A Reliable Automated Sampling System for On-Line and Real-Time Monitoring of CHO Cultures
- Quantification of the automated liquid handling and analysis
- Optimization and adaptation of an HPLC method for amino acid measurement
- Application on a mammalian cell culture fed batch
- Comparison to other real-time monitoring systems
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. HPLC Methods
2.3. Biochemical Methods
2.5. The Automated Sampling System
2.6. Software and Data Management
2.8. Technical Run
3.1. System Performance
3.2. Monitoring—HPLC Autosampler Required
3.3. Monitoring—HPLC Direct Transfer
3.4. Monitoring—Bio HT Direct Transfer
4.1. Automated Sampling in General
- Does the system impact the sterility of the bioprocess?—No.
- How much volume and how much dead volume are drawn from the bioreactor?—The sample volume is approx. 3.5 mL, of which about 1 mL is dead volume.
- Does the system support sample processing (i.e., dilution, cell removal etc.)?—Yes.
- Does the automated procedure impact the analytical result (i.e., dilution effects)?—There is a constant dilution factor observable, which can be included in final calculations.
- How is the communication between process, sampling system, and analyzers realized?—The communication was achieved with one software (Lucullus PIMS) that coordinated the sample draw as well as the transfer of the sample to the analyzer and the initiation of the measurement.
4.2. Assessment of the Presented Liquid Handling System
- The system was tested for CHO cells with a maximum viable cell density of about 12∙106 cells/mL, resulting in a required adaptation of the POT to reach the desired volume of permeate after filtration. Currently, the trend is towards high cell density cultivations in perfusion mode, which were not tested in this contribution. The actual filter area in the filtration module is 3.14 cm2, which is filtering 2.5 mL of cell suspension. An increase of the filter area might have a positive impact on the permeate volume.
- During the operation of the system (especially when applying the Protein A HPLC method), it was observed that the precolumn had to be changed approximately 8–10 times during one cultivation run. The required changing frequency increased with progress of the process. This leads to the conclusion that the load of particles of the samples after the automated filtration is higher than after manual cell removal. A reason could be the filtration procedure per se or the applied pore size of the filter of 0.45 µm. Possible solutions to overcome this problem are (i) the use of a membrane with 0.22 µm pore size or (ii) the introduction of a prefiltration step or a dual filtration.
- 1–2% of the samples resulted in empty vials after the filtration step.
4.3. Comparison with In-Line Sensors
- Depending on the preprocessing and the applied analytical method, systematic deviations were observed. They were mainly caused by dilution effects and can be assumed to be constant. On the other hand, the random error seems to be significantly reduced.
- An existing HPLC method for amino acid analysis was successfully adapted in a way that it can now be applied for full automated on-line monitoring.
- The automated sampling and analytic system was successfully tested in mammalian cell culture fed-batch processes. The monitoring of various analytes was performed without significant errors or system failures. The higher measurement frequency and strongly reduced random errors resulted in a larger information content per experiment.
- The accuracy of the system is an order of magnitude better than the compared methods.
Conflicts of Interest
|Sample||VCC [106 Cells/mL]||TCC [106 Cells/mL]||Viability [%]||Debris * [106 Cells/mL]|
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|Correlation||Slope||Offset||Confidence Interval Slope (95%)||R2|
|Off-line vs on-line||0.8409||0.0056||0.8216–0.8602||0.9981|
|Off-line vs on-line calibrated via Numera||0.9696||0.0133||0.9474–0.9918||0.9981|
|Off-line vs on-line considering DF||0.9973||0.0067||0.9745–1.0202||0.9981|
|Reference Method (HPLC)||Automated Sampling and On-Line HPLC||Raman Spectroscopy|
|cv(RMSE) [-]||cv(RMSE) [-]||cv(RMSE) [-]|
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Hofer, A.; Kroll, P.; Barmettler, M.; Herwig, C. A Reliable Automated Sampling System for On-Line and Real-Time Monitoring of CHO Cultures. Processes 2020, 8, 637. https://doi.org/10.3390/pr8060637
Hofer A, Kroll P, Barmettler M, Herwig C. A Reliable Automated Sampling System for On-Line and Real-Time Monitoring of CHO Cultures. Processes. 2020; 8(6):637. https://doi.org/10.3390/pr8060637Chicago/Turabian Style
Hofer, Alexandra, Paul Kroll, Matthias Barmettler, and Christoph Herwig. 2020. "A Reliable Automated Sampling System for On-Line and Real-Time Monitoring of CHO Cultures" Processes 8, no. 6: 637. https://doi.org/10.3390/pr8060637