Isolation and characterization of a monomethioninesulfoxide variant of interferon alpha-2b.
OBJECTIVE
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.
Gitlin
G
G
Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.
Tsarbopoulos
A
A
Patel
S T
ST
Sydor
W
W
Pramanik
B N
BN
Jacobs
S
S
Westreich
L
L
Mittelman
S
S
Bausch
J N
JN
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D013058:Mass Spectrometry; D008715:Methionine / Q000031:analogs & derivatives* / Q000737:chemistry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011994:Recombinant Proteins; D013056:Spectrophotometry, Ultraviolet
D000998:Antiviral Agents; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D008715:Methionine; C013111:methionine sulfoxide