Evaluation of disulfide scrambling during the enzymatic digestion of bevacizumab at various pH values using mass spectrometry

Wang Chou Sung, Chiung Wen Chang, Sheng Yu Huang, Ting Yu Wei, Yi Li Huang, Yu Hua Lin, Han Min Chen, Sung-Fang Chen

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Disulfide linkages play an important role in protein stability and activity. Thus, it is critical to characterize disulfide bonds to ensure the quality and function of protein pharmaceuticals. There are, however, problems associated with maintaining disulfide linkages in the conventional procedures that are used to digest a protein. In order to preserve enzyme activity during the digestion of a protein, it is commonly carried out at neutral to basic environment which increases the possibilities of disulfide bond scrambling. However, it is not easy to differentiate whether the scrambled disulfide linkages are initiated by the sample itself or whether they are induced during the protease digestion process. In this study, the optimum pH for minimizing disulfide bond rearrangements during the digestion process was determined. Three sets of proteases, trypsin plus Glu-C, Lys-C and thermolysin were used, followed by dimethyl labeling and mass spectrometry for a bevacizumab (Avastin) disulfide linkage analysis. No disulfide linkage scrambling was detected at pH 6 when Lys-C or trypsin plus Glu-C were used as enzymes. When thermolysin was applied, some scrambled disulfide bonds were identified at pH 5, 6 and 7. Nevertheless, there was less disulfide bond scrambling at a lower pH. All correct disulfide bonds on bevacizumab could be identified using this approach. The results demonstrated that by choosing the proper enzymes, using a lower pH environment for the digestion could reduce the degree of artifact disulfide scrambling.

Original languageEnglish
Pages (from-to)1188-1194
Number of pages7
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1864
Issue number9
DOIs
Publication statusPublished - 2016 Sep 1

Fingerprint

Disulfides
Mass spectrometry
Digestion
Mass Spectrometry
Thermolysin
Trypsin
Bevacizumab
Proteins
Peptide Hydrolases
Enzymes
Protein Stability
Enzyme activity
Artifacts
Labeling
Proteolysis

Keywords

  • Dimethyl labeling
  • Disulfide bond scrambling
  • Mass spectrometry
  • Monoclonal antibody
  • RADAR

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Evaluation of disulfide scrambling during the enzymatic digestion of bevacizumab at various pH values using mass spectrometry. / Sung, Wang Chou; Chang, Chiung Wen; Huang, Sheng Yu; Wei, Ting Yu; Huang, Yi Li; Lin, Yu Hua; Chen, Han Min; Chen, Sung-Fang.

In: Biochimica et Biophysica Acta - Proteins and Proteomics, Vol. 1864, No. 9, 01.09.2016, p. 1188-1194.

Research output: Contribution to journalArticle

Sung, Wang Chou ; Chang, Chiung Wen ; Huang, Sheng Yu ; Wei, Ting Yu ; Huang, Yi Li ; Lin, Yu Hua ; Chen, Han Min ; Chen, Sung-Fang. / Evaluation of disulfide scrambling during the enzymatic digestion of bevacizumab at various pH values using mass spectrometry. In: Biochimica et Biophysica Acta - Proteins and Proteomics. 2016 ; Vol. 1864, No. 9. pp. 1188-1194.
@article{00fded0d1b5245b8934b66199f5ea2e4,
title = "Evaluation of disulfide scrambling during the enzymatic digestion of bevacizumab at various pH values using mass spectrometry",
abstract = "Disulfide linkages play an important role in protein stability and activity. Thus, it is critical to characterize disulfide bonds to ensure the quality and function of protein pharmaceuticals. There are, however, problems associated with maintaining disulfide linkages in the conventional procedures that are used to digest a protein. In order to preserve enzyme activity during the digestion of a protein, it is commonly carried out at neutral to basic environment which increases the possibilities of disulfide bond scrambling. However, it is not easy to differentiate whether the scrambled disulfide linkages are initiated by the sample itself or whether they are induced during the protease digestion process. In this study, the optimum pH for minimizing disulfide bond rearrangements during the digestion process was determined. Three sets of proteases, trypsin plus Glu-C, Lys-C and thermolysin were used, followed by dimethyl labeling and mass spectrometry for a bevacizumab (Avastin) disulfide linkage analysis. No disulfide linkage scrambling was detected at pH 6 when Lys-C or trypsin plus Glu-C were used as enzymes. When thermolysin was applied, some scrambled disulfide bonds were identified at pH 5, 6 and 7. Nevertheless, there was less disulfide bond scrambling at a lower pH. All correct disulfide bonds on bevacizumab could be identified using this approach. The results demonstrated that by choosing the proper enzymes, using a lower pH environment for the digestion could reduce the degree of artifact disulfide scrambling.",
keywords = "Dimethyl labeling, Disulfide bond scrambling, Mass spectrometry, Monoclonal antibody, RADAR",
author = "Sung, {Wang Chou} and Chang, {Chiung Wen} and Huang, {Sheng Yu} and Wei, {Ting Yu} and Huang, {Yi Li} and Lin, {Yu Hua} and Chen, {Han Min} and Sung-Fang Chen",
year = "2016",
month = "9",
day = "1",
doi = "10.1016/j.bbapap.2016.05.011",
language = "English",
volume = "1864",
pages = "1188--1194",
journal = "Biochimica et Biophysica Acta - Proteins and Proteomics",
issn = "1570-9639",
publisher = "Elsevier",
number = "9",

