Effect of methionine oxidation of a recombinant monoclonal antibody on the binding affinity to protein A and protein G.
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.
Protein Analytics, Process Sciences Department, Abbott Bioresearch Center, 100 Research Drive, Worcester, MA 01605, USA.
Georgeen
G
Gaza-Bulseco
Sagar
S
Faldu
Karen
K
Hurkmans
Chris
C
Chumsae
Hongcheng
H
Liu
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism*; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008715:Methionine / Q000737:chemistry* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000378:metabolism*; D020122:tert-Butylhydroperoxide / Q000737:chemistry
D000911:Antibodies, Monoclonal; C029152:G-substrate; D007074:Immunoglobulin G; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D020122:tert-Butylhydroperoxide; D008715:Methionine