Effect of methionine oxidation of a recombinant monoclonal antibody on the binding affinity to protein A and protein G
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.
Conference Paper: On-Line Statistics In ManufacturingElectro International, 1991; 05/1991
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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.Journal of the American Society for Mass Spectrometry 12/2008; 20(3):525-8. DOI:10.1016/j.jasms.2008.11.011 · 3.19 Impact Factor
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ABSTRACT: Optical biosensor technology continues to be the method of choice for label-free, real-time interaction analysis. But when it comes to improving the quality of the biosensor literature, education should be fundamental. Of the 1413 articles published in 2008, less than 30% would pass the requirements for high-school chemistry. To teach by example, we spotlight 10 papers that illustrate how to implement the technology properly. Then we grade every paper published in 2008 on a scale from A to F and outline what features make a biosensor article fabulous, middling or abysmal. To help improve the quality of published data, we focus on a few experimental, analysis and presentation mistakes that are alarmingly common. With the literature as a guide, we want to ensure that no user is left behind.Journal of Molecular Recognition 01/2009; 23(1):1-64. DOI:10.1002/jmr.1004 · 2.34 Impact Factor