Takashi Mano

Immobilization of hybridoma cells with alginate and urethane polymer and improved monoclonal antibody production

SummaryHybridoma cells producing anti-α-amylase monoclonal antibody were entrapped in calcium alginate and the gels were then coated with urethane polymer. The urethane coating improved gel strength and prevented cell leakage. This immobilization method enabled direct air bubbling in the serum-free medium and a very high cell concentration (3×107 cells/ml gel) was obtained. By using a fluidized-bed reactor, effective removal of the medium in addition to sufficient oxygen supply could be achieved without any special devices and a very high concentration of the monoclonal antibody was continuously obtained.

A general framework for the assessment of extractive fermentations

Abstract A mathematical framework was developed for the assessment of batch, repeated-batch and repeated fed-batch extractive fermentations of acetone and butanol or ethanol. Several useful expressions were derived for determining the noninferior set defined in the two-objective function space, which is concerned with the productivity and the concentration of the metabolic products. The computer simulation based on the experimental data revealed the possibility of attaining a significant improvement for extractive fermentations as compared with conventional fermentations without extraction.

Kinetic expression for human cell growth in a suspension culture system

Abstract The effects of lactate and ammonium concentrations on the growth of HL-60 and RPMI 8226 human cells were kinetically evaluated in a suspension culture system. The specific growth rates of the cells were expressed as the following equation with the terms of the inhibitory effects owing to lactate, ammonium, and glucose. mu;=mu; m S {(K s +S)(1+ P 1 k 1 + P 2 k 2 )+ S 2 k 3 } On the basis of this equation, it was possible to stimulate the culture processes of the human cells.

Overexpression of CHOP alone and in combination with chaperones is effective in improving antibody production in mammalian cells

Secretory capacities including folding and assembly are believed to be limiting factors in the establishment of mammalian cell lines producing high levels of recombinant therapeutic proteins. To achieve industrial success, it is also important to improve protein folding, assembly, and secretory processes in combination with increasing transcription and translation. Here, we identified the expression of CHOP/Gadd153 and GRP78, which are unfolded protein response (UPR)-related genes, correlated with recombinant antibody production in stable CHO cells. Subsequently, CHOP overexpression resulted in increasing recombinant antibody production in some mammalian cell lines, and in addition a threefold further enhancement was obtained by combining expression with UPR-related genes or ER chaperones in transient assays. Overexpression of CHOP had no effect on the biochemical characteristics of the product. These results suggest overexpression of CHOP and its combinations may be an effective method to efficiently select a single cell line with a high level of antibody production in the development of cell lines for manufacturing.

New immobilization method of mammalian cells using alginate and polyacrylate

Abstract Mouse-mouse hybridoma cells were immobilized in polyacrylate-alginate gels. The immobilized hybridoma cells were cultured semi-continuously using a fluidized bed reactor, and allowed continuous antibody production without any gel destruction for one month. It has been proved that the polyacrylate-alginate gels were tolerant against physical stress. The composition of the gels suitable for cell growth and antibody production was given as follows; viscosity of alginate at 1% solution: 60–100 cP, alginate concentration: 0.8%, and polyacrylate concentration: 0.2%. In the semi-continuous culture using gels prepared under suitable conditions, the viable cell number was estimated as 2.5×10 7 cells/ml-gel, and the antibody production rate was 2.2 mg/ml-gel/d, at maximum.

Continuous culture of RPMI 8226 human cells

Abstract Continuous culture of RPMI 8226 human hematopoietic cells was performed. The viable cell number and glucose, lactate and ammonium concentrations became constant within 3–4 days at a constant dilution rate. The viable cell number decreased at low and high dilution rates. The growth and product yields slightly depended on the dilution rate, except for product yield for lactate based on cell number. Growth characteristics of these cells at various dilution rates could be expressed by equations considering the maintenance energy in growth yield. Maximum specific growth rate could be evaluated from the wash-out profile and the known inhibition constants.