Protein-based therapeutics such as mAbs have become emerging drugs in modern medicine. Most of the approved therapeutic proteins are glycoproteins. Glycosylation is an essential critical quality attribute (CQA) due to the influence that glycoforms have on the safety, efficacy, and pharmacokinetics/pharmacodynamics (PK/PD) of biotherapeutics. Here, we applied an LC–MS/MS-based glycoproteomics approach to characterize Fc glycans of an NISTmAb reference material (RM) 8671 (sample B) and a β-1,4-galactosidase-treated NISTmAb (sample A). Overall, 48 glycan compositions were identified and quantified. The glycan structure with the highest abundance was FA2, with a relative abundance of 52% in sample A and 38% in sample B. Over 50% of the identified glycans presented at levels smaller than 0.1%. Important glycan attributes were further derived using the quantitative results. The galactosylation level of modified NISTmAb was found to decrease by ∼10% when compared to the galactosylation level of NISTmAb. There was no significant difference between the two samples in the levels of sialylation, fucosylation, and high mannose. Moreover, unglycosylated peptides were also observed at a level of 1–2%.
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