November 1-5, 2015
About 25 years ago, we first demonstrated the use of inclined settlers for selective removal of dead cells and cell debris and complete recycle of live and productive cells back to perfusion bioreactors with murine hybridoma cells (Batt et al., 1990) and Chinese hamster ovary cells (Searles et al., 1994). These devices have been scaled up as lamellar settlers (Probstein and Yung, 1979) and applied successfully over the last two decades for commercial manufacture of several therapeutic biologics in high cell density continuous perfusion bioreactor cultures of recombinant mammalian cells by different biotech companies. However, the rectilinear scale up of inclined settlers into lamellar settlers is not very efficient and creates a large footprint. Further, this powerful cell retention technology has not so far been demonstrated or applied for successful retention of microbial cells to our knowledge.
As microbial secretory expression systems have now become well developed for some larger microbial cells, such as yeast Saccharomyces cerevisiae and Pichia pastoris cells, it is now possible to develop high cell density continuous perfusion cultures of microbial yeast cells. While the previously demonstrated inclined or lamellar settlers can be used for such perfusion bioreactors for microbial cells, the size and footprint requirements of such inefficiently scaled up devices can be quite large in comparison to the bioreactor size. Faced with this constraint, we have now developed novel patent-pending compact cell settlers that can be coupled more efficiently with microbial perfusion bioreactors.
We have constructed several prototypes with minor design variations in stainless steel 316 and attached these compact cell settlers to computer-controlled bioreactors (5 liter and 50 liters) growing the yeast cells. Early results from these devices attached to 5 liter perfusion bioreactors demonstrate complete sedimentation of the larger live yeast cells and their immediate recycle back to the bioreactor, while the harvest stream at the top of these cell settlers is a significantly clarified liquid, containing less than 10% of cell concentration, (i.e. OD) of the bioreactor OD. Further the size of cells and debris in the harvest stream is strikingly smaller under microscopic observation, suggesting that we have now successfully demonstrated for microbial yeast cells the unique advantage of inclined settlers over all other cell retention devices in removing selectively the dead cells and cell debris from the perfusion bioreactor. We are now carrying more quantitative cell size analysis in more extended operation of microbial yeast perfusion bioreactors at 5 liter and 50 liter scale. The latest results from these on-going experiments will be presented.
Batt, B.C., R.H. Davis, and D.S. Kompala, Biotechnology Progress, 6: 458-464, 1990 Searles, J.A., P.W. Todd, and D.S. Kompala, Biotechnology Progress 10: 198-206, 1994. Probstein, R.F. and D. Yung, US Patent 4151084, April 1979.