Online Collision-Induced Unfolding of Therapeutic Monoclonal Antibody Glyco-Variants through Direct Hyphenation of Cation Exchange Chromatography with Native Ion Mobility–Mass Spectrometry
Post-translational modifications (PTMs) not only substantially increase structural heterogeneity of proteins, but can also alter the conformation or even biological function. Monitoring of these PTMs is particularly important for therapeutic products, including monoclonal antibodies (mAbs), since their efficacy and safety may depend on the PTM profile. In this study we aimed at the combination of cation exchange chromatography (CEX) separation with collision induced unfolding (CIU) to monitor the unfolding pattern of separated mAbs and mAb charge variants and thereby, pick up (subtle) conformational differences. The results highlight the benefits of combining CEX with CIU for an in-depth characterization providing identification of mAb charge variants together with information on their resistance to gas-phase unfolding, and thus contributing for a better understanding of the correlation between primary structure modifications and HOS of proteins.
Guusje van Schaick, Elena Domínguez-Vega, Jérôme Castel, Manfred Wuhrer, Oscar Hernandez-Alba, and Sarah Cianférani. Analytical Chemistry 2023 95 (8), 3932-3939. https://www.doi.org/10.1021/acs.analchem.2c03163
