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 pH6 when Lys-C or trypsin plus Glu-C were used as enzymes. When thermolysin was applied, some scrambled disulfide bonds were identified at pH5, 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.
authors.affiliation: National Health Research Institutes, National Institute of Infectious Diseases and Vaccinology, Miaoli, Taiwan.
authors.forename: Wang-Chou
authors.initials: WC
authors.lastname: Sung
authors.affiliation: National Taiwan Normal University, Department of Chemistry, Taipei, Taiwan.
authors.forename: Chiung-Wen
authors.initials: CW
authors.lastname: Chang
authors.affiliation: Mithra Biotechnology Inc., New Taipei City, Taiwan.
authors.forename: Sheng-Yu
authors.initials: SY
authors.lastname: Huang
authors.affiliation: National Taiwan Normal University, Department of Chemistry, Taipei, Taiwan.
authors.forename: Ting-Yu
authors.initials: TY
authors.lastname: Wei
authors.affiliation: National Taiwan Normal University, Department of Chemistry, Taipei, Taiwan.
authors.forename: Yi-Li
authors.initials: YL
authors.lastname: Huang
authors.affiliation: National Taiwan Normal University, Department of Chemistry, Taipei, Taiwan.
authors.forename: Yu-Hua
authors.initials: YH
authors.lastname: Lin
authors.affiliation: Catholic Fu-Jen University, Department of Life Science, Taipei, Taiwan.
authors.forename: Han-Min
authors.initials: HM
authors.lastname: Chen
authors.affiliation: National Taiwan Normal University, Department of Chemistry, Taipei, Taiwan. Electronic address: sfchen@ntnu.edu.tw.
authors.forename: Sung-Fang
authors.initials: SF
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D020533:Angiogenesis Inhibitors / Q000737:chemistry*; D000068258:Bevacizumab / Q000737:chemistry*; D055162:Biocatalysis; D004220:Disulfides / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D013058:Mass Spectrometry; D012996:Solutions; D013820:Thermolysin / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry*
chemical_list: D020533:Angiogenesis Inhibitors; D004220:Disulfides; D012996:Solutions; D000068258:Bevacizumab; D014357:Trypsin; D013820:Thermolysin
keywords: Dimethyl labeling; Disulfide bond scrambling; Mass spectrometry; Monoclonal antibody; RADAR