Characterization of monoclonal antibody charge variants under near-native separation conditions using nanoflow sheath liquid capillary electrophoresis-mass spectrometry

Background: Monoclonal antibodies (mAbs) undergo multiple post-translational modifications (PTMs) during production and storage, resulting for instance in charge and oxidized variants. PTMs need to be assessed as critical quality attributes to assure protein quality and safety. Capillary zone electrophoresis (CZE) enables efficient charge-based separation. The CZE method developed by He et al. (2011) is currently applied routinely in the pharmaceutical industry for profiling charge heterogeneity of mAbs. However, as the method relies on a non-volatile background electrolyte (BGE), it cannot be directly hyphenated with mass spectrometry (MS), hampering the identification of separated charge variants. Results: This study presents a CZE-UV/MS method using a neutral static capillary coating of hydroxypropyl methylcellulose combined with a volatile BGE at pH 5.0 to allow for MS-compatible mAb charge variant separations. The effect of several parameters, including pH and concentration of the BGE, applied voltage, and injected mAb concentrations on separation performance was investigated using a panel of commercially available mAbs. The optimized method was evaluated with IgG₁ and IgG₄ mAbs of varying pI (7.4–9.2) and degrees of heterogeneity. Basic and acidic variants were separated from the parent mAb using a BGE of 50 mM acetic acid adjusted to pH 5.0 with ammonium hydroxide. The relative abundances of charge variants determined with the new method showed a good correlation with the corresponding relative levels obtained with the method of He et al. CZE-MS coupling was accomplished using the nanoCEasy, a low-flow sheath liquid interface, which enabled the identification and quantitation of basic, acidic, and incomplete pyroglutamate variants, and glycoforms of the tested mAbs. Significance: This manuscript describes a new CZE-MS method that permits heterogeneity assessment of mAbs under MS-compatible conditions, providing charge variant separation.