MS‐Based Glycome Characterization of Biotherapeutics With N‐ and O‐Glycosylation

ABSTRACT With the increasing FDA approvals of glycoprotein‐based biotherapeutics including monoclonal antibodies, cytokines, and enzyme treatments, the significance of glycosylation in modulating drug efficacy and safety becomes central. This review highlights the crucial role of mass spectrometry (MS) in elucidating the glycome of biotherapeutics that feature N‐ and O‐glycosylation, directly addressing the challenges posed by glycosylation complexity and heterogeneity. We have detailed the advancements and application of MS technologies including MALDI‐TOF MS, LC‐MS, and tandem MS in the precise characterization of glycoprotein therapeutics. Emphasizing MS‐based strategies for detecting immunogenic glycans and ensuring batch‐to‐batch consistency, this review highlights targeted approaches for glycoprotein, glycopeptide, and glycan analysis tailored to meet the stringent analytical and regulatory demands of biopharmaceutical development.