Adenovirus vectors are attractive delivery systems for vaccines and gene therapy applications. It is essential to have scalable and cost-efficient production technologies to enable manufacturing of safe and efficacious clinical grade virus. Commonly, in early stage studies anchorage dependent cells cultured in T-flasks, roller bottles or cell factories are used for production. In these cases scale-up is limited by the surface available for the cells to grow. A solution for larger scale cultivation of a variety of anchorage dependent cell lines is growing the cells on microcarriers. Another alternative is to use suspension adapted cells which may facilitate scale-up, but can on the other hand lead to lower productivity per cell as compared to adherent cells. We have established a process for adenovirus production using suspension HEK293 cells in serum free cell culture medium in a single use stirred-tank bioreactor. A recombinant adenovirus expressing Green Fluorescent Protein was used as a model system. The HEK293 cells were adapted to a number of media candidates for subsequent evaluation of cell growth and adenovirus productivity. Based on this data the chemically defined serum free medium was selected for further process development at bioreactor scale in order to optimize process conditions. HEK293 cells were inoculated at cell densities of 0,25x106 cells/mL or lower at 5-10 L scale in a single-use bioreactor system. At a cell density of 0,5-1x106 cells/mL the cultures were infected with adenovirus using a multiplicity of infection of 5-20. Harvest was performed 36-48 hrs post infection and viral lysate was obtained after cell lysis and concentration. Infectious viral titer was determined using standard TCID-50 and a more time saving technique. The results demonstrate that an efficient and stable process can be developed for adenovirus production in single use bioreactor systems. Furthermore, the tools and techniques used in this study can be applied for other suspension cell lines and viral vectors that are currently being evaluated for clinical use.

Production of recombinant adenovirus in a single use bioreactor system

253rd National Meeting (2017)

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