口服六价重配轮状病毒减毒活疫苗(Vero细胞)DNA残留检测方法的建立及验证  

Establishment and verification of DNA residue detection method of oral hexavalent reassortant live attenuated rotavirus vaccine(Vero cells)

作　　者：刘艳[1] 何鹏[1] 李娟娟 申瑷琳 肖瑶 彭小欢 郭旭梦 姚瑞宁 胡忠玉[1] 施金荣 LIU Yan;HE Peng;LI Juanjuan;SHEN Ailin;XIAO Yao;PENG Xiaohuan;GUO Xumeng;YAO Ruining;HU Zhongyu;SHI Jinrong(National Institutes for Food and Drug Control,Key Laboratory of Verification Methods and Standardization of Biotechnology Products,Ministry of Health,Beijing 100050,China;不详)

机构地区：[1]中国食品药品检定研究院,卫生部生物技术产品检定方法及其标准化重点实验室,北京100050 [2]武汉生物制品研究所有限责任公司,湖北武汉430207

出　　处：《中国生物制品学杂志》2024年第12期1505-1511,共7页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2020YFC0842100)。

摘　　要：目的 利用磁珠分离法结合荧光染色法,建立口服六价重配轮状病毒(rotavirus,RV)减毒活疫苗(Vero细胞)原液及成品中DNA残留量的检测方法,并进行验证。方法 采用宿主细胞残留DNA样本前处理试剂盒(磁珠法)提取样品中的残留Vero细胞DNA,再利用荧光染色对样品中残留DNA进行测定,采用直线回归拟合标准曲线并对样品中的DNA残留量进行分析。对建立的方法进行线性范围、精密度、准确度、专属性、耐用性、定量限验证。对6个血清型的单价原液和成品中的残留宿主细胞DNA进行测定,并采用3种不同方法(荧光染色法、qPCR法、分子杂交法)进行检测,比较检测结果的一致性。结果 该方法检测口服六价重配RV减毒活疫苗(Vero细胞)DNA残留量的定量限为1.25 ng/mL,标准品浓度在1.25~80 ng/mL范围内线性关系良好,线性相关系数(r)为1.00;单价原液重复检测6次的加标回收率在93.8%~132.1%之间,成品重复检测6次的加标回收率在66.0%~90.0%之间;同一人员对单价原液和成品重复检测6次的相对标准差(relative standard deviation,RSD)在5.5%~16.1%之间,不同人员对单价原液和成品重复检测6次的RSD分别为7.7%和15.6%;用干扰溶液与RNase-free Water分别稀释标准品至25μg/mL,Vero细胞DNA残留量检测结果的回收率在98.0%~138.8%之间,RSD <12%;单价原液和成品不同孵育时间检测结果的RSD分别<16.3%和8.6%;不同时间和不同实验室对6个不同血清型的单价原液和3批成品中DNA残留量检测结果的RSD <7.1%和6.1%;3种不同方法对同一份样品的检测结果一致性较好。结论 磁珠分离法和荧光染色法相结合可用于口服六价重配RV减毒活疫苗(Vero细胞)DNA残留的含量测定,同时也可作为工艺过程中对中间品等关键步骤的评价手段。Objective To establish a method for the detection of DNA residues in the stock solution and finished product of oral hexavalent reassortant live attenuated rotavirus(RV)vaccine(Vero cells)by magnetic bead separation method combined with fluorescent staining method,and to verify the method.Methods The residual Vero cell DNA in the sample was extracted by the host cell residual DNA sample preparation kit(magnetic bead method),and then the residual DNA in the sample was determined by fluorescence staining,and the standard curve was fitted by linear regression and the residual amount of DNA in the sample was analyzed.The established method was validated for the linear range,precision,accuracy,robustness,specificity,and limit of quantification(LOQ),and the residual host cell DNA in the monovalent stock solutions and finished products of six serotypes was determined.Three different methods(fluorescent staining,qPCR and molecular hybridization)were used for detection,and the consistency of detection results was compared.Results The LOQ for the determination of DNA residue content in oral hexavalent reassortant live attenuated RV vaccine(Vero cells)by this method was 1.25 ng/mL,and the linearity was good at the standard concentration in the range of 1.25-80 ng/mL,with the linear correlation coefficient(r)of 1.00.The spike recoveries were 93.8%-132.1%for six repeated tests of the monovalent stock solution and 66.0%-90.0%for the finished product.The relative standard deviations(RSDs)of the same operator for six repeated tests of the monovalent stock solution and finished product were between 5.5%and 16.1%,and the RSDs of different personnel for monovalent stock solution and finished product in six repeated tests were 7.7%and 15.6%,respectively.When the standard was diluted to 25μg/mL with interference solution and RNase-free Water respectively,the recoveries of DNA residue detection results of Vero cells were between 98.0%and 138.8%,RSD<12%.The RSDs of the test results of the monovalent stock solution and finished product

关 键 词：DNA残留 荧光染色法 磁珠分离法 VERO细胞 轮状病毒 

分 类 号：R917[医药卫生—药物分析学]