Current and future trends in reversed-phase liquid chromatography-mass spectrometry of therapeutic proteins

Reversed-phase liquid chromatography (RPLC) is one of the most widely used chromatographic approaches for the characterization of therapeutic proteins because it is able to answer several analytical queries and allow the fine characterization of the structural heterogeneity of these drugs. However, some issues may possibly occur when analysing therapeutic proteins by RPLC-MS, such as severe band broadening and peak tailing, strong adsorption, possible protein denaturation/degradation, limited MS sensitivity and poor chromatographic selectivity. The present review focuses on the latest advances in RPLC that have been adopted to overcome these issues, including the application of cutting-edge chromatographic conditions, alternative mobile phase compositions and critical discussions on recently commercialized bioinert instruments and RPLC stationary phases.