title: In vitro bioassay with enhanced sensitivity for human granulocyte colony-stimulating factor.
abstract: A method for the determination of human granulocyte colony-stimulating factor (hG-CSF) activity, based on stimulation of cellular proliferation, was developed using a subclone of the murine myeloid leukemia cell line NFS-60, with an improved sensitivity for hG-CSF, as indicator. The optimal range for quantitative analysis of hG-CSF was about 4-60 pg ml-1. The stimulatory effect was measured by a colorimetric microassay: the optical density of formazan, which is produced by viable cells from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), was obtained by reading plates in a multi-channel photometer. The assay was designed as a five-dose parallel line test, employing three or four doses for potency determinations, which fulfil pharmacopoeial requirements for assay validity. Inter-assay relative standard deviation (RSD) varied between 5.2 and 12.0%. Most assay experiments revealed potencies within limits of error of 90-110% and the mean index of precision value was 0.057. The recently developed yeast cell-derived International Standard (88/502) served as a reference for activity of rhG-CSF. Specificity of the assay was demonstrated by absence of response upon exposure to a panel of biomolecules, including recombinant human interleukin-3, and by the suppression of growth stimulation in the presence of neutralizing anti hG-CSF antibodies. Potency readings of unglycosylated rhG-CSF were dependent on pH of assay medium with higher relative activities observed at pH 6.6 than at 7.4. Moreover, SDS-PAGE analysis of the carbohydrate-deficient preparation, following incubation at physiological pH, revealed several high molecular weight rhG-CSF bands and decreased monomeric form. The method described was found suitable for potency assessments of pharmaceutical formulations of hG-CSF.
authors.affiliation: Division of Pharmacology, Medical Products Agency, Uppsala, Sweden.
authors.forename: U
authors.initials: U
authors.lastname: Hammerling
authors.forename: R
authors.initials: R
authors.lastname: Kroon
authors.forename: L
authors.initials: L
authors.lastname: Sjödin
mesh_terms: D000818:Animals; D002455:Cell Division / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D005069:Evaluation Studies as Topic; D005562:Formazans; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism* / Q000494:pharmacology; D006361:Heating; D006801:Humans; D015479:Leukemia, Myelomonocytic, Acute / Q000473:pathology; D051379:Mice; D010783:Photometry; D011994:Recombinant Proteins / Q000032:analysis / Q000494:pharmacology; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate; D013778:Tetrazolium Salts; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D005562:Formazans; D011994:Recombinant Proteins; D013778:Tetrazolium Salts; D016179:Granulocyte Colony-Stimulating Factor; C079782:MTT formazan; D012967:Sodium Dodecyl Sulfate