Comprehensive Glycosylation Characterization of Recombinant Human Erythropoietin by Electron-Activated Dissociation Mass Spectrometry

Abstract Recombinant human erythropoietin (rhEPO) is a glycoprotein that acts as the main hormone involved in regulating red blood cell production to treat anemia caused by chronic kidney disease or chemotherapy, which has three N-glycosylation sites and one O-glycosylation site. It contains a variety of different glycosylation modifications, such as sialyation, O-acetylation on sialic acids, etc, which causes a big challenge for the glycosylation analysis of rhEPO. In this study, a liquid chromatography-mass spectrometry (LC-MS) method combined with electron-activated dissociation (EAD) technology was used in qualitative and quantitative characterization of rhEPO N-glycosylation and O-glycosylation in just one injection. The usage of EAD not only generated abundant MS/MS fragment ions of glycopeptides and improved the MS/MS sequence coverage, but also preserved the glycans structures in the MS/MS fragment ions and the integrity of the glycosidic bond between the glycans and peptides. Three N-glycosylation sites (N24, N38 and N83) and one O-glycosylation site (S126) of rhEPO samples were successfully identified. Among them, the glycosylation ratios of N24, N38 and N83 sites were 82.7%, 100% and 100% respectively, and 15, 10 and 12 different N-glycans could be identified at the glycopeptide level. The total average number of sialic acids, N-hydroxyacetylneuraminoic acid and O-acetylation on sialic acid were 7.28, 4.21 and 0.66 at the Intact protein level, respectively. For O-glycosylation site S126, O-glycosylation ratios analyzed at the intact protein level and the glycopeptide level were 80.2% and 80.3%, respectively, and two O-glycans were identified, including Core1_S1 and Core1_S2. This study also compared the difference between the glycans and their relative contents in batch-to-batch rhEPO samples. The results proved that the workflow using EAD fragmentation in LC-MS method could be effectively applied for characterizing the glycosylation analysis of rhEPO samples and batch-to-batch consistency analysis, which would help to reasonably guide the optimization of rhEPO production process.