Yoshihito Shirai

Continuous production of monoclonal antibody with immobilized hybridoma cells in an expanded bed fermentor

SummaryContinuous production of monoclonal antibody was achieved in serum-free medium by hybridoma cells immobilized by calcium alginate. The cells were cultivated in an expanded bed fermentor under mild flow conditions which reduced destruction of the immobilized gel particles. Monoclonal antibody was produced continuously for more than 40 days.

Oxygen uptake rate of immobilized growing hybridoma cells

SummaryThe specific oxygen uptake rate of hybridoma cells immobilized in calcium alginate gel particles was measured, and the observed data was compared with those of non-immobilized cells. The uptake rate of the immobilized cells coincided with that of the non-immobilized hybridoma cells just after immobilization, but increased with cell growth. On the other hand, the cellular glucose consumption rate decreased slightly during the experiments. The increased oxygen uptake rate by immobilized cells was closely related to the formation of cell colonies in the gel particles.

Formation of effective channels in alginate gel for immobilization of anchorage-dependent animal cells

SummaryThe conditions for formation of effective channels in alginate gels for growth of anchorage-dependent animal cells were examined. Many channels were formed in the gels by adding a low concentration solution of a high molecular weight polymer of alginate to a high concentration solution of divalent cations. It is recommended that an alginate with a high molecular weight and a low mannuronic acid/guluronic acid ratio be gelled by contact with strontium ions for the cultivation of immobilized anchorage-dependent cells because the gels produced have many channels and are mechanically strong.

Growth kinetics of hybridoma cells in high density culture

The growth kinetics of a mouse-mouse hybridoma cell, 4C10B6, in a high density culture were compared with those in a low density culture. Lower glucose and glutamine consumption rates were observed in the high density culture, resulting in lower lactate and ammonium production rates. On the other hand, the specific oxygen consumption rate did not decrease in the high density culture. Higher growth yields against glucose and glutamine were obtained in the high density culture than in the low density one.

Continuous production of erythropoietin with immobilized animal cells

SummaryErythropoietin, a glycoprotein that is a physiological stimulator of erythrocyte production, was produced continuously for more than 32 days by three kinds of anchorage-dependent animal cells immobilized in alginate gel particles. Gelation caused by divalent cations added to an alginate solution containing cells resulted in the formation of clearly vacant spaces (referred to here as channels) with prolate ellipsoidal shapes inside the gel particles. Each channel originated from a cell and extended towards the center of the gel particle. The animal cells grew well three-dimensionally in the channels but proliferated little outside the channels. Most of the channels had been filled with cells 2 weeks after immobilization. The cell concentration in the gel particles reached more than 1×107 cells/g gel. The alginate immobilization method was useful for high-concentration cultivation of the anchorage-dependent cells.

Continuous separation of α-cyclodextrin and glucose using a simulated moving-bed adsorber

Abstract α-Cyclodextrin and glucose were continuously separated by a simulated moving-bed adsorber made up of four or 12 columns packed with the sodium ion form of a cation exchanger. In both cases, the components were successfully separated. The effect of the number of columns constituting the adsorber on the recovery of α-cyclodextrin was examined theoretically. The use of more than eight columns resulted in almost the same recovery.

Effect of the content of divinylbenzene in ion-exchange resins on the chromatographic separation of α-cyclodextrin and glucose

In the chromatographic separation of alpha-cyclodextrin and glucose, the parameters relating to the adsorption equilibria and rate processes of the solutes were evaluated for the sodium forms of cation-exchange resins with contents of divinylbenzene (DVB) of 2, 4, 6 and 8%. By using these parameters, the chromatograms of the solutes were then calculated. It is concluded that the resin with 6% DVB is most suitable for the chromatographic separation of the solutes on a preparative scale.

Analysis of ice crystallization in continuous crystallizers based on a particle size-dependent growth rate model

Abstract To predict the ice crystal size distribution in MSMPR type crystallizers easily, the population balance equation with respect to the crystal size was solved under steady state conditions with a size-dependent growth rate model, that is G = g −1 / r + g 0 . The kinetic parameters g −1 and g 0 obtained from the measurement of crystal size distribution in a dextran solution were consistent with those estimated by assuming that the growth process of ice crystals is governed by heat transfer process. We confirmed that the kinetic parameter β, determined from experiments in a batch crystallizer, can be adopted as the parameter for the nucleation rate per crystal in an MSMPR type crystallizer.

Continuous Production of Monoclonal Antibody by Hybridoma Cells Immobilized in Hardened Alginate Gel Particlesa

Animal cells are now widely used to produce various kinds of physiologically active components that microorganisms can never produce. The cultivation of animal cells is much more difficult than that of microorganisms because of their low proliferation rates, their complicated nutritional demands, their low tolerance to mechanical stresses, and their strong growth inhibition by waste products produced during their growth, among other reasons. Moreover, productivity of a useful component by animal cells is much lower than that of microorganisms in a fermentor. Thus, it is essentially required to raise the cell density in the fermentor in order to improve the productivity. To cultivate animal cells in high density, a part of the medium is removed and fresh medium is supplied simultaneously to dilute the waste metabolite accumulated. While this is occurring, the cells should be maintained in the fermentor. Therefore, in high density culture of animal cells, the animal cells should be separated from the medium. However, the efficient separation of animal cells is not easy because of their small size. A possible way to overcome these difficulties is to employ the immobilization of animal cells in suitable materials. Immobilization of the cells by entrapment in particles makes separation of the medium and the cells easy and still enables the cells to grow to high concentrations. Moreover, the cells are protected from mechanical stresses by materials for immobilization that act as an artificial cell wall. Animal cells are highly sensitive to toxic compounds and to changes in the environment caused by chemical reactions. Because adverse conditions such as high temperatures and extreme changes in pH must be avoided, methods available for their immobilization are limited. Natural polymers made from seaweed can be used because of their mild gelation reaction and general suitability for use with cell. Nilsson et a1.24 developed methods for animal cell immobilization with agarose gel and succeeded in producing several useful agents. Lim et u I . . ~ have successfully established a microencapsulation method with polylysine and alginate for producing monoclonal antibody and others on a large scale. Alginate gels are also useful for the immobilization of animal cells because they are easily produced and do not inhibit cell growth. Many researchers have developed

Effects of Conditioned Medium on the Growth Kinetics and the Monoclonal Antibody Productivity of Hybridoma Cells

Monoclonal antibody is one of the most important and promising materials that can be produced by animal cells. Monoclonal antibodies have been widely used for diagnostics and therapeutic purposes for immunological and virus diseases [1, 2]. It is still expected to cure patients of cancers [1]. Monoclonal antibody is usually produced by hybridoma cells in industry. Therefore, the growth and monoclonal antibody production should be examined from an engineering point of view.