byproducts during the recombinant production of asymmetric IgG-like bsAbs. • Monitoring and removal of homodimers pose great challenges to analytical and purification teams, respectively. • Protein L binds to the variable region of the kappa LC without interfering with the antigen binding site. • Protein L chromatography can be a useful tool for monitoring/separating homodimers during the production of asymmetric bsAbs. [ABSTRACT FROM AUTHOR]

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. • A Protein A chromatography process was developed to remove half antibody byproduct associated with bsAb production. • Including 500 mM NaCl in Protein A mobile phase can significantly improve resolution between half antibody and the bsAb. • A salt-containing wash at intermediate pH is the key step for half antibody clearance by Protein A chromatography. • Having the majority of half antibody removed at the capture stage improves the overall robustness of the downstream process. [ABSTRACT FROM AUTHOR]

KappaSelect is an affinity medium that specifically binds to the constant region of the kappa light chain (LC). Obviously, KappaSelect can be used to separate antibody species containing the kappa LC constant region from those lacking that region. However, it is not clear whether this resin can readily separate species containing one kappa LC constant region from those containing two kappa LC constant regions although the former are assumed to bind weaker than the latter. In this work, we demonstrated that antibody species with two kappa LC constant regions binds to the KappaSelect resin much tighter than species with only one kappa LC constant region. Consequently, these two species can be readily separated using this resin. This information not only enriches our knowledge with KappaSelect but also has important practical value. In addition to the sample case used in the current study, there are other cases in which the target protein contains one kappa LC constant region whereas a byproduct contains two kappa LC constant regions. Our finding provides a convenient means for removing such two kappa LC binding site containing byproduct in these cases. • Antibody species with two κ LC constant regions bind to KappaSelect much tighter than species with one κ LC constant region. • KappaSelect can readily separate antibody species containing one κ LC constant region from those containing two such regions. • In certain cases, KappaSelect provides a convenient means for separating the target from contaminating byproducts. • The novel information provided in this study can potentially expand KappaSelect's application. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. • LC-MS analysis of the protease-enriched fraction revealed 352 HCPs, including 25 proteases. • Among the four proteases selected as candidates, cathepsin B gave cleavage pattern similar to the observed fragmentation. • In consistent with previous observation, cathepsin B mediated cleavage was inhibited by proteases inhibitor Leupeptin. • Cathepsin B was identified as the protease responsible for the fragmentation of the CHO-expressed bsAb. [ABSTRACT FROM AUTHOR]

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. • The half-antibody separation capability of four subdomain-specific affinity resins was assessed. • Among the tested resins, three of them exhibit the potential to separate half-antibody from the intact bsAb. • The half-antibody separation potential of these resins relies on the property of the two mAbs from which the bsAb is derived. • These subdomain-specific affinity resins can potentially provide better half-antibody separation than Protein A resin. [ABSTRACT FROM AUTHOR]

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. • A key feature of WuXiBody bsAb platform is the replacement of one parental mAb's CH1/CL region with the TCR constant domain. • LC-missing species are potential byproducts associated with bsAbs containing engineered Fab arm, including WuXi Bodies. • While the LC-missing byproduct can be removed by MMC ImpRes under bind-elute mode, the binding capacity is relatively low. • Upon switching to weak partitioning mode, high throughput, good yield and byproduct clearance are simultaneously achieved. [ABSTRACT FROM AUTHOR]

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. • Diamond MMC Mustang is a relatively new mixed-mode resin, which mediates both cation exchange and hydrophobic interactions. • We evaluated MMC Mustang using Capto MMC ImpRes, an established mixed-mode resin with similar properties, as a benchmark. • In comparison with Capto MMC ImpRes, Diamond MMC Mustang exhibits comparable loading capacity and resolution. • In addition, Diamond MMC Mustang shows good lot-to-lot consistency. [ABSTRACT FROM AUTHOR]

