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.

反相液相色谱（RPLC）是用于表征治疗性蛋白质的最广泛使用的色谱方法之一，因为它能够回答多个分析问题，并能够对这些药物的结构异质性进行精细表征。但是，通过RPLC-MS分析治疗性蛋白质时，可能会出现一些问题，例如严重的谱带展宽和峰拖尾，强吸附，可能的蛋白质变性/降解，有限的MS敏感性和较差的色谱选择性。本综述着重介绍为克服这些问题而采用的RPLC最新进展，包括应用最先进的色谱条件，