In vitro bioassay for human erythropoietin based on proliferative stimulation of an erythroid cell line and analysis of carbohydrate-dependent microheterogeneity

Abstract

The human erythroleukemia cell line TF-1 was employed for the determination of proliferative stimulation induced by recombinant human erythropoietin (rhEpo). Potencies of various intact and sugar-trimmed rhEpo preparations were estimated using the International Standard for Human r-DNA-derived Epo (87/684) as a reference for activity. The cellular response was measured in a multi-channel photometer using a colorimetric microassay, based on the metabolism of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan, by viable cells. The linear part of the log dose-response relationship encompassed 2.5–90 pM and activity of rhEpo preparations was measured at doses between 3 and 60 pM. The assay was designed as a parallel line test, using three or four concentrations for potency determinations, which fulfills pharmacopoeial requirements for assay validity. Inter-assay relative standard deviation varied between 4.1% and 12.6% and most assays revealed potencies with limits of error within 87–113%. In order to acquire an additional means for an efficient probing of physiologically relevant features of rhEpo, a luminiscence-dependent Western detection system, based on a combined isoelectric focusing/sodium dodecyl sulphate-polyacrylamide gel electrophoresis separation, was established. As opposed to conventional electrophoresis the two dimensional approach enabled the disclosure of minor truncations in the rhEpo-attached glycan moieties using picomolar quantities of the hormone. Moreover, the separated isoforms of rhEpo were quantified by computer-assisted densitometry and compared with the 87/684 standard. Accordingly, results obtained by the cellular response were balanced against the general pattern observed and the relative amounts of separated rhEpo isomers as determined by the quantitative Western analysis. The method described should be suitable for potency assessments of pharmaceutical formulations of rhEpo.