Different VH3-binding Protein A Resins Show Comparable VH3-binding Mediated Byproduct Separation Capabilities Despite Having Varied Dynamic Binding Capacities Towards A VH3 Fab.

Background: Protein A resins have been widely used for product capture during mAb, bispecific antibody (bsAb), and Fc-fusion protein purification. While Protein A ligands mainly bind the Fc region, many of them can also bind the VH3 domain. During mAb/bsAb purification, certain truncated byproducts may contain the same Fc region as the product but fewer numbers of the VH3 domain. In such a scenario, VH3-binding Protein A resins provide a potential means for byproduct separation based on the difference in VH3-binding valency. As the ligands of different VH3-binding Protein A resins are derived from distinct domains of the native Protein A, it would be interesting to know whether they possess comparable capabilities for separating species with the same Fc region but different numbers of VH3 domain.

Objective: This study aims to explore the potential of different VH3-binding Protein A resins for separating antibody species with the same Fc region but different numbers of VH3 domain.

Methods: The VH3 Fab was released from a VH3-containing mAb by papain digestion. Post digestion, the released VH3 Fab was purified sequentially using CaptureSelect CH1-XL and MabSelect SuRe affinity chromatography. The purified VH3 Fab was used as the load material to assess the dynamic binding capacity (DBC) of five VH3-binding Protein A resins (i.e., Amshpere A3, Jetted A50, MabCaptureC, MabSelect and MabSelect PrismA). The potential of VH3-binding Protein A resins for separating species having the same Fc region but different numbers of VH3 domain was evaluated using an artificial mixture composed of the product and a truncated byproduct, which contained one and zero VH3 domain, respectively (both species contained the same Fc region). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to monitor Fab purification and separation of species containing the same Fc region but different numbers of VH3 domain.

Results: When loaded with an isolated VH3 Fab, different VH3-binding Protein A resins showed varied DBCs. Nevertheless, when these Protein A resins were used to separate a truncated byproduct, which contained the Fc region only without any VH3 domain, from the product, which included one VH3 domain in addition to the Fc region, they showed comparable capabilities for separating these two species.

Conclusion: Although different VH3-binding Protein A resins showed varied DBCs towards a VH3 Fab, they exhibited comparable capabilities for separating species with the same Fc region but different numbers of VH3 domain.