Use of a stable-isotope-labeled reporter peptide and antioxidants for reliable quantification of methionine oxidation in a monoclonal antibody by liquid chromatography/mass spectrometry.
Accurate quantification of methionine oxidation in therapeutic proteins by liquid chromatography/mass spectrometry (LC/MS) is challenging due to the potential artifacts introduced during sample preparation and analysis in the peptide mapping workflow. In this study, a systematic approach for optimization of the peptide mapping procedure to achieve reliable quantification of endogenous methionine oxidation in monoclonal antibodies was developed.



The approach is based on usage of a stable-isotope-labeled reporter peptide, identical in sequence to the tryptic peptide of an IgG1 monoclonal antibody containing the methionine residue most prone to oxidation. This approach was applied to evaluating various desalting procedures, and tested on nanoLC/MS, microLC/MS and UPLC/MS for the peptide mapping analysis of a model monoclonal antibody IgG1 sensitive to oxidation.



Several steps in the peptide mapping procedure with LC/MS detection at which protein oxidation occurred were identified and optimized using the reference stable-isotope-labeled peptide. Thus, reliable quantification of methionine oxidation in the target monoclonal antibody was validated.



The methodology which utilizes the reference stable-isotope-labeled reporter peptide is applicable to monoclonal antibody oxidation analysis and could be extended to other biotherapeutics once oxidation-prone methionine(s) in the protein sequence are identified. Copyright © 2016 John Wiley & Sons, Ltd.
Analytical Science and Technology Department, Global Manufacturing Science and Technology, Sanofi, 45 New York Ave., Framingham, MA, 01701, USA.
Pilsoo
P
Kang
BioFormulations Development Department, Biotherapeutics Development, Sanofi, 1 The Mountain Rd., Framingham, MA, 01701, USA.
Tanya
T
Mezhebovsky
Proteomics and Mass Spectrometry Core Facility, Cornell University, 139 Biotechnology Building, 526 Campus Road, Ithaca, NY, 14853, USA.
Wei
W
Chen
Proteomics and Mass Spectrometry Core Facility, Cornell University, 139 Biotechnology Building, 526 Campus Road, Ithaca, NY, 14853, USA.
James A
JA
McCardle
Proteomics and Mass Spectrometry Core Facility, Cornell University, 139 Biotechnology Building, 526 Campus Road, Ithaca, NY, 14853, USA.
Sheng
S
Zhang
Department of Biopharmaceutical Development, Pharmaceutical Science and Technology - Exton, Eisai Inc., 210 Welsh Pool Road, Exton, PA, 19341, USA.
Eric P
EP
Sullivan
Department of Biopharmaceutical Development, Pharmaceutical Science and Technology - Exton, Eisai Inc., 210 Welsh Pool Road, Exton, PA, 19341, USA.
Philip M
PM
Sass
Department of Biopharmaceutical Development, Pharmaceutical Science and Technology - Exton, Eisai Inc., 210 Welsh Pool Road, Exton, PA, 19341, USA.
Eric
E
Routhier
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000975:Antioxidants; D002853:Chromatography, Liquid; D007554:Isotopes; D013058:Mass Spectrometry; D008715:Methionine; D010455:Peptides
D000911:Antibodies, Monoclonal; D000975:Antioxidants; D007554:Isotopes; D010455:Peptides; D008715:Methionine