title: A novel reporter gene assay for recombinant human erythropoietin (rHuEPO) pharmaceutical products.
abstract: 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 24h and more sensitive with EC50 being 0.077IU/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.
authors.affiliation: National Institutes for Food and Drug Control, Beijing 100050, China.
authors.forename: Yushuai
authors.initials: Y
authors.lastname: Yang
authors.affiliation: National Institutes for Food and Drug Control, Beijing 100050, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Beijing Institute for Drug Control, Beijing 100035, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Yu
authors.affiliation: National Institutes for Food and Drug Control, Beijing 100050, China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Li
authors.affiliation: National Institutes for Food and Drug Control, Beijing 100050, China.
authors.forename: Xinchang
authors.initials: X
authors.lastname: Shi
authors.affiliation: National Institutes for Food and Drug Control, Beijing 100050, China.
authors.forename: Xi
authors.initials: X
authors.lastname: Qin
authors.affiliation: National Institutes for Food and Drug Control, Beijing 100050, China.
authors.forename: Chunming
authors.initials: C
authors.lastname: Rao
authors.affiliation: National Institutes for Food and Drug Control, Beijing 100050, China. Electronic address: wangjz_nifdc2014@163.com.
authors.forename: Junzhi
authors.initials: J
authors.lastname: Wang
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods* / Q000592:standards; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004921:Erythropoietin / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis* / Q000235:genetics; D008807:Mice, Inbred BALB C; D011401:Promoter Regions, Genetic / Q000187:drug effects; D017467:Receptors, Erythropoietin / Q000819:agonists / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D015203:Reproducibility of Results; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism; D014162:Transfection
chemical_list: C554270:EPO protein, human; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin; D008156:Luciferases
keywords: Biological activity; In vitro bioassay; Recombinant human erythropoietin; Reporter gene assay; Reticulocyte assay