基于U937-NF-κB-Luc细胞系的阿达木单抗生物学活性快速检测方法的建立及验证  

Development and verification of a rapid detection method for adalimumab biological activity based on U937-NF-κB-Luc cell line

作　　者：郭莎[1] 贾哲 龙彩凤 黄璟 贺鹏飞[1] 高洁[1] 于传飞[1] 徐刚领 刘万卉[2] 王兰[1] GUO Sha;JIA Zhe;LONG Caifeng;HUANG Jing;HE Pengfei;GAO Jie;YU Chuanfei;XU Gangling;LIU Wanhui;WANG Lan(National Institutes for Food and Drug Control,NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,NMPA Key Laboratory for Quality Research and Evaluation of Biological Products,Beijing 102629,China;不详)

机构地区：[1]中国食品药品检定研究院国家卫生健康委员会生物技术产品检定方法及标准化重点实验室国家药品监督管理局生物制品质量研究与评价重点实验室,北京102629 [2]烟台大学药学院,山东烟台264005

出　　处：《中国生物制品学杂志》2024年第1期79-85,共7页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2021YFF0600804);2020年国家药品标准提高课题(2020S08)。

摘　　要：目的建立基于U937-NF-κB-Luc细胞系的阿达木单抗生物学活性快速检测方法,并进行验证。方法以U937-NF-κB-Luc细胞系为效应细胞,通过荧光素酶发光原理建立阿达木单抗生物学活性检测方法,并优化方法的肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)浓度(以160 ng/mL为初始浓度,进行2倍系列稀释,共10个稀释度)、抗体起始浓度(起始终浓度设为2000 ng/mL,进行2倍系列稀释,共20个稀释度)、抗体稀释倍数(1.5、2、3、4倍)、细胞接种量(8×10^(3)、2×10^(4)、4×10^(4)、6×10^(4)个/孔)、孵育时间(0.5、1、2、3 h),验证方法的专属性、准确性、精密性及线性范围。采用优化的方法及基于L929细胞的TNF-α中和活性法分别检测5批阿达木单抗的相对效价。结果阿达木单抗国际标准品的剂量-效应曲线呈典型的S型,且所得数据符合四参数方程式:y=(A-D)/[1+(x/C)^(B)]+D,R~2>0.99。确定最适TNF-α浓度为5 ng/mL,抗体起始浓度为800 ng/mL,抗体稀释倍数为2倍,细胞接种量为2×10^(4)个/孔,诱导时间为2 h。阿达木单抗等3种TNF-α靶点的治疗性单抗均可获得较好的剂量-效应曲线,其他非TNF-α靶点的治疗性单抗未呈现该曲线;效价理论值对数与其实测值对数的直线回归方程的斜率为1.037,相对偏倚均在±12%范围内;各效价样品的相对效价测定值的几何变异系数(geometric coefficient of variation,GCV)均<20%;理论效价在64%~156%范围内,与检测值呈良好的线性关系,拟合直线回归方程为y=1.0374 x-0.0237,R~2=0.9984。两种方法检测5批阿达木单抗的相对效价检测结果差异无统计学意义(t=1.198,P=0.2651)。结论建立的基于U937-NF-κB-Luc细胞系的阿达木单抗生物学活性检测方法具有良好的专属性、准确性及精密性,且耗时短(仅需3 h),可作为阿达木单抗生物学活性的快速评价方法。Objective To develop and verify a rapid detection method for the biological activity of adalimumab based on U937-NF-κB-Luc cell line.Methods Using U937-NF-κB-Luc cell line as the detection cells,a method for detecting the biological activity of adalimumab was developed based on luciferase luminescence principle.The method was optimized for the concentration of tumor necrosis factor-α(TNF-α)(160 ng/mL as initial concentration,2 times serial dilution,10dilutions),the initial concentration of antibody(2000 ng/mL,2 times serial dilution,20 dilutions),the dilution multiple of antibody(1.5,2,3,4 times),the inoculation amount(8×10^(3),2×10^(4),4×10^(4),6×10^(4)cells/well)and the incubation time(0.5,1,2,3 h),and verified for the specificity,accuracy,precision and linear range.The relative potency of five batches of adalimumab was detected by using the optimized method and TNF-αneutralization activity method based on L929cells respectively.Results The dose-response curve of adalimumab international standard showed a typical S-type,and the data complied with the four-parameter equation y=(A-D)/[1+(x/C)B]+D,R2>0.99.The optimum concentration of TNF-αwas 5 ng/mL,the initial concentration of antibody was 800 ng/mL,the dilution ratio for adalimumab was 1∶2,the inoculation amount was 2×10^(4)cells/well,and the induction time was 2 h.Three therapeutic monoclonal antibodies of TNF-αtarget,such as adalimumab,obtained good dose-response curves,while therapeutic monoclonal antibodies of other non-TNF-αtargets did not show this curve.The linear regression equation of the logarithmic value of theoretical potency and the logarithmic value of the corresponding measured potency had a slope of 1.037,and the relative bias was within the range of±12%.The geometric coefficient of variation(GCV)of the relative titer measured value of each sample was less than20%.The theoretical potency ranged from 64%to 156%,showing a good linear relationship with the measured values,and the fitting linear regression equation was y=1.0374 x-0.0

关 键 词：阿达木单抗 U937-NF-κB-Luc细胞 肿瘤坏死因子-α 生物学活性 

分 类 号：R944[医药卫生—药剂学]