Department of Chemical and Materials Engineering, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan.
J Biosci Bioeng. 2010 Aug;110(2):187-93. doi: 10.1016/j.jbiosc.2010.02.004. Epub 2010 Mar 11.
Recombinant adeno-associated virus (rAAV) is one of the most promising vectors for human gene therapy. However, the production systems that are currently available have a limited capacity and cannot provide sufficient quantities of rAAV for preclinical or clinical trials. Many novel methods for improving rAAV production have been developed, but few researchers have focused on the culture process. In this study, we use a fed-batch culture system to enhance rAAV yield in the baculovirus/insect cell system. When the insect cells were co-infected with MOI=5 of Bac-GFP at a ratio of 1:9:9 (Bac-GFP: Bac-Rep: Bac-VP), the fed-batch culture achieved optimal rAAV yields. In batch culture, the optimal cell density for producing rAAV was found to be 1x10(6) cells/ml, and the highest rAAV yield (1.22x10(8) IVP/ml, 122 IVP/cell) occurred at day 5 post-infection. In the fed-batch culture, rAAV yield reached 2.13x10(8) IVP/ml at day 4 post-infection, and the highest rAAV yield was 2.40x10(8) IVP/ml (240 IVP/cell) at day 5 post-infection. The cost of the batch and fed-batch cultures is similar; however, the rAAV yield was 2.6-fold higher in the fed-batch culture system compared with that in the batch culture system. Therefore, here we demonstrated an economical and efficient strategy for rAAV production.
重组腺相关病毒（rAAV）是用于人类基因治疗最有前途的载体之一。然而，目前可用的生产系统容量有限，无法为临床前或临床试验提供足够数量的 rAAV。已经开发了许多提高 rAAV 产量的新方法，但很少有研究人员关注培养过程。在这项研究中，我们使用分批补料培养系统来提高杆状病毒/昆虫细胞系统中的 rAAV 产量。当昆虫细胞以 MOI=5 的比例共同感染 Bac-GFP（Bac-GFP: Bac-Rep: Bac-VP=1:9:9）时，分批补料培养可实现最佳 rAAV 产量。在分批培养中，发现生产 rAAV 的最佳细胞密度为 1x10(6) 个细胞/ml，最佳 rAAV 产量（1.22x10(8) IVP/ml，122 IVP/细胞）出现在感染后第 5 天。在分批补料培养中，感染后第 4 天 rAAV 产量达到 2.13x10(8) IVP/ml，感染后第 5 天最高 rAAV 产量达到 2.40x10(8) IVP/ml（240 IVP/细胞）。分批培养和分批补料培养的成本相似；然而，分批补料培养系统中的 rAAV 产量比分批培养系统高 2.6 倍。因此，我们在这里展示了一种经济高效的 rAAV 生产策略。
