对斑点杂交方法检测治疗性单克隆抗体制品中残留宿主细胞DNA含量的影响因素研究

Research on factors affecting Dot-Blot detection of residual host cell DNA in therapeutic monoclonal antibodies

目的:对斑点杂交方法检测重组生物制品中残留宿主细胞DNA含量试验中的6个影响因素进行评价。方法:对不同的样品稀释液,样品中是否含有蛋白,含蛋白样品是否采用蛋白酶K处理,采用不同方法预处理的模板DNA作为标记用模板,采用不同体积的离心管进行探针标记,分别采用不同的杂交温度,共计6个因素对检测结果的影响进行评价。结果:在使用随机标记的探针通过斑点杂交方法检测重组生物制品中残留宿主细胞DNA含量试验中:标准品和样品稀释中使用水进行稀释时结果可信度较高;样品中含有蛋白会降低检测的灵敏度;在含蛋白样品中加入蛋白酶K处理可以在一定程度上提高检测的灵敏度;使用超声处理的模板DNA作为探针标记的模板时,检测灵敏度较高,同时背景较低;探针标记过程中应采用0.2 mL薄壁PCR管作为容器以保证试验成功率;最适的杂交温度为42℃左右。结论:在斑点杂交方法检测治疗性单克隆抗体制品中残留宿主细胞DNA含量中,采用水作为稀释液,对含蛋白样品使用蛋白酶K消化,标记用模板DNA使用超声处理,使用0.2 mL薄壁PCR管作为标记反应管和在42℃杂交时,结果的稳定性和灵敏度有较大提高。

Objective： To evaluate six factors that possibly affect detection sensitivity of Dot - Blot applied to determinating the residual host cell DNA in recombinant bioproducts. Methods：Evaluated how 6 factors including different sample diluents, protein contained in samples, protease K treatment of prtein-containing samples, differently pretreated template DNA for probe labeling, vials with different volume used for probe labeling and different temperature for hybridization, took effect on the detection results. Results：In the assays which used randomly labeled probe for detection of residual host cell DNA in recombinant bioproducts ： the results of assays that took water for injection as diluent were more reliable than the results of assays that took fish sperm DNA as diluent ; If there was protein in sample, the detection sensitivity would decrease ; Protease K treatment would increase the detection sensitivity in the test of protein-containing samples;when used the supersonic treated template for probe labeling, the detection sensitivity and background of assay results would be greatly elevated;in the selection of vials for probe labeling,0. 2 mL PCR tubes with thin wall were preferred;the preferred hybridization temperature was about 42 ℃. Conclusion： In the Dot - Blot test for determination of residual host cell DNA content in therapeutic monoclonal antibodies, taking water as dilueuts, digesting protein-containing sample with protease K, treating template DNA with ultrasonic, taking 0.2 mL PCR tube as labeling reaction tube and hybridization at 42 ℃ can improve the repeatability and sensitivity in some degree.