均相时间分辨荧光法测定阿达木单抗结合活性

Measurement of binding activity of adalimumab by homogeneous time-resolved fluorescence method

目的建立一种基于均相时间分辨荧光(HTRF)技术的分析方法,用于快速、简便、稳定地检测阿达木单抗与TNF-α抗原的结合活性。方法将检测试剂、梯度稀释的标准品及待测样品加入96孔板,孵育后在酶标仪上读取荧光信号值,用软件对信号值和抗体浓度进行四参数曲线拟合,根据标准品及待测样品的EC_(50)值计算样品的相对结合活性;同时对方法的专属性、准确性、精密度、线性与范围及稳定性指示能力进行验证;此外对该方法与常规酶联免疫吸附法(ELISA)进行了对比研究。结果建立的HTRF检测法呈典型的“S”型剂量-效应曲线;方法验证结果显示,本法专属性强,相对结合活性的检测范围为50%~150%,在此范围内线性良好,相关系数在0.99以上,斜率接近1.0;平均回收率范围为92.5%~104.8%;重复性及不同人员之间精密度的CV均不超过15%;本法可反映加热破坏的单抗样品的结合活性变化趋势;对比研究显示,HTRF法与ELISA法测得的结果等效。结论建立了一种新的检测阿达木单抗与TNF-α结合活性的测定方法,该法经验证符合要求,可代替ELISA法用于该类单抗药物结合活性的测定,适用于细胞克隆筛选、原液或成品质量放行检测及稳定性研究等。

Objective To establish a test method for rapid,simple and stable measurement of TNF-αbinding activity of adalimumab based on homogeneous time-resolved fluorescence(HTRF)technology.Methods Testing regents,serially diluted reference standard and sample were added into a 96-well plate,followed by fluorescence reading with a microplate reader after a timed incubation.The curve of signal and antibody concentration was fitted by software in four-parameters model,and the relative binding activity of the sample was calculated as per the EC_(50)of reference standard and sample.Meanwhile,the method was validated regarding specificity,accuracy,precision,linearity,range and stability indicating property.In addition,the HTRF method was compared with ELISA method.Results The established HTRF assay showed a typical“S”shape dose-response curve.This HTRF assay is specific to anti-TNF-αmonoclonal antibody,and its testing range was 50%-150%,in which the linearity was regressed well with r;≥0.95 and slope around 1.0.The mean recovery of the HTRF assay was 92.5%-104.8%,and the CVs of repeatability and intermediate precision between two different analyses were less than 15%.In addition,the HTRF assay showed ability to detect and reflect the change trend of binding activity of heat-treated adalimumab samples.Comparative study indicated that the binding activity result obtained by HTRF assay was equivalent to that obtained by ELISA method.Conclusion A new assay for the determination of TNF-αbinding activity of adalimumab has been established.This HTRF assay meets the requirements of method validation,and could replace ELISA method for the determination of TNF-αbinding activity of such anti-TNF-αantibody drugs.It is suitable for cell line screening,releasing drug substance or drug product and stability study.