Characterization and monitoring of charge variants of a recombinant monoclonal antibody using microfluidic capillary electrophoresis-mass spectrometry

Abstract

Rigorous characterization of biotherapeutics, and monoclonal antibodies in particular, is a challenging task in terms of ensuring safety, efficacy, and potency of a therapeutic agent because of structural heterogeneity during cell culture, purification and storage. In this work, we used microfluidic capillary electrophoresis-mass spectrometry to analyze intact monoclonal antibody and assess the root cause of increases in acidic and basic variants under stress at high temperature. The antibody was analyzed at multiple levels, including its intact state under native conditions, and subunit and peptide levels. The normal and degraded antibodies at different time points were characterized and compared with each other. We concluded that the basic variants in the unstressed sample were produced C-terminal amidation, while the acidic variants were produced by deamidation. In stressed samples, change in the acidic and main peaks were caused by deamidation, and changes in the basic peaks were caused by both deamidation and oxidation. These results demonstrate that microfluidic capillary electrophoresis-mass spectrometry (CE-MS) is a powerful direct and generic tool for separation and identification of charge heterogeneity of biotherapeutics.