Mass Spectrometry-Based Comparative Evaluation of Forced Glycation Profiles of Therapeutic Monoclonal Antibodies

Background/Objectives: Glycation, a non-enzymatic post-translational modification, can increase structural heterogeneity in therapeutic monoclonal antibodies (mAbs) and is considered a critical quality attribute in biosimilar (BS) development. Despite extensive studies on glycation in individual mAbs, comparative reports on originator (OR) and BS mAbs under forced glycation conditions remain limited. This study aimed to comparatively evaluate the glycation profiles of one OR and three BS anti-tumor necrosis factor-alpha (anti TNF-α) mAb products using integrated mass spectrometry-based methods. Methods: Forced glycation was induced by incubating mAbs with 200 mM D-glucose at 37 °C for 7 days. Intact mass analysis and peptide mapping were used to assess glycation extent and site distribution, respectively. Results: Intact mass analysis revealed a consistent mass increase of approximately 486 Da across all major glycoform species for each mAb product, indicating predominant formation of the tri-glycated mAb population under the applied stress conditions. The overall glycation levels were comparable at the intact level, ranging from 67% to 73% among the OR and BS mAb products. Peptide mapping identified nine glycated lysine (K)-containing peptides, among which three major glycation hotspots (LC:V5 K145/K149, LC:V7 K183, and HC:V7 K250/K252) showed elevated occupancies (~10-16%). These sites collectively accounted for the dominant intact level mass shift. The remaining glycated peptides exhibited only minimal modification levels (<2%). Despite their distinct manufacturing processes, all tested mAb products showed nearly identical site-specific glycation profiles. Conclusions: Forced glycation susceptibility in the studied mAbs was driven by a limited set of structurally preferred K hotspots, leading to highly comparable glycation profiles across OR and BS mAb products. The combined intact mass and peptide mapping strategy provides a robust analytical platform for comparative glycation assessment in BS characterization.