Viral vectors have become increasingly used for gene transfer of specific tissue or cell type modifications. Several viruses have been investigated for their use in cell and gene therapy with adeno-associated virus (AAV) as the main vector for gene therapies. We show how an efficient and scalable cell culture process for AAV production was developed by evaluation and optimization of each process step. HEK293T cells were successfully adapted to suspension culture in a serum-free medium without animal-derived components. Triple plasmid transfection using polyethylenimine (PEI) was optimized for different parameters and conditions using a design of experiments (DoE) strategy in shake flasks. We also developed a Biacore™ surface plasmon resonance -based method for total virus capsid titer quantitation. XDR-10 single-use bioreactor process was designed and optimized based on the shake flask results. Furthermore, a second process developed in WAVE™ bioreactors gave similar productivities. Thus, we developed a scalable, robust and reproducible production of AAV from small-scale shake flask production up to 20 L in single-use bioreactor systems.
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