Increasing Efficiency in Protein and Cell Production by Combining Single-Use Bioreactor Technology and Perfusion - An off-the-shelf, single-use perfusion system. - BioPharm International


Increasing Efficiency in Protein and Cell Production by Combining Single-Use Bioreactor Technology and Perfusion
An off-the-shelf, single-use perfusion system.

BioPharm International Supplements
Volume 24, Issue 5, pp. s4-s11


Figure 2: Glucose, glutamine and lactate concentrations over the course of the culture.
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).

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.

Figure 3: Biorector pH control and comparison between online and offline pH data.
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 4: Dissolved oxygen concentration (DO), gas flow, percentage of pure oxygen (O2) in gas stream, rocking rate and angle.
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.

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