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,

Citrate is a commonly used buffer in pharmaceutical formulations which forms complexes with adventitious metals such as Fe 3+ . Fe 3+ -citrate complexes can act as potent photosensitizers under near-UV and visible light exposure, and recent studies reported evidence for the photo-production of a powerful reductant, carbon dioxide radical anion ( • CO 2 − ), from Fe 3+ -citrate complexes (Subelzu, N.; Schöneich, N., Mol. Pharm. 2020, 17, 4163−4179). The mechanisms of • CO 2 − formation are currently unknown but must be established to devise strategies against • CO 2 − formation in pharmaceutical formulations which rely on the use of citrate buffer. In this study, we first established complementary evidence for the photolytic generation of • CO 2 − from Fe 3+ -citrate through spin trapping and electron paramagnetic resonance (EPR) spectroscopy, and subsequently used spin trapping in conjunction with tandem mass spectrometry (MS/MS) for mechanistic studies on the pathways of • CO 2 − formation. Experiments with stable isotope-labeled citrate suggest that the central carboxylate group of citrate is the major source of • CO 2 − . Competition studies with various inhibitors (alcohols and dimethyl sulfoxide) reveal two mechanisms of • CO 2 − formation, where one pathway involves β-cleavage of a sterically hindered alkoxyl radical generated from the hydroxyl group of citrate.

Fragmentation of therapeutic monoclonal antibodies represents a critical quality attribute. Here, we report a novel visible light-induced heavy chain fragmentation of IgG1 mediated by an Fe(III)-containing histidine (His) buffer. Based on non-reducing sodium dodecylsulfate–polyacrylamide gel electrophoresis and mass spectrometry analysis, IgG1 fragments with apparent molecular weights of ∼130, ∼110, and ∼22 kDa were detected in photo-irradiated samples and were mechanistically rationalized with an oxidative cleavage at Thr259. Specifically, the reactions are proposed to involve the generation of an intermediary alkoxyl radical, which undergoes β-cleavage to yield a glycyl radical. The latter either converts into Gly or adds oxygen and follows a peroxyl radical chemistry. The cleavage process requires the presence of His, while only negligible yields of cleavage products are formed when His is replaced by acetate, succinate, or phosphate buffer. Importantly, the fragmentation can be prevented by ethylenediaminetetraacetic acid (EDTA) only when the EDTA concentrations are in significant excess over the concentrations of Fe(III) and proteins, suggesting a strong binding between Fe(III) and IgG1.

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