Isolation and characterization of a monomethioninesulfoxide variant of interferon alpha-2b

• Published in: Pharmaceutical research 1996-05, Vol.13 (5), p.762-769
• Main Authors: Gitlin, G, Tsarbopoulos, A, Patel, S T, Sydor, W, Pramanik, B N, Jacobs, S, Westreich, L, Mittelman, S, Bausch, J N
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antiviral Agents - chemistry; Antiviral Agents - isolation & purification; Antiviral Agents - pharmacology; Cells, Cultured; Chromatography, High Pressure Liquid; Circular Dichroism; Electrophoresis, Polyacrylamide Gel; Humans; Interferon alpha-2; Interferon-alpha - chemistry; Interferon-alpha - isolation & purification; Interferon-alpha - pharmacology; Mass Spectrometry; Methionine - analogs & derivatives; Methionine - chemistry; Molecular Sequence Data; Peptide Mapping; Recombinant Proteins; Spectrophotometry, Ultraviolet
• ISSN: 0724-8741
• DOI: 10.1023/A:1016059902645

To isolate and characterize a monomethioninesulfoxide variant of the commercially available therapeutic protein interferon alpha-2b. The methionine (Met)-oxidized variant was isolated by reverse-phase high performance liquid chromatography and characterized by SDS-PAGE, peptide mapping and mass spectrometric analysis of the trypsin/V8-generated peptide fragments. The biological and immunological activities of the isolated variant were also evaluated. The rHuIFN alpha-2b variant was found to contain a Met sulfoxide residue at position 111 of the rHuIFN alpha-2b molecule. The far-UV CD spectra showed a slight loss of alpha-helical content and an increase in the beta-sheet contribution. The CD spectra indicate that both chromatographic conditions and Met oxidation contribute to the observed secondary structure changes. Both interferon alpha-2b main component and its methionine-oxidized variant showed different reactivity to monoclonal antibodies employed in immunoassays for the protein. A monomethioninesulfoxide rHuIFN alpha-2b variant was found to be present in the rHuIFN alpha-2b bulk drug substance in solution. The Met(111) residue was identified as Met sulfoxide by comparative tryptic/V8 mapping and mass spectrometric analysis. Nevertheless, the oxidation of the Met(111) residue did not seem to have a detectable effect on the biological activity of the molecule.