Chemical Modifications in Aggregates of Recombinant Human Insulin Induced by Metal-Catalyzed Oxidation: Covalent Cross-Linking via Michael Addition to Tyrosine Oxidation Products

PURPOSE: To elucidate the chemical modifications in covalent aggregates of recombinant human insulin induced by metal catalyzed oxidation (MCO). METHODS: Insulin was exposed for 3 h at room temperature to the oxidative system copper(II)/ascorbate. Chemical derivatization with 4-(aminomethyl) benzenesulfonic acid (ABS) was performed to detect 3,4-dihydroxyphenylalanine (DOPA) formation. Electrospray ionization-mass spectrometry (ESI-MS) was employed to localize the amino acids targeted by oxidation and the cross-links involved in insulin aggregation. Oxidation at different pH and temperature was monitored with size exclusion chromatography (SEC) and ESI-MS analysis to further investigate the chemical mechanism(s), to estimate the aggregates content and to quantify DOPA in aggregated insulin. RESULTS: The results implicate the formation of DOPA and 2-amino-3-(3,4-dioxocyclohexa-1,5-dien-1-yl) propanoic acid (DOCH), followed by Michael addition, as responsible for new cross-links resulting in covalent aggregation of insulin during MCO. Michael addition products were detected between DOCH at positions B16, B26, A14, and A19, and free amino groups of the N-terminal amino acids Phe B1 and Gly A1, and side chains of Lys B29, His B5 and His B10. Fragments originating from peptide bond hydrolysis were also detected. CONCLUSION: MCO of insulin leads to covalent aggregation through cross-linking via Michael addition to tyrosine oxidation products. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11095-012-0755-z) contains supplementary material, which is available to authorized users.