Effect of UVC Irradiation on the Oxidation of Histidine in Monoclonal Antibodies
Yuya Miyahara, Koya Shintani, Kayoko Hayashihara-Kakuhou, Takehiro Zukawa, Yukihiro Morita, Takashi Nakazawa, Takuya Yoshida, Tadayasu Ohkubo, Susumu Uchiyama
Abstract
Abstract We oxidized histidine residues in monoclonal antibody drugs of immunoglobulin gamma 1 (IgG1) using ultraviolet C irradiation (UVC: 200–280 nm), which is known to be potent for sterilization or disinfection. Among the reaction products, we identified asparagine and aspartic acid by mass spectrometry. In the photo-induced oxidation of histidine in angiotensin II, 18 O atoms from H 2 18 O in the solvent were incorporated only into aspartic acid but not into asparagine. This suggests that UVC irradiation generates singlet oxygen and induces [2 + 2] cycloaddition to form a dioxetane involving the imidazole C γ − C δ2 bond of histidine, followed by ring-opening in the manner of further photo-induced retro [2 + 2] cycloaddition. This yields an equilibrium mixture of two keto-imines, which can be the precursors to aspartic acid and asparagine. The photo-oxidation appears to occur preferentially for histidine residues with lower p K a values in IgG1. We thus conclude that the damage due to UVC photo-oxidation of histidine residues can be avoided in acidic conditions where the imidazole ring is protonated.