Quantification of low levels of methionine oxidation in erythropoietin drug substance Development of a reference standard to quantify the oxidised form of erythropoietin

摘要

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. A simple routine method to monitor and quantify the oxidised methionine-54 (Met-54) variant in recombinant human erythropoietin (rhEPO) drug substance is lacking. Peptide mapping has been highlighted as an approach to quantify product-related impurities in protein products [2], although we are aware of only one current application [3]. The European Pharmacopoeia (Ph.Eur.) monograph on Erythropoietin concentrated solution [4] includes a tryptic mapping procedure as an identification assay, which resolves the methionine-oxidised peptide from the native form but which is unwieldy for routine use in this application. The paper describes the optimisation of Ph. Eur.’s method separation to resolve the 23-mer peptides arising from the native and oxidised forms of rhEPO. A mixture of synthetic peptides was developed as a reference standard to support peak identification and quantification in the assay presented herein, and also future assays sufficiently sensitive to be applied to drug product. This paper describes the method development and the evaluation of the feasibility to develop the corresponding reference standard.