Removal of Antibody-Related Fragments During Asymmetric Bispecific Antibody Purification

Bispecific antibodies (bsAbs) can bind two different epitopes or targets. BsAb expression during cell culture is accompanied with high levels of impurities, product- related or process-related impurities. The product-related impurities, especially fragments can reduce the stability and safety of therapeutic protein, which must be removed as much as possible. Fragments removal by chromatography during the downstream process could be a challenge. In this work, we summarizes the downstream fragment removal strategies of asymmetric bsAbs in our laboratory. Due to weaker binding ability with protein A, some fragment species can be removed by affinity chromatography under high loading capacity condition, or by adding wash step to get rid of them. Anion exchange chromatography (AEX) was found to be efficient to reduce fragments through the UV peak-cutting method. According to the difference of charge and hydrophobicity between the fragment and the target antibody, the UV peak-cutting method accompanied with flow-through anion exchange chromatography (FT-AEX) or bind-elute hydrophobic interaction chromatography (BE-HIC) can also play an obvious effect on fragment removal. This study discussed various fragments reduction means by affinity chromatography, anion exchange chromatography and hydrophobic interaction chromatography, which supply many options to achieve the fragment reduction in future.