Evaluation of the combined effects of pegylation and glycosylation on the stability of erythropoietin using a stability-indicating SE-HPLC

摘要

Abstract Recombinant human erythropoietin (rhEPO) is a commonly used biopharmaceutical for the treatment of anemia-associated disorders. Epogen; glycosylated erythropoietin (G-EPO) has short half-life and poor stability. Pegylated Epogen (Peg-G-EPO) was introduced to the market to overcome these limitations. The combined effects of pegylation and glycosylation on the stability of Peg-G-EPO was studied. Determination of Peg-G-EPO in the presence of its degradation products was achieved using SE-HPLC. The assay was validated according to ICH guidelines over concentration range of 50.00–320.00?μg/mL (r 0.9999). A mobile phase of 50?mM phosphate buffer (pH 6.5) with 300?mM sodium chloride and 20% ethanol was employed. Isocratic elution was carried out at 0.5?mL/min over run time of 30?min. Peg-G-EPO was found stable towards mechanical agitation only at low concentrations while it was stable towards repeated freeze/thaw; regardless of the concentration. Effect of temperature and pH were also investigated and Peg-G-EPO was found stable within narrow ranges. Results indicated formation of small molecular weight and very high molecular weight aggregates that have been filtered-off the column. Although Peg-G-EPO was found relatively more stable than its non-pegylated but glycosylated version, results indicated the need for careful stability-assessment of Peg-G-EPO. Highlights ? Effects of pegylation and glycosylation on stability of erythropoietin was investigated. ? Stress-induced degradation of pegylated epogen was performed. ? Stability-indicating SE-HPLC was used for determination of pegylated epogen.