Abstract
Oxidation of methionine (Met) residues of a recombinant fully human monoclonal antibody after exposure to light was investigated and compared with chemically induced oxidation using tert-butyl-hydroperoxide (tBHP). Met256 and Met432 in the Fc region in the samples exposed to light or incubated with tBHP were oxidized. The Fc mass spectra of the antibody exposed to light showed mainly peaks with a molecular weight (MW) increase of 32 Da, however the sample treated with tBHP showed peaks with increase of only 16 Da. These results suggested that either oxidation of one Met residue (either Met256 or Met432) catalyzed the oxidation of the second Met residue on the same heavy chain (HC) or Met residues of one HC were preferentially oxidized when the antibody was exposed to light, while Met256 and Met432 were randomly oxidized when the antibody was incubated with tBHP.
Article PDF
Avoid common mistakes on your manuscript.
References
Wang, W.; Singh, S.; Zeng, D. L.; King, K.; Nema, S. Antibody Structure, Instability, and Formulation. J. Pharm. Sci. 2007, 96(1), 1–26.
Liu, H.; Gaza-Bulseco, G.; Faldu, D.; Chumsae, C.; Sun, J. Heterogeneity of Monoclonal Antibodies. J. Pharm. Sci. 2008, 97(1), 2426–2447.
Liu, H.; Gaza-Bulseco, G.; Xiang, T.; Chumsae, C. Structural Effect of Deglycosylation and Methionine Oxidation on a Recombinant Monoclonal Antibody. Mol. Immuno. 2008, 45(3), 701–708; Epub 2007 Aug. 24.
Gaza-Bulseco, G.; Faldu, S.; Hurkmans, K.; Chumsae, C.; Liu, H. Effect of Methionine Oxidation of a Recombinant Monoclonal Antibody on the Binding Affinity to Protein A and Protein G. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2008, 870(1), 55–62; Epub 2008 Jun. 5.
Shen, J. F.; Kwong, Y. M.; Keck, G. R.; Harris, J. R. The Application of tert-Butylhydroperoxide Oxidization to Study Sites of Potential Methionine Oxidization in a Recombinant Antibody; Techniques in Protein Chemistry VII, Marshak, D. R., Eds, Academic Press: New York, 1996; p.275.
Chumsae, C.; Gaza-Bulseco, G.; Sun, J.; Liu, H. Comparison of Methionine Oxidation in Thermal Stability and Chemically Stressed Samples of a Fully Human Monoclonal Antibody. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2007, 850(1/2), 285–294; Epub 2006 Dec. 19.
Lam, X. M.; Yang, J. Y.; Cleland, J. L. Antioxidants for Prevention of Methionine Oxidation in Recombinant Monoclonal Antibody HER2. J. Pharm. Sci. 1997, 86(11), 1250–1255.
Lefranc, M. P.; Pommie, C.; Kaas, Q.; Duprat, E.; Bosc, N.; Guiraudou, D.; Jean, C.; Ruiz, M.; Da Piedade, I.; Rouard, M.; Foulquier, E.; Thouvenin, V.; Lefranc, G. IMGT Unique Numbering for Immunoglobulin and T cell Receptor Constant Domains and Ig Superfamily C-Like Domains. Dev. Comp. Immunol. 2005, 29(3), 185–203.
Deisenhofer, J. Crystallographic Refinement and Atomic Models of a Human Fc Fragment and Its Complex with Fragment B of Protein A from Staphylococcus aureus at 2.9- and 2.8-A Resolution. Biochemistry. 1981, 20(9), 2361–2370.
Saphire, E. O.; Stanfield, R. L.; Crispin, M. D.; Morris, G.; Zwick, M. B.; Pantophlet, R. A.; Parren, P. W.; Rudd, P. M.; Dwek, R. A.; Burton, D. R.; Wilson, I. A. Crystal Structure of an Intact Human IgG: Antibody Asymmetry, Flexibility, and a Guide for HIV-1 Vaccine Design. Adv. Exp. Med. Biol. 2003, 535, 55–66.
Sauer-Eriksson, A. E.; Kleywegt, G. J.; Uhlen, M.; Jones, T. A. Crystal Structure of the C2 Fragment of Streptococcal Protein G in Complex with the Fc Domain of Human IgG. Structure. 1995, 3(3), 265–278.
Shields, R. L.; Namenuk, A. K.; Hong, K.; Meng, Y. G.; Rae, J.; Briggs, J.; Xie, D.; Lai, J.; Stadlen, A.; Li, B.; Fox, J. A.; Presta, L. G. High Resolution Mapping of the Binding Site on Human IgG1 for Fc γ RI, Fc γ RII, Fc γ RIII, and FcRn, and Design of IgG1 Variants with Improved Binding to the Fc γ R. J. Biol. Chem. 2001, 276(9), 6591–6604; Epub 2000 Nov. 28.
Roopenian, D. C.; Akilesh, S. FcRn: The Neonatal Fc Receptor Comes of Age. Nat. Rev. Immunol. 2007, 7(9), 715–725; Epub 2007 Aug. 17.
Author information
Authors and Affiliations
Corresponding author
Additional information
Published online November 27, 2008
Rights and permissions
About this article
Cite this article
Liu, H., Gaza-Bulseco, G. & Zhou, L. Mass spectrometry analysis of photo-induced methionine oxidation of a recombinant human monoclonal antibody. J Am Soc Mass Spectrom 20, 525–528 (2009). https://doi.org/10.1016/j.jasms.2008.11.011
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1016/j.jasms.2008.11.011