Development of New Immobilization Method and Continuous Production of Bioproducts in Immobilized Mammalian Cell System

A novel immobilization method for animal cells, in which the alginate gel was covered with urethane polymer was developed. By this method, gel particles became resistant to physical stress, cell leakage was minimized and a high concentration of cells could be obtained. Use of the immobilized hybridoma cells in a fluidized-bed reactor improved the production of the monoclonal antibody. The production of monoclonal antibody increased with culture time and finally reached 300 mg/ml gel per day on day 17, which was eight times higher than the value obtained by the repeated-batch culture in spinner flasks. Using this method, we were able to introduce air by bubbling, which is the most effective oxygen transfer method. This bubbling caused free cells to die. In the case of the alginate gels, cell leakage was evident but the free cells soon died. In contrast, cells did not leak from the polymer layer and thus gave very good results which were obtained for hybridoma growth and monoclonal antibody production. In the immobilized cell culture with gel entrapment, diffusion of oxygen seems to be poor compared with the suspension culture. Therefore, we attempted to improve the oxygen supply by using pure oxygen gas instead of air. After reaching a stationary phase, oxygen gas containing 0–5% carbon dioxide was introduced at 10 d. By this change, although glucose concentration in the bioreactor was almost the same the lactate concentration decreased to about two-thirds of that under air. Furthermore, the antibody production rate increased 2-fold. It became feasible to obtain a high concentration of monoclonal antibody continuously for a long period.