荧光染色法检测重组乙型肝炎疫苗中残留DNA含量

Determination of residual DNA content in recombinant hepatitis B vaccine by fluorescent staining

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1．25～80ng／ml，r^2≥0．999，最低检测限为1．25ng／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10ng／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 determine the residual DNA contents in intermediate products of purification, bulk and final bulk of recombinant hepatitis B （HB） vaccine （S. cerevisiae） by fluorescent staining. Methods The residual DNA contents in intermediate products of purification（harvested liquid of butyl-agarose chromatography, BA enrichment liquid）, bulk and final bulk of recombinant HB vaccine（S, cerevisiae ） was determined by fluorescent staining in Appendix ⅨB of Chinese Pharmacopoeia （Volume Ⅲ, 2010 edition）, based on which the method was verified for accuracy and precision, and used for determination of 10 batches of BA enrichment liquid, bulk and final bulk. Results The linear range, r2 value and minimum detection limit of the method were 1.25 - 80 ng / ml, not less than 0. 999 and 1.25 ng / ml respectively. The mean recovery rates of DNA standard in BA enrichment liquid, bulk and final bulk were 105. 8%, 101. 4% and 108. 5% respectively, indicating high accuracy of the method. The CVs of repeat determination results at various time points were 1.6% - 6. 56%, while those of the same batch of samples in various tests was 1.4% - 3. 6% indicating high precisions in intra-and inter-assays of the method. The DNA contents were high in 10 batches of BA enriched liquid while decreased significantly in bulk. However, the DNA contents in final bulks, except those in individual batches, were less than 10 ng / dose. Conclusion Fluorescent staining may be used for routine determination of residual DNA contents in intermediate products of purification, bulk and final bulk of recombinant HB vaccine （S. cerevisiae）, which is simple, rapid and highly automatic, and the samples need no special treatment before determination.