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.