Structural analysis of the glycosylation of gene-activated erythropoietin (epoetin delta, Dynepo) |
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Authors: | Llop Esther Gutiérrez-Gallego Ricardo Segura Jordi Mallorquí Joaquim Pascual José A |
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Affiliation: | a Bioanalysis Research Group, Neuropsycho-pharmacology Program, IMIM-Hospital del Mar, Barcelona Biomedical Research Park (PRBB), 08003 Barcelona, Spain b Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park (PRBB), 08003 Barcelona, Spain |
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Abstract: | Recently, a novel recombinant human erythropoietin (epoetin delta, Dynepo) has been marketed in the European Union for the treatment of chronic kidney disease, cancer patients receiving chemotherapy, and so forth. Epoetin delta is engineered in cultures of the human fibrosarcoma cell line HT-1080 by homologous recombination and “gene activation.” Unlike recombinant erythropoietins produced in other mammalian cells, epoetin delta is supposed to have a human-type glycosylation profile. However, the isoelectric focusing profile of epoetin delta differs from that of endogenous erythropoietin (both urinary and plasmatic). In this work, structural and quantitative analysis of the O- and N-glycans of epoetin delta was performed and compared with glycosylation from recombinant erythropoietin produced in Chinese hamster ovary (CHO) cells. From the comparison, significant differences in the sialylation of O-glycans were found. Furthermore, the N-glycan analysis indicated a lower heterogeneity from epoetin delta when compared with its CHO homologue, being predominantly tetraantennary without N-acetyllactosamine repeats in the former. The sialic acid characterization revealed the absence of N-glycolylneuraminic acid. The overall sugar profiles of both glycoproteins appeared to be significantly different and could be useful for maintaining pharmaceutical quality control, detecting the misuse of erythropoietin in sports, and establishing new avenues to link glycosylation with biological activity of glycoproteins. |
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Keywords: | Erythropoietin Epoetin delta Glycosylation 2-DE Mass spectrometry MALDI Sialic acid |
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