Meghrous Jamal, Aucoin Marc G, Jacob Danielle, Chahal Parminder S, Arcand Normand, Kamen Amine A
Animal Cell Technology Group, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montréal, Québec, Canada.
Biotechnol Prog. 2005 Jan-Feb;21(1):154-60. doi: 10.1021/bp049802e.
Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell system at various scales is presented. Shake flask studies were conducted to assess conditions to be used in bioreactors. Two insect cell lines, Trichoplusia ni (H5) and Spodoptera frugiperda (Sf9), were compared for their ability to produce rAAV-2 after infection with recombinant baculoviruses coding for the essential components of the vector. The effect of varying the ratio between individual baculoviruses and the effect of the overall multiplicity of infection (MOI), as well as the cell density at infection, were also examined. Infectious rAAV-2 particles were proportionally produced when increasing the individual MOI of BacRep virus up to 1.6. When equal amounts of each virus were used, a leveling effect occurred beyond an overall MOI of 5 and a maximum titer was obtained. Increasing the cell density at infection resulted in higher yields when infecting the cells in fresh medium; however, for the production of bioactive particles, an optimal peak cell density of approximately 1 x 10(6) cells/mL was observed without medium exchange. Infection in 3- and 20-L bioreactors was done at an overall MOI of 5 with a ratio of the three baculoviruses equal to 1:1:1. Under these conditions and infecting the cells in fresh medium, a total of approximately 2.2 x 10(12) infectious viral particles (bioactive particles) or 2.6 x 10(15) viral particles were produced in a 3-L bioreactor. Without replacing the medium at infection, similar titers were produced in 20 L. Our data demonstrates the feasibility of rAAV-2 production by BEVS at various scales in bioreactors and indicates that further optimization is required for production at high cell densities.
介绍了使用杆状病毒/昆虫细胞系统在不同规模下生产重组腺相关病毒载体的方法。进行了摇瓶研究以评估生物反应器中使用的条件。比较了两种昆虫细胞系，即粉纹夜蛾（H5）和草地贪夜蛾（Sf9），在感染编码载体必需成分的重组杆状病毒后产生rAAV-2的能力。还研究了改变单个杆状病毒之间的比例、总体感染复数（MOI）以及感染时的细胞密度的影响。当将BacRep病毒的单个MOI增加至1.6时，可按比例产生感染性rAAV-2颗粒。当使用等量每种病毒时，在总体MOI超过5时会出现平稳效应，并获得最大滴度。在新鲜培养基中感染细胞时，增加感染时的细胞密度会导致更高的产量；然而，对于生物活性颗粒的生产，在不更换培养基的情况下观察到最佳峰值细胞密度约为1×10⁶个细胞/毫升。在3升和20升生物反应器中以总体MOI为5、三种杆状病毒的比例为1:1:1进行感染。在这些条件下并在新鲜培养基中感染细胞，3升生物反应器中总共产生了约2.2×10¹²个感染性病毒颗粒（生物活性颗粒）或2.6×10¹⁵个病毒颗粒。在感染时不更换培养基的情况下，20升中产生了类似的滴度。我们的数据证明了杆状病毒表达系统在生物反应器中不同规模下生产rAAV-2的可行性，并表明在高细胞密度下生产需要进一步优化。
