Native CZE-MS analysis of antibodies and hemoglobin using protein-adapted CZE and ESI conditions for detailed proteoform characterization

Capillary zone electrophoresis-mass spectrometry (CZE-MS) under native-like conditions is a selective and efficient tool for studying the proteoforms of proteins or protein complexes. Recently, it has been shown that a neutral capillary coating in combination with an ammonium acetate-based electrolyte with near-neutral pH can result in a high electrophoretic resolution between different proteoforms of an antibody, allowing for detailed characterization of the drug product. Here, we extend the CZE-MS method and demonstrate its flexibility for the analysis of protein constructs having different isoelectric points (pIs) by adapting the pH value of the electrolyte depending on the pI of the protein, the desired separation performance, and analysis time. Depending on the applied sheath liquid composition, the method allows for protein analysis under denaturing or native-like conditions. Several antibodies were analyzed by CZE-MS with a systematic evaluation of the separation of typical charge variants. The high separation performance enables the detection of between 45 (bevacizumab) and 100 proteoforms (adalimumab), including variants not resolvable by MS and proteoforms not reported before. Native CZE-MS analysis of hemoglobin enables the separation of different hemoglobin variants, such as glycated, oxidized, and glutathionylated variants, as well as hemoglobin complexes with different iron oxidation states, while preserving non-covalent interactions of the complex. The alternative application of a denaturing sheath liquid leads to dissociation into monomers, thereby allowing a more reliable assignment of modifications and their localization to the respective subunit.