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

  1. Get@NRC: Improving adeno-associated vector yield in high density insect cell cultures (Opens in a new window)
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Journal titleJournal of Gene Medicine
Pages157167; # of pages: 11
SubjectAnimal; Baculovirus; bio; Biotechnology; Cell Culture; Cells; Gene Therapy; insect cell; methods; therapy
AbstractBackground: 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 × 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. Results: 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. 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.
Publication date
AffiliationNational Research Council Canada (NRC-CNRC); NRC Biotechnology Research Institute
Peer reviewedYes
NRC number52747
NPARC number16225346
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Record identifier617cb24e-cb37-4278-a7a9-e9bb0834364e
Record created2010-11-05
Record modified2016-05-09
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