Discrimination of granulocyte colony-stimulating factor isoforms by high-performance capillary electrophoresis.
Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein which acts primarily to stimulate the proliferation, differentiation and activation of committed progenitor cells of the neutrophil-granulocyte lineage into functionally mature neutrophils. The traditional biological assays employed to detect G-CSF are a myeloid bone marrow colony assay, a factor-dependent cell line specific for G-CSF and commercially available immunoassays. However, these methods will not distinguish between glycosylated and non-glycosylated forms of the molecule. In this study high-performance capillary electrophoresis (HPCE) was used to analyse glycosylated and non-glycosylated recombinant human granulocyte colony-stimulating factor (r-met-hG-CSF). Glycosylated G-CSF preparations contained human serum albumin (HSA), added as a protein carrier. Glycosylated and non-glycosylated G-CSFs were prepared in 40 mM Na2HPO4 buffer, pH 2.5, containing hydroxypropylmethylcellulose (HPMC) or 50 mM Na2HPO4 buffer, pH 9.0. Glycosylated G-CSF could be separated into two distinct glycoform populations at the lower pH studied. Differences in migration time and peak shape between glycosylated and non-glycosylated G-CSF were demonstrated. HPCE analysis of G-CSF produced using a baculovirus expression vector system revealed a further distinct G-CSF glycoform and demonstrated the resolving power of the technique.
Somerville
L E
LE
Department of Haematology, The Queen's University of Belfast, Royal Victoria Hospital, Northern Ireland, UK.
Douglas
A J
AJ
Irvine
A E
AE
D019075:Electrophoresis, Capillary / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis; D006031:Glycosylation; D016179:Granulocyte Colony-Stimulating Factor / Q000032:analysis*; D006801:Humans; D020033:Protein Isoforms / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis
D006023:Glycoproteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor