Near UV and Visible Light Photo-Degradation Mechanisms in Citrate Buffer: One-Electron Reduction of Peptide and Protein Disulfides promotes Oxidation and Cis/Trans Isomerization of Unsaturated Fatty Acids of Polysorbate 80

Abstract

Citrate is a common buffer for slightly acidic pH ranges of protein formulations. In the presence of iron, citrate buffer undergoes photo-degradation induced by near UV and visible light. Recent studies (Subelzu and Schöneich, Mol. Pharm. 2020, 17, 4163-4179) have documented that such photo-degradation results in the formation of carbon dioxide radical anion (•CO2−), a strong reductant which reduces Fe3+, O2, and disulfide bonds. In the present study we show that near UV and visible light photo-degradation of citrate in the presence of iron can induce reductive peptide and protein disulfide cleavage as well as free radical damage of a surfactant, polysorbate 80 (PS80). Reductive disulfide cleavage is most likely caused by efficient electron transfer from carbon dioxide radical anions to disulfides, resulting in the generation of thiol/thiolate and thiyl radicals. The latter can react with mono- and polyunsaturated fatty acids of PS80 to cause cis/trans isomerization and/or oxidation. Representative products generated by cis/trans isomerization and oxidation of oleic acid esters have been detected by HPLC-MS analysis. Further evidence for reductive disulfide cleavage was obtained through the analysis of free thiols. The oxidation of PS80 can also be the consequence of reactive oxygen species (ROS) generation through the reduction of O2 by carbon dioxide radical anions and/or intermediary Fe2+.