Article

Removing half antibody byproduct by Protein A chromatography during the purification of a bispecific antibody

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Abstract

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.

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... In addition to direct product capture, both types of chromatography are capable of separating various product-related impurities under appropriate conditions [10]. For example, both Protein A and Protein L chromatographies can effectively separate half-antibody from the intact bsAb [11,12]. Under optimized conditions, both chromatographies also showed great potential for removing aggregates [13][14][15]. ...
... For example, Protein A chromatography provided limited resolution between half-antibody and the full-length bsAb under typical linear pH gradient elution without salt additive, and complete separation was achieved when 500 mM sodium chloride (NaCl) was added to the mobile phase [11]. Salt additives are also required for attaining good aggregate removal by Protein A and Protein L chromatography [13][14][15]. ...
... Protein A binding capacity and hence results in asymmetric Protein A binding between the two heavy chains), salt additive significantly improved the resolution between heterodimer and homodimer [16]. According to previous studies, both kosmotropic and chaotropic salts, when used as mobile phase additives, could improve the resolution of Protein A and Protein L chromatography [11,[14][15][16]18]. ...
Article
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Protein A and Protein L affinity chromatographies are extensively used in mAb and bispecific antibody (bsAb) purification. In addition to product capture, they are both capable of separating certain product-related by-products and aggregates under appropriate conditions. For both types of chromatography, previous studies suggested that adding a salt additive to the mobile phase can significantly improve the resolution between product and by-products/aggregates. Nevertheless, the effects of different salt additives on antibody elution in Protein A and Protein L chromatography have not been compared. In the current study, we compared the effects of three salt additives, sodium chloride (NaCl), calcium chloride (CaCl2), and arginine hydrochloride (Arg·HCl), on antibody elution in Protein A and Protein L chromatography. Interestingly, while NaCl suppressed antibody elution in both types of chromatography, CaCl2, and Arg·HCl promoted antibody elution in Protein A chromatography but suppressed antibody elution in Protein L chromatography. In addition, we evaluated the effect of each salt gradient on aggregate removal by Protein L chromatography. The information provided by the current study should be useful to the selection of conditions/additives for improving by-product removal by Protein A and Protein L chromatography.
... Consistently, we showed that Protein A chromatography provides certain degree of resolution between half-antibody and the intact bsAb under linear pH gradient elution. 27 Furthermore, we demonstrated that complete separation between half-antibody and the full-length bsAb can be achieved by including salt additive (i.e., 500 mM NaCl) to the mobile phase ( Figure 2a). 27 According to the previous studies, both kosmotropic and chaotropic salts, when used as mobile phase additives, can improve resolution and selectivity of Protein A chromatography. ...
... 27 Furthermore, we demonstrated that complete separation between half-antibody and the full-length bsAb can be achieved by including salt additive (i.e., 500 mM NaCl) to the mobile phase ( Figure 2a). 27 According to the previous studies, both kosmotropic and chaotropic salts, when used as mobile phase additives, can improve resolution and selectivity of Protein A chromatography. 20,28 Besides Protein A, several other antibody-binding affinity media (e.g., Protein L and CaptureSelect FcXP) also possess the capabilities of separating half-antibody. ...
Article
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Bispecific antibodies (bsAbs) can simultaneously bind two different antigens or epitopes. Their dual‐targeting capability enables novel mechanisms of action, gaining therapeutic advantages over conventional monospecific mAbs. In recent years, the number of bsAbs grows rapidly and bsAbs under development are available in diverse formats. In particular, Fc‐containing IgG‐like bsAbs, which represent the major group, can be constructed in asymmetric or symmetric format. For asymmetric ones, whose assembly requires multiple distinct chains, although numerous strategies have been developed to promote desired chain pairing, product‐related variants such as free chains, half molecules and mispaired species are usually present at various levels. For symmetric ones, increased level of aggregates and truncating variants is often associated with their production. In general, bsAbs pose greater challenges to the downstream team than regular mAbs. In the past few years, our team successfully developed the downstream process for over 70 bsAbs in greater than 30 different formats and accumulated substantial experience. This review introduces general strategies that we have used while purifying these challenging molecules.
