Abstract
Bispecific antibodies hold great potential in next-generation biotherapeutics simultaneously bind to two targets. However, the perspective of bispecific has biotherapeutics has been hindered the byproducts at high levels, including high molecular weight (HMW), low molecular weight (LMW) variants at very high levels. In addition, the inevitable expression of homodimers in the host cells further contributes to the obstacles in the commercial development of bispecific antibodies as therapeutics. These above-mentioned byproducts, which closely resemble the target in physicochemical properties, pose several challenges for downstream processes in terms of purification. Here, we have introduced the Knob-into-hole design to avoid mispairing, where mixed-mode resin Capto Adhere was used to capture the target protein at pH 7.8 ± 0.1, with a subsequent AEX as a polishing step. Overall, the results of the two-step chromatography purification method show that the final product purity improved to 98% for SEC-HPLC and 98% for RP-HPLC, with residual HCP controlled at 10 ppm and an excellent recovery rate of 75%. This study offers a non-Protein A capture platform, which offers a simplified, streamlined, and competitive alternative to conventional affinity chromatography.