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Optrode Dual connects Single-UseBioreactors to Conventional ControllersMonitoring of CHO Cell Cultivation with Chemical Optical SensorsStephan Kaiser, Franziska Fietz, Nina Steiger, and Dieter EiblInstitute for Biotechnology, Department of Life Sciences and Facility Management, Zurich Universityof Applied Sciences, Switzerland The Optrode Dual prototype has been applied for measurements in suspension adapted CHO cell
culture. The transmitter converts the reading of chemical optical sensors integrated in the
cultivation bag into electrochemical signals, which are transferred to a conventional controller. CHO
cell culture was successfully conducted in this set-up for 5 days. Correct functioning of the Optrode
Dual throughout the whole cultivation period was verified even though operational errors occurred.

Most controllers are designed to work with electrochemicalsensors for oxygen and pH monitoring. The Optrode Dualwas developed to convert the reading of chemical opticalsensors into an electrochemical signal (ECS) which can betransferred to the controller. This way the functionality ofconventional controllers can be expanded to work withoptical sensors for measurement of the important cultureparameters oxygen and pH. The device also allows fail-safecalibration of optical sensors via 2D barcode control. In thefollowing tests a prototype of the Optrode Dual wasevaluated for its functionality in combination with theez-control® by Applikon. Chemical optical sensorsintegrated in a cultivation bag were read out and their Fig. 1: The Optrode Dual is connected to the ez-control® by Applikon. Measurements signal successfully converted and transferred to the were taken in a 20 L cultivation bag with integrated optical sensors running on aBioWave 20 SPS. The sensors are connected with the Optrode Dual and data controller. The combination of the systems of different inoculated with 0.5 x 106 cells mL-1 with a starting volume of 2.4 L. Approx. 48 hours and 72 hours after inoculation 3L, respectively 5 L of fresh culture medium were added.
The Optrode Dual prototype was connected to the Angle and speed of movement of the cultivation bag were ez-control® (Applikon, Netherlands) which was connected adapted to the filling volume of the bioreactor, to ensure via Ethernet cable to a PC for data collection (Fig. 1). Data optimal mixing. Samples of about 5 mL volume were taken recording for dissolved oxygen and pH was realized with four times a day. Cell count and determination of cell the software BioXpert (version 2.93.122b2) with a viability were performed automatically with NucleoCounter sampling rate of 1 min. Calibration of the optical sensors NC-100 (chemometec, Denmark). Furthermore, pH was integrated in a 20 L cultivation bag was performed with the measured off-line with a pH meter (Mettler Toledo, Optrode Dual . Therefore, a specific barcode was generated with the software QR Code Generator in manual mode. Thecultivation bag was placed on a BioWave 20 SPS platform (Wave Biotech, Switzerland). Suspension adapted CHOcells (CHO XM111-10, obtained from Fussenegger et al., Functionality of the Optrode Dual during cultivation of ETH University, Zurich) were used for cultivation, which suspension adapted CHO cells was tested in this set-up.
have a tetracycline regulated promotor for SEAP (secreted The change of cell density and viability throughout the alkaline phosphatase) expression. However, only a growth whole cultivation period is shown in Fig. 2. The initial experiment was performed without product formation. The density of 0.5 x 106 cells mL-1 increased to a value of 2.11 x cultivation bag was filled 3 h prior to inoculation with 1.5 L 106 cells mL-1 within 48 hours, showing a growth rate of chemical defined medium (CHO Master HP1, Cell Culture 0.03 h-1. 25 hours after inoculation cell growth was Technologies) containing tetracycline (2.5 mg L-1) and stagnating. The bioreactor movement rate of 15 rpm was Pluronic F68 (2 mg L-1). The medium was conditioned to 37 raised to 19 rpm and gassing of 0.2 slpm (standard liter °C and aerated till it was saturated. The bioreactor was per minute) was set to 0.4 slpm (0.1 vvm to 0.2 vvm) Fig. 2: Cell density and viability during CHO cell cultivation in a 20 L cultivation bag.
Fig. 3: Online measurement data for dissolved oxygen and pH; offline measured pH The arrows indicate addition of HP-1 cell culture medium.
values are also shown for comparison. The arrows indicate finalizing therecalibration of the optical pH sensor (a), the increase in supply rate (b and c), andthe termination of CO which reestablished growth in the culture. After 48 hours fresh medium was added to the bioreactor and another increased from 75 % to 80 % due to decreasing cell growth.
period of exponential growth could be monitored with a The last increase in DO was recorded after 110 hours doubling rate of 23 hours. 72 hours after inoculation of cultivation time, when CO2 supply was turned off. pH was another 5 L of medium were added to the bioreactor.
measured offline to verify the online sensor reading.
Maximum cell density of 2.88 x 106 cells mL-1 was reached During the whole cultivation acceptable differences of after 100 hours of cultivation. In the last 24 hours cell below 0.2 pH units could be detected with offline density was decreasing and reached 2.15 x 106 determined values always being higher. This was probably cells mL-1 with 98 % viability when the cultivation was caused by the time difference between sampling and terminated. Online recorded data for dissolved oxygen measurement, during which pH regulating CO2 might have (DO) and pH are shown in Fig. 3. During the first 6 hours of escaped the medium and caused pH to increase.
cultivation a constant pH value of 8.22 was measured,because the calibration of the sensor was not conducted correctly due to an operational error. The barcode was read The test described here was designed to evaluate correct by the Optrode Dual but measurement was not restarted.
functioning of the Optrode Dual for monitoring CHO cell Furthermore, the CO2 supply was not functioning correctly, culture. The device was successfully connected to the so only after 10 hours the pH value could be adjusted to optical sensors in the cultivation bag and accurate 7.2. Because of the increased streaming of CO2 into the measurement data could be obtained. During tests no bag the dissolved oxygen level decreased from 92 % to 77 functional and technical errors occured for the whole 5 day % and was rising again after gassing was correctly adjusted period. The Optrode Dual proved to be a reliable tool for to 0.2 slpm with 10 % CO2. A further increase in the DO level measuring culture parameters with optical sensors and was recorded after 24 hours when gassing and movement transferring data to a conventional controller. It is time of the bioreactor were changed. To determine the weight of saving as it can be easily connected to the electrochemical the bioreactor during medium addition it was taken off the inputs of the controller and no further changes of controlling system, this is why DO reading stopped at controller settings are necessary. With the Optrode Dual it hours 48 and 72. At the same time the pH value rose with is possible to save costs as the purchase of new process adding the slightly basic medium for 0.1 and 0.3 pH units, analysis tools for reading optical sensors might not be respectively. After 77 hours of cultivation another increase necessary, and non-invasive monitoring of pH and oxygen in DO could be investigated, caused by the second increase in gassing to 0.5 slpm. From hour 100 on DO Bring to light what‘s inside. Ask our experts: PreSens Precision Sensing GmbH

Source: http://www.presens.de/uploads/tx_presensapplicationnotes/121030_APP_OptDual_CHO_web.pdf

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