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Bulletin of the Korean Chemical Society (BKCS)

ISSN 0253-2964(Print)
ISSN 1229-5949(Online)
Volume 34, Number 9
BKCSDE 34(9)
September 20, 2013 

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*
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 oC and 40 oC) 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 oC and pH 9, respectively.
2663 - 2670
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