Suspension HEK-293 cells at 1 x 10^6 cells/mL were transfected with PEIpro in 20 mL working volume shake flasks. Complexes were generated by mixing 1 µg of total plasmid DNA/mL cell culture and 2 µg PEIpro/ mL cell culture. Mixture was vortexed for 5 s and incubated at room temperature for 10–15 min. After incubation the volume of transfection was adjusted to 10% total volume with medium and transferred drop-by-drop into the shake flasks. The flasks were incubated at 37°C and 5% CO2.

Despite major advances in developing capacities and alternative technologies to egg-based production of influenza vaccines, responsiveness to an influenza pandemic threat is limited by the time it takes to generate a Candidate Vaccine Virus (CVV) as reported by the 2015 WHO Informal Consultation report titled "Influenza Vaccine Response during the Start of a Pandemic". In previous work, we have shown that HEK-293 cell culture in suspension and serum free medium is an efficient production platform for cell culture manufacturing of influenza candidate vaccines. This report, took advantage of, recombinant DNA technology using Reverse Genetics of influenza strains, and advances in the large-scale transfection of suspension cultured HEK-293 cells. We demonstrate the efficient generation of H1N1 with the PR8 backbone reassortant under controlled bioreactor conditions in two sequential steps (transfection/rescue and infection/production). This approach could deliver a CVV for influenza vaccine manufacturing within two-weeks, starting from HA and NA pandemic sequences. Furthermore, the scalability of the transfection technology combined with the HEK-293 platform has been extensively demonstrated at >100L scale for several biologics, including recombinant viruses. Thus, this innovative approach is better suited to rationally engineer and mass produce influenza CVV within significantly shorter timelines to enable an effective global response in pandemic situations.