Erythropoietin (EPO) is a highly glycosylated protein hormone that stimulates the production of red blood cells. EPO exhibits significant heterogeneity due to its multiple sites of glycosylation; each site can bear highly branched sialylated N-glycan structures. In vivo studies using glycoengineered EPO have shown links between the safety and efficacy of a therapeutic EPO and several glycosylation-associated critical quality attributes, with sialic acid being important. Patents for EPO therapeutics are approaching expiration and there is much interest in developing biosimilars.
This application example shows how the characterization of EPO N-glycans is facilitated by preparing N-glycan samples for direct analysis by HILIC ESI-QTof MS analysis. GlycoWorks RapiFluor-MS labeling not only streamlined sample preparation, but also enhanced the sensitivity of N-glycan detection by fluorescence and MS, making it possible to elucidate the complicated N-glycan profile of recombinant human EPO (rhEPO).