单抗制品中CHO细胞宿主蛋白残留双抗夹心ELISA检测方法的建立

Development of a double antibody sandwich ELISA for detection of residual host cell protein of CHO cells in monoclonal antibody products

目的建立单抗制品中CHO细胞宿主蛋白（host cell protein,HCP）残留双抗夹心ELISA检测方法。方法确定双抗夹心ELISA方法的最佳工作条件,并对该方法的精密度和准确度进行验证,确定线性范围及最低检出限;用建立的方法与市售试剂盒分别检测低、中、高浓度HCP标准品及抗TNF-α单抗原液、抗VEGF单抗纯化工艺样品中的HCP。结果建立的双抗夹心ELISA法最佳抗体包被浓度为1.5μg/ml,酶标抗体浓度为1μg/ml,160 r/min,37℃孵育2.5 h;TMB 37℃孵育15 min;以630 nm为参比波长,450 nm为测定波长。该方法的线性范围为3.125~100 ng/ml,最低检测限为3.125 ng/ml;试验内变异系数（CV）和试验间CV均小于15%,回收率为85.3%~115.1%。与商品化试剂盒检测结果基本一致,可用于检测纯化工艺样品。结论已成功建立单抗制品中CHO细胞HCP残留双抗夹心ELISA检测方法,该方法可代替市售试剂盒用于CHO细胞HCP残留检测,可使检测成本降低30倍。

Objective To develop a double antibody sandwich ELISA method for detection of residual host cell protein（HCP）of CHO cells in monoclonal antibody（Mc Ab）products. Methods The working condition for double antibody sandwich ELISA was optimized, and the developed method was verified for precision and accuracy and determined for lin-ear range and minimum detection limit. The standard HCPs at low, moderate and high concentrations as well as HCPs in bulk of Mc Ab against TNF-α and purification process sample of Mc Ab against VEGF were determined by the developed method, using the commercial kit as control. Results The optimal working concentrations of coating and enzyme-labeled antibodies were 1. 5 and 1. 0 μg / ml respectively. The samples and enzyme-labeled antibody were added simultaneously and oscillated at 160 r / min, and incubated at 37 ℃ for 2. 5 h, then added with substance TMB and incubated at 37 ℃for 15 min. The reference and detection wavelengths were 630 and 450 nm respectively. The linear range of developed method was 3. 125 ~ 100 ng / ml, while the minimum detection limit was 3. 125 ng / ml. Both the CVs in intra- and interassays were less than 15%, while the recovery rate was 85. 3% ~ 115. 1%. The determination results by the developed method was basically consistent with that by commercial kit, indicating that the method might be used for determination of purification process samples. Conclusion A double antibody sandwich ELISA method for detection of HCP of CHO cells in Mc Ab products instead of commercial kit was successfully developed, of which the cost decreased by 30 folds.