A generic platform to couple affinity chromatography with native mass spectrometry for the analysis of therapeutic monoclonal antibodies.

Affinity chromatography coupled with native mass spectrometry has emerged as a powerful tool for the analysis of therapeutic monoclonal antibodies (mAbs). Exploiting the specific interactions between mAbs and their ligands, these methods not only provide orthogonal means to study the highly complex mAb attributes, but also offer insights on their biological relevance. Despite the great promise, application of affinity chromatography - native mass spectrometry in routine mAb characterization has been limited, largely due to the complicated experimental set up. In this study, we introduced a generic platform to facilitate the online coupling of different affinity separation modes with native mass spectrometry. Built upon a recently introduced native LC-MS platform, this new strategy can accommodate a wide range of chromatographic conditions, and therefore, allow greatly simplified experimental set up and facile swapping of affinity separation modes. The utility of this platform was demonstrated by successful online coupling of three affinity chromatography methods (protein A, FcγRIIIa, and FcRn) with native mass spectrometry. The developed protein A-MS method was tested both in a "bind-and-elute" mode for rapid mAb screening and in a high-resolution resolving mode to study mAb species with altered protein A affinity. The FcγRIIIa-MS method was applied to achieve glycoform-resolved analyses of both IgG1 and IgG4 subclass molecules. The FcRn-MS method was demonstrated in two case studies, where specific post-translational modifications and Fc mutations were known to alter FcRn affinities.