Enhancement of Monoclonal Antibody Production Related to Metabolic Behavior in High Density Hybridoma Culture

Abstract

The influences of dissolved oxygen (DO) concentration and culture pH on specific monoclonal antibody (MoAb) productivity of hybridoma cells were examined. Perfused, fluidized-bed, porous collagen microsphere system (Verax) as high density cell culture system was used for this experiment. To compare with the data of the suspension culture using stirred-vessel perfusion culture system, we analyzed the properties of cellular metabolic behaviors by controlling environmental conditions. We used mouse-mouse hybridoma producing IgG1 type MoAb. By using the Verax system, we succeeded in long-term perfusion culture of over 300 days. We monitored the concentrations of substrates (glucose, glutamine) and metabolites (ammonia, lactate) under various DO concentrations and culture pH, and determined optimum DO concentration and culture pH. By controlling these factors, we succeeded in enhancement of monoclonal antibody productivity. I Nl'RODUCfIONMoAb is useful for biochemistry, chemical industry and medical science. For the commercial scale production of MoAb, cells must be grown efficiently at high density with high MoAb productivity. In hybridoma culture, it has been reported that environmental factors such as 00 concentration have important effects on cell growth and MoAb production (1,2,3). We have been investigating about high density culture using fluidized-bed, microsphere culture system (Verax). But there are little previous studies on the effects of environmental factors on this kind of culture systems. Therefore these experiments were carried out to demonstrate the effects of environmental factors such as DO concentration and culture pH on MoAb production. In addition,the correlations between cellular metabolic behaviors and cell growth and also MoAb production were investigated. 408 MATERIALS AND METHODS Cell Line and MediumA mouse-mouse hybridoma producing an IgGl kappa antibody was used in this work. E-RDF medium was purchased from Kyokuto Seiyaku Kogyo (Tokyo. Japan) (4). Fetal calf serum (FCS) was purchased from Commonwealth Serum Laboratories (Melbourne. Australia). Cell Culture SystemPerfusion culture was carried out with two kinds of culture systems. As the suspension culture system. we used a stirred vessel named AXELLON (2L working volume. 5L total volume) equipped with a helical screw agitator and a bubble-free oxygenation system (Kirin Brewery. Yokohama. Japan) (5). The oxygenation was performed with a coiled gas-permeable silicon tube. Perfusion culture was performed with a rotating stainless steel membrane filter system. Benchmark GX(Membrex. Fairfield. NJ. USA) and was done after 4 days. Perfusion rate was 0.5/day. E-RDF medium containing 3% FCS was used. DO concentration was controlled at 5ppm and agitation rate was kept at 40rpm.As the fluidized-bed. porous collagen microsphere system. we used Verax System One (6.7). We measured DO concentration. culture pH and cuI ture temperature at the outlet of the reactor. Cells were immo~ilized in microsphere with E-RDF containing 1% FCS. About 1X10 cells wer...