运用ELISA对利妥昔单抗及其生物类似药在方法学上进行相似性验证

Method Validation of Methodology Similarity between Rituximab and Its Biosimilar by ELISA

目的验证受试品(抗人CD20嵌合单克隆抗体,biosimilar,B)与参比品(利妥昔单抗,rituximab,美罗华,reference,R)在方法学上的类似性。方法受试品和参比品的测定采用双抗夹心酶联免疫吸附分析法(ELISA),检测结果用Watson LIMSTM V.7.3.0.01(Thermo Scientific公司)软件进行处理,方法类似性比较采用均衡实验设计并用Allfit软件中平方和的统计方式比较标准曲线类似性,方法从准确度、精密度、特异性、灵敏度、选择性、稀释线性及稳定性等方面进行系统验证。结果受试品与参比品标准曲线平衡实验结果进行类似性比较,统计结果P=0.0507无统计学差异(P>0.05);系统性验证结果表明,受试品和参比品批内准确度和精密度分别在5.6%~7.7%,-9.0%^-0.8%内,受试品和参比品批间准确度和精密度分别在6.5%~7.2%,-7.3%^-3.2%内,受试品和参比品的总误差≤13.9%,满足方法学接受标准;选择性、特异性、稀释线性和稳定性在方法学接受范围内。结论经平衡实验设计和系统验证的ELISA法,满足生物类似物方法学接受标准,可用于利妥昔单抗生物类似药药动学研究。

OBJECTIVE To validate the methodology similarity of the biosimilar(recombinant anti-human CD20 chimeric monoclonal antibody,B)and the reference(rituximab,R).METHODS The biosimilar and the reference were measured by a sandwich enzyme-linked immunosorbent assay(ELISA),and the concentration of the analytes were back-calculated with Watson LIMSTM V.7.3.0.01(Thermo Scientific Inc.).The comparison of similarity between B and T were investigated with a balanced experiment design,and the results of the standard curves were evaluated with a statistic method,square of sum,using the statistic tool,Allfit.Systematic validation of the method from accuracy,precision,specificity,sensitivity,selectivity,dilution linearity,and stability etc.aspects.RESULTS The results of the standard curves from the balanced designed experiments of the biosimilar and the reference were compared,and the statistical result was P=0.0507,which showed no significant statistic difference(P>0.05).The systematic validation results showed that the intra-assay accuracy and precision of the method between the biosimilar and the reference were within the range of 5.6%-7.7%and-9.0%--0.8%,respectively;the inter-assay accuracy and precision of the method between the B and the T were within the range of 6.5%-7.2%and-7.3%--3.2%,respectively;the total error of the method was not more than 13.9%,which met the methodological acceptance criteria.The results of selectivity,specificity,dilution linearity,and stability were within the acceptance criteria.CONCLUSION The ELISA method,which was systematically validated with the balanced experimental design meets the acceptance criteria of biosimilar methodology requirement,and could be used to support the pharmacokinetic study of rituximab and its biosimilar.