用于肠道病毒71型灭活疫苗纯度分析的毛细管区带电泳法研究

Development and application of capillary zone electrophoresis for purity analysis of enterovirus type 71 inactivated vaccine

目的：建立毛细管区带电泳（capillary zone electrophoresis,CZE）方法,用于肠道病毒71型（Enterovirus 71,EV71）灭活疫苗纯度检测及分析。方法：以样品检测峰高度、分离度、信噪比为判断标准,对不同运行缓冲液、样品缓冲液、毛细管长度、上样时间等进行选择优化,确定毛细管区带电泳条件,同时采用HPLC法分离及免疫共沉淀法对各样品检测峰进行鉴定。利用建立的CZE方法对7批EV71灭活疫苗原液进行检测,采用面积归一法,计算疫苗原液纯度。结果：当运行缓冲液为pH 8.2硼酸缓冲体系、盐浓度为150 mmol·L^-1、SDS浓度为10 mmol·L^-1时,上样时间为100 s、毛细管有效长度为50 cm时,主检测峰高度最高、信噪比最优和分离度大于1.5。以含SDS 10 mmol·L^-1的150 mmol·L^-1pH 8.2硼酸盐缓冲液为运行缓冲液,5 mmol·L^-1硼酸盐缓冲液为上样缓冲液,以长50 cm、内径为50μm的无涂层石英毛细管作为色谱分离柱,对EV71灭活疫苗原液纯度进行检测,可见2个样品检测峰,经HPLC法及免疫共沉淀法鉴定,样品检测峰1为EV71病毒颗粒,迁移时间6.98 min、样品检测峰2为杂质,迁移时间8.42 min;连续进样6次,2个样品检测峰的峰面积、峰起时间、峰止时间和保留时间的相对标准偏差（relative standard deviation,RSD）均〈2.0%。应用该方法对7批EV71灭活疫苗原液纯度进行检测,为95.3%-99.4%,均大于95%。结论：初步建立了可用于EV71灭活疫苗原液纯度测定的毛细管区带电泳方法,为该疫苗的质量研究、工艺过程控制及稳定性研究奠定了基础。

Objective： To establish a capillary zone electrophoresis（ CZE） method for analyzing purity of enterovirus 71 inactivated vaccine. Methods： Based on the peak height,signal-to-noise ratio（ S /N） and resolution,the running buffer,sample buffer,sample loading condition and length of uncoated silica capillary were optimized for establishing the CZE method. The separated peaks were identified by co-immunoprecipitation and high performance liquid chromatography（ HPLC）. The purity of enterovirus 71 inactivated vaccines was detected by the developed CZE method and determined by using the area normalization method. Results： A pH 8. 2 borate buffer containing 10 mmol·L^-1sodium dodecyl sulfate and 150 mmol·L^-1sodium chloride was used as running buffer,an uncoated silica capillary with length of 50 cm and diameter of 50 μ m was used as the chromatographic column,and sample loading time was 100 s. Under this condition,the capillary electrophoretogram was optimal; the peak andS /N were the highest and resolution was higher than 1. 5. The purity of EV71 inactivated vaccine was evaluated by CZE with 5 mmol·L^-1borate buffer solution as sample buffer. Two peaks were observed,peak 1 was identified as EV71 with a migration time of 6. 98 min and peak 2 as impurity proteins with a migration time of 8. 42 min by HPLC and co-immunoprecipitation assay. Relative standard deviations calculated from means of peak area,start time,stop time and migrate time were 2. 0%,respectively. The developed CZE was applied to detect purity of 7 batches of inactivated EV71 vaccine,and the result showed a purity of 95. 3% - 99. 4%. Conclusion： The CZE method used for purity detection of EV71 inactivated vaccine has been established,which is helpful for the quality control and stability study of the vaccine.