Biology Reference
In-Depth Information
22. The inlet filters (0.2 μm) placed in the exhaust cooler and
aeration system serve to allow gas flow while maintaining the
vessel under sterile conditions after sterilization. The exit of the
exhaust cooler should have two filters attached. One is main-
tained open during sterilization to release pressure formed dur-
ing sterilization. The other is maintained clamped as a backup if
the other is damaged due to humidity release. The exhaust
cooler should be placed on the same side of the water jacket
tubes. In this way all the water connections are made in the
same side of the bioreactor leaving the rest of the space free for
easy handling all the other connections and sampling.
23. Use T associations or equivalent between connections, case
the bioreactor ports are not enough to have all the connec-
tions necessary.
24. For the sampling system, it is necessary a sampling probe
inside the bioreactor with silicone tubing at the bottom touch-
ing the vessel base. This will allow removing the water used
during sterilization and can be also used as harvesting exit.
25. Perform a hold-up test to assure the entire bioreactor vessel
and connecting bottles are properly sealed. Connect the gas
supply to the air inlet filter of the aeration system and close all
vessels exits. Start the airflow and set the pressure inside the
vessel to 0.5 bar (this value is dependent on the vessel used).
See if the airflow goes to zero when the desired pressure is
reached inside the vessel and let the vessel under pressure a
while to see if it is maintained. To depressurize the vessel,
switch off the airflow and then open the tubing of exhaust
cooler. Perform the hold-up test also in the sampling system
and in the base bottle by connecting the gas supply to the inlet
filter of the bottle. To be sure that the vessel is properly sealed,
pressure should be maintained after the airflow reach zero for
at least 1 h. If the airflow does not reach this value, check the
sealing of all connections, probes, and vessel.
26. Before autoclaving the bioreactor, cover tubing, filter ends,
probes, and stirrer system with aluminum foil and tape them
with steam indicator tape. Clamp the aeration system if a
sparger with direct contact to the water inside the bioreactor
is connected, to avoid it exit during autoclaving. Open the
clamp from the exhaust cooler to allow pressure release during
sterilization.
27. After sterilization perform an hold-up test to make sure that
all the connections are fit properly (some of the cable ties can
be broken during autoclaving). If not, sterilize the vessel again.
28. The exhaust cooler has to be connected to the water supply as
soon as possible to avoid the water condensation and exit fil-
ters damaging.
