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For monoclonal antibodies (mAbs) produced in mammalian cells, viral safety is a critical concern. The downstream process, in addition to removing other impurities, needs to ensure robust clearance (removal or inactivation) of potential endogenous and adventitious viruses. In general, Protein A and polishing chromatography steps all can provide certain level of virus removal. Chromatographic removal combined with virus inactivation and nanofiltration usually provides adequate virus clearance across the overall downstream process. This article reviews the virus clearance capability of commonly used column chromatography, with attention to possible interference of virus-mAb interaction on virus removal. In addition, the potential of using viral surrogate as a safe alternative to live virus for assessing viral clearance is briefly discussed. • Virus-mAb interactions negatively impact viral removal by Protein A and other types of column chromatography. • Including interaction-disrupting additives to Protein A wash buffer can improve this step's viral clearance capacity. • Virus surface charge distribution is more relevant than pI in determining its interaction with IEX resins. • Viral surrogates are potential safe alternatives to live viruses for assessing a unit operation's viral removal capability. [ABSTRACT FROM AUTHOR]

Abstract: LL-37 is a human antimicrobial peptide that has been shown to possess multiple functions in host defense. In this report, the peptide was expressed as a fusion with a thioredoxin–SUMO dual-tag. Upon SUMO protease mediated cleavage at the SUMO/peptide junction, LL-37 with its native N-terminus was generated. The released peptide was separated from the dual-tag and cleavage enzyme by size-exclusion chromatography. Mass spectrometry analysis proves that the recombinant peptide has a molecular weight as theoretically expected for its native form. The produced peptide displayed antimicrobial activity against Escherichia coli K-12. On average, 2.4mg peptide was obtained from one liter of bacterial culture. Thus, the described approach provides an effective alternative for producing active recombinant LL-37 with its natural amino acid sequence in E. coli. [Copyright &y& Elsevier]

Abstract Aggregation is a major concern for therapeutic monoclonal antibody (mAb), as aggregates reduce drug efficacy and safety. In addition to aggregation control, aggregate removal by downstream processing is crucial. Hydrophobic and mixed-mode resins are widely used for aggregate removal in different cases, but they are seldom compared side by side. In this study, the aggregate removing capability of eight resins belonging to different chromatographic types was demonstrated by a case study. This work, by providing multiple options for aggregate removal, allows more flexibility to be gained in downstream processing. Highlights • The aggregate removal capability of eight resins belonging to different chromatographic types was demonstrated. • In consistent with published results, adding polyethylene glycol (PEG) to the mobile phase can significantly improve the resolution for CHT, Capto MMC and ion exchange (IEX) columns. • All studied resins can effectively reduce aggregates from approximately 14% to less than 2% while maintaining good recovery under typical or optimized conditions. • For Capto adhere and POROS XS columns, step gradient elution was successfully developed based on linear gradient elution results. [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]

While purifying a regular monospecific antibody, we found that the Protein A step yield was much lower than expected. Further studies revealed that the antibody formed large-size aggregates that did not bind to the Protein A resin, hence leading to dropped recovery. In an attempt to solve this low yield issue, we found that mildly acidic pH or ammonium sulfate treatment can partially convert the aggregates into monomers. In addition, when acidic pH treated culture harvest was processed by Protein A chromatography, the yield was restored to the normal range, suggesting that the monomers recovered from aggregates regained Protein A binding capability. Thus, low pH treatment of culture harvest can be potentially used as a general approach for improving Protein A step yield in cases where non-binding antibody aggregates are formed through noncovalent interactions. • SEC-HPLC analysis of culture harvest of a monospecific antibody disclosed formation of large- and small-size aggregates. • The large-size aggregates are predominant and did not bind to the Protein A resin, leading to lower-than-expected step yield. • Upon mildly acidic pH or ammonium sulfate treatment, the non-binding aggregates were partially converted into monomers. • Low pH treatment of culture harvest restored normal Protein A yield, suggesting recovered monomers regained binding ability. [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]

