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Open AccessArticle

Fab-Arm Exchange Combined with Selective Protein A Purification Results in a Platform for Rapid Preparation of Monovalent Bispecific Antibodies Directly from Culture Media

Antibody Discovery and Protein Engineering, AstraZeneca, One MedImmune Way, Gaithersburg, MD 201878, USA
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Pharmaceutics 2020, 12(1), 3; https://doi.org/10.3390/pharmaceutics12010003
Received: 5 November 2019 / Revised: 5 December 2019 / Accepted: 12 December 2019 / Published: 18 December 2019
(This article belongs to the Special Issue Recombinant Therapeutic Proteins for Drug Delivery)
Bispecific antibody (bsAb) applications have exponentially expanded with the advent of molecular engineering strategies that have addressed many of the initial challenges, including improper light chain pairing, heterodimer purity, aggregation, and pharmacokinetics. However, the lack of high-throughput methods for the generation of monovalent bsAbs has resulted in a bottleneck that has hampered their therapeutic evaluation, as current technologies can be cost-prohibitive and impractical. To address this issue, we incorporated single-matched point mutations in the CH3 domain to recapitulate the physiological process of human IgG4 Fab-arm exchange to generate monovalent bsAbs. Furthermore, we utilized the substitutions H435R and Y436F in the CH3 domain of IgG1, which incorporates residues from human IgG3, thus ablating protein A binding. By exploiting this combination of mutations and optimizing the reduction and reoxidation conditions for Fab arm exchange, highly pure monovalent bsAbs can be rapidly purified directly from combined culture media using standard protein A purification. This methodology, reported herein for the first time, allows for the high-throughput generation of monovalent bsAbs, thus increasing the capacity for evaluating monovalent bsAb iterations for therapeutic potential. View Full-Text
Keywords: monovalent bispecific antibodies; Fab-arm exchange; protein A binding; high-throughput bispecific generation monovalent bispecific antibodies; Fab-arm exchange; protein A binding; high-throughput bispecific generation
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Steinhardt, J.; Wu, Y.; Fleming, R.; Ruddle, B.T.; Patel, P.; Wu, H.; Gao, C.; Dimasi, N. Fab-Arm Exchange Combined with Selective Protein A Purification Results in a Platform for Rapid Preparation of Monovalent Bispecific Antibodies Directly from Culture Media. Pharmaceutics 2020, 12, 3.

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