Equivalence assessment of biotherapeutics with N- and O-glycosylation sites by sequential intact glycoform mass spectrometry (IGMS)

Glycosylation is a crucial attribute for biotherapeutics with significant impacts on quality, stability, safety, immunogenicity, pharmacokinetics, and efficacy. Therefore, to ensure consistent glycosylation, a systematic review of biotherapeutics is absolutely required including the variable glycan structure (micro-heterogeneity) and different occupancy at individual site (macro-heterogeneity) from drug design to upstream and downstream bioprocesses. Various methods have been used for glyco-characterization of biotherapeutics at the glycan, glycopeptide, and intact protein levels. In particular, intact protein analysis is considered a facile and rapid glycoform monitoring approach used throughout the product development lifecycle to determine suitable glycosylation lead candidates and reproducible product quality. However, intact glycoform characterization of diverse and complex biotherapeutics with multiple N- and O-glycosylation sites can be very challenging. To address this, a robust analytical platform that enables rapid and accurate characterization of a biotherapeutics with highly complex multiple glycosylation using two-step intact glycoform mass spectrometry has been developed. We used darbepoetin alfa, a second-generation EPO bearing multiple N- and O-glycosylation sites, as a model biotherapeutics to obtain integrated information on glycan heterogeneity and site occupancy through step-by-step MS of intact protein and enzyme-treated protein. In addition, we performed a comparative assessment of the heterogeneity from different products, confirming that our new method can efficiently evaluate glycosylation equivalence. This new strategy provides rapid and accurate information on the degree of glycosylation of a therapeutic glycoprotein with multiple glycosylation, which can be used to assess glycosylation similarity between batches and between biosimilar and reference during development and production.