Production of Recombinant Adeno-Associated Viral Vectors Using a Baculovirus/Insect Cell Suspension Culture System: From Shake Flasks to a 20-L Bioreactor Jamal Meghrous, † Marc G. Aucoin, †,‡ Danielle Jacob, † Parminder S. Chahal, † Normand Arcand, † and Amine A. Kamen* ,† Animal Cell Technology Group, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montre ´al, Que ´bec, Canada, H4P 2R2, and De ´partement de ge ´nie chimique, E Ä cole Polytechnique de Montre ´al, Campus de l’Universite ´ de Montre ´al, Montre ´al, Que ´bec, Canada 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 ∼1 × 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 ∼2.2 × 10 12 infectious viral particles (bioactive particles) or 2.6 × 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. Introduction Recombinant adeno-associated viruses (rAAV) are at- tractive vehicles for gene transfer and have been widely investigated for their ability to transduce target cells in vitro and in vivo. They are also promising vectors for human gene therapy. AAV vectors are derived from a replication-deficient member of the parvovirus family, which are naturally replication-deficient and dependent on a helper virus, usually adenovirus or herpesvirus, for productive infection. AAVs are small (25-30 nm) non- pathogenic viruses having a 4.8-kb single-stranded DNA genome (1). No significant immunological responses against transduced cells or the vector-derived gene product have been reported. Their ability to transduce both dividing and nondividing cells and their ability to produce stable long-term gene expression make AAV vectors valuable and interesting alternatives to other gene delivery systems. Recombinant AAV production requires three essential elements. The first component is the rAAV vector, harboring the DNA transgene flanked by AAV inverted terminal repeats (ITR), the only cis-component required for the formation of the viral particle. The two other elements, which can be supplied in trans, include the AAV rep and cap genes, which code for replication and structural proteins, respectively. Currently, two methods are commonly used for the production of these vectors: transient transfection of cells or the use of stable cell lines containing the required genes. The transient transfection method generally consists of cotransfecting HEK293 cells with three to four plasmids, harboring the transgene sequence, the rep and cap genes, and a final plasmid containing adenovirus helper genes. Transient transfec- tion method is appropriate to generate and test multiple rAAV vector constructs but is limited by the inability to achieve high transfection efficiency of mammalian cells in culture. Generation of stable cell lines is suitable for large-scale production of rAAV and has focused mainly on the transformation of HeLa cells; however, the gen- eration of such cell lines is tedious and time-consuming. Furthermore, the production of rAAV vectors is roughly about 10 7 infectious viral particles per milliliter (IVP/ mL) for both methods, which is a limitation for clinical applications. About 10 12 to 10 14 rAAV vector particles are required for clinical human doses (2). The baculovirus expression vector system (BEVS) has been used extensively for high-level production of highly * To whom correspondence should be addressed. E-mail: amine.kamen@cnrc-nrc.gc.ca. † Biotechnology Research Institute. ‡ E Ä cole Polytechnique de Montre ´al. 154 Biotechnol. Prog. 2005, 21, 154-160 10.1021/bp049802e CCC: $30.25 © 2005 American Chemical Society and American Institute of Chemical Engineers Published on Web 11/30/2004