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Integration of Chemical-Optical Oxygen and pH Sensors in Existing Control Units
Cultivation monitoring in a glass bioreactor system with subsequently integrated sensor spots
Cedric Schirmer1, Vivian Ott1, Gernot T. John2, Dieter Eibl1
1Zurich University of Applied Sciences, School of Life Sciences and Facility Management, Institute of Chemistry and Biotechnology, Wädenswil, Switzerland
2PreSens Precision Sensing GmbH, Regensburg, Germany
The use of chemical-optical sensors for monitoring a cultivation using bioreactors with an already existing control unit requires the input of additional measuring signals. For this purpose, the digital signal often must be converted into an analog signal. This can be done by acquiring the online measurement data of the pH and dissolved oxygen (DO) sensor spot signals with the software PreSens Measurement Studio 2 and then converting them via a Complementary Straight Binary (CSB) Converter and transmitting them to the control unit. The signals sent to the control unit can be further used to monitor and control pH and DO profiles. The exact and correct functioning of the experimental setup could be verified in experiments with Chinese Hamster Ovary (CHO) and Escherichia coli cells and should also be suitable for use in combination with other bioreactor systems and control units from different manufacturers.
Chemical-optical sensors enable non-invasive monitoring of dissolved oxygen DO and pH in bioreactors and have proven themselves in single-use systems over the last few years. If the sensor spots are exposed to a light signal transmitted through polymer fibers, a measuring signal is generated which can be evaluated in digital form by the corresponding software. Many control units are still designed for operation with traditional electrochemical probes. In order to be able to use the chemical-optical sensors for cultivation monitoring, PreSens offers OEM solutions like Electro-Optical Modules (EOM) and stand-alone solutions for DO and pH measurements that can be integrated in control units. Both products enable the readout of sensor spot signals via polymer optical fiber cables connected to the sensor spots. The systems can be connected to a computer running the PreSens Measurement Studio 2 software, which allows for easy handling, calibration and readout of the sensor spot signals, as well as the recording of measurement data. When using a software extension and integrating a 2-channel CSB converter, the digital measurement signal can be transferred in analog form to a universal control unit with available analog inputs. The experimental setup was verified through cultivation with E. coli (data not shown) and CHO cells.
Materials & Methods
The experimental setup shown in Figure 2 was used to transmit the sensor spot signals to the control unit. In the first step, the pH (SP-LG1-SA) and the DO (SP-PSt3-YAU) sensor spots were placed in the lower part on the inner surface of the stirred glass bioreactor. Polymer fiber cables were indirectly connected to the sensor spots over the glass bioreactor wall from the outside (1). In order to generate measurement signals and transmit them, an EOM-pH-LG1-mini and a stand-alone Fibox 4 (both from PreSens, Germany) were coupled to the other ends of the polymer fiber cables connected to the pH and DO sensor spots (2). The measurement signals acquired by the EOM-pH-LG1-mini and the Fibox 4 were transmitted via USB interface to a computer running the PreSens Measurement Studio 2 software (3). The software was used to collect and record the data in a 30-second interval (adjustable in a range from 1 s to several hours) and to calibrate the sensor spots. For converting the digital signals, a 2-channel CSB converter (PreSens, Germany) was also connected to the computer via USB (4). For this purpose, output channels were assigned to the individual sensors in the PreSens Measurement Studio 2 software to ensure data transmission to the respective channel of the 2-channel CSB converter. The analog signals, ranging between 4 to 20 mA and made available at the respective output channel of the CSB converter, were further transmitted to analog inputs of the control unit (KLF, Bioengineering, Switzerland) and used for the process control of pH and DO (5).
CHO cell cultivation
The CHO cell cultivations were performed with the CHO cell line XM111-10 (CCOS no. 837). This cell line has a tetracycline-regulated promoter that regulates the expression of the secreted alkaline phosphatase (SEAP) gene, which codes for secretory alkaline phosphatase . In the experiments performed, the focus was on mass propagation in batch mode, so that the synthesis of SEAP was prevented by supplementing the ChoMaster® HP-1 medium (Cell Culture Technologies, Switzerland) with 2.5 mg L-1 tetracycline. Furthermore, 0.2 % of Pluronic F-68 was added to the medium. The bioreactor was inoculated with a viable cell density of 0.5 x 106 cells mL-1 at a culture volume of 600 mL and cultivated at 37 °C with 0.44 vvm surface aeration (process air). In order to maintain a DO ≥ 40 % and pH ≤ 7.2, oxygen and CO2 were sparged as required. During the 4 day cultivation period, a sample was taken every 12 hours to determine cell growth. Furthermore, pH was measured externally with each sampling and recalibrated if necessary.
Figure 3A shows an example of viable cell density trend during a CHO cell cultivation. With an initial inoculation of 0.4 x 106 cells mL-1, a maximum cell density of 3.6 x 106 cells mL-1 was achieved after a 3 day cultivation period, which is comparable with previous results . Within the first 1.5 days of cultivation, an exponential growth phase with a specific growth rate of 0.042 h-1 was observed. The resulting pH fluctuations between 7.08 and 7.25 during this period (Fig. 3B) were due to the lack of a suitable CO2 mass flow controller, combined with the 30-second measuring interval of the chemical-optical sensor spot. The subsequent decrease of the pH value, which lasted until day 2, and its subsequent increase, until day 2.6, occurred as anticipated through lactate formation and catabolisation. Thereafter, the pH value was maintained between 6.87 and 7.25 until the end of cultivation, with its increased fluctuation range owed to the reasons mentioned above. The DO fluctuated between 30 % and 45 % during the entire cultivation. As expected, the viability decreased from 96 % to 2 % due to the depletion of glucose and lactate from day 3 on, resulting in the termination of the cultivation on day 4.
The integration of chemical-optical pH and oxygen sensor spots in an existing control unit was successfully evaluated using CHO cells in a stirred bioreactor system. The experimental setup, consisting of an EOM-pH-LG1-mini module and the stand-alone Fibox 4 in combination with the PreSens Measurement Studio 2 software and a 2-channel CSB converter, used for process control, provided precise measurements throughout the investigations. No functional or technical problems could be detected during the entire test period. The integration and configuration of the experimental setup was realized within a few minutes and is suitable for incorporation into various commercially available bioreactor systems and control unit combinations. It is worth mentioning that the aproach used, not only works for CHO cells, but also for E. coli cells (data not shown).
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 Schirmer C, Nussbaumer T, Schöb R, Pörtner R, Eibl R, Eibl D. Development, Engineering and Biological Characterization of Stirred Tank Bioreactors. In: Yeh M-K, Chen Y-C, editors. Biopharmaceuticals, InTech; 2018, p. 87–108. doi:10.5772/intechopen.79444.