Non-affinity platform for processing knob-into-hole bispecific antibody

Abstract

Bispecific antibodies hold significant potential as next-generation biotherapeutics owing to their ability to simultaneously bind to two targets. However, the development of bispecific antibodies as biotherapeutics has been hindered by the high levels of byproducts produced, including both high molecular weight and low molecular weight variants. In addition, the inevitable expression of homodimers in host cells presents further obstacles to the commercial development of bispecific antibodies as therapeutics. These byproducts, which share similar physicochemical properties with the target, pose several challenges for downstream purification processes. In this study, we present a non-protein A purification platform that employ a one-step polishing chromatography to purify bispecific antibodies. Mixed-mode Capto adhere resin was used to capture the target protein at pH 7.90 ± 0.10, followed by anion exchange chromatography as a polishing step. Overall, the results of this two-step chromatography purification method demonstrated at final product purity of 98% as assessed by size-exclusion high-performance liquid chromatography (SEC-HPLC) and 98% by reversed-phase-high-performance liquid chromatography (RP-HPLC), with residual host cell proteins controlled at 10 ppm and an excellent recovery rate of approximately 60%. This study presents a non-protein A capture platform, offering a simplified, streamlined, and competitive alternative to conventional affinity chromatography.

Graphical Abstract

References

[1] BrinkmannU, KontermannRE. The making of bispecific antibodies. Mabs, 2017, 9: 182-212

[2] BrownMR, BurnhamMS, JohnsonSA, LuteSC, BrorsonKA, RoushDJ. Evaluating the effect of in-process material on the binding mechanisms of surrogate viral particles to a multi-modal anion exchange resin. J Biotechnol, 2018, 267: 29-35

[3] ChenSW, ZhangW. Current trends and challenges in the downstream purification of bispecific antibodies. Antib Ther, 2021, 4: 73-88

[4] ChenT, HanJ, GuoG, WangQ, WangY, LiY. Monitoring removal of hole-hole homodimer by analytical hydrophobic interaction chromatography in purifying a bispecific antibody. Protein Expr Purif, 2019

[5] ChenSW, HoiKM, MahfutFB, YangY, ZhangW. Effective flow-through polishing strategies for knob-into-hole bispecific antibodies. Bioresourc Bioprocess, 2022

[6] ChenSW, HoiKM, MahfutFB, YangY, ZhangW. Excellent removal of knob-into-hole bispecific antibody byproducts and impurities in a single-capture chromatography. Bioresourc Bioprocess, 2022

[7] CoffmanJL, KramarczykJF, KelleyBD. High-throughput screening of chromatographic separations: I. method development and column modeling. Biotechnol Bioeng, 2008, 100: 605-618

[8] GuoG, HanJ, WangY, LiY. A potential downstream platform approach for WuXiBody-based IgG-like bispecific antibodies. Protein Expr Purif, 2020

[9] KatejaN, KumarD, SethiS, RathoreAS. Non-protein A purification platform for continuous processing of monoclonal antibody therapeutics. J Chromatogr A, 2018, 1579: 60-72

[10] KelleyB, ToblerSA, BrownP, CoffmanJL, GodavartiR, IskraT, SwitzerM, VunnumS. Weak partitioning chromatography for anion exchange purification of monoclonal antibodies. Biotechnol Bioeng, 2008, 101: 553-566

[11] LabrijnAF, JanmaatML, ReichertJM, ParrenPWHI. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov, 2019, 18: 585-608

[12] LiY. A brief introduction of IgG-like bispecific antibody purification: Methods for removing product-related impurities. Protein Expr Purif, 2019, 155: 112-119

[13] LiY. IgG-like bispecific antibody platforms with built-in purification-facilitating elements. Protein Exp Purif, 2021

[14] Li Y, Wang Y, Shen P, Zhou W (2020) A roadmap for IgG-like bispecific antibody purification. In: Approaches to the Purification, Analysis and Characterization of Antibody-Based Therapeutics, pp 167–179.

[15] LiX, LiuW, LiH, WangX, ZhaoY. Capture and purification of an untagged nanobody by mixed weak cation chromatography and cation exchange chromatography. Protein Expr Purif, 2022

[16] LiangX, HeQ, QinG, LiG, LiQ, TanH, WangZ, FanM, XuD. Effectively removing the homodimer in bispecific antibodies by weak partitioning mode of anion exchange chromatography. J Chromatogr B, 2023

[17] MariaS, JouclaG, GarbayB, DieryckW, LomenechA-M, SantarelliX, CabanneC. Purification process of recombinant monoclonal antibodies with mixed mode chromatography. J Chromatogr A, 2015, 1393: 57-64

[18] MasudaY, OginoY, YamaichiK, TakahashiY, NonakaK, WakamatsuK. The prevention of an anomalous chromatographic behavior and the resulting successful removal of viruses from monoclonal antibody with an asymmetric charge distribution by using a membrane adsorber in highly efficient, anion‐exchange chromatography in flow‐through mode. Biotechnol Progr, 2020

[19] O’ConnorE, AspelundM, BartnikF, BergeM, CoughlinK, KambaramiM, SpencerD, YanH, WangW. Monoclonal antibody fragment removal mediated by mixed mode resins. J Chromatogr A, 2017, 1499: 65-77

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