Opposing effects of arginine on Fc- and VH3-mediated antibody-Protein A interactions.

MabCaptureC is a new Protein A resin recently launched by Thermo Fisher. In addition to Fc-binding, MabCaptureC also possesses VH3-binding capability. In consistency with previous reports, we found that elution of a VH3-containing antibody from MabCaptureC requires conditions harsher than those normally used due to the existence of VH3-mediated binding in addition to the regular Fc-mediated binding. In addition, even though arginine is widely known for having a promoting effect on antibody elution from Protein A columns, at 100 mM it unexpectedly suppressed elution of the VH3-containing antibody from this resin, reducing the yield from 79.3% to 4.6%. A comprehensive study using multiple commercial Protein A resins with distinct binding specificities (i.e., Fc-binding only, Fc- and VH3-binding, and VH3-binding only) revealed that the suppressive effect of arginine on elution is specific to VH3-containing antibody from Protein A resins with VH3-binding capability. Results from the current study strongly suggest that arginine exhibits an opposing effect on Fc- and VH3-mediated binding: it weakens the former while strengthens the latter. Leveraging the opposing effect of arginine on Fc-binding and VH3-binding, we successfully separate a VH3 mAb from a non-VH3 mAb using MabCaptureC with arginine-containing elution buffer. Furthermore, by applying a similar approach, we effectively removed the homodimer byproducts associated with the production of an asymmetric bispecific antibody (bsAb) whose two parental mAbs' VH regions have different origins (i.e., VH1 and VH3), which demonstrates the practical value of the current finding.