In-depth size and charge variants characterization of monoclonal antibody with native mass spectrometry

Although the reversed-phase liquid chromatography (RPLC) is the most used separation front for mass spectrometry, many other separation modes are critical for enabling characterization of the protein therapeutics. Specifically, chromatographic separations under native conditions, such as those based on size exclusion chromatography (SEC) and ion-exchange chromatography (IEX), are used for characterizing important biophysical properties of protein variants in drug substance and drug product. Because most native state separation modes use non-volatile buffers with high salt concentration, optical detection has been traditionally used. However, there is an increasing need to understand and identify the optical underlying peaks by mass spectrometry for structure elucidation. For size variant separation by SEC, the native MS helps to understand the nature of the high molecular weight species, as well as clipping sites for low molecular weight fragments. For charge variant separation by IEX, native MS can reveal the post-translational modifications or other important factors contributing to charge heterogeneity at the intact level. Here, we demonstrate the power of native MS by direct coupling of SEC and IEX eluent to a time-of-flight mass spectrometer to characterize bevacizumab and NISTmAb. Our studies exemplify the effectiveness of native SEC-MS for characterizing bevacizumab's high molecular weight species at less than 0.3% (based on SEC/UV peak area%) and analyzing the fragment pathway with single amino acid difference for its low molecular weight species at less than 0.05%. Good IEX charge variant separation was obtained with consistent UV and MS profiles. The identity of separated acidic and basic variants were elucidated by native MS at intact level. We successfully differentiated several charge variants including glycoform variants that have not been reported before. In addition, native MS allowed identification of higher molecular weight species as late eluted variants. Overall, the SEC and IEX separation combined with high resolution and high sensitivity native MS, which is significantly different from the traditional RPLC-MS workflows, can be an effective tool that offers valuable insights for us to understand protein therapeutics at native state.