Cysteine-mediated redox strategy for enhancing desired heterodimer formation in KiH-based antibody purification

Although Knobs-into-Holes (KiH) technology effectively promotes heterodimer formation in bispecific antibodies, product-related impurities, including half-antibody species, are frequently generated during manufacturing. Owing to their similar physicochemical properties, these impurities are often difficult to separate from the target antibody using conventional chromatographic methods. Here, we present a cysteine-mediated redox strategy that improves product quality by reassembling these product-related impurities into desired heterodimers, rather than removing them. We implemented two process strategies: a cysteine-supplemented wash step during Protein A affinity chromatography (PA) and a cysteine-mediated batch reaction prior to polishing chromatography. Design of experiments (DoE) identified cysteine concentration as the critical process parameter controlling heterodimer reassembly ( p < 0.05) across both strategies. The cysteine-integrated process consistently improved reversed-phase high-performance liquid chromatography (RP-HPLC) purity while preserving the structural integrity of the target antibody. Peptide mapping and disulfide bond analysis revealed that the cysteine-integrated process promotes inter-chain disulfide bond rearrangement, leading to correct heterodimer assembly, thereby reducing mispaired species and increasing the abundance of correctly assembled heterodimers compared with the conventional process. Remarkably, the cysteine-integrated process achieved approximately a 1.9-fold higher cumulative yield than the conventional approach at an equivalent RP-HPLC purity (>90%). The process was successfully scaled up to a 200 L pilot scale without compromising process performance, demonstrating its scalability and robustness. Collectively, these findings establish the cysteine-mediated redox strategy as a practical, scalable, and manufacturing-compatible approach for improving both product quality and process efficiency in the production of heterodimeric bispecific antibodies.