Sensitive removal of cells from the bioreactor, the addition of fresh media and the removal of cell debris for purification combined are a significant aid to cancer research. Carrie Bracco reports.
During the drug development process, many different cell lines are used to produce specific therapeutic protein. There are hundreds of permanent cell lines available to the researcher. Often the best cell line for protein production is not the best suited for large-scale suspension culture. These cell lines especially benefit from perfusion. Removing the metabolic waste produced by the dividing cells, and adding fresh nutrients to the bioreactor, promote a healthier environment.
Another added benefit to perfusion is that scientists can often increase the productivity of the cell line and, consequently, reduce the size of the bioreactor required to produce the quantities needed for clinical trials and final production. The smaller size bioreactor has many benefits, including lower capital cost and less space required in the pilot and production facilities. Depending on the stability of the cell line, the perfusion system can be harvested for a long period of time, resulting in higher yield versus production costs of batch culture.
The Sorvall and Centritech systems are used in the perfusion of cell cultures. These systems can sensitively remove the cells from the bioreactor, while fresh media is added and the supernatant, with cell debris, is removed for purification.
The concept of the Centritech systems allows the bioreactor feed to be pumped into the system, then the cells are drawn to the bottom of the rotor by centrifugal force. The cells are drawn back into the reactor and the supernatant and cell debris are collected. The whole system operates in a totally closed pre-sterilised bladder. This can be programmed to run continually or intermittently to turn over a fixed volume from the bioreactor.
In this case study a perfusion system using a bioreactor and the Centritech Lab II on a culture of mouse myeloma cells (NSO) is described.
Cell culture
A NSO cell line producing recombinant protein was studied. The cell line was a weak one and was viable for two to three days only. These cells were inoculated to a 10 litre bioreactor. The bioreactor was connected to the Centritech Lab II and the cell separation was performed using the pump mode.
The 10 litre bioreactor was perfused a half a volume for the first day and the full volume for five days. Cell density measurements were made daily.
Results
The perfusion with the Sorvall Centritech Lab II system increased the cell density and maintained viability of this very weak cell line. Before perfusion with Centritech Lab II system, the NSO cell line died within a few days. The test resulted in a cell density above 4 million/ml for three days (Fig. 1).
Also, the Centritechi is the only perfusion technology that removes dead cells from the bioreactor. The rotor speed of the Centritech can be reduced to separate the dead cells from the cell concentrate that is concentrated and processed back to the bioreactor. The Centritech can be optimised to remove the dead cells periodically throughout the cell culture production run.
Conclusion
The use of the Centritech Lab II perfusion system resulted in increasing the productivity of a difficult cell line. The increasing cell density resulted in an increase of product. The system's sensitive operation did not damage the sensitive cells, and maintained viability in the bioreactor. The increased productivity can eventually lead to using a smaller bioreactor. In this case, the plan was to increase the productivity of the cell line and keep the bioreactor the same size. The Centritech Lab II perfusion system gently processed from bioreactor volume up to two volumes per day.
Unlike other technologies the Centritech systems are extremely gentle on the cells, which makes it an ideal perfusion system for difficult or sensitive cell lines. Several modes of operation are available to eliminate the need for extremely sensitive cells from being processed through the peristaltic pump. Extremely low g-force separates the cells from the media. The system is versatile and can accommodate a variety of applications and cell culture needs.
Perfusion is often so effective in increasing the product yields that it is possible to decrease the size of the bioreactor needed for the process.
Scalability
The Centritech systems are simple to scale up. There are two systems available for perfusion, the Lab II for laboratory or pilot production and the Cell instrument for production volumes. Since the parameters that control the separation are g-force, time and flow rate, filter capacity or square footage issues are not a concern. The separation occurs in the same manner in the Lab II as in the larger Cell instrument. The size of the system would depend on the volume that will be processed in one day. Both systems use a pre-sterilised disposable insert.
ENQUIRY No 93
Carrie Bracco is with Kendro Laboratory Products, Newtown, CT, USA. www.kendro.com
Acknowledgments: Kendro Laboratory Products would like to acknowledge R&D Systems for data obtained from a Centritech Lab II evaluation performed at its facility in Minneapolis, MN, USA. R&D Systems produces and markets cytokines and adhesion molecules, corresponding antibodies, human and mouse ELISAs, as well as genes and probes.
