Characterization of mAb charge heterogeneity by CE based methods coupled to mass spectrometry

The characterization of charge heterogeneity is of utmost importance for the efficacy and safety of monoclonal antibodies (mAbs) in pharmaceutical development and quality control. Capillary electrophoresis (CE) as an analytical technique includes separation modes such as capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF), which provide high selectivity to charge heterogeneity of proteins and mAbs and are frequently applied in the scientific and biopharmaceutical sectors. However, these separation methods lack electrospray-mass spectrometry (ESI-MS) compatibility due to the required chemical composition with high amounts of salts or ampholytes which are necessary to achieve high separation performance. The identification of protein charge species and further localization of post-translational modifications (PTMs) by CE-MS methods requires furthermore high sensitivity to detect occurring low abundant species. In this thesis, different ways are presented to hyphenate ESI-interfering CE methods online to MS using a developed nanoflow sheath liquid CE-MS interface with focus on sensitivity, flexibility, and ease of use. The first part describes the development of this CE-MS interface based on 3D-printed parts. The second part describes the coupling of a preparative iCIEF-UV instrument to MS for the charge variant characterization of intact mAbs with high accuracy and high precision. The last part describes the utilization of a two-dimensional CZE-MS setup for the characterization of mAb charge heterogeneity on the peptide level. Therefore, the first dimension was used for intact mAb charge variant separation and the second dimension was used for an in-capillary digest with pepsin. In this way, PTMS of stressed and unstressed mAbs could be localized and sequence coverages >90% could be achieved. The presented developments and setups demonstrate a large potential for the in-depth characterization of mAb charge variants using CE-based methods.