Application Note USD3011. Influenza Virus Production with Adherent VERO Cells in PadReactor Mini Bioreactor

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1 Application Note USD311 Influenza Virus Production with Adherent VERO Cells in Reactor Mini Bioreactor

2 Introduction Single-use solutions are found in many applications as they offer straightforward alternatives to classical glass or stainless steel systems. These ready-to-use systems require neither cleaning nor sterilization steps and facilitate the scale-up from bench scale to large scale productions. Within the range of its Reactor bioreactor systems, Pall Life Sciences has introduced the Reactor Mini bioreactor, as a scale-down model, perfectly suitable for feasibility studies, research, process development and small volume production of proteins, cells and viruses from both cells in suspension and in adherence on microcarriers (Figure 1). The gentle mixing linked to an efficient gas transfer allows Reactor Mini bioreactor to host suspension and anchorage-dependent cell cultures (e.g. on microcarriers). Critical cell culture parameters such as ph, DO, and temperature are controlled and monitored by the Reactor Mini controller. In this application note, we present results for the production of influenza virus with adherent VERO cells in a 16 L Reactor Mini system at a working volume of 13 L. Material and Methods Reactor Mini single-use bioreactor with classical ph and DO probes 5 ml Shake Flask Adherent VERO cells OptiPro medium - serum free (Gibco, Belgium) added by mm Glutamine Influenza virus H1N1 Cytodex-1 (GE Healthcare Bio-Sciences, Sweden) at 3 g/l final concentration Glucose and lactate meters Figure 1 Reactor Mini system The cells were seeded at.x1 6 cells/ml in a 13 L working volume and compared to a 5 ml Shake Flask ( 1 ml working volume). Cell culture set-points for the Reactor Mini bioreactor were of for the ph, 5% for the DO with a constant air flow at 35 L/min (equivalent to.5 vvm) in the head space and stirring speed set at 5 RPM (vs. 13 RPM for the ). Both cultures were infected on day post-inoculation. Prior to infection, stirring was stopped to allow the microcarriers settlement (15 min) before medium removal. Viruses were added to the microcarriers containing cells at a.1 MOI with fresh medium containing mm glutamine and 5 µg/ml trypsin final concentrations. During the culture, medium samples were taken to follow the cell growth, the ph, the metabolites consumption, and the viral production. At the end of the culture, the influenza virus titers were quantified by TCID5 test and HA assay.

3 Results and Interpretations Figure Glucose and lactate concentrations for Reactor Mini bioreactor and control..5 Glucose concentration (g/l) Medium exchange + infection Lactate concentration (mm) Day of culture Reactor Mini Reactor Mini Lac Lac Cells were seeded at 1,8 cells/ml (equivalent to ~16, cells/cm²) and grew in an exponential phase during days to reach 676, cells/ml (~5, cells/cm²). During that period, the cell viability was above 95%. Also, during these days, the glucose concentration in the Reactor Mini system decreased rapidly (below the limit of detection of the meter) while the lactate was at 18.7 mm (Figure ). As comparison, the glucose consumption and the lactate production rates were lower in. Post-infection, cells presented cytopathic effects (Figure 3) with a rapid decrease of the viability. Supernatant samples were collected at various hours post-infection (hpi) to assess the amount of virus produced. During the viral production period, glucose decreased rapidly to become negligible on day 7 while the lactate remained constant (Figure ). Table 1 Historic of the influenza samples from the Reactor Mini bioreactor and cultures. Sample # Hours post-infection (hpi)

4 Figure 3 Microscopic observations of the growth and viral production (cytopathic effect). Cells prior infection (8 hours post-inoculation) Cells in Reactor Mini Bioreactor hpi Cells in Reactor Bioreactor Mini 8 hpi Cells in hpi Cells in 8 hpi The infectious titers as well as the hemagglutinin content of each sample were quantified by respectively TCID5 and HA assay (Figure ). Based on these two tests, it clearly appeared that the production was very low compared to the bioreactor. The HA contents were negligible except for the two last samples were the value was log HA/1 µl vs. 3 and 6 log HA/1 µl for the two last Reactor Mini bioreactor samples. Additionally, the TCID5 titers were 1 Log lower in the than in the bioreactor after 8 hours of viral production.

5 Figure Influenza infectious titers (TCID5/mL) and HA content (log HA/1µL) in the different Reactor Mini bioreactor and samples. Log TCID 5/mL hpi.7 3 hpi hpi 8 hpi hpi 17 hpi hpi.9 1 hpi hpi.9 66 hpi Log HA/1 µl Influenza sample Log TCID5 Log HA titers Conclusions We demonstrated the potential of the disposable Reactor Mini system for adherent cell culture. Here, the agitation speed was set based on preliminary tests performed with Dextran based carriers (see Application Note USD31). For a 13 L working volume with 3 g/l of Cytodex-1, we demonstrated that the Cytodex-1 are in good suspension from RPM. The set-point of 5 RPM, selected to avoid extra sedimentation of microcarriers during the cell growth, demonstrated efficient mixing for nutrients and oxygen distribution through the liquid meanwhile reducing excessive shear stress. In parallel, we highlighted the flexibility of the system to first, enable good cell growth and second, support viral production. The infectious titers as well as the HA contents are significantly higher in the Reactor Mini bioreactor than in the which renders the system attractive for many viral production applications. Visit us on the Web at us at bioreactors@pall.com Corporate Headquarters Port Washington, NY, USA toll free (USA) phone biopharm@pall.com European Headquarters Fribourg, Switzerland +1 () phone LifeSciences.EU@pall.com Asia-Pacific Headquarters Singapore phone sgcustomerservice@pall.com International Offices Pall Corporation has offices and plants throughout the world in locations such as: Argentina, Australia, Austria, Belgium, Brazil, Canada, China, France, Germany, India, Indonesia, Ireland, Italy, Japan, Korea, Malaysia, Mexico, the Netherlands, New Zealand, Norway, Poland, Puerto Rico, Russia, Singapore, South Africa, Spain, Sweden, Switzerland, Taiwan, Thailand, the United Kingdom, the United States, and Venezuela. Distributors in all major industrial areas of the world. To locate the Pall office or distributor nearest you, visit The information provided in this literature was reviewed for accuracy at the time of publication. Product data may be subject to change without notice. For current information consult your local Pall distributor or contact Pall directly. 15, Pall Corporation. Pall,, and Reactor are trademarks of Pall Corporation. indicates a trademark registered in the USA and TM indicates a common law trademark. Filtration.Separation.Solution. is a service mark of Pall Corporation. /15, PDF, GN USD311