重组人抗TNFα单抗制品中CHO宿主细胞蛋白质残留夹心ELISA检测方法的建立及验证  

Development and validation of a sandwich ELISA for residual host cell protein of CHO cells in recombinant human anti-TNFαmonoclonal antibody

作　　者：邓春平 梅雄 陈航 何水华 刘翠华 DENG Chun-ping;MEI Xiong;CHEN Hang;HE Shui-hua;LIU Cui-hua(Bio-Thera Solutions,Ltd,Guangzhou 519085,Guangdong Province,China)

机构地区：[1]百奥泰生物制药股份有限公司,广东广州519085

出　　处：《中国生物制品学杂志》2020年第8期925-928,933,共5页Chinese Journal of Biologicals

基　　金：国家“重大新药创制”科技重大专项资助项目(2013ZX09401001);广东省科技计划项目资助项目(2012B010900029)。

摘　　要：目的建立检测重组人抗TNFα单抗制品中CHO宿主细胞蛋白质(host cell protein,HCP)残留的夹心ELISA法,并进行验证。方法采用二维电泳-Western blot(2D-Western blot)法筛选兔抗CHO HCP多克隆抗体(产品号:AB000103-A、AB000103-C)及羊抗CHO HCP多克隆抗体(批号:3G-0016-PA)中识别重组人抗TNFα单抗特异性CHO HCP覆盖率最高的一种作为检测抗体,建立检测样品中残余HCP的夹心ELISA法,并验证方法的线性、基质干扰、稀释线性、灵敏度、精密度及准确性。结果以覆盖率达57%的AB000103-C号兔抗CHO HCP多克隆抗体作为检测抗体;样品基质干扰HCP检测,样品需稀释6倍;HCP标准品浓度在3.33~810 ng/mL范围与A450值曲线拟合良好,R2=1,试验内及试验间精密性验证的CV均小于12%,检测限及定量限分别为2.4和20 ng/mL;20、150及300 ng/mL的加标样品回收率分别为99.5%、97.1%及100.3%。结论成功建立了重组人抗TNFα单抗HCP残留的夹心ELISA检测方法,该方法灵敏度高,准确性、精密性及线性良好,适用于重组人抗TNFα单抗制品的残留HCP检测。Objective To develop and validate a sandwich ELISA for residual host cell proteins(HCP)of CHO cells in recombinant human anti-TNFαmonoclonal antibody.Methods The coverage of product-specific CHO HCP population of recombinant human anti-TNFαmonoclonal antibody recognized by rabbit(Cat.NO AB000103-A and AB000103-C)and goat(Cat.NO 3 G-0016-PA)anti-CHO HCPs polyclonal antibodies was determined by two-dimensional electrophoresis with Western blot(2 D-Western blot).The polyclonal antibody with the highest coverage was selected as the detection antibody,based on which a sandwich ELISA method for detection of residual HCP in product was developed and validated for linearity,matrix interference,dilution linearity,sensitivity,precision and accuracy.Results Rabbit anti-CHO HCP polyclonal antibody with a coverage of 57%was screened as the detection antibody.Since the matrix of samples interferred in the detemination of HCP,the samples needed to be diluted by 6 folds.The linear range of the developed sandwich ELISA was 3.33~810 ng/mL(R2=1).Both the CVs in intra-and inter-assays were less than 12%,while the detection limit and quantitative limit in validation for sensitivity were 2.4 and 20 ng/mL respectively.The recovery rates of spike samples at concentrations of 20,150 and 300 ng/mL were 99.5%,97.1%and 100.3%respectively.Conclusion A sandwich ELISA method was successfully developed,which showed high sensitivity,accuracy,precision and good linearity,and was suitable for detection of residual HCP in recombinant human anti-TNFαmonoclonal antibody.

关 键 词：重组人抗TNFα单抗 CHO细胞 宿主细胞蛋白质 酶联免疫吸附测定 

分 类 号：R392-33[医药卫生—免疫学]