Scalable Purification of Adeno-Associated Virus Serotype 1 (AAV1) and AAV8 Vectors, Using Dual Ion-Exchange Adsorptive Membranes
Scalable Purification of Adeno-Associated Virus Serotype 1 (AAV1) and AAV8 Vectors, Using Dual Ion-Exchange Adsorptive Membranes
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DOI：
10.1089/hum.2009.006
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发表时间：
2009-09-01
影响因子：
4.2
通讯作者：
Ozawa, Keiya
中科院分区：
文献类型：
--
作者：
Okada, Takashi;Nonaka-Sarukawa, Mutsuko;Ozawa, Keiya
以腺相关病毒（AAV）为基础的载体的体内基因转导依赖于生产高滴度载体的繁琐程序。为了满足最终产品的规格要求，需要有效清除诸如宿主细胞蛋白和空载体颗粒等杂质的纯化步骤。因此，开发替代的、现实的方法来促进可扩展的病毒恢复程序对于促进体内研究至关重要。然而，传统的树脂填充床层析纯化方法存在许多局限性，包括压力变化、孔隙扩散缓慢和床体积大。本研究采用一次性高性能阴离子和阳离子交换膜吸附剂对重组病毒进行了有效的纯化。等电聚焦分析发现，空粒子的等电点明显高于包装病毒粒子的等电点。因此，用膜吸附剂纯化AAV载体是成功的，并且允许更高水平的基因在体内转移，而没有明显的毒性或炎症迹象。获得的AAV载体库的电子显微镜显示高度纯化的病毒粒子，空颗粒少至0.8%。此外，膜吸附剂可以在转导的培养上清中恢复AAV载体。此外，携带两性包膜的逆转录病毒载体的离子交换富集是成功的。这种使用一次性膜吸附剂的快速和可扩展的病毒纯化方案特别有希望用于体内实验和临床研究。
In vivo gene transduction with adeno-associated virus (AAV)-based vectors depends on laborious procedures for the production of high-titer vector stocks. Purification steps for efficient clearance of impurities such as host cell proteins and empty vector particles are required to meet end-product specifications. Therefore, the development of alternative, realistic methods to facilitate a scalable virus recovery procedure is critical to promote in vivo investigations. However, the conventional purification procedure with resin-based packed-bed chromatography suffers from a number of limitations, including variations in pressure, slow pore diffusion, and large bed volumes. Here we have employed disposable high-performance anion- and cation-exchange membrane adsorbers to effectively purify recombinant viruses. As a result of isoelectric focusing analysis, the isoelectric point of empty particles was found to be significantly higher than that of packaged virions. Therefore, AAV vector purification with the membrane adsorbers was successful and allowed higher levels of gene transfer in vivo without remarkable signs of toxicity or inflammation. Electron microscopy of the AAV vector stocks obtained revealed highly purified virions with as few as 0.8% empty particles. Furthermore, the membrane adsorbers enabled recovery of AAV vectors in the transduced culture supernatant. Also, the ion-exchange enrichment of retroviral vectors bearing the amphotropic envelope was successful. This rapid and scalable viral purification protocol using disposable membrane adsorbers is particularly promising for in vivo experimentation and clinical investigations.