(582ah) Effect of Agitation Rate in Hybridoma Cell Culture On Monoclonal Antibody Production

In the medical field, the use of monoclonal antibody has attracted much attention in late years. It can be used not only for the diagnosis and treatment of cancer and other deceases but also for the purification processes of drug medicine which needs high purity. In cultivation of animal cells using large tanks, understanding about effect of agitation on cell growth and antibody production is needed for improvement of production efficiency. In the cultivation using an industrial scale tank, mixing by stirring is necessary for homogenizing cell population, oxygen, nutrient and temperature in culture broth. However, animal cells are vulnerable to shear stress by agitation. In this study, we used a 10(AAT)CloneA as a model hybridoma cell. 10(AAT)CloneA was cultured in Jar-fermenter (total volume 0.5 L) . Here we investigated the effect of agitation rate  on the efficiency of antibody production.

  10(AAT)CloneA (ECACC) was adapted to serum free medium(invitrogen) and batch culture in jar-fermenter. In each culture, temperature and pH were controlled at 37℃,7.4 ,respectively. We set the seven patterns of agitation rate, and examined the effect of agitation rate on the cells growth and antibody production. As the reference data, static cultures were carried out ,using a polystyrene petri dish with a diameter of 60mm(IWAKI), at 37 ℃,5%CO₂. We took a sample during cultivation once a day .We measured viable cell density and total cell density. Antibody concentration was also measured by Sandwich ELISA. We investigated the change of the protein expression inside cells with the variation of agitation rate, by using 2-dimensional electrophoresis and image analysis.

The viable cell density increased until 4 days and then decreased in cultivations at any agitation rate. This indicated cell proliferation was not severely damaged up to 360 rpm agitation rate. On the other hand, the antibody production in which agitation rate is more than 60 rpm, was only 60~70 % production of petri dish culture. The antibody production at 30 rpm agitation rate was more than antibody production in petri dish culture. Oxygen volumetric mass transfer coefficient (k_La) are 6.6(h^-1) in the jar-fermenter at 30 rpm agitation rate and 2.9(h^-1) in the petri dish culture, respectively. These results suggested that oxygen was supplied sufficiently and cells growth and antibody production are not severely damaged in culture broth at 30 rpm of agitation rate. To investigate the change of the protein expression inside cells caused by the difference in agitation rate, cells samples were obtained from cultures at 30 rpm and at 300 rpm, and petri dish cultivation, and their proteins were analyzed by protein analysis based on 2-dimensional electrophoresis and image analysis. From these results, it is considered that the amount of antibody production at 300 rpm of agitation was lower than production at 30 rpm because the nutrients needed for antibody production might be exhausted by the expression of proteins of major metabolic pathway. Many of proteins expressed inside cells in culture of petri dish were not expressed inside cells in culture of Jar-fermenter,  which suggested that the metabolism of cells in petri dish is much different from the one in the jar-fermenter.