Analysis of the structure and stability of erythropoietin by pH and temperature changes using various LC/MS

Seong Hun Chang, Hyun Jung Kim, Chan Wha Kim

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

The purpose of stability testing is to provide evidence about how the quality of a drug varies with time under the influence of a variety of environmental factors. In this study, erythropoietin (EPO) was analyzed under different pH (pH 3 and pH 9) and temperature (25 °C and 40 °C) conditions according to current Good Manufacturing Practice (cGMP) and International Conference on Harmonisation (ICH) guidelines. The molecular weight difference between intact EPO and deglycosylated EPO was determined by SDS-PAGE, and aggregated forms of EPO under thermal stress and high-pH conditions were investigated by size exclusion chromatography. High pH and high temperature induced increases in dimer and high molecular weight aggregate forms of EPO. UPLC-ESI-TOF-MS was applied to analyze the changed modification sites on EPO. Further, normal-phase high-performance liquid chromatography was performed to identify proposed glycan structures and high pH anion exchange chromatography was carried out to investigate any change in carbohydrate composition. The results demonstrated that there were no changes in modification sites or the glycan structure under severe conditions; however, the number of dimers and aggregates increased at 40 °C and pH 9, respectively.

Original languageEnglish
Pages (from-to)2663-2670
Number of pages8
JournalBulletin of the Korean Chemical Society
Volume34
Issue number9
DOIs
Publication statusPublished - 2013 Sep 20

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Keywords

  • Erythropoietin
  • HPAEC-PAD
  • NP-HPLC
  • SEC-HPLC
  • UPLC-ESI-Q TOF-MS/MS

ASJC Scopus subject areas

  • Chemistry(all)

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