Novo Nordisk Pharmatech - Increase specific influenza production with recombinant human insulin

Vaccines must be made in large quantities rapidly and cost-effectively. Aziza Manceur, Sonia Tremblay and Sven Ansorge from the Vaccine Program at the National Research Council Canada, with help from Novo Nordisk Pharmatech, discuss how production can be boosted by adding insulin to HEK293 cells to increase influenza virus yield.

Major changes to current bioprocesses are difficult and very expensive to implement. It has been demonstrated using HEK293 cells that adding animal-origin-free human insulin to off-the-shelf, chemically defined media can be used as a supplement to increase VCD, productivity and specific viral yield.

Mammalian cells are considered an alternative to eggs for the production of influenza vaccines. HEK293SF-3F6 is a suspension GMP cell line that grows in serum-free media. Using this cell line, influenza production has been achieved in shake flasks, microbioreactors and 3-7L bioreactors. In this project, insulin was added to the cultures in order to accelerate the bioprocess and boost influenza production.

Trial by media

HEK293SF-3F6 cells were grown in two types of media: one developed specifically for this cell line (IHM-03), and another in a commercially available media (CD293). Cells were infected with H1N1/A/Puerto Rico/08/34 or H3N2/A/Aichi/8/68 in a 24-well microbioreactor cassette. Next, 5-100mg/L insulin was added. After 48 hours, supernatants were collected and haemagglutinin (HA) was quantified. HA is a highly expressed glycoprotein at the surface of the virus and it was quantified by dot blot using pan-HA monoclonal antibodies (mAb) developed in-house. As opposed to strain-specific antibodies, pan-HA mAbs have successfully probed more than 40 different influenza strains including influenza A and B types.

Adding insulin meant that maximal cell density was reached sooner. Overall, a concentration of 25mg/L insulin provided an increase in influenza yield, regardless of the media or viral strain used: the yield of H1N1/A/Puerto Rico/08/34 in IHM-03 was increased almost twofold, while the production of H3N2/A/Aichi/8/68 increased by 150% in CD293 media. A concomitant activation of signalling pathways associated with cell survival (PI3K-Akt pathway) was observed.

Stimulating effect

Insulin is known to stimulate cell proliferation and it has cell survival effects. It also acts on cellular signalling pathways that are exploited by the influenza virus. Adding 25mg/L of insulin is an effective way of increasing influenza production and can easily be implemented in a vaccine bioprocess. Evaluation of the effect of insulin on the production of other viruses and viral vectors is in progress.

Novo Nordisk Pharmatech is the leading supplier of recombinant insulin, which is sourced directly from Novo Nordisk, the world's largest producer. Its recombinant insulin is a key component in serum-free growth media for mammalian cells and approved by regulatory bodies worldwide, including FDA and EMA. The company's products are manufactured to cGMP standards, and have distinguished record for global regulatory compliance, consistent high quality, extensive regulatory documentation, continuous availability, secure global supply chain, and high levels of service and support.

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Adding insulin, which is known to stimulate cell proliferation, is an effective means of boosting influenza production.
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