Recombinant adeno-associated virus (rAAV) are the most promising vectors for gene therapy. However, large-scale rAAV production remains a challenge for the translation of rAAV-based therapeutic strategies to the clinic. The baculovirus expression vector system (BEVS) has been engineered to produce high rAAV titers in serum-free suspension cultures of insect cells.
The typical approach of rAAV production in BEVS has been based on a synchronous infection with three baculoviruses at high multiplicity of infection (MOI) [>3 plaque forming units (pfu)/cell]. An alternative approach is to co-infect at low MOI (0.1 pfu/cell). Both strategies (high and low MOI) were compared at a cell density of 1.0 × 106 cells/ml in shake-flask experiments. To increase the rAAV titer, a low MOI combined with an initial cell density at infection of 5.0 × 106 cells/ml, in fed-batch mode, was evaluated. Subsequently, the production strategy was validated in 3-l bioreactor runs.
An increase of 210% in the rAAV titer (4.7 × 1011 enhanced transduction units/l) was observed when using low MOI, an effect primarily caused by the increase in cell density. The fed-batch approach resulted in a seven-fold increase of rAAV yield. Controlled operations in bioreactor contributed to further increase the rAAV yield (2.8 × 1014 vector genomes/l) by 25% in comparison to the shake flask results.
This high yield production process using low MOIs and a feeding strategy successfully addresses several limitations of current rAAV production in insect cells and contributes to position the BEVS system as one of the most efficient for large-scale manufacturing of rAAV vectors. Copyright © 2010 John Wiley & Sons, Ltd. The copyright in Jimmy A. Mena's, Johnny Montes', Parminder S. Chahal's and Amine A. Kamen's contributions belongs to the Crown in right of Canada and such copyright material is reproduced with the permission of the Research Council of Canada.