... 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 an appropriate pre-elution wash [1,2]. On the other hand, for impurities that bind stronger, their separation can be achieved by choosing a suitable elution condition, under which the target antibody is selectively eluted while the impurities remain bound to the column [3,4]. ...
... Thus, for this AEX step, which employs wash for impurity clearance, yield and eluate quality are highly sensitive to the loading density under fixed wash and elution conditions. High sensitivity of wash-enabled impurity clearance to column loading density has been observed in other cases [1,2]. In general, a fixed wash condition only offers effective impurity clearance and good yield at the same time within a narrow loading density range. ...
Article
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.
... For Protein A and Protein L affinity chromatography, several previous studies showed that adding salt to the mobile phase could significantly improve resolution [16][17][18][19]. Thus, we applied this strategy to Praesto 70 CH1. ...
Article
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CaptureSelect CH1-XL and Praesto 70 CH1 are two affinity media that specifically bind to the CH1 domain of an antibody. In the current work, we first demonstrated that these two CH1-specific affinity media bound to different monoclonal antibodies (mAbs) with varied strengths under identical conditions. We previously had observed the same on a Protein L-conjugated resin and showed that such a property could facilitate homodimer removal in asymmetric bispecific antibody (bsAb) purification. Next, using Praesto 70 CH1, we showed that a small difference in binding between two mAbs could be significantly exaggerated by adding sodium chloride to the mobile phase, further demonstrating this resin can potentially play a role in bsAb purification. Finally, with a concrete bsAb case study, we showed that, like Protein L, Praesto 70 CH1 could separate the target heterodimer from the homodimer by-product. Homodimers are common product-related impurities associated with the recombinant production of asymmetric bsAbs, which can be difficult to remove. Their removal, even a partial one, at the capture stage is a big advantage as it can alleviate the purification burden on subsequent polishing steps and render the overall process more robust. Therefore, Praesto 70 CH1’s unique property is highly desirable, and this affinity resin can be a better alternative than Protein A for product capture in asymmetric bsAb purification.
... A similar principle applies here as halfmers can just bind with one domain, while full antibodies bind with two HC. This phenomenon can be utilized on ProA where half antibodies can be washed off with a suitable pH in the presence of high salt concentrations before the final low pH elution of the complete bsAb (25). ...
... In particular, differential Protein A affinity chromatography has been proposed to be able to separate the target bsAb from the undesired heavy chain mispaired products through the use of a pH gradient or multi-step pH elutions (Tustian et al. 2016;Lindhofer et al. 1995;Smith et al. 2015;Zwolak et al. 2017;Zwolak et al. 2017a, b;Skegro et al. 2017;Ollier et al. 2019), particularly for bsAbs generated with modifications made to alter the Protein A-binding affinity between target bsAb and that of mispaired products. The use of salts has also been reported to improve the separation resolution between the target bsAb and binding mispaired homodimers (Tustian et al. 2016) as well as ½ antibodies (Chen et al. 2020). With regard to resin selection, engineered Protein A affinity ligands that lack VH binding, such as MabSelect SuRe (Tustian et al. 2016), have been proposed to be useful in preventing a reduced avidity difference between the target bsAb and undesired bound homodimers due to VH3-Protein A interactions (Sasso et al. 1991(Sasso et al. , 1989. ...
... In particular, bsAbs are often associated with a higher level of impurities and byproducts, including aggregates, fragments and mispaired products (Garber 2014;Taki et al. 2015;Andrade et al. 2019;Michaelson et al. 2009;Michaelson et al. 2009;Jakobsen et al. 2011, Klein et al. 2012, with the generally lower titers of bsAbs often translating to relatively higher host cell protein (HCP) levels. We (Chen et al. 2022), along with others (Tustian et al. 2016(Tustian et al. , 2018Lindhofer et al. 1995;Smith et al. 2015;Zwolak et al. 2017a, b;Zwolak et al. 2017a, b;Skegro, et al. 2017;Ollier et al. 2019;Chen et al. 2020;Zhang et al. 2021), have reported the effective use of Protein A as a capture step to effectively remove such impurities and byproducts. An effective polishing strategy is, however, irreplaceable, as > 1% of high molecular weight (HMW) species and > 100 ppm HCP often remains after the first Protein A capture step. ...
