目的 建立一个可靠的、可供临床应用检测的rAAV2-KAL残余HEK293细胞宿主蛋白(host cell protein,HCP)的测试方法。方法 Western blot 方法鉴定rAAV,ELISA方法确定最佳线性范围及最低检测限,验证准确度及精密度。本实验连续生产并用二次氯化铯纯化3批rAAV2-KAL,使用ELISA方法检测rAAV2-KAL所含有的HCP含量,验证超速离心纯化工艺中的适用性。结果 建立的ELSA 方法的最佳线性范围为4~200 ng·mL-1,最低检测为4 ng·mL-1;不同浓度的 HEK293细胞蛋白抗原回收率77.0%~115.0%,准确度较好;变异系数为8.2%~15.4%均低于 20%,显示准确度和精密度均良好;制备的 3 批rAAV2-KAL经过二次CsCl纯化后,HCP含量均低于100 ng·mL-1,显示该纯化工艺可有效去除HCP。结论 本实验成功建立 HEK293细胞残余蛋白含量检测的双抗体夹心法 ELSA 方法,可用于rAAV 病毒载体中HEK293宿主细胞蛋白残余含量的检测。
Abstract
OBJECTIVE To develop a reliable method to measure host cell protein(HCP)content of HEK293 cells in rAAV2-KAL vectors for clinic application. METHODS The rAAV2-KAL vectors used for this study was purified by CsCl ultracentrifugation twice,then were subjected to by Western blot. The optimal linear range, minimum detection limit,and the accuracy and precision were verified by ELISA. Three batches of rAAV2-KAL were prepared by CsCl method. The change of HCP content during purification was determined to verify the method suitability and reliabilities. RESULTS The optimal linear range of the method developed was 4-200 ng·mL-1,while the minimum detection limit of 4 ng·mL-1. The recovery rates of HEK293 cell protein at various concentrations were at range of 77.0%-115.0%,with a coefficient of variation of less than 20%. The HCP contents in three batches of rAAV2-KAL were less than 100 ng·mL-1, all data taken together indicated that HCP was effectively removed by CsCl ultracentrifugation. CONCLUSION A reliable ELISA assay for residual host cell protein of HEK293 cells is successfully developed,which might be used for determination of HCP content in CsCl ultracentrifugation purified rAAV2-KAL for clinical application.
关键词
重组腺相关病毒 /
宿主细胞蛋白 /
联免疫吸附试验测定
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Key words
recombinant adeno-associated virus /
HEK293 cell host protein /
ELISA
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中图分类号：
R917
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参考文献
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脚注
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基金
国家自然科学基金资助项目(81271692);国家863资助项目(2012AA020810)
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