基于离子交换色谱法检测结核治疗性DNA疫苗纯度的方法建立  

Development of a method for determination of purity in tuberculosis therapeutic DNA vaccine based on ion exchange chromatography

作　　者：孔雯雯 周飞燕 符美娟 杨丽菲 梁艳 吴雪琼 倪世明 黄明 KONG Wenwen;ZHOU Feiyan;FU Meijuan;YANG Lifei;LIANG Yan;WU Xueqiong;NI Shiming;HUANG Ming(Guangzhou Baiyunshan Baidi Bio-Technology Co.,Ltd.,Guangzhou 511495,China;Tuberculosis Key Laboratory,The 8th Medical Center of PLA General Hospital,Beijing 100091,China)

机构地区：[1]广州白云山拜迪生物医药有限公司,广东广州511495 [2]中国人民解放军总医院第八医学中心结核病重点实验室,北京100091

出　　处：《药学研究》2020年第11期628-631,共4页Journal of Pharmaceutical Research

基　　金：国家科技重大专项资助(No.2018ZX10731301-005-003)。

摘　　要：目的建立并验证结核治疗性DNA疫苗纯度的高效液相色谱测定方法。方法采用TSK gel DNA-NPR(4.6 mm×75 mm,2.5μm)色谱柱,流动相:以20 mmol·L^-1三羟甲基氨基甲烷(pH=9)为流动相A,以20 mmol·L^-1三羟甲基氨基甲烷-1 mol·L^-1氯化钠(pH=9)为流动相B,梯度洗脱,柱温:25℃;流速:0.5 mL·min^-1;检测波长:260 nm。结果质粒DNA在0.5~2.5μg的范围内与峰面积呈良好线性关系,R^2=0.9998,该法专属性、精密度和耐用性良好,检测限为0.001 mg·mL^-1,供试品在24 h内稳定。结论该方法符合检测结核治疗性DNA疫苗纯度的要求,适用于该产品的质量控制。Objective To establish and validate a method of area normalization for detection of purity in the tuberculosis therapeutic DNA vaccine by high performance liquid chromatography(HPLC).Methods Chromatography was performed on TSK gel DNA-NPR(4.6 mm×75 mm,2.5μm),using 20 mmol·L^-1 Tris buffer(pH=9)as mobile phase A,and 20 mmol·L^-1 Tris-1 mol·L^-1 sodium chloride solution(pH=9)as mobile phase B,with gradient elution at a flow rate of 0.5 mL·min^-1.The detection wavelength was 260 nm at 25℃.Results The plasmid DNA content showed good linear relationship to peak area within a concentration range of 0.5~2.5μg(R^2=0.9998).The method showed good specificity,precision and durability,of which the minimum detection limit was 0.001 mg·mL^-1.Test solution was stable within 24 h.The purity in three batches of tuberculosis therapeutic DNA vaccine were above 95%.Conclusion This method conformed to requirement of the purity testing of tuberculosis therapeutic DNA vaccine content and purity,and be applied to quality control.

关 键 词：结核DNA疫苗 离子交换色谱法 高效液相色谱法 纯度 

分 类 号：R927.1[医药卫生—药学]