In downstream processing of protein therapeutics, ion exchange (IEX) chromatography is a powerful tool for removing byproducts whose isoelectric point (pI) is appreciably different from that of the product. Although in theory for a given case cation exchange (CEX) and anion exchange (AEX) chromatography should be equally effective for separation, in reality they may show different effectiveness. In the current work, with a case study, we demonstrated that AEX is more effective than CEX chromatography at removing the associated byproducts. In addition, we screened AEX resins and loading conditions to achieve best separation. Finally, we demonstrated that effective separation was achieved with the selected resin/condition, and chromatography performance was comparable between runs conducted at low and high load densities, suggesting that the developed process was relatively robust. The procedure described in this work can be used as a general approach for selecting resin and loading condition that allow for effective and robust removal of byproduct that binds weaker than the product to the selected type of column. • For byproducts whose pIs are different from that of the product, IEX chromatography is a promising tool for their removal. • For a given case, CEX and AEX chromatography may exhibit different effectiveness at removing the associated byproducts. • An approach was developed for identifying AEX loading conditions under which byproducts flow through and product binds. • AEX performance was not sensitive to load density under the selected conditions, which improves process robustness. [ABSTRACT FROM AUTHOR]

For recombinantly produced monoclonal antibody (mAb), charge variants including acidic and basic species are common heterogeneities. For characterization purpose, sufficient amount of acidic and basic species with high purity is needed. In this work, we developed an approach that allows for continuous separating and collecting of acidic and basic charge variants. First, with batch-mode cation exchange (CEX) chromatography, the load density and linear salt gradient elution conditions under which good separation of both charge variants can be achieved were determined. Next, a stepwise elution protocol was developed based on the linear gradient elution. Finally, acidic and basic charge variants were persistently produced under stepwise elution using a customized twin-column continuous chromatography system. This approach allows acidic and basic charge variants with high purity (i.e., >90%) to be efficiently generated in sufficient amount, which greatly facilitates the necessary characterization of these mAb variants. • Full characterization of antibody charge variants is highly desirable. • A prerequisite for charge variants characterization is an effective and efficient method for enriching these species. • CEX chromatography is an effective tool for separating mAb charge variants. • Acidic and basic charge variants can be simultaneously obtained using a twin-column continuous chromatography system. [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]

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]

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]

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]

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]

In downstream processing of monoclonal antibody (mAb), post Protein A neutralization and subsequent intermediate depth filtration are critical steps for host cell protein (HCP) clearance. Previous studies have shown that adding caprylic acid (CA) during neutralization can further improve HCP removal by promoting their precipitation. In this study, we replaced CA with its sodium salt – sodium caprylate (SC). For the five mAbs studied, SC has been shown to be equally effective as CA at precipitating HCPs. As the salt form has a higher solubility, SC stock solution with relatively high concentration can be easily prepared, which facilitates its adding to the Protein A elution pool. Thus, this study not only confirms the effectiveness of CA/SC-induced HCP precipitation but also provides a more convenient way to integrate this method into the downstream process. • Sodium caprylate (SC) induced precipitation is an effective means for host cell protein (HCP) clearance. • SC has been shown to be equally effective as caprylic acid (CA) at precipitating HCPs. • SC stock solution with relatively high concentration can be easily prepared as the salt form has much higher solubility. • Replacement of CA with SC provides a more convenient way to integrate this HCP removing method into the downstream process. [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]

We previously developed a method that can significantly improve Protein A chromatography's capability of removing antibody aggregates. That particular method requires polyethylene glycol (PEG) and 400 mM or more of calcium chloride/sodium chloride to be added to wash and elution buffers. Consequently, Protein A chromatography performed using this method has relatively high concentration of salt in its eluate. The high salt content prevents the neutralized eluate from binding to ion exchange columns without conductivity adjustment. In the current study we demonstrated that mixed-mode chromatography can be used as a subsequent step to Protein A chromatography with high-salt-containing eluate. As mixed-mode ligand mediates salt-tolerant adsorption, it allows the neutralized Protein A eluate to be directly loaded without the need of conductivity adjustment, and thus enables a smooth and convenient connection between capture and polishing steps. In this work we also showed that the mixed-mode chromatography, performed in bind-elute mode, removed most of PEG in the Protein A eluate. • Protein A capture step optimized for aggregate removal can be conveniently connected with mixed-mode chromatography. • Mixed-mode chromatography allows high-salt-containing Protein A eluate to be directly loaded without conductivity adjustment. • Capto MMC and CHT chromatography can effective remove antibody aggregates. • Capto MMC and CHT columns under bind-elute mode removed most of PEG in the Protein A eluate. [ABSTRACT FROM AUTHOR]