title: Quantification of low levels of methionine oxidation in erythropoietin drug substance.
abstract: 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.
authors.affiliation: Laboratory of Molecular Structure, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Herts EN6 3QG, UK, carmen.romera@nibsc.org.
authors.forename: C
authors.initials: C
authors.lastname: Romera
authors.affiliation: Laboratory of Molecular Structure, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Herts EN6 3QG, UK.
authors.forename: A F
authors.initials: AF
authors.lastname: Bristow
authors.affiliation: Laboratory of Molecular Structure, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Herts EN6 3QG, UK.
authors.forename: C
authors.initials: C
authors.lastname: Jones
mesh_terms: D004921:Erythropoietin / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D008715:Methionine / Q000032:analysis* / Q000378:metabolism*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D012015:Reference Standards
chemical_list: D011994:Recombinant Proteins; D004921:Erythropoietin; D008715:Methionine
keywords: erythropoietin; methionine oxidation; peptide mapping; peptides; reference standard; reversed-phase-liquid chromatography