Improving Stepwise Assembly of a Bispecific F(ab′)2 from Two Different Fab′ Molecules

An upstream process was developed for large-scale assembly of bispecific F(ab′)2 from two different Fab′ molecules. We identified a novel product-related structural variant that is difficult to remove downstream and modified our process to decrease its generation during the assembly step. The resulting multistep process includes affinity capture, an on-column cysteine uncapping step, batch reoxidation and pyridylation, and assembly of the two Fab′ molecules into a F(ab′)2. Optimizations include selection of hinge domain-specific reducing agent, column chromatography conditions, and reaction pH. We selected L-reduced Glutathione to target hinge cysteines for reduction, uncapping the Fab′ while minimizing off-target reduction. We scaled up the improved method for assembling bispecific F(ab′)2 with similar assembly efficiency but significantly improved product quality, generating a pool with bispecific F(ab′)2 for downstream processing.