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目的采用荧光染色法检测重组乙型肝炎疫苗(酿酒酵母)纯化工艺中间产品、原液及半成品中残留DNA含量。方法采用《中国药典》三部(2010版)附录ⅨB荧光染色法检测重组乙型肝炎疫苗(酿酒酵母)纯化工艺主要中间产品[丁基琼脂糖层析收集液(以下简称BA富集液)]、原液及半成品中残留DNA含量,对该方法的准确度、精密性进行验证,并用建立的方法检测10批BA富集液、原液及半成品中残留DNA含量。结果该方法线性范围1.25~80 ng/ml,r2≥0.999,最低检测限为1.25 ng/ml;DNA标准品在BA富集液、疫苗原液及半成品中的平均回收率分别为105.8%、101.4%和108.5%,准确度较好;不同时间点重复测定同一批样品的变异系数(CV)在1.6%~6.56%之间,不同试验间重复测定同一批样品的CV在1.4%~3.6%之间,试验内和试验间精密性较好;10批BA富集液中DNA浓度均较高,经过纯化后,原液中DNA含量明显降低,半成品中除个别批号外,均基本达到低于10 ng/剂的要求。结论荧光染色法可用于重组乙型肝炎疫苗(酿酒酵母)纯化工艺中间产品、原液及半成品中残留DNA含量的常规监测,具有简便、快速、自动化程度高等特点,测定前样品无需进行处理。
Objective To detect the content of residual DNA in the intermediate products, stock solutions and semi-finished products of recombinant hepatitis B vaccine (Saccharomyces cerevisiae) by fluorescence staining. Methods The main intermediates of purification process of recombinant hepatitis B vaccine (Saccharomyces cerevisiae) were determined by fluorescence spectrophotometry (Appendix IXB) of “Chinese Pharmacopoeia” (2010 edition) [butyl sepharose chromatography (hereinafter referred to as BA enrichment)] , The original DNA and semi-finished products in the residual DNA content of the method to verify the accuracy and precision, and established the method to detect 10 batches of BA enrichment solution, the original liquid and semi-finished products residual DNA content. Results The linear range of this method was 1.25-80 ng / ml, r2 ≥ 0.999, and the lowest detection limit was 1.25 ng / ml. The average recoveries of DNA standards were 105.8% and 101.4% respectively in BA enrichment solution, vaccine stock solution and semi- And 108.5% respectively. The coefficient of variation (CV) of the same batch of samples was repeatedly measured at different time points between 1.6% and 6.56%. The CV of the same batch of samples was repeatedly measured between 1.4% and 3.6% , The precision in the experiment and in the experiment was better. The concentration of DNA in the 10 batches of BA-enriched solution was high. After purification, the DNA content in the stock solution was significantly reduced. Except for individual lot numbers, the semi-finished products basically reached below 10 ng / Agent requirements. Conclusion Fluorescent staining can be used to routinely monitor the residual DNA content of the recombinant hepatitis B vaccine (Saccharomyces cerevisiae) purification intermediate products, stock solutions and semi-finished products. The method has the advantages of simple, rapid and high degree of automation. The sample before treatment needs no treatment.