Genetic Engineering & Biotechnology News

SEP15 2017

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36 | SEPTEMBER 15, 2017 | GENengnews.com CO N T I N U O U S B I O P R O C E S S I N G Tu t o r i a l Aziza P. Manceur, Ph.D., Sonia Tremblay, and Sven Ansorge, Ph.D. In this study, our goal was to 1) evaluate the effect of insulin on HEK293SF-3F6 cell growth in two types of media, 2) boost influenza production with the addition of different concentrations of insulin, and 3) quantify influenza using a robust method. Effect of Insulin on Cell Growth HEK293SF-3F6 is a suspension-adapted cell line (a GMP cell bank is available). HEK293SF-3F6 cells have successfully been used for the production of influenza virus, virus-like particles, lentiviral vectors, adenovirus and adeno-associated virus. HEK293SF-3F6 can be cultured in in-house media (IHM-03) and in commercially-avail- able serum-free media (Hyaline), but weak growth profiles are obtained in chemically defined media. Growth limitations observed in a chemically defined media (CD293 media) were alleviated by insulin (Figure 1). Growth kinetics were improved by insulin in an in- house media (IHM-03) (Figure 2). Effect of Insulin on Influenza Production in HEK293SF-3F6 Cells Cell-culture-based vaccines are a valuable alternative to egg-produced vaccines: the equivalent of 1,500 influenza vaccine doses can be produced in a 1-L bioreactor within 48 hours using HEK293 cells. Insulin is a strong activator of the Pl3K/ Akt pathway, which plays a key role in in- fluenza production (Figure 3). Addition of insulin to cell culture was found to increase the yield of influenza virus in a 24-well mi- crobioreactor (Figure 4). Some key findings of the experiment: • Increase in total HA was not due to increased cell density. • Similar results were obtained with H3N2 A/Aichi/2/68 treated with 5 and 25 mg/L insulin, respectively, at time of infection. • Akt activity is increased by influenza in- fection and insulin addition in HEK293SF-3F6 cells (Figure 5). Influenza Can be Quantified with PAN-HA Antibodies in a Dot-Blot Assay Quantification of influenza is an enduring challenge: Insulin Increases Influenza Virus Yield Evaluating the Effect of Insulin on HEK293 Cells Figure 1. By adding 10–20mg/L insulin every 72 hours, the maximal viable cell density of HEK293SF-3F6 cells was increased by 4-fold in CD293 media. Figure 2. By adding 10–20mg/L insulin every 72 hours, the maximal viable cell density of HEK293SF-3F6 cells was reached three days sooner in IHM-03, a media designed specifically for HEK293SF-3F6 cells. Figure 3. (A) Overview of signaling pathways modulated by influenza infection: the replication of influenza virus hijacks the cellular machinery and involves multiple signaling pathways, including Akt. (B) Akt is activated by viral NS-13. B A Figure 4. Addition of 25–100 mg/L insulin at time of infection Increases the yield of influenza H1N1 A/Puerto Rico 8/34 by almost 2-fold In CD293 media (A), without affecting the total cell count at harvest (B). The virus was added at an MOI of 0.01 at 35 o C in the presence of 1 µg/mL trypsin- TPCK. Hemagglutinin (HA) concentration was measured by dot-blot assay using pan-HA antibodies developed in house. A B ➜

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