A pharmacopoeial monograph under development for recombinant human erythropoietin (rhEPO) drug substance is likely to contain a specification limit for the proportion of the methionine-oxidised variant. Methionine oxidation has no effect on the folded structure and global thermodynamic stability of rhEPO but can decrease biological activity [1]. We describe here the development of a reference standard, a calibrated mixture of the native and oxidised tryptic peptides which contain methionine-54, and an optimised peptide mapping procedure to support this assay. The approach may be developed for analysis of drug product or generalised for other assays in which product-related impurities are quantified by peptide mapping.
Keywords: erythropoietin; methionine oxidation; peptide mapping; peptides; reference standard; reversed-phase-liquid chromatography.