Kimihiko Hamamoto

High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge.

The influence of centrifugal force on the growth of cells was examined by exposing the cells of the mouse-human hybridoma X87 line to centrifugal force (100–500 G) for ten minutes twice a day and comparing the static culture with that of unexposed cells. In this experiment, both cell proliferation and specific antibody productivity were independent of the centrifugal effect, and gave the same results as in the case of no exposure to centrifugal force. High density cultivation of the mouse-human hybridoma X87 line was obtained by a perfusion system where the cells were separated from the culture medium by continuous centrifugation. In the serum-free culture, the maximum viable cell density exceeded 107 cells/ml, and monoclonal antibody was stably produced for 37 days. The results in this culture were equivalent to those obtained by intermittent centrifugal cell separation from the culture medium, and separation by gravitational settlement.

High cell density perfusion culture of hybridoma cells recycling high molecular weight components

We have developed a high cell density and high product concentration culture system recycling high molecular weight components. The production of monoclonal antibodies in high concentration was performed by this culture system with mouse human hybridoma H2 and V6 cells in serum-free defined media.The concentration of IgG after 48 days culture of H2 cells in ITES-eRDF reached 2 mg/ml and the purity of IgG in culture fluid was 61%. In addition, high molecular weight components in serum-free media, such as transferrin or BSA, could be reduced to 5% of the original concentration.

Large-scale perfusion culture process for suspended mammalian cells that uses a centrifuge with multiple settling zones

A high-cell-density perfusion culture process, using a novel centrifuge, was developed. The centrifuge has spiral multiple settling zones to separate cells from culture medium. Because of the multiple zones, the separation area can be efficiently increased without enlarging the diameter of the centrifuge. The centrifuge used in this study had a separation capacity of 2600 ml culture medium min−1 at 100g of the centrifugal force. A new cell separation and withdrawal method was also developed. The cells separated in the centrifuge can be withdrawn easily from the centrifuge with no cell clogging by feeding a liquid carrier such as a perfluorocarbon into the centrifuge and pushing the cells out with the liquid carrier. By this culture process, monoclonal antibodies were produced with mouse-human hybridoma X87X at a cell density of about 8 × 106 cells ml−1 for 25 days. This centrifuge culture shows promise as a large-scale perfusion culture process.

Large Scale Suspension Perfusion Culture Process with a Compact Centrifuge for Mammalian Cell Recycle

We have established an industrially competitive and original suspension perfusion culture process meeting the commercial production of pharmaceuticals. The system basically consists of usual culture vessel with an agitator and a compact external centrifuge for cell recycle, which facilitates expansion of production with scale-up potential highly increased. The cells separated in the centrifuge can be withdrawn easily from the centrifuge as the cells are kept viable and the system can continuously work for a long period of time with no cell clogging by feeding a liquid carrier such as perfluorocarbon into the centrifuge and pushing the cells out with the liquid carrier. Further, it can be widely applied not only to anchorage-independent hybridoma cells but also to 293 cells which are intrinsically anchorage-dependent cells but have been allowed to adapt to the suspension culture. These have been veri lied by the following results: I) Maximum separation capacity of the centrifuge attained so far is about 1.6 Umin at 100 G in hybridoma cells and large scale-up can be expected, 2) long operated-Umin at 100 G in hybridoma cells and large scale-up can be expected, 2) long operated-perfusion culture of about 4 months has been performed at (1-2)x107 cells/ml with hybridoma cens, 3) perfusion culture of 293 cells producing recombinant Protein C has maintained (1-2)x107 cells/ml over 2 months.