Triethylenetetramine prevents insulin aggregation and fragmentation during copper catalyzed oxidation

Metal catalyzed oxidation via the oxidative system Cu2+/ascorbate is known to induce aggregation of therapeutic proteins, resulting in enhanced immunogenicity. Hence, inclusion of antioxidants in protein formulations is of great interest. In this study, using recombinant human insulin (insulin) as a model, we investigated the ability of several excipients, in particular triethylenetetramine (TETA), reduced glutathione (GSH) and ethylenediamine tetraacetic acid (EDTA), for their ability to prevent protein oxidation, aggregation, and fragmentation. Insulin (1 mg/ml) was oxidized with 40 μM Cu2+ and 4 mM ascorbic acid in absence or presence of excipients. Among the excipients studied, 1 mM of TETA, EDTA, or GSH prevented insulin aggregation upon metal catalyzed oxidation (MCO) for 3 h at room temperature, based on size exclusion chromatography (SEC). At lower concentration (100 μM), for 72 h at +4 °C, TETA was the only one to inhibit almost completely oxidation-induced insulin aggregation, fragmentation, and structural changes, as indicated by SEC, nanoparticle tracking analysis, light obscuration particle counting, intrinsic/extrinsic fluorescence, circular dichroism, and chemical derivatization. In contrast, GSH had a slight pro-oxidant effect, as demonstrated by the higher percentage of aggregates and a more severe structural damage, whereas EDTA offered substantially less protection. TETA also protected a monoclonal IgG1 against MCO-induced aggregation, suggesting its general applicability. In conclusion, TETA is a potential candidate excipient for inclusion in formulations of oxidation-sensitive proteins.

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