Negrete Alejandro, Yang Linda C, Mendez Andres F, Levy Justin R, Kotin Robert M
Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, US National Institutes of Health, Bethesda, MD 20892, USA.
J Gene Med. 2007 Nov;9(11):938-48. doi: 10.1002/jgm.1092.
The versatility of recombinant adeno-associated vector (rAAV) as a gene delivery system is due to the vector's ability to transduce different cell types as well as dividing and non-dividing cells. Large-scale production of rAAV remains one of the major challenges for continued development of pre-clinical and clinical studies, and for its potential commercialization. The baculovirus expression vectors (BEVS) and insect cells represent a potential method to produce rAAV economically at large scale. This technology uses three different BEVs (Bac-Rep, Bac-GFP, and Bac-VP) each at a multiplicity of infection (MOI) of 3. We reported previously the production of rAAV at 40 L scale using a stirred-tank bioreactor (STB). However, production in larger volumes is limited by the stability of the BEVs and amount of BEVs needed to achieve the target MOI of 3 per BEV. Here, the production parameters were optimized and the baculovirus stability was determined.
The stability of the three types of baculovirus used to produce rAAV was determined for six expansion passages by protein expression analysis. To economize baculovirus, MOI and cell density at time of infection (TOI) were evaluated initially at small scale and then applied to the 10 L scale.
An MOI = 0.03 and TOI cell density of 1 x 10(6) cells/mL produced high titer rAAV without comprising yield. To confirm the scalability of the process, rAAV was produced in a 10 L STB using the optimized parameters obtaining a 10x increase in yield ( approximately 1 x 10(14) rAAV DNAse-resistant particles per liter).
These findings contribute to the process development for large-scale production of rAAV for gene therapy applications and its commercialization.
重组腺相关病毒（rAAV）作为一种基因传递系统具有多功能性，这归因于该载体能够转导不同类型的细胞以及分裂和非分裂细胞。rAAV的大规模生产仍然是临床前和临床研究持续发展及其潜在商业化的主要挑战之一。杆状病毒表达载体（BEVS）和昆虫细胞代表了一种大规模经济生产rAAV的潜在方法。该技术使用三种不同的BEVS（Bac-Rep、Bac-GFP和Bac-VP），每种的感染复数（MOI）均为3。我们之前报道了使用搅拌罐生物反应器（STB）在40 L规模上生产rAAV。然而，更大体积的生产受到BEVS稳定性以及达到每个BEV目标MOI为3所需的BEVS量的限制。在此，对生产参数进行了优化并确定了杆状病毒的稳定性。
通过蛋白质表达分析确定用于生产rAAV的三种杆状病毒在六个传代扩增中的稳定性。为了节约杆状病毒，最初在小规模评估感染时的MOI和细胞密度（TOI），然后应用于10 L规模。
MOI = 0.03且TOI细胞密度为1×10⁶个细胞/mL时可产生高滴度的rAAV且不影响产量。为了确认该工艺的可扩展性，使用优化参数在10 L STB中生产rAAV，产量提高了10倍（每升约1×10¹⁴个抗DNA酶的rAAV颗粒）。
这些发现有助于基因治疗应用中rAAV大规模生产的工艺开发及其商业化。
