The importance of glycosylation towards optimal drug bioactivity in biotherapeutics design has been increasingly underscored. In biotherapeutic products, structure, specific modifications, and abundance of glycans can significantly affect the quality, immunogenicity, and stability of drugs. Therefore, detailed glycomic analysis is a key step in assessing quality control and adjusting the glycosylation pathway of a biotherapeutic protein. However, the inherent structural diversity and complexity of glycosylation significantly make glyco-analysis extremely challenging. Here, we presented an analytical strategy that facilitates the rapid identification of glycosylation in biopharmaceutics using isomer-sensitive LC/MS/MS. To aid this process, we built a glycan database of diagnostic fragment peaks that can be used to quickly annotate MS/MS data in a user-independent manner. Diagnostic fragments obtained from MS/MS spectra of archetypal glycans bearing common bioactive motifs of biotherapeutics were included in the DB for use in predicting the presence or absence of specific glycan structures. The DB was designed to contain glycans that can be expressed in representative subclass of biotherapeutic glycoproteins including mAbs, cytokines, and enzymes. DB entries include not only typical glycans found in biotherapeutics, but also atypical glycans such as sialic acid O-acetylation, mannose phosphorylation, polylactosaminylation, NeuGc sialylation, HexNAc sulfation, and galactose-alpha-1,3-galactose. Our strategy of glycan structure DB was applied to structurally characterize glycans from biotherapeutics produced by various cell-based expression systems. We were successful in identifying atypical and/or contaminant glycosylation on various types of biotherapeutic glycoproteins using screening of diagnostic ion. Our strategy enables rapid, yet highly accurate identification of both desired and undesired biotherapeutic glycosylation.

Structure-specific characterization bioactive glycan motifs in therapeutic glycoproteins

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