Article
Full-text available
Bispecific antibodies (bsAbs), though possessing great therapeutic potential, are extremely challenging to obtain at high purity within a limited number of scalable downstream processing steps. Complementary to Protein A chromatography, polishing strategies play a critical role at removing the remaining high molecular weight (HMW) and low molecular weight (LMW) species, as well as host cell proteins (HCP) in order to achieve a final product of high purity. Here, we demonstrate using two knob-into-hole (KiH) bsAb constructs that two flow-through polishing steps utilising Capto Butyl ImpRes and Capto adhere resins, performed after an optimal Protein A affinity chromatography step can further reduce the HCP by 17- to 35-fold as well as HMW and LMW species with respect to monomer by ~ 4–6% and ~ 1%, respectively, to meet therapeutical requirement at 30–60 mg/mL-resin (R) load. This complete flow-through polishing strategy, guided by Design of Experiments (DoE), eliminates undesirable aggregation problems associated with the higher aggregation propensity of scFv containing bsAbs that may occur in the bind and elute mode, offering an improved ease of overall process operation without additional elution buffer preparation and consumption, thus aligning well with process intensification efforts. Overall, we demonstrate that through the employment of (1) Protein A chromatography step and (2) flow-through polishing steps, a final product containing < 1% HMW species, < 1% LMW species and < 100 ppm HCP can be obtained with an overall process recovery of 56–87%. Graphical Abstract
... In particular, differential Protein A affinity chromatography has been proposed to be able to separate the target bsAb from the undesired heavy chain mispaired products through the use of a pH gradient or multi-step pH elutions (Tustian et al. 2016;Lindhofer et al. 1995;Smith et al. 2015;Zwolak et al. 2017;Zwolak et al. 2017a, b;Skegro et al. 2017;Ollier et al. 2019), particularly for bsAbs generated with modifications made to alter the Protein A-binding affinity between target bsAb and that of mispaired products. The use of salts has also been reported to improve the separation resolution between the target bsAb and binding mispaired homodimers (Tustian et al. 2016) as well as ½ antibodies (Chen et al. 2020). With regard to resin selection, engineered Protein A affinity ligands that lack VH binding, such as MabSelect SuRe (Tustian et al. 2016), have been proposed to be useful in preventing a reduced avidity difference between the target bsAb and undesired bound homodimers due to VH3-Protein A interactions (Sasso et al. 1991(Sasso et al. , 1989. ...
Article
Full-text available
Bispecific antibodies (bsAbs) are therapeutically promising due to their ability to bind to two different antigens. However, the bsAb byproducts and impurities, including mispaired homodimers, half-antibodies, light chain mispairings, antibody fragments and high levels of high molecular weight (HMW) species, all pose unique challenges to their downstream processing. Here, using two knob-into-hole (KiH) constructs of bsAbs as model molecules, we demonstrate the excellent removal of bsAb byproducts and impurities in a single Protein A chromatography under optimized conditions, including hole–hole homodimer mispaired products which are physicochemically very similar to the target bsAbs and still present even with the use of the KiH format, though at reduced levels. The removal occurs through the incorporation of an intermediate low-pH wash step and optimal elution conditions, achieving ~ 60% monomeric purity increase in a single Protein A step, without the introduction of sequence-specific bsAb modifications to specifically induce differential Protein A binding. Our results also suggest that the higher aggregation propensity of bsAbs may cause aggregation during the column process, hence an optimization of the appropriate loading amount, which may be lower than that of monoclonal antibodies (mAbs), is required. With the use of loading at 50% of 10% breakthrough (QB10) at 6-min residence time, we show that an overall high monomer purity of 92.1–93.2% can be achieved with good recovery of 78.4–90.6% within one capture step, which is a significant improvement from a monomer purity of ~ 30% in the cell culture supernatant (CCS). The results presented here would be an insightful guidance to all researchers working on the purification process development to produce bispecific antibodies, especially for knob-into-hole bispecific antibodies. Graphical Abstract
... The risks of mispairing are mitigated with a common LC approach; the only concern is the heterodimerization purity, of which there have been many good approaches resulting in >95% heterodimerization, with minimal risk of heterogeneity [66,67]. There are manufacturing protocols that can be used to remove residual half-antibodies since Fcs can be engineered such that half-antibodies bind protein A with lower affinity than full-length antibodies [68]. In some cases, the sequences and biophysical properties between the two half antibodies are sufficiently different that half-antibodies can be removed by chromatographic methods. ...
