Near UV and Visible Light Induce Iron-Dependent Photodegradation Reactions in Pharmaceutical Buffers: Mechanistic and Product Studies

Near UV (lambda = 320-400 nm) and visible light (lambda = 400-800 nm) can lead to the oxidation of pharmaceutical proteins, which can affect efficiency and promote immunogenicity. However, no concise mechanism has been established for the photo-oxidation of pharmaceutical proteins under near UV and visible light. Here, we show that carboxylic acid buffer-Fe3+ complexes can function as photosensitizers, causing peptide degradation via the formation of various radicals and oxidants. Three pharmaceutical relevant carboxylic acid buffers (citrate, acetate, and succinate) were tested under near UV and visible light. Oxidation reactions were monitored for model peptides containing readily oxidizable amino acids, such as methionine- or leucine-enkephalin and proctolin peptide. Oxidation products were evaluated by RP-HPLC coupled to UV or fluorescent detection and RP-HPLC-MS/MS. Specifically for citrate buffer, the light-induced formation of H2O2, center dot OH, center dot CO2- and formaldehyde was demonstrated. The peptides displayed oxidation of Met, hydroxylation of Tyr and Phe, as well as the formation of novel products from Tyr. Experiments with O-18(2) resulted in the incorporation of O-18 into various reaction products, consistent with a metal- catalyzed activation of O-2 into reactive oxygen species. The addition of EDTA and DTPA did not prevent the oxidation of the peptides and, in some cases, enhanced the oxidation. Our results demonstrate that pharmaceutical buffer-Fe3+ complexes, exposed to UV and visible light, can promote various pathways of oxidation reactions in pharmaceutical formulations.