基于报告基因的重组人Ⅱ型肿瘤坏死因子受体抗体融合蛋白生物学活性测定方法的建立、验证及应用  

Establishment,verification and application of biological activity assay method of recombinant human TNF receptorⅡFc fusion protein based on reporter gene

作　　者：范文红[1] 安怡芳 裴德宁[1] 韩春梅[1] 郭莹[1] 周勇[1] FAN Wenhong;AN Yifang;PEI Dening;HAN Chunmei;GUO Ying;ZHOU Yong(National Institutes for Food and Drug Control,Beijing 100050,China)

机构地区：[1]中国食品药品检定研究院,北京100050 [2]华润医药控股有限公司分析与质控中心,北京100120

出　　处：《中国生物制品学杂志》2025年第1期73-79,共7页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2021YFF0600804)。

摘　　要：目的建立简便快捷的重组人Ⅱ型肿瘤坏死因子受体(recombinant human tumor necrosis factor receptorⅡ,rhTNFRⅡ)抗体融合蛋白(rhTNFRⅡ-Fc)制品生物学活性测定方法,并对方法进行验证及应用,为该类制品工艺稳定性评价奠定基础。方法利用HEK293T-NF-κB-Luc转基因细胞,不同浓度rhTNFRⅡ-Fc融合蛋白能够不同程度抑制TNF-α对转基因细胞中核因子(nuclear factor,NF)-kB的转录激活作用,通过荧光素酶检测系统(Bright-GloTM-Luciferase Assay System)对rhTNFRⅡ-Fc进行生物学活性检测,并对该方法进行试验参数的优化及专属性、准确性、线性及精密性验证。以rhTNFRⅡ-Fc融合蛋白生物学活性测定国际标准品作为参比品,采用该方法测定原研药依那西普及国内3家不同企业(A、B、C)各1批rhTNFRⅡ-Fc融合蛋白产品的比活性。结果rhTNFRⅡ-Fc融合蛋白在该方法中存在量效关系,且符合四参数曲线方程,R^(2)>0.99;方法优化后确定细胞铺板浓度为4×10^(5)个/mL,TNF-α作用浓度为10 ng/mL,rhTNFRⅡ-Fc稀释起始浓度为200 ng/mL,1∶1.5倍稀释,作用时间为4~5 h,样品稀释液为DMEM+1%FBS。仅依那西普产品能够抑制TNF-α对HEK293T-NF-κB-Luc细胞中NF-κB的转录激活,而其他几种融合蛋白类制品均无对TNF-α的诱导抑制作用;5个不同稀释组回收率样本经3次测定,回收率平均值分别为(101.54±4.63)%、(99.67±6.41)%、(101.20±5.58)%、(101.44±6.80)%、(100.72±6.15)%,相对标准偏差(relative standard deviation,RSD)均小于7%,线性拟合的R^(2)=0.9998;精密性验证日间和板间RSD均小于8.0%。该方法检测原研药依那西普及A、B、C企业rhTNFRⅡ-Fc融合蛋白的比活性分别为2.01×10^(6)、2.59×10^(6)、1.56×10^(6)、2.04×10^(6)IU/mg。结论利用HEK293T-NF-κB-Luc转基因细胞成功建立了rhTNFRⅡ-Fc融合蛋白生物学活性测定方法,该方法简便快捷,专属性强,准确性高,精密性良好,可用于不同企业该类制品的生物学活性常Objective To establish a simple and rapid method for the determination of biological activity of recombinant human tumor necrosis factor receptorⅡ(rhTNFRⅡ)Fc fusion protein(rhTNFRⅡ-Fc)products,and to verify and apply the method,in order to lay a foundation for the process stability evaluation of this kind of products.Methods Using HEK293TNF-κB-Luc transgenic cells,different concentrations of rhTNFRⅡ-Fc fusion protein can inhibit the transcriptional activation of nuclear factor(NF)-kB induced by TNF-αin transgenic cells to different degrees.The biological activity of rhTNFRⅡ-Fc was detected by Bright-Glo~(TM)-Luciferase Assay System,the experimental parameters were optimized,and the specificity,accuracy,linearity and precision of the method were validated.Using the international standard for biological activity assay of rhTNFRⅡ-Fc fusion protein products as the reference,this method was used to determine the specific activity of rhTNFRⅡ-Fc fusion protein products from three different domestic enterprises(A,B and C)and the original drug Etanercept.Results rhTNFRⅡ-Fc fusion protein had a dose-effect relationship in this method and conformed to the four-parameter curve equation with R^(2)greater than 0.99.After optimization,the cell seed plate density was determined to be 4×10^(5)/mL,the action concentration of TNF-αwas 10 ng/mL,the initial dilution concentration of rhTNFRⅡ-Fc was 200 ng/mL,1∶1.5times the dilution,the action time was 4-5 h,and the sample diluting solution was DMEM+1%FBS.Only Etanercept inhibited the activation of NF-κB transcription in HEK293T-NF-κB-Luc cells induced by TNF-α,while other fusion proteins had no inhibition on the induction of TNF-α.The recovery samples of five different dilution groups were measured three times,and the average recoveries were(101.54±4.63)%,(99.67±6.41)%,(101.20±5.58)%,(101.44±6.80)%and(100.72±6.15)%,respectively,with the relative standard deviations(RSDs)lower than 7%and the linear fitting R^(2) of 0.9998.The RSDs of verification for in

关 键 词：重组人Ⅱ型肿瘤坏死因子受体抗体融合蛋白 核因子-ΚB 报告基因 生物学活性 荧光素酶检测系统 国际标准品 

分 类 号：R917[医药卫生—药物分析学]