Structural Analysis of Modified Forms of Recombinant IFN-β Produced under Stress-Simulating Conditions

Abstract

The present study focused on the investigation of the chemical stability of recombinant human interferon-beta (rhIFN-beta) tested in vitro by chemical treatments that simulate stress-induced conditions that may occur during handling, storage or ageing of protein samples. Mild oxidation and/or alkylation of the recombinant protein showed that the four methionines occurring in the interferon displayed different chemical susceptibility in that Met36 and Met117 were fully modified, whereas Met1 showed only little modification and Met62 was completely resistant. Moreover, incubation of rhIFN-beta under alkaline conditions resulted in the formation of a covalent dimeric species stabilised by an intermolecular disulphide bridge involving the free SH group of Cys17 from each polypeptide chain. Analysis of biological activity of the different IFN-beta derivatives showed that rhIFN-beta fully retains its specific activity following mild oxidation treatments whereas reaction with a high concentration of alkylating agents or incubation under alkaline conditions strongly reduce its specific antiviral activity.

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

“…The data of liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS) analysis of undigested untreated IFNβ1a (Figure 5A) are in agreement with previously reported results (31) : the spectrum of the protein represents a mixture of multiple glycoforms (mainly fucosylated and sialylated protein) where the most abundant isoform contains one mono-fucosylated, biantennary structure carrying two sialic acid residues, featuring a total molecular weight of 22376 Da. The spectrum of the oxidized protein represents a single broad peak with a maximum at 22460.5 Da resulting from an overlay of multiple heterogeneously oxidized protein isoforms (Figure 5B), similar to results published for another oxidized protein (32) .…”

“…Met1 has been found susceptible to oxidation already by other authors (7, 31) . The b2 + ion in Figure S1, where the peptide M 1 SYNLLGFLQR 11 is displayed, shows the incorporation of one oxygen atom (+ 16 Da) into the sequence Met1Ser2: here, Ser is less prone to oxidation than Met, hence the target of oxidation is likely Met1.…”

“…Met36 is located in a solvent exposed domain of IFNβ1a (7, 31) . Figure S5a and S5b represent the MS/MS spectra matching the sequence M 36 NFDIPEEIK 45 , in which either Met36 or Phe38 is oxidized.…”

“…Met62 is a buried residue (7, 31) . It was resistant to oxidation by hydrogen peroxide (31) , however, in conditions of MCO, we detected MS/MS spectra for the sequence M 62 LQNIFAIFR 71 containing the oxidized Met62 (Figure S7).…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…RhIFN-β1 has known modifications, such as complex glycosylation, deamidation and succinimide, as well as oxidation [208,229,244,245]. Here, CZE-MS is demonstrated to successfully characterize and quantitate individual molecular proteoforms with high-resolution accurate mass analysis.…”