title: Development of a sample preparation method for monitoring correct disulfide linkages of monoclonal antibodies by liquid chromatography-mass spectrometry.
abstract: The number and positions of disulfide linkages in a therapeutic monoclonal antibody (mAb) play a crucial role in forming and stabilizing a correct mAb structure that is critical to its function. Peptide mapping by liquid chromatography-mass spectrometry (LC-MS) analysis of enzymatically digested mAb under nonreducing condition is a powerful method for disulfide linkage characterization to ensure mAb drug function and quality. However, the development of a robust sample preparation method with improved digestion efficiency and minimized disulfide scrambling for disulfide linkage analysis is essential but challenging. In this study, a sample preparation method for analysis of correct disulfide linkages in therapeutic mAbs was developed. Instead of common trypsin digestion, Lys-C plus trypsin was used in this approach to improve digestion efficiency. In addition, lower digestion temperature (25 °C) and lower digestion pH (pH 6.8) were also examined to minimize disulfide scrambling. Our results showed that Lys-C plus trypsin digestion at pH 6.8 and 25 °C is a better sample preparation condition for all therapeutic mAbs tested in this study because of a better digestion efficiency (all expected disulfide linkages can be confidently observed) and minimal disulfide scrambling.
authors.affiliation: Protein Mass Spectrometry, Sterile Product and Analytical Development, and Bioprocess Development, Merck Research Laboratories, Kenilworth, NJ 07033, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Protein Mass Spectrometry, Sterile Product and Analytical Development, and Bioprocess Development, Merck Research Laboratories, Kenilworth, NJ 07033, USA.
authors.forename: Huijuan
authors.initials: H
authors.lastname: Li
authors.affiliation: Protein Mass Spectrometry, Sterile Product and Analytical Development, and Bioprocess Development, Merck Research Laboratories, Kenilworth, NJ 07033, USA.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Shameem
authors.affiliation: Protein Mass Spectrometry, Sterile Product and Analytical Development, and Bioprocess Development, Merck Research Laboratories, Kenilworth, NJ 07033, USA. Electronic address: wei.xu2@merck.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Xu
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism*; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000378:metabolism; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D004220:Disulfides; D010455:Peptides; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase
keywords: Disulfide linkage; Disulfide scrambling; LC–MS; Nonreducing; Peptide mapping; Therapeutic mAbs