报告基因法测定重组人白细胞介素-2产品生物学活性的研究

Biological activity determination of recombinant human interleukin-2 products using reporter gene assay

目的:建立测定重组人白介素-2(recombinant human interleukin-2,rhIL-2)制品生物学活性的报告基因法(reporter gene assay,RGA)。方法:根据ICH Q2和《中华人民共和国药典》 2020年版指导原则,构建慢病毒转染表达荧光素酶(luciferase,Luc)的C8166细胞,进行rhIL-2生物学活性测定方法学优化与验证,优化内容包括孵育时间、细胞数量、待测样品的初始浓度和稀释比例;验证内容包括特异性、线性和准确性、精密度和耐用性。结果:优化后的检测条件为:孵育时间为24 h;细胞接种密度为1×10~5细胞·孔^(-1);待测样品以10 000 IU·mL^(-1)为起始浓度1∶3稀释10个梯度。荧光信号值强度与待测样品浓度拟合四参数曲线,计算相对效价。选用不同作用类型的细胞因子进行特异性验证均符合应用要求。采用50%,75%,100%,125%和150%的5个浓度点验证线性和部分准确性,实测与理论效价线性回归分析相关系数R~2=0.993 1,各浓度点的回收率均在80%~120%之间。标准品和供试品按5个不同的比例进行混合,测定供试品的加标回收率均在84%~111%之间,均符合质量控制要求。精密度验证主要考察日内和日间精密度及不同人员的重复性,日内、日间的精密度和不同人员之间的重复性相对标准偏差(relative standard deviation,RSD)均<10%。选取第5,26和78代次的细胞测定荧光信号值,信噪比(S/N)无统计学差异,表明不同代次细胞均可达到检测要求。结论:建立的rhIL-2生物学活性RGA方法适用性强,可应用于质量控制放行检测中。

Objective:To establish a cell-based reporter gene assay(RGA) for bioactivity determination of recombinant human interleukin-2(rhIL-2).Methods:A C8166 cell line that stably expressed signal transducer and activator of transcription 5(STAT5) was transfected with a lentivirus expressing luciferase.The bioassay was further optimized and validated according to the guideline of ICH Q2 and Pharmacopoeia of the People's Republic of China(ChP,2020).The assay performance characteristics of the established RGA were validated including specificity,linearity,accuracy,precision,and robustness.Results:The parameters of bioassay based on RGA were validated by optimized conditions:the incubation time was 24 h,the cells were seeded in 96-well plate with 1×10~5 cells per well,the initial concentration of rhIL-2 was 10 000 IU·mL~(-1) by dilution ratio of 1∶3 for 10 gradients.After incubation,the luminescence(Luc) signals were recorded.The relative bioactivity of the sample was calculated according to 4-parameter mode,which was fitted by Luc intensity to the dose-response curve.Various types of recombinant human cytokines were selected to verify the specificity of bioassays that comply with quality control requirements.The linearity and accuracy verification was performed by 5 potency concentrations,which were 50%,75%,100%,125% and 150%,respectively.The measured potency to expected potency was evaluated by linear regressing model analysis(R~2=0.993 1).The recovery rate range of relative bioactivities of 5 potency concentrations was 80%~120%.The recovery range of the mixture of the reference and tested samples in 5 different proportions was 84%~111%.The repeatability of intra-day,inter-day and different people was accessed in a batch of rhIL-2 by calculating the relative bioactivity.Accessing precision was expressed by RSD%,all of which were less than 10%.The luciferase was stably expressed by S/N(Signal Noise ratio),which had no statistical difference in C8166-Luc cells between 5,26 and 78 passages.This result evidenced that different passages of C8166-Luc cells can meet the quality control requirements.Conclusion:The developed bioassay of rhIL-2 based on RGA has trong applicability and can be used in quality control release test.