A new analytical strategy has exposed a subtle layer of antibody damage that standard tests have struggled to resolve: the “handed” forms of methionine oxidation in therapeutic antibodies. Researchers from the National Institutes of Natural Sciences and collaborators combined methyl-based NMR spectroscopy, selective enzymatic reduction, and LC-MS peptide mapping to distinguish the R and S stereoisomers of methionine sulfoxide in the Fc region of IgG1 antibodies.
The team focused on two conserved Fc methionines, Met252 and Met428, both of which can influence antibody stability and receptor interactions when oxidized. NMR revealed multiple oxidation-dependent signals, while treatment with methionine sulfoxide reductase A – an enzyme that selectively reduces the S-form – helped assign the stereochemistry of the resulting products. LC-MS independently confirmed the identities and relative abundances of the oxidized species. The data also showed that the more solvent-exposed Met252 oxidizes more readily than the more buried Met428.
“Oxidation of methionine residues is a well-known issue in antibody therapeutics, but the stereochemical diversity of these modifications has been difficult to analyze,” said co-author Koichi Kato in a press release. “Our integrated NMR and LC-MS strategy allows us to visualize these subtle structural differences at atomic resolution.”
Because Fc oxidation can alter receptor binding, stability, and in vivo persistence, the authors say the approach could strengthen stability testing and manufacturing control for antibody drugs.
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