测定人血清中利妥昔单抗及其生物类似物浓度ELISA方法的建立及验证  

The determination of rituximab and its biosimilar in human serum by ELISA

作　　者：李黎 陈杰 李琨 贾志君 邹佳 陈方 董立厚 欧伦 董菁 宋海峰 LI Li;CHEN Jie;LI Kun;JIA Zhi-jun;ZOU Jia;CHEN Fang;DONG Li-hou;OU Lun;DONG Jing;SONG Hai-feng(Institute of Lifeomics,Academy of Military Medical Science,Military Academy of Sciences,Beijing 102206,China;Beijing United-Power Pharma Tech Co.,Ltd.,Beijing 102206,China;Chia Tai Tianqing Pharmaceutical Group Co.,Ltd.,Nanjing 201203,China)

机构地区：[1]军事科学院军事医学研究院生命组学研究所,北京102206 [2]军科正源(北京)药物研究有限责任公司,北京102206 [3]正大天晴药业集团股份有限公司,南京201203

出　　处：《中国新药杂志》2020年第9期993-1000,共8页Chinese Journal of New Drugs

基　　金：国家“重大新药创制”科技重大专项资助项目——生物类似药(Biosimilar)技术评价相关支撑体系的研究(2015ZX09501008)。

摘　　要：目的:建立和验证测定人血清中利妥昔单抗和生物类似物的酶联免疫吸附分析(ELISA)方法,用于生物类似药的药动学评估。方法:在96孔板中包被rat anti Rituximab (MCA2260)以捕获人血清中的抗CD20人鼠嵌合单抗(TQB2303)或者利妥昔单抗,加入生物素标记MCA2260与被捕获TQB2303或者利妥昔单抗结合,以链霉亲和素-辣根过氧化物酶偶联试剂作为检测试剂。从选择性、特异性、精密度和准确度、稀释线性、钩状效应和平行性等方面分别验证了本方法在定量分析人血清中利妥昔单抗和TQB2303浓度时的可靠性。结果:本方法检测利妥昔单抗和TQB2303的灵敏度为19. 53 ng·m L^-1;用2个待测物5个浓度水平验证样品的数据,统计计算方法的批间精密度和准确度分别在10. 1%~22. 4%和1. 8%~6. 1%范围内;方法的选择性、特异性和2~2 000倍稀释范围内稀释线性均符合指导原则的要求;方法在1 000. 00~1 000 000. 00ng·m L^-1浓度范围内无钩状效应,受试药物TQB2303的稳定性可以支持从样品采集到样品测定的整个过程。此外本实验用真实样品考察了方法的平行性,平行性结果显示本方法不存在真实样品稀释非线性的情况。结论:成功建立并验证了能够测定人血清中利妥昔单抗和TQB2303浓度的ELISA方法,满足生物类似药的药动学评估和申报需要。Objective:To develop and validate the determination of rituximab and its biosimilar in human serum by enzyme-linked immunosorbent assay(ELISA)in support of the pharmacokinetics study of the biosimilar.Methods:Rat anti rituximab(MCA2260)antibody was coated on the 96-well plates as to capture test article TQB2303 or rituximab in human serum.Then the biotin-labeled MCA2260 was added to the plate to bind the captured drug,after which the streptavidin-horseradish peroxidase conjugate was applied as the detection reagent.The reliability of this method in determination of rituximab and TQB2303 concentrations in human serum was verified in terms of selectivity,specificity,accuracy,precision,dilution linearity,hook effect and parallelism.Results:The sensitivity of rituximab and TQB2303 was 19.53 ng·m L^-1.The inter-batch precision and accuracy ofthe assay obtained by calculating data from validation samples at five levels were within the range of 10.1%~22.4%and 1.8%~6.1%,respectively.The selectivity,specificity and dilution linearity of 2~2000 folds all met the requirements of the guidelines.No hook effect was observed in the assay range of 1000.00 to 1000000.00 ng·m L^-1.The stability of TQB2303 met the requirement to support the duration of all samples from collection to determination.In addition,the parallelism of the method was examined with clinical samples and the results show the linear recovery of the analytes.Conclusion:An ELISA method for determining the concentration of rituximab and TQB2303 in human serum was established and validated.It can be successfully used in support of the pharmacokinetic evaluation for the application of TQB2303 as a biosimilar.

关 键 词：酶联免疫吸附分析 抗CD20单抗 生物类似药 交叉验证 

分 类 号：R969.1[医药卫生—药理学]