Background & Aim Cell and Gene Therapies have the potential to revolutionize medicine in the 21st Century. Gene therapy clinical trials often require high titer vector preparations to adequately deliver the therapeutic transgene, in great excess of research-level production utilized in many laboratories. Bioprocessing for the therapeutic agents for these therapies still face many challenges during scale up. To assess manufacturing large scale production of AAV9 in adherent HEK293 cells, we evaluated AAV9 production utilizing the small scale iCELLis Nano bioreactor. This fixed-bed benchtop bioreactor was used to develop a transient transfection process. Following a production phase, >1016 vector genomes were isolated from crude harvest of the bioreactor. Here, we will present a case study which illustrates the challenges and solutions to scale up process steps required to develop manufacture iCELLis process for scalable production of AAV9. After implementation of a large-scale production bioreactors, we offer the client scalability and speed to the market Methods, Results & Conclusion To assess manufacturing large scale production of AAV9 in adherent HEK293 cells, we evaluated AAV9 production utilizing the small scale iCELLis Nano bioreactor. This fixed-bed benchtop bioreactor was used to develop a transient transfection process, utilizing PEI as a transfection agent. Based on conditions developed in the pilot iCELLis Nano bioreactor system, we scaled this production to the large scale iCELLis 500 bioreactor (200 m2 and 333 m2 surface area). A HEK293 cell seed train was utilized, using the Xpansion® 200 bioreactor (Pall Corporation) to generate sufficient cell numbers for seeding the bioreactor. We have developed the upstream manufacturing process for the production of the FDA approved drug Zolgensma. Simplification of seed train, replacing N-1 inoculation source to iCELLis 500-333 m2 with 2 Xpansion 200 bioreactors instead of 30 Cellstack10. Parameters determined for growth and transient transfection in the iCELLis Nano bioreactor are transferable and scale linearly to the iCELLis 500-333m2 bioreactor. Transient transfection strategy was developed for manufacturing scale following a production run in the iCELLis bioreactor with a fixed bed area of 333 m2, replacing 185 Hyperstacks 36 a vector titer of >1016 vector genomes was obtained from the crude harvest, reaching client desired target for yield per batch. Cell and Gene Therapies have the potential to revolutionize medicine in the 21st Century. Gene therapy clinical trials often require high titer vector preparations to adequately deliver the therapeutic transgene, in great excess of research-level production utilized in many laboratories. Bioprocessing for the therapeutic agents for these therapies still face many challenges during scale up. To assess manufacturing large scale production of AAV9 in adherent HEK293 cells, we evaluated AAV9 production utilizing the small scale iCELLis Nano bioreactor. This fixed-bed benchtop bioreactor was used to develop a transient transfection process. Following a production phase, >1016 vector genomes were isolated from crude harvest of the bioreactor. Here, we will present a case study which illustrates the challenges and solutions to scale up process steps required to develop manufacture iCELLis process for scalable production of AAV9. After implementation of a large-scale production bioreactors, we offer the client scalability and speed to the market To assess manufacturing large scale production of AAV9 in adherent HEK293 cells, we evaluated AAV9 production utilizing the small scale iCELLis Nano bioreactor. This fixed-bed benchtop bioreactor was used to develop a transient transfection process, utilizing PEI as a transfection agent. Based on conditions developed in the pilot iCELLis Nano bioreactor system, we scaled this production to the large scale iCELLis 500 bioreactor (200 m2 and 333 m2 surface area). A HEK293 cell seed train was utilized, using the Xpansion® 200 bioreactor (Pall Corporation) to generate sufficient cell numbers for seeding the bioreactor.