Glycosylation

Recombinant proteins are increasingly used as therapeutics, and they form a large subset of the best-selling drugs. Their characterisation, from development to post-marketing quality control, is challenging due to their size and inherent heterogeneity caused notably by post-translational modifications.

Glycosylation is probably the most prominent PTM in terms of occurrence and alteration of protein function. Adequate glycosylation is critical for therapeutic proteins regarding binding, solubility and stability, safety, and for pharmacokinetics and pharmacodynamics properties.

This study sheet presents the physicochemical characterisation of glycosylated proteins and highlights the approaches used to assess N‑ and O‑glycosylation. It describes peptide mapping strategies supported by specific enzymatic digestions, glycan labelling methods (2‑AB, RapiFluor‑MS, APTS), and complementary workflows such as permethylation, β‑elimination, acid hydrolysis, and the use of monosaccharide and sialic acid profiling. The full document details how these analytical techniques enable site‑dependent glycosylation assessment.