}

TY - JOUR

T1 - Evaluation of disulfide scrambling during the enzymatic digestion of bevacizumab at various pH values using mass spectrometry

AU - Sung, Wang Chou

AU - Chang, Chiung Wen

AU - Huang, Sheng Yu

AU - Wei, Ting Yu

AU - Huang, Yi Li

AU - Lin, Yu Hua

AU - Chen, Han Min

AU - Chen, Sung-Fang

PY - 2016/9/1

Y1 - 2016/9/1

N2 - Disulfide linkages play an important role in protein stability and activity. Thus, it is critical to characterize disulfide bonds to ensure the quality and function of protein pharmaceuticals. There are, however, problems associated with maintaining disulfide linkages in the conventional procedures that are used to digest a protein. In order to preserve enzyme activity during the digestion of a protein, it is commonly carried out at neutral to basic environment which increases the possibilities of disulfide bond scrambling. However, it is not easy to differentiate whether the scrambled disulfide linkages are initiated by the sample itself or whether they are induced during the protease digestion process. In this study, the optimum pH for minimizing disulfide bond rearrangements during the digestion process was determined. Three sets of proteases, trypsin plus Glu-C, Lys-C and thermolysin were used, followed by dimethyl labeling and mass spectrometry for a bevacizumab (Avastin) disulfide linkage analysis. No disulfide linkage scrambling was detected at pH 6 when Lys-C or trypsin plus Glu-C were used as enzymes. When thermolysin was applied, some scrambled disulfide bonds were identified at pH 5, 6 and 7. Nevertheless, there was less disulfide bond scrambling at a lower pH. All correct disulfide bonds on bevacizumab could be identified using this approach. The results demonstrated that by choosing the proper enzymes, using a lower pH environment for the digestion could reduce the degree of artifact disulfide scrambling.

AB - Disulfide linkages play an important role in protein stability and activity. Thus, it is critical to characterize disulfide bonds to ensure the quality and function of protein pharmaceuticals. There are, however, problems associated with maintaining disulfide linkages in the conventional procedures that are used to digest a protein. In order to preserve enzyme activity during the digestion of a protein, it is commonly carried out at neutral to basic environment which increases the possibilities of disulfide bond scrambling. However, it is not easy to differentiate whether the scrambled disulfide linkages are initiated by the sample itself or whether they are induced during the protease digestion process. In this study, the optimum pH for minimizing disulfide bond rearrangements during the digestion process was determined. Three sets of proteases, trypsin plus Glu-C, Lys-C and thermolysin were used, followed by dimethyl labeling and mass spectrometry for a bevacizumab (Avastin) disulfide linkage analysis. No disulfide linkage scrambling was detected at pH 6 when Lys-C or trypsin plus Glu-C were used as enzymes. When thermolysin was applied, some scrambled disulfide bonds were identified at pH 5, 6 and 7. Nevertheless, there was less disulfide bond scrambling at a lower pH. All correct disulfide bonds on bevacizumab could be identified using this approach. The results demonstrated that by choosing the proper enzymes, using a lower pH environment for the digestion could reduce the degree of artifact disulfide scrambling.

KW - Dimethyl labeling

KW - Disulfide bond scrambling

KW - Mass spectrometry

KW - Monoclonal antibody

KW - RADAR

UR - http://www.scopus.com/inward/record.url?scp=84976608261&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84976608261&partnerID=8YFLogxK

U2 - 10.1016/j.bbapap.2016.05.011

DO - 10.1016/j.bbapap.2016.05.011

M3 - Article

VL - 1864

SP - 1188

EP - 1194

JO - Biochimica et Biophysica Acta - Proteins and Proteomics

JF - Biochimica et Biophysica Acta - Proteins and Proteomics

SN - 1570-9639

IS - 9

ER -