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Isolation and characterization of a monomethioninesulfoxide variant of interferon alpha-2b
G Gitlin1, A Tsarbopoulos, S T Patel
1Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.
Purpose:
To isolate and characterize a monomethioninesulfoxide variant of the commercially available therapeutic protein interferon alpha-2b.
Methods:
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.
Results:
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.
Conclusions:
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.