Conference Dates
June 6-11, 2010
Abstract
Recombinant adeno-associated virus (rAAV) has become a promising candidate vector for gene therapy. Anchorage-dependent cells are traditionally used to produce rAAV. However, mass production to meet demands for clinical trials requires large-scale and cost-effective manufacturing processes. The key advantages of rAAV vectors are a broader tissue tropism through the use of different serotypes and a good safety profile.
In the present work, a serotype 6-derived rAAV was produced by transfection of suspension HEK293 cells in serum-free medium using three plasmids: one encoding the GFP gene flanked by ITR sequences, the second encoding the replication and capsid genes, and a third one encoding adenovirus genes required by the AAV. Polyethylenimine (PEI) was employed as transfection agent. rAAV 6 production was investigated by evaluating the titres of genomic (Vg) and infectious viral particles (IVP) obtained in shake flask and bioreactor experiments. The transfections in shake flasks were carried out at 1, 1.5, 2 and 3x106 cells/mL at a PEI:DNA ratio of 2:1 (2 µg/mL of PEI and 1 µg/mL of DNA) and samples were evaluated at 48 hours post-transfection. Transfection at 3x106 cells/mL resulted in 2.5- and 3.5-fold increase in IVP and Vg, respectively, when compared with transfection at 1x106 cells/mL. When different PEI:DNA ratios were tested, IVP varied from 1.19x108 IVP/mL to 1.03x109 IVP/mL whereas Vg varied from 3.03x109 Vg/mL to 3.34x1011 Vg/mL. The results indicated that the individual concentrations of PEI and DNA have a more pronounced effect on IVP and Vg titres than the ratio between both of them. To evaluate process scalability, rAAV 6 was produced in stirred bioreactors with 2 L working volume. Using a PEI:DNA ratio of 2.67:1 (1.6 µg/mL of PEI and 0.6 µg/mL of DNA) and carrying out transfection at 1x106 cells/mL, at 48 hours post-transfection IVP and Vg values were higher than 2x108 IVP/mL and 7x1010 Vg/mL, respectively. Adopting the methodology used in this work, assuming that similar virus titres can be obtained upon further scale-up of the process, a 1000-L bioreactor could produce a total amount of 1x1014 IVP and 1x1016 Vg particles. Assuming an overall recovery of 50% in capture, purification and concentration steps of rAAV 6, these quantities should be sufficient for use in large clinical trials.
Recommended Citation
Érica A. Schulze, "PRODUCTION OF SEROTYPE 6-DERIVED RECOMBINANT ADENO-ASSOCIATED VIRUS IN SERUM-FREE SUSPENSION CULTURES OF HEK 293 CELLS" in "Vaccine Technology III", John G. Auniņš,Merck, USA; Barry C. Buckland, BiologicB, USA; Kathrin U. Jansen, Pfizer, USA; Paula Marques Alves, IBET, Portugal Eds, ECI Symposium Series, (2010). https://dc.engconfintl.org/vaccine_iii/23