Rapid and comprehensive monoclonal antibody Characterization using microfluidic CE-MS.

The identification and control of monoclonal antibody (mAb) critical quality attributes (CQAs) is a key component of quality by design (QbD). In this work, rapid peptide mapping and native intact charge variants analysis have been developed to comprehensively characterize and monitor mAb CQAs using a microfluidic capillary electrophoresis - mass spectrometry (CE-MS) platform. The ultrafast peptide mapping simultaneously analyzed multiple CQAs, including protein primary structure, oxidation, deamidation, succinimide, C-terminal lysine (Lys) clipping, N-terminal cyclization, and glycosylation. The microfluidic CE-MS based peptide mapping acquired results comparable to conventional but lengthy liquid chromatography - MS (LC-MS) based approach. The native intact analysis resolved mAb charge variants with a comparable resolution as commonly achieved using capillary isoelectric focusing (cIEF). Charge variants' identities were assigned based on characteristic mass shifts, knowledge learned from peptide mapping, and changes in electrophoretic mobility. Major mAb glycoforms of each charge variants were resolved and identified in the deconvoluted mass spectra. Furthermore, a model simulation was performed to reconstruct intact deconvoluted mass spectra using peptide mapping results. The reconstructed and experimentally determined intact deconvoluted mass spectra were highly correlated, suggesting that our data collected at the peptide level and intact level were consistent and highly comparable. Overall, the microfluidic CE-MS based peptide mapping and native intact charge variants analysis are high-throughput methods that have great potential to support biopharmaceutical development.