Scalable Production of Adenovirus Vectors - Adenovirus: Methods and Protocols - page 178

the protocols described in this heading. Table 1 represents the
main operational and hydrodynamic conditions for cell culture and
AdV infection in STB. All cell manipulations during bioreaction
are performed under sterile conditions in a portable laminar flow
wood. Parameters such as temperature, stirring speed, pH and pO 2
must be monitored and recorded throughout the all bioprocess
including pH and pO 2 calibration ( see Note 15 ).

3.3.1 Bioreactor
Preparation

1. Calibrate pH probe with standard buffer solutions to pH 4.01
and pH 7.00 ( see Note 16 ).
2. Check pO 2 probe response ( see Note 17 ).
3. Set the aeration system with 0.2 μm vent filters for inlet gas
flow ( see Note 18 ): use direct sparging of air or oxygen with a
ring sparger placed below the stirrer for the production of
HAdV using HEK 293 cells, while for the production of CAV-2
using MDCK-E1 cells in microcarriers perform aeration
through the headspace.
4. Before use, check that glass vessel is not damaged. To ensure
the reactor is airtight, check all rubber rings and, if necessary,
grease them.
5. Add MilliQ water to bioreactor vessel up to the level of probes
after bioreactor assembly ( see Note 19 ).
6. Assemble the bioreactor and tighten all fittings ( see Fig. 3 ).
Assemble the necessary impeller(s): 6-blade rushton turbine
impellers for HEK 293 cells or 3-blade impellers for MDCK-E1
cells ( see Note 20 ). Insert pH, pO 2 , and temperature probes
( see Note 21) . Set exhaust cooler in place with 2 × 0.2 μm vent
filters for outlet gas flow ( see Note 22 ). Assemble all the bioreac-
tor addition system and add entry connections for ( see Note 23 ):
(a) Culture medium.
(b) Cell inoculation.
(c) Virus infection/medium exchange at infection.
(d) Extra entry (for safety).
7. Assemble the sampling/harvesting system with a sampling
probe inside the bioreactor with silicone tube to reach the ves-
sel base ( see Note 24 ). Add an extra sampling probe to the
bioreactor of CAV-2 vectors with proper height to perform the
medium exchange at infection without removing the settled
microcarriers. Connect the base bottle, secure all connections
with cable ties and perform an hold-up test ( see Note 25 ).
8. Prepare connecting bottles for medium, cell and virus inocula-
tion in duplicates.
9. Autoclave bioreactor and connecting bottles at least 1 h,
121 °C for sterilization ( see Note 26 ).