Development of a sub-hour on-line comprehensive cation exchange chromatography x RPLC method hyphenated to HRMS for the characterization of lysine-linked antibody-drug conjugates.

This study details the development of on-line two-dimensional liquid chromatography (2D-LC) methods combining cation-exchange chromatography (CEX) and reversed-phase liquid chromatography (RPLC) for the separation of the charge variants of a lysine-linked antibody-drug conjugate (ADC). This combination gives an excellent example of the potential benefits of 2D-LC approaches for the analysis of such complex protein formats. CEX is considered the reference technique for the separation of protein charge variants but its retention mechanism usually requires the use of a high concentration of non-volatile salts, which impedes its compatibility with MS detection. In this context, the use of an on-line 2D-LC-MS approach not only allows on-line desalting and indirect coupling of CEX with mass spectrometry (MS) detection but it also provides increased and complementary information within a single analysis. The first part of this study was devoted to the choice of stationary phases and the optimization of chromatographic conditions in both dimensions. Based on the results obtained in 1D-CEX with ultraviolet detection (UV) and 1D-RPLC with UV and high-resolution mass spectrometry (HRMS) detections, an on-line comprehensive two-dimensional liquid chromatography method combining CEX and RPLC was developed. The last part of this study was devoted to the identification of the separated species using HRMS detection and in the comparison of three ADC samples exposed to different durations of thermal stress.