Fluorogenic tagging methodology applied to characterize oxidized tyrosine and phenylalanine in an immunoglobulin monoclonal antibody.

Metal-catalyzed oxidation (MCO) of proteins is of primary concern in the development of biotherapeutics as it represents a prominent degradation pathway with potential undesired biological and biotherapeutic consequences. We developed a fluorogenic derivatization methodology to study the MCO of IgG1 using a model oxidation system, CuCl2/L-ascorbic acid. Besides the oxidation of Met, Trp and His residues, we detected significant oxidation of Phe and Tyr in IgG1. The fluorogenic derivatization method provides an alternative approach for the rapid detection of oxidized Tyr and Phe as their benzoxazole derivatives by fluorescence spectrometry and size exclusion chromatography coupled to fluorescence detection.