Improving adeno-associated vector yield in high density insect cell cultures.
BACKGROUND
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.


METHODS
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 x 10(6) 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 x 10(6) cells/ml, in fed-batch mode, was evaluated. Subsequently, the production strategy was validated in 3-l bioreactor runs.


RESULTS
An increase of 210% in the rAAV titer (4.7 x 10(11) 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 x 10(14) vector genomes/l) by 25% in comparison to the shake flask results.


CONCLUSIONS
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.
Mena
Jimmy A
JA
Animal Cell Technology Group, Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada.
Aucoin
Marc G
MG
Montes
Johnny
J
Chahal
Parminder S
PS
Kamen
Amine A
AA
D000818:Animals; D019149:Bioreactors; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics*; D005822:Genetic Vectors / Q000096:biosynthesis*; D007313:Insecta / Q000166:cytology*