Affordable Access

Publisher Website

Effect of methionine oxidation of a recombinant monoclonal antibody on the binding affinity to protein A and protein G

Authors
Journal
Journal of Chromatography B
1570-0232
Publisher
Elsevier
Publication Date
Volume
870
Issue
1
Identifiers
DOI: 10.1016/j.jchromb.2008.05.045
Keywords
  • Recombinant Monoclonal Antibody
  • Methionine Oxidation
  • Mass Spectrometry
  • Protein A
  • Protein G
Disciplines
  • Biology

Abstract

Abstract Oxidation of methionine (Met) residues is one of the most common protein degradation pathways. Two Met residues, Met256 and Met432, of a recombinant fully human monoclonal IgG1 antibody have been shown to be susceptible to oxidation. Met256 and Met432 are located in the antibody CH 2–CH 3 interface and in close proximity to protein A and protein G binding sites. The effect of oxidation of these susceptible Met residues on the binding to protein A and protein G was investigated in the current study. Incubation of the antibody with 5% tert-butyl hydroperoxide (tBHP) resulted in a nearly complete oxidation of Met256 and Met432, while incubation with 1% tBHP resulted in mixed populations of the antibody with different degrees of Met oxidation. Oxidation of Met256 and Met432 resulted in earlier elution of the antibody from protein A and protein G columns when eluted with a gradient of decreasing pH. Analysis by ELISA and surface plasmon resonance (SPR) revealed decreased binding affinity of the oxidized antibody to protein A and protein G. It is therefore concluded that oxidation of the Met256 and Met432 residues of the recombinant monoclonal antibody altered its interaction with protein A and protein G resulting in a decrease in binding affinity.

There are no comments yet on this publication. Be the first to share your thoughts.