Identification of Oxidation Sites and Covalent Cross-Links in Metal Catalyzed Oxidized Interferon Beta-1a: Potential Implications for Protein Aggregation and Immunogenicity

Oxidation via Cu2+/ascorbate of recombinant human interferon beta-1a (IFNβ1a) leads to highly immunogenic aggregates, however it is unknown which amino acids are modified and how covalent aggregates are formed. In the present work we mapped oxidized and cross-linked amino acid residues in aggregated IFNβ1a, formed via Cu2+/ascorbate catalyzed oxidation. Size exclusion chromatography (SEC) was used to confirm extensive aggregation of oxidized IFNβ1a. Circular dichroism and intrinsic fluorescence spectroscopy indicated substantial loss of secondary and tertiary structure, respectively. Derivatization with 4-(aminomethyl)benzenesulfonic acid was used to demonstrate, by fluorescence in combination with SEC, the presence of tyrosine (Tyr) oxidation products. High performance liquid chromatography coupled to electrospray ionization mass spectrometry of reduced, alkylated, and digested protein was employed to localize chemical degradation products. Oxidation products of methionine, histidine, phenylalanine (Phe)...