CAST scientist Annika van der Zon recently introduced a near-native separation method for characterizing charge variants of intact monoclonal antibodies (mAbs) using capillary zone electrophoresis (CZE) coupled with mass spectrometry (MS). In this study we used a nanoflow sheath liquid interface, known as nanoCEasy applied thanks to the collaboration via the Uniiversity of Aalen.
The CZE-MS method, employs a neutral static capillary coating made of hydroxypropyl methylcellulose, combined with 50 mM acetic acid at pH 5.0, to create MS-compatible conditions for separating mAb charge variants. Currently, the pharmaceutical industry uses the EACA method of He et al. (2011) method to routinely profile charge variants, but this method relies on a non-volatile background electrolyte (BGE), making it incompatible with MS and hindering the identification of separated charge variants.
The MS-compatible CZE method we introduce allows to obtain similar charge variant profiling as the EACA method but allows for MS analysis. The CZE-MS coupling, enabled by nanoCEasy’s low-flow sheath liquid interface, successfully identified and quantified basic and acidic variants, incomplete pyroglutamate variants, and glycoforms of the mAbs tested. This CZE-MS method provides a powerful tool for assessing mAb heterogeneity and achieving charge variant separation.
