荧光定量PCR法测定重组人/门冬胰岛素原料中宿主DNA的残留量

目的：利用wako DNA 提取试剂盒结合荧光定量聚合酶链式反应（PCR）技术，建立重组人/门冬胰岛素原料中宿主DNA 残留量测定的方法并进行验证，用于对该产品进行质量控制。方法：通过wako DNA提取试剂盒提取重组人/门冬胰岛素原料中的宿主残留DNA，再利用SYBRGreen 染料法对样品和标准DNA 进行定量PCR 测定，根据标准曲线对样品中的DNA 残留量进行分析。对建立的方法进行引物特异性以及结果准确性和精密性验证，同时对本室留样的3 批重组人胰岛素原料和3 批重组门冬胰岛素原料中的残留DNA进行测定。结果：该方法检测酿酒酵母菌基因组DNA的最低准确定量质量浓度可达0.137 8 ng·mL^-1，DNA 质量浓度在0.137 8~137 800 ng·mL^-1 范围内，其质量浓度的对数值与Ct 值呈良好线性关系，标准曲线的相关系数（r）为0.994；DNA 提取试剂盒提取不同量的加标样品回收率均在50%~200% 范围内；该方法检测3 批重组人胰岛素原料和3 批重组门冬胰岛素原料中的DNA 残留量均小于10 ng·剂量^-1，符合进口药品注册标准中关于重组人/门冬胰岛素原料中宿主DNA 残留量的要求。结论：wako DNA 提取试剂盒能解决重组人/门冬胰岛素原料中样品前处理的技术难点，与定量PCR 法结合能够简便、快速、准确地对重组人/门冬胰岛素原料中残留的DNA 进行定量测定。

Objective: To establish validated test method for determination of residual host cell DNA in recombinant human/aspart insulin substances by using DNA extractor kit combined with quantitative polymerase chain reaction (PCR). Methods: The residual host cell DNA was extracted by wako DNA extractor kit. SYBRGreen method combined with qualitative PCR was employed to determine the samples and standard DNA. The residual host cell DNA was analyzed refer to the standard curve. The developed method was confirmed by primer specifity,results accuracy and precision, and applied for determination of 3 batches of recombinant human insulin substances and 3 batches of recombinant aspart insulin substances. Results: The minimum quantitative limit of residual host cell DNA by the developed method was 0. 137 8 ng·mL^-1,while the linear range was 0. 137 8-137 800 ng·mL^-1,with a correlation coefficient (r)of 0. 994. The designed primers were specific to the DNA templates. The recovery rates of spiked samples containing various level of DNA were in the range of 50%-200%. The residual host cell DNA determined by this method were no more than 10 ng per dose,which were comformed to the acceptance criteria for the control of residual host cell DNA adopted by imported drug registration standards. Conclusion: The application of wako DNA extractor kit successfully make a breakthough for the sample pretreatment during residual DNA assay. The quantitative PCR method is simple,rapid and accurate for quatification of residual host cell DNA in recombinant human/aspart substances.

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