For recombinant proteins produced in Chinese hamster ovary (CHO) cells, fragmentation is a common phenomenon that results in generation of product-related low-molecular-weight (LMW) species. Recently while purifying a bispecific antibody (bsAb), we observed that the target protein experienced cleavage at a couple of potential sites, leading to truncated products. Further studies suggest that the cleavage can likely be attributed to residual CHO cell protease activity. In order to maximally remove potential protease(s) that contribute fragmentation, we optimized Protein A chromatography by adding sodium caprylate (SC) to the wash buffer. Upon optimization, fragmentation of Protein A eluate happened to a much lesser degree as compared to that of eluate from unoptimized process, and the increased sample stability is in accordance with significantly reduced host cell protein (HCP) level. Taken together, the data suggest that SC wash during Protein A chromatography is an effective means for removing HCPs including endogenous protease(s) that are responsible for target antibody fragmentation. • A bsAb under purification experienced fragmentation, which resulted in generation of low-molecular-weight byproducts. • Further studies suggest that cleavage can be attributed to residual CHO cell protease activity. • Protein A chromatography was optimized by adding sodium caprylate to the wash buffer to remove potential protease(s). • Fragmentation of Protein A eluate was minimized upon optimization, confirming effective removal of responsible protease(s). [ABSTRACT FROM AUTHOR]

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. • Single-arm antibody species is a potential byproduct associated with the production of asymmetric bispecific antibody. • Unlike MabSelect SuRe LX, PrismA resin displays certain resolution between the target bsAb and the single-arm byproduct. • Fibro PrismA, which is based on cellulose fiber matrix, has the same Protein A ligand as the PrismA chromatography resin. • Fibro PrismA exhibits further improved resolution, allowing more efficient clearance of the single-arm byproduct. [ABSTRACT FROM AUTHOR]

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. • WuXiBody, a bsAb platform developed by WuXi Biologics, enables any mAb pair to be assembled into a bispecific construct. • A key feature of WuXiBody is the replacement of one parental mAb's CH1/CL region with the TCR constant domain. • MMC ImpRes mixed-mode chromatography is a powerful tool for WuXiBody purification. • MMC performed under pH-salt dual gradient elution more effectively removes byproducts than performed under mono gradient. [ABSTRACT FROM AUTHOR]

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. • A certain impurity may bind weaker or tighter to a particular type of column than the target antibody. • For impurities bind weaker, they can be removed by pre-elution wash under appropriate conditions. • For impurities bind stronger, they can be separated by selecting a suitable condition that selectively elutes the product. • Here we compared the relative robustness of byproduct removal by wash and by elution with the same set of impurities. [ABSTRACT FROM AUTHOR]

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. • WuXiBody, a bsAb platform developed by WuXi Biologics, enables any mAb pair to be assembled into a bispecific construct. • A key feature of WuXiBody is the replacement of one parental mAb's CH1/CL region with the TCR constant domain. • LC-missing species are potential byproducts associated with bsAbs containing engineered Fab arm, including WuXiBodies. • WuXiBody's unique design facilitates removal of LC-missing byproducts by IEX or mixed-mode chromatography. • As shown in two cases, mixed-mode resin Capto MMC ImpRes effectively removes LC-missing byproducts and aggregates. [ABSTRACT FROM AUTHOR]

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. • WuXiBody, a bsAb platform developed by WuXi Biologics, enables any mAb pair to be assembled into a bispecific construct. • A key feature of WuXiBody is the replacement of one parental mAb's CH1/CL region with the TCR constant domain. • WuXiBody's unique design not only ensures desired chain pairing but also facilitates removal of product-related impurities. • Protein A followed by AEX and mixed-mode chromatography can be a platform approach for WuXiBody purification. [ABSTRACT FROM AUTHOR]

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. • Half antibody, hole-hole homodimer and aggregates are common by-products associated with the production of KiH-based bsAb. • Mixed-mode resin Capto MMC ImpRes can effectively remove the above mentioned impurities under appropriate conditions. • BsAb and the hole-hole homodimer, which are different by <1 kDa in size, can be differentiated by non-reducing SDS-PAGE. • Analytical HIC is a powerful tool for monitoring hole-hole homodimer in bsAb purification. [ABSTRACT FROM AUTHOR]

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. • Homodimer and heterodimer may not be readily differentiated using SEC-HPLC as the two species are usually close in size. • Hole-hole homodimer is a common by-product in production of bsAb whose HC heterodimerization is promoted by KiH technology. • Hole-hole homodimer is usually more hydrophobic than heterodimer as its Fc region is less well folded than that of the latter. • Analytical HIC is a powerful tool for monitoring hole-hole homodimer in bsAb purification. [ABSTRACT FROM AUTHOR]