PLATFORM FOR THE GROWTH AND PROPOGATION OF HEK293 CELLS AND ADENOVIRUS VIRAL VECTOR AMPLIFICATION

Background & Aim Industrialization of viral vector production for gene therapy applications demands consistent processes utilizing robust production platforms and raw materials that can meet quality standards and regulatory compliance. Typical vector production schemes involve propagating seed trains and viral amplification in cultures utilizing serum containing media. Although serum-containing media systems deliver high viral vector yields, the serum component has certain disadvantages such as performance variability, high costs, and potential of introducing adventitious agents. Utilizing serum-free chemically-defined medium for growth and amplification addresses these concerns. Here we describe a highly efficient and economical single-use bioreactor platform for producing gene therapy viral vectors with flexibility of using serum-containing or serum-free media. Methods, Results & Conclusion We evaluated multiple serum-free chemically-defined media formulations for adenovirus vector production in the single-use fixed-bed iCELLis® bioreactor. These media were evaluated for ease of adaptation and growth characteristics with HEK293 cells previously adapted to serum-free growth conditions. Process parameters such as multiplicity of infection (MOI) and duration of infection (DOI) were optimized in a shake flask to maximize vector amplification. The process was then transferred into the iCELLis Nano bioreactor. The HEK293 cells were maintained under adherent conditions and growth was monitored until the cell densities reached approximately 200,000 cells/cm2. Complete medium exchange was performed in iCELLis Nano bioreactor without cell removal, followed by viral infection at MOI 50. Harvest was performed 72 hours post infection. Viral titers were quantitated using an adenovirus in-vitro infectivity assay. Viral productivity was generally consistent between the shake flask and the iCELLis Nano bioreactor system and between serum-containing or serum-free chemically-defined medium. These studies demonstrate the iCELLis bioreactor as a versatile scalable technology, and support its use for production of gene therapy vectors in processes using serum-containing or serum-free media.