title: Triethylenetetramine prevents insulin aggregation and fragmentation during copper catalyzed oxidation.
abstract: Metal catalyzed oxidation via the oxidative system Cu(2+)/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 (1mg/ml) was oxidized with 40 μM Cu(2+) and 4mM ascorbic acid in absence or presence of excipients. Among the excipients studied, 1mM of TETA, EDTA, or GSH prevented insulin aggregation upon metal catalyzed oxidation (MCO) for 3h 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.
authors: Division of Drug Delivery Technology, Leiden University, Leiden, The Netherlands.
authors: Riccardo
authors: R
authors: Torosantucci
authors: Daniel
authors: D
authors: Weinbuch
authors: Robin
authors: R
authors: Klem
authors: Wim
authors: W
authors: Jiskoot
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D019540:Area Under Curve; D001205:Ascorbic Acid / Q000737:chemistry; D001596:Benzylamines / Q000737:chemistry; D002384:Catalysis; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry*; D004492:Edetic Acid / Q000737:chemistry; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D007328:Insulin / Q000737:chemistry*; D008027:Light; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors; D014266:Trientine / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D001596:Benzylamines; D007074:Immunoglobulin G; D007328:Insulin; D011994:Recombinant Proteins; D003300:Copper; D004492:Edetic Acid; D005978:Glutathione; D001205:Ascorbic Acid; D010100:Oxygen; D014266:Trientine