Determination of Site-specific N- & O-glycan heterogeneity on Darbepoetin Alfa by Mass Spectrometry

Recombinant erythropoietin (EPO) as a regulator for formation of red blood cells is well known glycosylated therapeutic protein. Darbepoetin alfa (NESP) is a heavily heterogeneous glycoprotein biotherapeutic containing one O-glycosylation and five N-glycosylation sites. The type, extent, and location of glycosylation on NESP mediate biological activity, plasma half-life, immunogenicity, and stability. Thus, the determination of site-specific glycan microheterogeneity is important to assess biotherapeutic quality and establish the equivalency of biosimilar EPOs. However, there is no method to monitor site-specific glycosylation on NESP. Here, we have developed a highly reproducible analytical platform for characterization site-specific glycosylation with microheterogeneity, which combines molecular size fractionation, multiple proteolysis, PGC nanoLC separation, in silico screening by accurate mass, and tandem MS analysis. We were successful in identifying all N- and O-glycosylation sites and quantifying glycoforms including 61 different N-glycopeptides and 8 different O-glycopeptides on NESP. This is the first comprehensive study to determine site-specific glycosylation with extreme glycan heterogeneity on NESP.