Methionine, tryptophan, and histidine oxidation in a model protein, PTH: Mechanisms and stabilization

Recent oxidation events on monoclonal antibody candidates prompted us to investigate the mechanism of oxidation of Met, Trp, and His residues and to search for suitable stabilizers. By using parathyroid hormone (1-34), PTH, as a model protein and various oxidants, aided by liquid chromatography, peptide mapping, and mass spectrometry, we identified and quantified the oxidation of these vulnerable residues. Whereas H2O2 and t-butyl hydroperoxide (t-BHP) primarily oxidized the two Met residues, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), and H2O2 + Fe(II) oxidized Met and Trp residues, with AAPH more capable of generating oxidized Trp species than the latter. H2O2 + Fe(III) generated results comparable to those with H2O2 + Fe(II), except that there was a lesser amount of hydroxylated Phe. Oxidation of the His residue in PTH occurred when copper was used instead of iron. AAPH, a free-radical generator, produced alkylperoxides, which simulated the oxidizing species from degraded polysorbate, commonly found in protein formulations. It is prudent to screen stabilizers by using H2O2, H2O2 + Fe(II), and AAPH because these agents represent potential assaults from the H2O2 commonly present in degraded polysorbate, the residue of aseptic agents and the metal from stainless steel surfaces, and alkylperoxides from degraded polysorbate, respectively. Free Met protected the Met residues in PTH from oxidation by H2O2 and H2O2 + Fe(II). Mannitol and EDTA were effective against H2O2 + Fe(II). Free Trp protected only the Trp residue in PTH from oxidation by AAPH, the combination of Trp and Met was effective against all three oxidant conditions. By using AAPH to generate oxidant, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and pyridoxine were also found to exhibit good free-radical scavenging activity and thus protected Trp in PTH against oxidation. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4485-4500, 2009