We developed a substitute for serum to produce fed-batch cultures of hybridoma cells in serum-free medium and confirmed that the cells could be successfully cultivated this way. Our substitute consisted of 12 components. The specific production rates of lactate and ammonia, which are harmful byproducts from the cells, were significantly reduced compared with a conventional serum-containing batch culture. This reduction led to a higher cell concentration and a longer production lifetime. As a result, the final concentration of monoclonal antibody was 400 mg/L, or five times greater than that in the conventional serum-containing batch culture. The developed substitute is expected to enable fed-batch cultivation in a serum-free condition.
Gas sparging directly into the culture-broth is not done in cell culture, except when the gas flow rate is very small, because much foaming occurs. During screening of defoaming methods, foam was observed to be broken up effectively when it made contact with a net fabricated from hydrophobic materials. Providing a highly efficient oxygen supply to suspension culture was tried using the new defoaming method. In a 5 l reactor equipped with the foam-eliminating net fabricated with polysiloxane, oxygen was transferred at 21 mmole/l.h equivalent to a consumption rate of 1 X 10(8) cells/ml, even at a low oxygen gas flow rate of 0.1 cm/s corresponding to a fourth of the gas flow rate when foam leaked through the net. Perfusion culture of rat ascites hepatoma cell JTC-1 was successfully carried out in the 5 l scale culture system with the net and a hydrophobic membrane for cell filtration. The viable cell concentration reached 2.7 X 10(7) cells/ml after twenty-seven days, in spite of the nutrient-deficient condition of the lower medium exchange rate, that is, a working volume a day, and viability was maintained at more than 90%. In a 1.21 scale culture of mouse-mouse hybridoma cell STK-1, viable cell concentration reached 4 X 10(7) cells/ml. These results showed that oxygen transfer by gas sparging with defoaming was useful for high density suspension culture. A foam-breaking mechanism was proposed.
For mammalian cell culture, getting a continuous supply of oxygen and extracting carbon dioxide are primary challenges even in the most modern biopharmaceutical manufacturing plants, due to the low oxygen solubility and excessive carbon dioxide accumulation. In addition, various independent flow and mass transfer characteristics in the culture tanks vessel make scale-up extremely difficult. One method for overcoming these and providing rational optimization is solving the fluid and mass transport equations by numerical simulation. To develop a simulation program, it is decisively important to know mass transfer coefficients of gaseous species in the culture tank. In this study, oxygen mass transfer coefficients are measured using a beaker with a sparger and impellers. In order to investigate the formulation of the mass transfer coefficients, the turbulent flow statistics is calculated by a CFD code for all cases, and the expressions of the mass transfer coefficients are established as functions of the statistics. Until now, the expression by Kawase is known in this field. This expression becomes a function only of energy dissipation rate epsilon. It does not coincide with the conventional experimental fact that mass transfer coefficient is proportional power 0.5 of impeller rotation speed. The new mass transfer coefficient is dependent on both of energy dissipation rate epsilon and turbulent flow energy k. It satisfies the relation of power of 0.5 of impeller rotation speed.
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