Abstract
BackgroundCell culture-based production of influenza vaccine remains an attractive alternative to egg-based production. Short response time and high production yields are the key success factors for the broader adoption of cell culture technology for industrial manufacturing of pandemic and seasonal influenza vaccines. Recently, HEK293SF cells have been successfully used to produce influenza viruses, achieving hemagglutinin (HA) and infectious viral particle (IVP) titers in the highest ranges reported to date. In the same study, it was suggested that beyond 4 × 106 cells/mL, viral production was limited by a lack of nutrients or an accumulation of toxic products.ResultsTo further improve viral titers at high cell densities, perfusion culture mode was evaluated. Productivities of both perfusion and batch culture modes were compared at an infection cell density of 6 × 106 cells/mL. The metabolism, including glycolysis, glutaminolysis and amino acids utilization as well as physiological indicators such as viability and apoptosis were extensively documented for the two modes of culture before and after viral infection to identify potential metabolic limitations. A 3 L bioreactor with a perfusion rate of 0.5 vol/day allowed us to reach maximal titers of 3.3 × 1011 IVP/mL and 4.0 logHA units/mL, corresponding to a total production of 1.0 × 1015 IVP and 7.8 logHA units after 3 days post-infection. Overall, perfusion mode titers were higher by almost one order of magnitude over the batch culture mode of production. This improvement was associated with an activation of the cell metabolism as seen by a 1.5-fold and 4-fold higher consumption rates of glucose and glutamine respectively. A shift in the viral production kinetics was also observed leading to an accumulation of more viable cells with a higher specific production and causing an increase in the total volumetric production of infectious influenza particles.ConclusionsThese results confirm that the HEK293SF cell is an excellent substrate for high yield production of influenza virus. Furthermore, there is great potential in further improving the production yields through better control of the cell culture environment and viral production kinetics. Once accomplished, this cell line can be promoted as an industrial platform for cost-effective manufacturing of the influenza seasonal vaccine as well as for periods of peak demand during pandemics.
Highlights
Cell culture-based production of influenza vaccine remains an attractive alternative to egg-based production
The perfusion system is a powerful means to observe the impact of HEK293SF cell metabolism on the influenza infection process and production kinetics
The objectives were to identify if the cell density effect observed beyond 4 × 106 cells/mL could be overcome by providing a constant supply of nutrients to the cell and eliminating toxic elements from the culture supernatant
Summary
Cell culture-based production of influenza vaccine remains an attractive alternative to egg-based production. Several cell-based processes using adherent cell lines, such as Vero or MDCK cells, for the production of influenza or other viral pathogens, are already welldocumented [1,2,3,4,5,6,7]. Adherent cell culture processes remain limited in cell density, due to microcarrier surface saturation. They are mostly performed with serum-containing media [5]. New influenza production processes were proposed with suspension cell lines. Contrary to adherent cell cultures, suspension cell cultures have the potential to be operated at high cell densities and can achieve higher virus titers [5]. Other suspension growing cell lines, including duck AGE1.CR cells [9], human PER.C6 cells [10] or the avian embryonic derived stem cells EB14 (chicken) and EB66 (duck) [11], have been evaluated for infection and production of the different A and B influenza strains
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