Oxidation of recombinant methionyl human granulocyte colony stimulating factor

The oxidation of methionine residues in recombinant methionyl human granulocyte colony stimulating factor with hydrogen peroxide has been investigated. Kinetic data of the oxidation were obtained by using reversed phase-high performance liquid chromatography. The stability-indicating capability of t...

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Bibliographic Details
Published in:Journal of pharmaceutical and biomedical analysis 1998-06, Vol.17 (2), p.283-289
Main Authors: Reubsaet, J.Léon E., Beijnen, Jos H., Bult, Auke, Hop, Ed, Scholten, Serge D., Teeuwsen, Jan, Underberg, Willy J.M.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Biological and medical sciences
Blood. Blood coagulation. Reticuloendothelial system
Chromatography, High Pressure Liquid
Cyanogen Bromide - chemistry
Filgrastim
Granulocyte Colony-Stimulating Factor - chemistry
Humans
Hydrogen Peroxide
Hydrogen-Ion Concentration
Mass Spectrometry
Medical sciences
Methionine - chemistry
Methionine residues
Molecular Sequence Data
Neutropenia
Oxidation
Oxidation-Reduction
Pharmacology. Drug treatments
Recombinant methionyl human granulocyte colony stimulating factor
Recombinant Proteins
Spectrophotometry, Ultraviolet
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Summary:The oxidation of methionine residues in recombinant methionyl human granulocyte colony stimulating factor with hydrogen peroxide has been investigated. Kinetic data of the oxidation were obtained by using reversed phase-high performance liquid chromatography. The stability-indicating capability of this system was confirmed with micellar electrokinetic capillary chromatography. In the pH range 1.9–7.5, the k obs value for the oxidation process is constant. Above pH 7.5, k obs tends to increase with increasing pH. In the pH range 1.9–11.8, four oxidation products were detected in RP-HPLC. Mass spectrometric analysis revealed that one mono-, one di- and two trioxidation products were formed. Using the cyanogen bromide cleavage method the nature of the oxidation products was determined. The mono-oxidation product is the protein with Met 121 oxidized, while the dioxidation product has oxidized Met 121 and Met 126 residues. The trioxidation products are the proteins with Met 121, Met 126 and Met 137 or Met 0, Met 121 and Met 126 oxidized.
ISSN:0731-7085
1873-264X
DOI:10.1016/S0731-7085(97)00199-4