Metabolic and Kinetic analyses of influenza production in perfusion HEK293 cell culture

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.

Use of uniform designs in combination with neural networks for viral infection process development

“…The big challenge comprising virus production is to find a set of values for parameters mentioned above in order to maximize the difference between viral production and degradation. [19][20][21] Whereas the processes of viral infection are influenced by many variables, the optimal combination of parameters varies greatly in different virus-host cell systems, and there are few phenomenological mathematical models that describe the processes of viral production. For this reason, it is useful to develop small scale experimental strategies that can deliver at relatively low cost optimization of these processes.…”

Influence of HEK293 metabolism on the production of viral vectors and vaccine

“…The majority of amino acids (AA) are consumed by HEK293 cells in serum-free medium, except for alanine, glycine, proline and cysteine which are generally accumulated [63,64]. Similarly to other cell lines, glutamine is the most consumed AA in HEK293 cell culture, followed by serine, asparagine/aspartate, leucine, valine and arginine [46,51,65].…”

“…As an example, in the case of adenovirus production asparagine is essential for the formation of virions, and the highest flux observed for AA consumption is for asparagine/aspartate [51]. On the contrary, for influenza production, highest flux is observed for proline in batch and serine and leucine in continuous feeding mode [64].…”

Real-time monitoring of adherent Vero cell density and apoptosis in bioreactor processes

“…Several works have already reported apoptosis induction just after virus infection in large-scale viral productions (Petiot et al 2011;Ravindra et al 2008;Chan and Abubakar 2003). Furthermore, the cell viability may affect the viral infection efficiency.…”