Insulin Improves Cell Growth and Influenza Yield in a HEK293 Suspension Cell Line

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In her “Ask the Expert” webcast on 21 June 2017, Aziza Manceur of the National Research Council Canada (NRC Canada) discussed the benefits of using insulin in bioprocessing.

Manceur’s Presentation
Canada’s NRC has research facilities across Canada covering disciplines such as aerospace, energy, mining, and medical devices. The NRC serves both the government and the private sector, working with Canadian and international clients. Manceur is based in Montreal with the cell culture scale-up team (in the human health and therapeutic group). The team works on process robustness and looks for industrially relevant strategies for producing viral vectors, antibodies, and recombinant proteins using suspension cells in large-scale bioreactors.

In one project, Manceur’s group tested insulin as a booster for cell growth and influenza production. The hormone supports cell growth and is widely used in bioprocessing. Already approved by regulatory agencies, the recombinant form available from Novo Nordisk is not animal derived. Insulin can stimulate the PI3K/AKT pathway, which is activated during influenza production.

For the experiment, a proprietary cell line (HEK293SF) adapted to suspension and serum-free conditions was grown in two different types of media: an NRC proprietary medium (IHM-03) and a chemically defined and protein-free Gibco medium (CD293) from Thermo Fisher Scientific. For each culture, either no insulin, 10 mg/L, or 20 mg/L insulin was added every 72 hours.

The proprietary medium is formulated specifically for the HEK2935F cell line. Even without insulin addition, the IHM-03 culture achieved ~6 × 106 cells/mL. When insulin was added, it reached that cell density three days sooner.

In the CD293 medium without insulin, cells reached a density of only 1.5 × 106 cells/mL. When insulin was added, the density achieved also was ~6 × 106 cells/mL. The amount of insulin added did not seem to make a difference in growth rate (results were about the same for each amount).

During the webcast, Manceur asked audience members what culture strategy they use, and most respondents said they use off-the-shelf commercial media either as-is or with supplements, additives, and feeds. Most did not develop their own media or platform. Manceur asked which methods were used to quantify influenza viruses, and the two main methods of choice were 50% tissue culture infective dose (TCID50) assays and single radial immunodiffusion (SRID) assays.

Manceur explained how the influenza virus replicates in mammalian cells and focused on associated signaling pathways, specifically the PI3K/Akt pathway and how insulin works as a strong Akt activator. She detailed the methods and equipment her team used in this experiment: microbioreactors, the two different media, and two different influenza strains. Then she showed their methods for quantifying influenza viruses. They generate monoclonal antibodies (MAbs) that recognize multiple strains of influenza. Manceur demonstrated detection of different strains by Western blot and dot blot.

Finally, Manceur showed that adding 25 mg/L insulin increased the yield of two different strains of influenza by twofold. NRC’s next steps will be to confirm the effects of insulin on influenza production at large scale and explore its effects with other cell lines and viral vectors.

Questions and Answers
Does higher cell density relate to more virus particles?
We quantified viral particles by HPLC and did see an increase in the number of viral particles. But we could not conclude that they were all influenza particles (would need to check samples with electron microscopy).

Are there any results or ideas for the effect on the B strains? I have not tested the effect of insulin on B strains yet, but it is something that I would like to do. My guess is that we would see a similar effect because I think insulin activates the Akt pathway, which basically increases cell survival. That should expand the window during which the virus can bud out of the cell.

Was the insulin quantity for the best result different or strain dependent? With the H3H2 strains, we needed slightly lower concentrations than for the H1N1 strain. It could be strain-specific; it also seems to depend on the media. For the proprietary medium, we needed a lower concentration of insulin than was needed for the chemically defined medium. I suggest trying two or three concentrations with the media and strains you are using to find the best concentration for your situation.

Given the cost of insulin, is its addition a viable option for biomanufacturing vaccines? The cost evaluation would be different for each system. But from a bioprocessing point of view, if adding insulin increases your yield twofold, then you could run half the number of cultures. A more concentrated product might make purification and concentration easier. So if you get at least a twofold increase in influenza by adding insulin, it probably would be cost effective.

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