A novel reporter gene assay for Recombinant Human Erythropoietin (rHuEPO) pharmaceutical products

Accurate determination of in vitro biological activity of therapeutic erythropoietin is essential in quality control of recombinant human erythropoietin (rHuEPO) pharmaceutical products. However, most of currently-used methods leave much to be desired so that a simpler, quicker and more accurate method is urgently needed. The bioassay described here utilizes a sub clone of UT-7/epo cell line stably transfected with luciferase gene under the control of sis inducible element and interferon γ-activated sequence element promoter. Active erythropoietin could induce the expression of luciferase by signaling through the erythropoietin receptor and the dose-response curve showed good linearity, yielding a coefficient of determination of 0.99 or higher. The optimized assay was simpler with the operation completed within 24 h and more sensitive with EC50 being 0.077 IU/mL. The accuracy estimates ranged from 81.7% to 102.4%, and both intra-assay and inter-assay precision was below 15.0%. The robustness of the assay was demonstrated by no effect of passage levels of the cells on the performance of the assay (p values: 0.772 for sample 1 and 0.943 for sample 2). Besides, Bland–Altman analysis showed a high consistency of the new assay with in vivo reticulocyte assay in results. These results suggested that the new reporter gene assay can be a viable supplement to the traditional reticulocyte assay and employed in potency determination of rHuEPO pharmaceutical products.