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

  • Gaza-Bulseco G
  • Faldu S
  • Hurkmans K
 et al. 
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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 CH2-CH3 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.

Author-supplied keywords

  • Animals
  • Antibodies, Monoclonal/*chemistry/*metabolism
  • CHO Cells
  • Chromatography, Affinity
  • Cricetinae
  • Cricetulus
  • Humans
  • Immunoglobulin G/*chemistry/*metabolism
  • Kinetics
  • Methionine/*chemistry/metabolism
  • Nerve Tissue Proteins/*metabolism
  • Oxidation-Reduction
  • Protein Binding
  • Recombinant Proteins/chemistry/metabolism
  • Staphylococcal Protein A/*metabolism
  • tert-Butylhydroperoxide/chemistry

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Authors

  • G Gaza-Bulseco

  • S Faldu

  • K Hurkmans

  • C Chumsae

  • H Liu

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