Development Of Highly Glycosylated Erythropoietin

In this study, we modified amino acid sequence of human erythropoietin (HuEPO) using genetic mutation, resulting in protein with increased glycosylation, enhanced in vivo half-life and promoted bioactivity. The researches include specific site mutation, potency analysis, screening and construction of recombinant cell line, cell culture process development, protein purification and characterization, bioassay in mice, PK study in rats.Additional 1-4 glycosylation sites were added to EPO molecule based on sequence analysis. The modified gene was introduced into CHO cells and expressed EPO analogs were isolated. In vivo potencies of analogs were evaluated by reticulocyte count in mice. Compared with rHuEPO, analogs showed enhanced erythropoietic activity and longer half-life. Duration of action of EPO analogs positively correlated with the number of glycosylated sites, addition of 3 and 4 glycosylation sites significantly increased t1/2 in rats comparing with the analogs with 1 and 2 mutation sites. Protein with 3 additional glycosylation sites N3 was purified and analyzed.Recombinant cells expressing N3 were cultured in bioreactor. Temperature, pH, DO, agitate, air flow, glucose content and nutrition feeding were controlled by software. After 28 days of cell culture, cell density reached 5×106,100 liter of condition media with an expression level of 80mg/L was collected. N3 protein was isolated from supernatant using ion exchange chromatography, HPLC and size exclusion chromatography. A molecule with MW of 47kDa, prity of 98%, pI 2.9-3.8, sialic acid content 15 mol/mol was purified. Analog N3 increases 3.3 times in elimination half-life and higher activity in comparison to rHuEPO.A highly glycosylated EPO analog with promoted bioactivity and prolonged half-life was found, an efficient in vivo bioassay method and process of production was developed. Protein was characterized and evaluated to be suitable for clinical applications.