A Reporter Gene Assay For Bioactivity Determination Of Recombinant Human Sgp130-Fc Fusion Protein

The recombinant human soluble gp130-Fc fusion protein（sgp130-Fc）is a specific inhibitor of IL-6 trans-signal transduction pathway and anti-inflammatory biologic agent for the treatment of ulcerative colitis.At present,its biological activity is determined by BAF3/gp130 cell proliferation inhibition method,which takes a long time and involves complicated operations.Besides,the proliferation of BAF3/gp130cells is only partially dependent on IL-6/sIL-6R compound（excessive cell culture densities can result in the loss of this dependence）,thus the uncertainty of cell culture also brings instability to the tests,which tends to cause large variability and poor reproducibility of experimental data.Reporter gene assay（RGA）based on the mechanism of actionhas been increasingly used in the field of drug biological activity detection due to its simplicity,specificity,high sensitivity and low variability.The aim of this study was to establish a reporter gene assay for rapid detection of the biological activity of sgp130-Fc.According to the mechanism of action of sgp130-Fc（specific inhibition of IL-6 trans-signaling pathway）,a plasmid vector,containing the specific DNA response element SIE of JAK/STAT3,Luciferase gene and hygromycin selection marker gene,was transfected into CHO-K1 cells,and a highly responsive and stable monoclonal cell line was screened,based on which a reporter gene assay was established and optimized,followed by method validation.In this study,a reporter cell line CHO/SIE-Luc was successfully constructed,and the stability of gene modification was evaluated by a novel digital PCR-based method.The results showed that the relative copy numbers of Luciferase gene in low,medium and high generation cells was basically consistent（0.093-0.095 copies/copy GAPDH）.Sgp130-Fc could inhibit the expression of Luciferase in CHO/SIE-Luc cells induced by IL-6/sIL-Rαcomplex,and the dose-response fitted the four-parameter equation with R² above 0.98.It has been verified that this method was only for IL-6trans-signaling pathway;intra-and inter-day variability of IC₅₀0 both were less than10%;the recovery rate was between 94.1%and 106.2%;R² of the linear fitting equation of the measured values versus the expected values was above 0.99;the dose-response curves of different generation cells were basically parallel;no significant difference was found between the test results of new assay and BAF3/gp130 proliferation assay（p=0.4960,t test,n=6）.The method in this study is specific,accurate,stable,rapid and easy to operate.It can be applied to the activity determination of sgp130-Fc,providing an important reference for the development and quality control of sgp130-Fc related products.