Genetic Engineering & Biotechnology News

SEP15 2017

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38 | 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 • Measures of infectivity are highly variable. • Physical methods to count viral particles can be confounded by the presence of nonviral particles. • Antibody-dependent methods to quantify surface proteins such as HA rely on strain- specific antibodies A pan-HA antibody cocktail was generated using a highly conserved peptide sequence found within the HA molecule (the fusion peptide) (Figures 6 and 7). Conclusion The HEK293SF-3F6 cell-line platform is an ideal candidate for the development of com- mercial manufacturing processes of virus and viral vectors for vaccines and gene/cell therapy. Influenza concentration in supernatant was measured using pan-HA antibodies produced in house using a peptide conjugate derived from the fusion peptide. Overall, a concentration of 25 mg/L of insulin provided an increase in influenza yield regardless of the media or viral strain used in HEK293SF-3F6 cells. A concomitant activation of signaling pathways associated with cell survival (PI3K-Akt pathway) was observed. Figure 7. Quantification by dot blot. A calibration curve ranging from 160 ng/mL to 20 µg/mL hemaggluttinin (HA) was obtained using a calibrating antigen of known concentration. Ten different samples produced in HEK293SF-3F6 cells were loaded in four dilutions and the concentration of HA was determined. Samples S4 and S5 were purified by sucrose-cushion, while the other samples were crude samples harvested after different treatments. Aziza P. Manceur Ph.D. (Aziza.Manceur@ cnrc-nrc.gc.ca), Sonia Tremblay, and Sven Ansorge, Ph.D., conduct research at the National Research Council in Montreal, Quebec, Canada. This bioprocess tutorial represents an edited and modified version of a poster presentation with the same title as the tutorial. Website: www.cnrc-nrc.gc.ca References and Acknowledgements 1. Proprietary of National Research Council (NRC) under Canadian Patent Document number 2252972 and US Patent: number 6,210,922, available for commercial licensing 2. O. Planz, Antiviral Research 98 (3), (June 2013). 3. Group Hale, http://www.virology.uzh.ch/research/ghale. html, accessed on August 24, 2017. Thank you to Magnus Franzmann and Clare Medlow (Novo Nordisk Pharmatech) for scientific input and for providing insulin. The results described do not represent an endorsement of any product by NRC. Figure 6. Western blots of influenza strains belonging to 13 different subtypes. Influenza viruses were produced in eggs and probed with mAb 11H12 (A) or mAb 10A9 (B). When mixed together, the two mAbs make a pan-HA cocktail. A B Figure 5. HEK293 cells were infected in a shake flask. The control was treated with 1 µg/mL trypsin- TPCK (without virus). Phospho- Akt was measured by flow cytometry after infection and representative results are shown (A–B) and summarized (C). The addition of insulin further activates Akt (D). Results were obtained by dividing the fluorescence signal obtained for the infected sample by the untreated control (n=2). B A C D Insulin Increases Influenza Virus Yield Continued from page 36

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