EASE OF HANDLING
It was possible to obtain very high cell densities without the need for sophisticated equipment, fed-batch strategies or process
optimization by using off-the-shelf equipment with powerful control capabilities. The L–glutamine and glucose concentrations
were used to initiate the perfusion mode and to determine the medium exchange rate. When the glutamine concentration dropped
below 1 g/L, which was 66 h after inoculation, addition of fresh medium started. In order to maintain the glutamine concentration
at above 0.5 g/L, the perfusion rate was increased step-wise. After 138 hours, the perfusion rate was increased to 1.5/d (0.315
kg/h), and after 163 hours, to an exchange rate of 3/d (0.63 kg/h) (see Figure 2).
Figure 2: Glucose, glutamine and lactate concentrations over the course of the culture.
The perfusion process was terminated after 9 days when the perfusion membrane started to block; the perfusion control system
detected the reduced harvest flow rate and shut down the pumps. Consequentially, the remaining substrate was consumed, lactate
accumulated, and the logarithmic growth phase ended. Typically, in a production setting, one would directly move to harvest
of the supernatant. In our set up, we wanted to test the limit and robustness of the bioreactor system. In the case of cell
production, e.g., for inoculation of a production bioreactor or large scale cell banking in cryobags, the culture would be
terminated before reaching the limit of the perfusion membrane.
The signals from the disposable optical DO and pH sensors that are integrated into the single use bioreactor bag were used
to control the respective process parameters. pH was controlled mainly by adjustment of CO2 in the gas stream. Disposable optical sensor technology is a rather new, but versatile, technology which is suitable even
for challenging applications like this high cell density perfusion culture. Regular off-line measurement using a conventional
pH meter served to check the precision of the optical pH sensor, and to determine if recalibration of the optical pH sensor
would be necessary to compensate for a potential drift. Also, changes in ionic strength might affect the accuracy of the sensor
readout. During culture, the ionic strength might be influenced by accumulation of lactate or ammonia or other metabolic by-products.
A comparison of the pH values obtained from the disposable optical sensors and the off-line values is shown in Figure 3. At
the beginning of the perfusion process, one recalibration of the optical pH sensor based on the off-line value was performed.
During the course of the high density culture, pH could be maintained at the set point despite a change of the perfusion rate,
which temporarily led to an increased pH in the bioreactor.
Figure 3: Biorector pH control and comparison between online and offline pH data.
HIGH OXYGEN DEMAND IS EASILY MET
Process parameters affecting DO in a rocking motion bioreactor are shown in Figure 4. The DO could be maintained throughout
the whole high cell density culture at approximately 40%. The rocking rate was increased manually from 19 rocks/min to 21
rocks/min, and finally to 23 rocks/min. The angle was increased from 6° to 7° and finally to 10°. As the rocking rate and
angle increased, the wave formation in the bag became stronger, hence increasing the surface exchange rate at the gas-liquid
interface and ultimately the oxygen transfer rate (OTR) of the bioreactor. The OTR could further be increased by adding pure
oxygen to the process gas. Since we worked with moderate rocking rates, which leave room for further increase for most common
cell lines, it can be concluded that we are still far away from the upper limit of the oxygen transfer capacity that can be
achieved in this type of bioreactors. It should be noted that the increase of the rocking angle from 7° to 10° at process
time 160 h reduced the requirement of pure oxygen in the process gas dramatically.
Figure 4: Dissolved oxygen concentration (DO), gas flow, percentage of pure oxygen (O2) in gas stream, rocking rate and angle.