High-Performance Native Separation of mAb Proteoforms by CZE-MS under Native and Denaturing nanoESI Conditions for Ultrasensitive Charge Variant Characterization

The monitoring of charge variants is crucial for quality control of therapeutic antibodies as variants can influence the efficacy or safety of a biopharmaceutical. Capillary zone electrophoresis (CZE) is a powerful tool for the efficient separation of charge variants using optimized electrolyte systems and is thus widely applied in the pharmaceutical industry. However, these electrolytes do not allow for coupling to mass spectrometry (MS), preventing direct identification of the separated variants. Here, we present a CZE-MS method for a detailed characterization of monoclonal antibody (mAb) charge variants and other proteoforms with a separation performance similar to that of the best existing non-MS-compatible separation method for charge variant analysis based on an electrolyte of ε-aminocaproic acid. A neutral static capillary coating in combination with an ammonium acetate-based electrolyte at physiological pH enables a powerful separation of mAb charge variants with subsequent hyphenation to MS. The CZE was coupled to MS using a nanoflow sheath liquid (SL) interface, providing ultrasensitive and highly flexible ionization. Different SLs have been tested to compare the ionization under denaturing as well as under nondenaturing nanoESI conditions, leading to slightly better quality for the denatured charge-deconvoluted spectra. 115 proteoforms could be detected for NISTmAb and 70 proteoforms could be detected for trastuzumab, including proteoforms that have not been found on the intact level so far. The presented method enables a fast and detailed heterogeneity assessment of therapeutic mAbs on the intact level and has great potential for proteoform-resolved structural and functional characterization.