Removal of homodimer species with MabSelect VH3 during the purification of an asymmetric bispecific antibody

By-products are continuously generated during the manufacturing process of bispecific antibodies, among which homodimers represent the most critical impurity. The effective removal of homodimers is essential, as their presence compromises product purity and may adversely affect therapeutic safety and efficacy. Affinity chromatography, which exploits the highly selective molecular interactions between target antibodies and immobilized ligands, remains the gold-standard purification technique for monoclonal (mAbs) and bispecific antibodies (bsAbs). In this study, we evaluated the separation efficiency of homodimers using three commercially available Protein A resins with distinct binding sites: MabSelect PrismA, MabSelect SuRe LX, and MabSelect VH3. Both the Fc and VH3 regions of the bispecific antibodies (bsAbs) and homodimers in this study have different binding capacities to the affinity resin. Comparative analysis revealed that MabSelect VH3, which relies exclusively on VH3 domain interactions, achieved the highest separation performance with final product purity exceeding 98.9 %. In contrast to MabSelect SuRe LX's single Fc-binding mechanism, separation efficiency was compromised by MabSelect PrismA's dual-binding (Fc and VH3) interactions. Additionally, the differential Fc affinities were identified as the dominant factor influencing resolution under concurrent Fc-binding and VH3 domain disparities between the bsAb and homodimer. These findings provide valuable insights for downstream process optimization in bsAb production, emphasizing the importance of strategic resin selection based on molecular interaction mechanisms.