摘要
目的建立重组抗VEGF单抗中CHO宿主细胞蛋白质(HCPs)残留的ELISA检测方法,并对方法进行验证。方法采用空载CHO细胞制备工艺特异HCPs,并进行二维电泳表征;采用二维电泳-Western blot(2D SDS-PAGE/Western blot)法对6种抗CHO HCPs多克隆抗体(C3、C6、C7、AB000103-A、AB000103-C、3G-0016-AF)识别重组抗VEGF单抗工艺特异性HCPs的覆盖率进行测定;选择覆盖率最高的多抗作为检测抗体,建立制品工艺特异性HCPs ELISA检测方法,并对方法的线性、基质干扰、稀释线性、灵敏度、精密度和准确性进行验证;此外对该方法与商业化通用检测方法进行了桥接对比研究。结果制备的工艺特异性HCPs蛋白质谱分布广泛;筛选得到覆盖率为59%的C6兔抗CHO HCPs多抗作为检测抗体,并建立了夹心ELISA检测法;该方法不受原液基质干扰,HCPs标准品在3.33~810 ng/ml浓度范围曲线拟合良好,原液在1~8倍范围内有良好的稀释线性;检测限0.075 ng/ml,定量限20 ng/ml;试验内和试验间变异系数均不超过10%;30、150及300 ng/ml浓度的HCPs标准品的回收率分别为107%、107%和95%;工艺特异性ELISA法测得的结果显著高于商业化通用检测方法。结论建立了重组抗VEGF单抗工艺特异性HCPs ELISA检测方法,该方法具有良好的检出率、灵敏度、精密度、准确性和线性,适用于该制品HCPs残留检测。
Objective To develop and validate an ELISA assay for testing residual CHO host cell proteins(HCPs)in recombinant anti-VEGF monoclonal antibody.Methods The process-specific HCPs were prepared using mock-transfected null CHO cells and then characterized by 2D-SDS PAGE.The coverage of the process-specific HCPs population of recombinant anti-VEGF monoclonal antibody recognized by six anti-CHO HCPs polyclonal antibodies(C3,C6,C7,AB000103-A,AB000103-C,3G-0016-AF)was evaluated by two-dimensional electrophoresis coupled with Western blot(2D SDS-PAGE/Western blot).The polyclonal antibody with the highest coverage was chosen as a testing antibody,based on which the process-specific ELISA for the measurement of residual HCPs in products was developed and validated with respect to the linearity,matrix interference,dilution linearity,sensitivity,precision and accuracy.In addition,a bridging comparison study was performed between the established process-specific ELISA and commercial generic detection method.Results The process-specific HCPs showed a broad spectrum of proteins.Rabbit anti-CHO HCPs polyclonal antibodies(C6)with coverage of 59%was screened as a testing antibody,and a sandwich ELISA was accordingly established.The ELISA method was not interfered by drug substance matrix;the curve of HCPs standard fit well in the range of 3.33-810 ng/ml;the drug substance showed a good dilution linearity in the range of 1-8.The limit of detection and quantitative of the method was 0.075 ng/ml and 20 ng/ml,respectively,both the CVs in intra-and inter-assays were less than 10%,while the recovery of HCPs standard at 30,150 and 300 ng/ml was 107%,107%and 95%,respectively.The results obtained by process-specific ELISA from drug substance were significantly higher than those obtained by commercial generic detection method.Conclusion A process-specific ELISA method has been successfully established,which shows appropriate coverage,sensitivity,accuracy,precision and linearity,and is suitable for detection of the residual HCPs in recombinan
作者
邓春平
梅雄
王星
刘翠华
DENG Chun-ping;MEI Xiong;WANG Xing;LIU Cui-hua(Bio-Thera Solutions,Ltd.,Guangzhou 519085,China;ArrayBridge Inc.,St.Louis,63110,USA)
出处
《中国医药生物技术》
2021年第6期506-511,共6页
Chinese Medicinal Biotechnology
基金
“重大新药创制”国家科技重大专项(2013ZX09401001)
广东省引进创新创业团队资助项目(2013Y116)。
