Analysis of NIST Monoclonal Antibody Reference Material Glycosylation Using the LC–MS/MS-Based Glycoproteomic Approach

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%.