慢病毒载体生产用质粒DNA构象检测毛细管凝胶电泳法的建立及验证  

Development and validation of capillary gel electrophoresis method to quantitate the content of plasmid DNA isoforms used for lentivirus production

作　　者：张丽霞 吴雪伶[1] 陈雪清 徐玲丽 赵鹏 孟淑芳[1] ZHANG Li-xia;WU Xue-ling;CHEN Xue-qing;XU Ling-li;ZHAO Peng;MENG Shu-fang(National Institutes for Food and Drug Control,Beijing 100050,China;Guangzhou Virotech Pharmaceutical Co.,Ltd,Guangzhou 510000,China;SCIEX,Shanghai 200335,China)

机构地区：[1]中国食品药品检定研究院,北京100050 [2]广州威溶特医药科技有限公司,广州510000 [3]上海爱博才思分析仪器贸易有限公司,上海200335

出　　处：《药物分析杂志》2021年第7期1189-1202,共14页Chinese Journal of Pharmaceutical Analysis

基　　金：国家药典委员会2020年药典标准提高课题(1041740404401)。

摘　　要：目的:建立可定量检测质粒DNA超螺旋构象相对百分含量的毛细管凝胶电泳法(CGE),并开展方法学验证研究及初步应用探讨。方法:选用涂层毛细管,采用荧光检测器CGE法分离质粒DNA,首先通过限制性内切酶和缺刻酶处理法确认质粒DNA超螺旋(covalently closed circular,ccc)、线性(linear)及开环(open circular,oc)这3种构象在电泳图谱中的位置,然后通过对荧光染料、样品上样浓度等条件进行优化,建立质粒DNA构象的检测方法。对所建方法进行专属性、准确性、线性、精密度、检测下限和定量下限验证,并通过将质粒DNA置于不同保存温度、反复冻融及紫外照射后再进行质粒DNA构象的检测,初步分析该方法应用的可行性。结果:采用总长度为40 cm、有效长度为30.2 cm涂层毛细管,毛细管和样品上样温度分别为20℃和4℃,质粒浓度为4 ng·μL^(-1)以1378.95 Pa,4 s上样分离,CGE法可准确地定量质粒DNA 3种构象所占的比例,且分离度良好。通过对3种常用的质粒保存液基质的检测结果显示,这些基质不会干扰检测结果,专属性较好。质粒DNA 3种构象的线性范围均在0.25^8 ng·μL^(-1),构象峰面积与上样浓度具有良好的线性关系,相关系数分别是0.9977、0.9994、0.9927。将线性化质粒及开环质粒分别以3%^50%添加至原质粒中,可检出的线性及开环构象的峰面积与添加浓度呈现良好的线性关系,相关系数分别为0.9999和0.9972,线性构象的回收率在104%-120%,准确性良好;开环构象的回收率稍差,为68%-94%。将质粒DNA分别以高(4μg·mL^(-1))、中(2μg·mL^(-1))、低(0.5μg·mL^(-1))3个浓度验证实验的重复性,结果显示,试验内重复性良好,质粒DNA超螺旋构象迁移时间的RSD在0.22%-0.54%,相对百分含量的RSD在0.38%-2.1%;中间精密度也较好,迁移时间的RSD在0.52%-1.1%;相对百分含量的精密度在质量浓度为2 ng·μL^(-1)和4 ng·μL^(-1)时,RSD值分别为3.6%、0.25%。�Objective:To establish and validate a kind of capillary gel electrophoresis(CGE)method which can quantitate the content of plasmid DNA isoforms,and explore its application.Methods:Firstly using the coated capillary,the plasmid DNA(p16E25)was separated by CGE method.Then the plasmid p16E25 was digested by restriction and nicking endonuclease respectively and separated by CGE to determine which migration peak was supercoiled closed circular(ccc),linear or open circular(oc)form.Secondly,CGE method for plasmid DNA isoforms separation was further optimized for fluorescent dyes and loading concentration and then validated for its the specificity,accuracy,linearity,precision,limit of detection(LOD)and limit of quantitation(LOQ).Finally,the method was used to detect the change of plasmid DNA isoforms after treatment for being kept at varied temperature,several freeze-thawed cycles and UV irradiation.Results:After optimization,the content percentage of plasmid DNA isoforms could be well quantitated by CGE method with LIF detector using following parameters,including coated capillary whose total length was 40 cm,the 30.2 cm effective length(inlet to window),loading the 4 ng·μL^(-1) of plasmid DNA at 4℃with capillary temperature at 20℃,and injection at 4 s at 1378.95 Pa.The established CGE method could accurately quantify the content percentage of three plasmid DNA isoforms(ccc,linear and oc isoforms)with good resolution.The method had good specificity asthere was no observed interference from substances which were often used as plasmid DNA excipients.Linear range of the method for each of plasmid isoforms was from 0.25 ng·μL^(-1) to 8 ng·μL^(-1).A good linearity was exhibited between content percentage and loading concentration of each isoform,and the correlation coefficient were 0.9977,0.9994 and 0.9927 for linear,oc and ccc isoforms of plasmid DNA,respectively.Spiking 3%-50%of linear or oc isoform plasmid to the original plasmid,the content percentage of the linear or plasmid exhibited good linearity,and the co

关 键 词：毛细管凝胶电泳 质粒DNA 慢病毒载体 拓扑结构 超螺旋构象 线性构象 开环构象 定量检测 

分 类 号：R917[医药卫生—药物分析学]