App Note - Real-time cell density measurement for PAT applications
Total and Viable Cell Density In-situ Monitoring for Biopharma R&D
- Industry: Biopharma R&D
- Application: Perfusion and batch production of mAb using CHO cells
- Hamilton products: Incyte and Dencytee
Improve Bioreactor Productivity through Real-Time Measurement
The biopharmaceutical industry is constantly trying to gain a deeper understanding of their processes to improve productivity. A good example of this are initiatives such as the 2004 Process Analytical Technology (PAT) from the FDA.
During the production of monoclonal antibodies (mAb) control of the bioreactor throughout all phases of cell growth is critical to increase yield. Over time these control parameters have changed from discrete off-line measurements to continuous in-situ measurements. Continuous measurement in the bioreactor is already common for variables such as pH and dissolved oxygen. Recently more complex measurement parameters such as Total Cell Density (TCD) and Viable Cell Density (VCD) have entered the market. Studying these new parameters allows further monitoring and automation such as precise regulation of the nutrient feed based on the growth rate of cell cultures.
The Cell Culture Research Team of the University of Bielefeld (Germany) is actively studying the applications of new technologies to bioprocesses understanding. Part of their studies focuses on testing innovative in-situ sensor technologies.
Figure 1: Set-up of the experiments and Incyte sensor’s detail.
In-situ Measurement of Total and Viable Cell Density
Bielefeld’s Cell Culture Research Team set about to compare the accuracy of in-situ versus offline cell density measurements. They conducted both perfusion and batch experiments using CHO cell cultures for monoclonal antibody production.
Their experiment involved a 2 L bioreactor with pH and pO2 variables kept constant throughout the runs. Cell growth was monitored inside the same bioreactor using two different cell density sensors in parellel (Fig. 1 and 2).
One Incyte sensor was used to measure viable cells based on permittivity. The total cell density was measured, as well, using a Dencytee sensor: the parameter was determined by an optical measurement of turbidity at 880 nm wavelength. Throughout each experiment, off-line cell culture measurements
were taken daily to provide comparison data.
Good correlation between off-line measurements and data obtained from the Incyte and Dencytee sensor were observed in the growth phase for all experiments. The viable cell density measurement from the Incyte sensor highlighted higher accuracy in the log phase. In the perfusion cell culture correlation between in-situ and off-line measurements carried through for the death phase as well.
Benefits of Incyte and Dencytee Cell Density Measurement
- Continuous, accurate measurement of the cell growth enabling real-time control
- Better insight about cell health based on the parallel measurement of TCD and VCD
- Reliable and stable measurements for longlasting continuous fermentations
By measuring both TCD and VCD in parallel, it has been possible to verify that the bioprocess conditions have been ideal: the viable cell density signal being was really close to the total cell density one.
The results of the experiments are presented in the graphs (figure 3 and 4). The continuous green and blue lines represent the continuous measurement of VCD and TCD with in-situ sensors. The discrete red dots represent the daily measurement with the off-line reference method. In order to facilitate the performance comparison, the TCD raw data have been scaled to the VCD ones.
The experiments demonstrated:
- The real potential of in-situ cell density measurement. Compared to the off-line reference method, the Incyte and Dencytee allow a continuous and accurate measurement of cell density providing instant information about the status of the cell culture.
- The potential for rich, real-time process data by parallel
measurement of the TCD and VCD signals and comparison
with each other.
- The capability to deliver reliable and stable measurements
independently from the process set-up. Either for shorter
batch-processes as well as for long-lasting perfusion processes,
the performances are the same.
Figure 2: Dencytee sensor’s detail
The results prove that Incyte and Dencytee Cell Density sensors are reliable tools to provide valuable measurement data for both R&D bioprocess as well as PAT tools in production.
Figure 3: Performance comparison of cell density measurement for the perfusion set-up.
Figure 4: Performance comparison of cell density measurement for the batch set-up.
Dr. Heino Büntemeyer, Anica Schmidt
Zellkulturtechnik, Technische Fakultät, Universität Bielefeld
33615 Bielefeld, Germany