Oxidation is a common degradation pathway that affects therapeutic proteins and peptides during production, purification, formulation, transportation, storage and handling of solid and liquid preparations. In the present work we review the scientific literature about structural and biological consequences of protein/peptide oxidation. Representative examples are discussed of specific products whose oxidation has been recently studied, including monoclonal antibodies, calcitonin, granulocyte colony-stimulating factor, growth hormone, insulin, interferon alpha and beta, oxytocin and parathyroid hormone. These examples illustrate that oxidation often leads to modifications of higher-order structures, including aggregate induction, and can generate products that are pharmacokinetically different, biologically less active and/or potentially more immunogenic than their native counterpart. It is therefore crucially important during the pharmaceutical development of therapeutic proteins and peptides to comprehensively characterize oxidation products and evaluate the impact of oxidation-induced structural modifications on the biological properties of the drug.
PurposeTo elucidate the chemical modifications in covalent aggregates of recombinant human insulin induced by metal catalyzed oxidation (MCO).MethodsInsulin 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.ResultsThe 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.ConclusionMCO of insulin leads to covalent aggregation through cross-linking via Michael addition to tyrosine oxidation products.Electronic supplementary materialThe online version of this article (doi:10.1007/s11095-012-0755-z) contains supplementary material, which is available to authorized users.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.