2.3 2 L stirred tank reactor cultures
All STR studies were conducted in 2 L UniVessel® SU single-use bioreactors (Sartorius AG, Gottingen, Germany) in conjunction with the Biostat® B universal benchtop controller (Sartorius AG, Gottingen, Germany). The STRs were run with GSK’s platform process operating conditions with defined inoculation and induction cell densities as well as concentrations of induction agents (i.e. doxycycline and sodium butyrate). In addition, the STRs were also operated with a controlled stirrer speed, temperature, pH and pO2. Agitation was provided by two 3-blade segment impellers (diameter = 54 mm) generating a down-flow flow characteristic. Data acquisition and process control was managed by an MFCS/Win 2.0 system (Sartorius AG, Gottingen, Germany). Temperature was controlled using a heating/cooling jacket. The pH was controlled around the set-point through the addition of sodium carbonate base and CO2 in the inlet air. The dissolved oxygen tension (DOT) was maintained at the set-point using a cascade of air, oxygen and nitrogen. In order to prime the DOT and pH patch sensors, the STRs were media filled a day prior to inoculation. Just before inoculation, the pH was measured using a blood gas analyser (BGA) (Siemens, Healthineers, Erlangen, Germany) and recalibrated if it deviated by more than 0.05 units from the target value. The STRs were inoculated using a diluted mid-exponential phase shake flask culture. Once the target induction cell density was reached, the cultures were induced to initiate the production of the LVV. Induction was carried out by the addition of Doxycycline (Sigma-Aldrich, St. Louis, Missouri, United States) and Sodium Butyrate (Sigma-Aldrich, St. Louis, Missouri, United States). The induction agents were diluted in complete culture medium prior to being added to the STR. The working volume was between 1.5 L to 1.8 L. The vector was harvested at a defined number of hours post-induction which has been determined as part of GSK’s platform process. Vector harvests were performed by spinning down the cell culture in a bench top centrifuge (Eppendorf, Hamburg, Germany) at 1000g for 5 min at room temperature. The vector supernatant was aliquoted into cryovials (Corning Inc. New York, United States) and stored in a -80 °C freezer. The STR cultures were performed in triplicate.