Laboratory of Molecular Virology and Gene Therapy, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Hum Gene Ther. 2011 Aug;22(8):1021-30. doi: 10.1089/hum.2010.250. Epub 2011 May 16.
The large amounts of recombinant adeno-associated virus (rAAV) vector needed for clinical trials and eventual commercialization require robust, economical, reproducible, and scalable production processes compatible with current good manufacturing practice. rAAV produced using baculovirus and insect cells satisfies these conditions; however, recovering rAAV particles from 200-liter bioreactors is more complicated than bench-scale vector preparations. Using a variety of processing media, we developed a reliable and routine downstream procedure for rAAV production that is scalable from 0.02- to 200-liter cultures. To facilitate the upstream process, we adapted the titerless infected-cell preservation and scale-up process for rAAV production. Single-use aliquots of cryopreserved baculovirus-infected insect cells (BIIC) are thawed and added to the suspension culture to achieve the desired ratio of BIIC to rAAV-producer cells. By using conditions established with small-scale cultures, rAAV was produced in larger volume cultures. Strikingly consistent rAAV yields were attained in cultures ranging from 10 liters to 200 liters. Based on the final yield, each cell produced 18,000 ± 6,800 particles of purified rAAV in 10-, 20-, 100-, and 200-liter cultures. Thus, with an average cell density of 4.32 × 10(6) cells/ml, ≥ 10(16) purified rAAV particles are produced from 100 to 200 liters. The downstream process resulted in about 20% recovery estimated from comparing the quantities of capsid protein antigen in the crude bioreactor material and in the final, purified product. The ease and reproducibility of rAAV production in 200-liter bioreactors suggest that the limit has not been reached, and 500-liter productions are planned.
需要大量的重组腺相关病毒 (rAAV) 载体用于临床试验和最终商业化，这就需要强大、经济、可重复和可扩展的生产工艺，并且与现行良好生产规范相兼容。使用杆状病毒和昆虫细胞生产的 rAAV 满足这些条件；然而，从 200 升生物反应器中回收 rAAV 颗粒比 bench-scale 载体制备更为复杂。我们使用各种加工介质，开发了一种可靠且常规的 rAAV 生产下游工艺，可从 0.02 升至 200 升培养规模。为了便于上游工艺，我们对 rAAV 生产的无滴度感染细胞保存和放大过程进行了适应性改造。无滴度感染的昆虫细胞（BIIC）的即用型等分试样被解冻，并添加到悬浮培养液中，以达到所需的 BIIC 与 rAAV 生产细胞的比例。通过使用小规模培养确定的条件，在更大体积的培养物中生产 rAAV。在从 10 升至 200 升的培养物中，rAAV 的产量均达到惊人的一致。根据最终产量，在 10 升、20 升、100 升和 200 升的培养物中，每个细胞分别产生 18000±6800 个纯化的 rAAV 颗粒。因此，以平均细胞密度 4.32×10(6)个细胞/ml 计算，从 100 升至 200 升培养物中可生产出 ≥10(16)个纯化的 rAAV 颗粒。从粗生物反应器材料和最终纯化产物中衣壳蛋白抗原的数量比较来看，下游工艺的回收率约为 20%。200 升生物反应器中 rAAV 的生产既简单又可重复，表明尚未达到生产极限，并且计划进行 500 升生产。
