Separation techniques for intact antibody analysis by mass spectrometry

Abstract The possibilities to directly couple different separation methods with mass spectrometry (MS) for the analysis of intact proteins has attracted significant attention over the last decade. While sample preparation is critical for MS analyses, coupling an efficient separation method before MS can significantly improve the ability to resolve protein heterogeneity and reveal intact masses or higher-order structure information (e.g., PTM or conformation changes). To that end, this review focuses on common online separation techniques coupled with MS detection to detect antibodies or large proteins with a focus on biopharmaceutical applications. An overview of liquid chromatography modes, such as reversed-phase, size-exclusion, mixed-mode, hydrophobic interaction, and ion-exchange chromatography will be discussed, along with electrophoretic separation, gas-phase separation using ion-mobility and other next-generation tools. Finally, the application space within the biopharmaceutical industry will be discussed along with how improvements for separation techniques before MS detection can ultimately help characterize charge-, size-, or hydrophobic variants of novel biotherapeutics throughout all stages of drug development. GRAPHICAL ABSTRACT