Deciphering complex charge heterogeneity in a glycosylated monoclonal antibody by integrated icIEF-MS and enzymatic digestion system.

Charge heterogeneity is a critical quality attribute of therapeutic monoclonal antibodies. However, structurally characterizing charge variants resolved by imaged capillary isoelectric focusing (icIEF) remains challenging for antibodies with complex charge profiles. Here, we established an integrated enzymatic digestion and direct icIEF-MS workflow, enabling the elucidation of charge heterogeneity origins in a highly glycosylated monoclonal antibody that exhibits an unusually complex, multi-fingered icIEF profile. icIEF revealed eight well-resolved charge variants. icIEF-MS analysis identified that the basic variants were primarily attributed to incomplete C-terminal lysine clipping, whereas the major acidic variants originated predominantly from sialylated glycan species. Sequential treatment with carboxypeptidase B, PNGase F, neuraminidase, and IdeS facilitated the systematic assignment of the charge variants and indicated that the sialylated glycans responsible for the acidic peak cluster were mainly located in the Fab region. Following neuraminidase treatment, the multi-fingered acidic profile largely collapsed into a single main peak, confirming sialylation as the dominant source of charge heterogeneity. Residual low-level acidic variants with reduced pI but no obvious intact-mass differences were attributed to deamidation. Overall, this work demonstrates that combining direct icIEF-MS with orthogonal enzymatic digestion strategies enables a more confident elucidation of complex antibody charge heterogeneity. This workflow provides a practical framework for in-depth characterization and quality assessment of complex charge variants in biopharmaceutical.