Mass spectrometric glycoform profiling of the innovator and biosimilar erythropoietin and darbepoetin by LC/ESI-MS

Mass spectrometric glycoform profiles of innovator and biosimilar erythropoietins. •Glycoform profiling of erythropoietin (EPO) products was done using LC/ESI-MS.•We revealed the characteristics of innovator and biosimilar EPO products.•All the EPO products tested in this study showed different glycoform profiles. The recent patent expirations of erythropoietin (EPO) have promoted the development of biosimilars. Two and one biosimilar EPO products were approved in 2007 in Europe and in 2010 in Japan, respectively. Glycosylation heterogeneity of EPO is very complex, and its pattern has a large impact on its in vivo activity. In this study, glycoform profilings of biosimilar and innovator EPO products were performed using LC/ESI-MS. Glycoforms of EPO were detected within the range of m/z 1700–3600 at the 10+–16+ charge states. The charge-deconvoluted spectra showed complex glycoform mass profiles at 28,000–32,000Da, and most of the observed peaks were assigned to the peptide (18,236Da)+glycans with the compositions of NeuAc10–14Hexn+3HexNAcnFuc3 (n=16–26) with or without some O-acetylations (+42Da) and attachment of NeuGc for NeuAc or oxidation (+16Da). Analysis of de-N-glycosylated EPO showed the distributions of O-glycans of NeuAc1–2Hex1HexNAc1 and site occupancy. Each EPO product showed a characteristic glycoform profile with respect to sialylation, glycan size, O-acetylation of sialic acids and O-glycosylation. Analysis of darbepoetin suggested that glycans of darbepoetin were highly sialylated and O-acetylated. LC/ESI-MS was shown to be useful to evaluate the similarity of the glycoform profiles of EPO.