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. Sep-Oct 2015;31(5):1315-22.
doi: 10.1002/btpr.2130. Epub 2015 Jul 15.

Improved Assembly of Bispecific Antibodies From Knob and Hole Half-Antibodies

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Improved Assembly of Bispecific Antibodies From Knob and Hole Half-Antibodies

Ambrose Jon Williams et al. Biotechnol Prog. .

Abstract

A process was developed for large-scale assembly of IgG1 and IgG4 bispecific antibodies from knob and hole half-antibodies. We optimized assembly conditions such as pH, temperature, stabilizers, and reducing agent. We also identified and exploited structural changes unique to knob and hole half-antibodies with the result of improving assembly outcome, specifically storing half-antibodies at higher pH will condition them to assemble more rapidly and produce fewer high molecular-weight species (HMWS). Application of heat to the assemblies resulted in an acceleration of assembly rate, with optimal formation of bispecific achieved at 37°C. IgG4 half-antibodies were unusually sensitive to temperature-dependent formation of HMWS in pre-assembly conditioning as well as during assembly. We selected l-histidine and Polyvinylpyrrolidone (PVP) as stabilizers to prevent HMWS formation in IgG4 , and achieved rapid and high-efficiency assemblies. Using optimized assembly conditions, we developed and scaled up a method for assembling bispecific antibody with 90% assembly efficiency over 6 h with minimal impact to product quality, generating a pool with bispecific antibody for downstream processing.

Keywords: assembly; bispecific antibody; half-antibody; knob and hole.

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