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

7 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

Fingerprint

Erythropoietin
Dimers
Temperature
Polysaccharides
Molecular weight
Size exclusion chromatography
High performance liquid chromatography
Chromatography
Thermal stress
Anions
Carbohydrates
Testing
Chemical analysis
Pharmaceutical Preparations

Keywords

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

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Analysis of the structure and stability of erythropoietin by pH and temperature changes using various LC/MS. / Chang, Seong Hun; Kim, Hyun Jung; Kim, Chan Wha.

In: Bulletin of the Korean Chemical Society, Vol. 34, No. 9, 20.09.2013, p. 2663-2670.

Research output: Contribution to journalArticle

@article{eb8a4e1843814122931bdfe15fab8f41,
title = "Analysis of the structure and stability of erythropoietin by pH and temperature changes using various LC/MS",
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.",
keywords = "Erythropoietin, HPAEC-PAD, NP-HPLC, SEC-HPLC, UPLC-ESI-Q TOF-MS/MS",
author = "Chang, {Seong Hun} and Kim, {Hyun Jung} and Kim, {Chan Wha}",
year = "2013",
month = "9",
day = "20",
doi = "10.5012/bkcs.2013.34.9.2663",
language = "English",
volume = "34",
pages = "2663--2670",
journal = "Bulletin of the Korean Chemical Society",
issn = "0253-2964",
publisher = "Wiley-Blackwell",
number = "9",

}

TY - JOUR

T1 - Analysis of the structure and stability of erythropoietin by pH and temperature changes using various LC/MS

AU - Chang, Seong Hun

AU - Kim, Hyun Jung

AU - Kim, Chan Wha

PY - 2013/9/20

Y1 - 2013/9/20

N2 - 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.

AB - 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.

KW - Erythropoietin

KW - HPAEC-PAD

KW - NP-HPLC

KW - SEC-HPLC

KW - UPLC-ESI-Q TOF-MS/MS

UR - http://www.scopus.com/inward/record.url?scp=84885414795&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84885414795&partnerID=8YFLogxK

U2 - 10.5012/bkcs.2013.34.9.2663

DO - 10.5012/bkcs.2013.34.9.2663

M3 - Article

AN - SCOPUS:84885414795

VL - 34

SP - 2663

EP - 2670

JO - Bulletin of the Korean Chemical Society

JF - Bulletin of the Korean Chemical Society

SN - 0253-2964

IS - 9

ER -