荧光定量PCR法测定重组人干扰素α2b原液中宿主DNA的残留  

Determination of residual host cell DNA in recombinant human interferon α2b substances by quantitative PCR

作　　者：苏喆[1] 周朝东[1] 黄哲甦[1] 尉然[2] 

机构地区：[1]天津市药品检验所生化室,天津300070 [2]天津华立达生物工程有限公司,天津300457

出　　处：《中国生化药物杂志》2016年第4期193-195,共3页Chinese Journal of Biochemical Pharmaceutics

摘　　要：目的建立重组人干扰素α2b原液中宿主DNA残留量测定的方法并进行验证,用于对该产品的质量控制。方法通过wako DNA提取试剂盒提取干扰素原液中的宿主残留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的验证,同时对企业提供的3批干扰素原液中的残留DNA测定。结果该方法检测假单胞菌基因组DNA的最低准确定量浓度可达12 fg/μL,DNA含量在12 fg/μL^120 ng/μL范围内线性良好,标准曲线的相关系数r=0.998;wako 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批干扰素原液的DNA残留量均低于标准限度,符合《中国药典》三部2010年版和2015年版中关于假单胞菌产重组人干扰素α2b原液中宿主DNA残留量的要求。结论 wako DNA提取试剂盒能解决干扰素原液中样品前处理的技术难点,与定量PCR法结合能够简便、快速、准确地对干扰素原液中残留的DNA定量测定。Objective To develop and verify a method for determination of residual host cell DNA in recombinant human interferon α2b substances,which is used for the quality control of the product. Methods The residual host cell DNA was extracted by wako DNA extractor kit and determined by SYBRGreen based q-PCR using standard DNA as control. The residual host cell DNA was analyzed according to the standard curve. The developed method was verified by primer specifity,results accuracy and precision and used for determination of 3 batches of interferon substances.Results The minimum quantitative limit of residual host cell DNA by the developed method was 12 fg / μL,while the linear range was 12 fg / μL-120 ng /μL,with a correlation coefficient（ r） of 0. 998. The designed primers were specific to the DNA templates. The recovery rates of spiked samples with different DNA quantity were between 50%-200%. The residual host cell DNA determined by this method were not more than the limit,which were complied with the requirements for residual host cell DNA in Chinese Pharmacopeia（ volume III,2010 edition and 2015 edition）. Conclusion The wako DNA extractor kit could successfully solved the technical difficulties of sample pretreatment during residual DNA assay. The q-PCR method was simple,rapid and accurate for quantitation of residual host cell DNA in interferon substances.

关 键 词：wako DNA提取试剂盒 荧光定量PCR 假单胞菌 干扰素 DNA残留 质量控制 

分 类 号：R917[医药卫生—药物分析学]