Discrimination of granulocyte colony-stimulating factor isoforms by high-performance capillary electrophoresis

• Published in: Journal of chromatography. B, Biomedical sciences and applications Biomedical sciences and applications, 1999-09, Vol.732 (1), p.81-89
• Main Authors: Somerville, Linda E, Douglas, Alastair J, Irvine, Alexandra E
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Electrophoresis, Capillary - methods; Fundamental and applied biological sciences. Psychology; Glycoproteins; Glycoproteins - analysis; Glycosylation; Granulocyte colony-stimulating factor; Granulocyte Colony-Stimulating Factor - analysis; Humans; Protein Isoforms - analysis; Proteins; Recombinant Proteins - analysis
• ISSN: 0378-4347; 1387-2273
• DOI: 10.1016/S0378-4347(99)00271-6

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 m M Na 2HPO 4 buffer, pH 2.5, containing hydroxypropylmethylcellulose (HPMC) or 50 m M Na 2HPO 4 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.