基于实验设计的报告基因法检测甘精胰岛素生物活性方法的建立及验证OA

Objective To establish and validate a reporter gene assay based on design of experiments(DOE)princi-ples for measuring the biological activity of insulin glargine.Methods Using HEK293 cells(HEK293/IRL-Luc)stably trans-fected with the insulin receptor gene,STAT5b response element,and luciferase reporter gene.Key assay parameters(cell densi-ty,drug dilution factor,and drug incubation time)were optimized via response surface methodology.The method was validated in accordance with ICH Q2(R2)and the General Chapters of the Chinese Pharmacopoeia(2020 Edition),assessing specificity,precision,accuracy,linearity and repeatability.Results The optimized assay conditions were as follows:cell density ranging from 1.4×105 to 4.6×105 cells per mL,drug incubation time of 16 hours,and two-fold serial dilutions of the drug starting from an initial concentration of 600 nmol•L-1.Validation results demonstrated high specificity;precision with repeatability(GCV≤10%)and intermediate precision(GCV<12%);accuracy with relative bias and its confidence intervals within±12%across the potency range of 64%-156%;and excellent linearity(R2＝0.9908).Conclusions The reporter gene assay optimized based on DOE and established in this study features straightforward operation and accurate results,enhances detection throughput and reproducibility,and demonstrates strong consistency with the mouse blood glucose method.It provides an in vitro alternative ap-proach compliant with the"3R"principles for the quality control of recombinant human insulin products.