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

2008; Elsevier BV; Volume: 870; Issue: 1 Linguagem: Inglês

10.1016/j.jchromb.2008.05.045

1873-376X

Georgeen Gaza‐Bulseco, Sagar Faldu, Karen Hurkmans, Chris Chumsae, Hongcheng Liu,

Enzyme Structure and Function

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.