Integrated Approach for Characterizing Bispecific Antibody/Antigens Complexes and Mapping Binding Epitopes with SEC/MALS, Native Mass Spectrometry, and Protein Footprinting.

Bispecific antibodies (BsAbs), with a unique mechanism of recognizing two different epitopes or antigens, have shown potential in various therapeutic areas. Molecular characterization of BsAbs' epitopes not only allows for detailed understanding of their mechanism of actions but also guides the design and selection of drug candidate molecules. In this study, we illustrate the practical utility of an integrated approach, including size exclusion chromatography with multiangle light scattering and native mass spectrometry (MS) for the biophysical characterization of complex formation of a BsAb with two target antigens, cluster of differentiation 3 (CD3) and B-cell maturation antigen (BCMA). MS-based protein footprinting strategies, including hydrogen/deuterium exchange MS, fast photochemical oxidation of proteins, and carboxyl group footprinting with glycine ethyl ester, were further applied to determine BsAb's binding epitopes. This combination approach provides molecular details on the binding mechanisms of BsAb to the two distinct antigens with rapid output and high resolution.