Article
Introduction: Antibody therapies have made huge strides in providing safe and efficacious drugs for autoimmune, cancer and infectious disease. These bispecific antibodies can be assembled from the basic building blocks of IgGs, resulting in dozens of formats. Areas covered: It is important to consider the manufacturability of these formats early in the antibody discovery phases. Broadly categorizing bispecific antibodies into IgG-like, fragment-based, appended and hybrid formats can help in looking at early manufacturability considerations. Expert opinion: Ideally, bispecific antibody manufacturing should contain a minimal number of steps, with processes that give high yields of protein with no contaminants. Many of these have been determined for the fragment-based bispecific blinatumomab and the IgG-like bispecifics from hybridomas. However, for new formats, these need to be considered early in the research and development pipeline. The hybrid formats offer an unusual alternative in generating high pure yields of bispecific molecules if the engineering challenges can be deciphered.
... In particular, differential Protein A affinity chromatography has been proposed to be able to separate the target bsAb from the undesired heavy chain mispaired products through the use of a pH gradient or multi-step pH elutions (Tustian et al. 2016;Lindhofer et al. 1995;Smith et al. 2015;Zwolak et al. 2017;Zwolak et al. 2017a, b;Skegro et al. 2017;Ollier et al. 2019), particularly for bsAbs generated with modifications made to alter the Protein A-binding affinity between target bsAb and that of mispaired products. The use of salts has also been reported to improve the separation resolution between the target bsAb and binding mispaired homodimers (Tustian et al. 2016) as well as ½ antibodies (Chen et al. 2020). With regard to resin selection, engineered Protein A affinity ligands that lack VH binding, such as MabSelect SuRe (Tustian et al. 2016), have been proposed to be useful in preventing a reduced avidity difference between the target bsAb and undesired bound homodimers due to VH3-Protein A interactions (Sasso et al. 1991(Sasso et al. , 1989. ...
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Bispecific antibodies (bsAbs) are therapeutically promising due to their ability to bind to two different antigens. However, the bsAb byproducts and impurities, including mispaired homodimers, half antibodies, light chain mispairings, antibody fragments and high levels of high molecular weight (HMW) species, all pose unique challenges to their downstream processing. Here, using two knob-into-hole (KiH) constructs of bsAbs as model molecules, we demonstrate the excellent removal of bsAb byproducts and impurities in a single Protein A chromatography under optimized conditions, including hole-hole homodimer mispaired products which are physicochemically very similar to the target bsAbs and still present even with the use of the KiH format, though at reduced levels. The removal occurs through the incorporation of an intermediate low pH wash step and optimal elution conditions, achieving ~ 60% improvement in monomeric purity in a single Protein A step, without the introduction of sequence specific bsAb modifications to specifically induce differential Protein A binding. Our results also suggest that the higher aggregation propensity of bsAbs may cause aggregation during the column process, hence an optimisation of the appropriate loading amount, which may be lower than that of monoclonal antibodies (mAbs), is required. With the use of loading at 50% of 10% breakthrough (QB10) at 6 min residence time, we show that an overall high monomer purity of 92.1–93.2% can be achieved with good recovery of 78.4–90.6% within one capture step, which is a significant improvement from a monomer purity of ~ 30% in the cell culture supernatant (CCS). The results presented here would be an insightful guidance to all researchers working on the purification process development to produce bispecific antibodies, especially for knob-into-hole bispecific antibodies.
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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.
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