Kinetic characterization of hybridoma growth, metabolism, and monoclonal antibody production rates.

Abstract

Monoclonal antibodies produced by hybridoma cells are one of the most important products of biotechnology. Optimal design and development of bioreactors require a quantitative understanding of cell growth, metabolism, and antibody production rates. This thesis is a comprehensive investigation of the influence of culture environment on these biological variables. Both the extent of cell growth and the final antibody concentrations were influenced by the inoculum size, but specific growth, metabolic, and antibody production rates were less sensitive to initial cell density. Short-term exposure to new serum concentrations influenced the growth rate in a Michaelian fashion, but did not alter the cell metabolism and antibody production rate. When cells were cultured in low serum-containing media for prolonged periods of time (6 months), they adapted and both growth and antibody titer were improved. However, for one cell line, adaptation to low serum resulted in a gradual loss of antibody productivity. We have determined that this loss is due to the appearance of a sub-population that has lower internal and surface antibody content. Cell growth was inhibited at 100% air saturation and at very low dissolved oxygen concentrations leading to an optimal range between 25 and 50% air saturation. We have also demonstrated that the cells used in this study could grow and produce antibody under total anaerobic conditions, which has important implications for the design of high density cultures. The antibody production rate was unaffected by the dissolved oxygen concentration. Cell growth and antibody production were optimal at pH 7.2 while the specific antibody production rate, though unaltered under alkaline conditions, was 2-3 fold higher under acidic conditions. Elevated media osmolarity also influenced the specific antibody production rate. Both ammonia and lactate inhibit growth, but do not accelerate cell death. Cell metabolism was influenced by lactate and ammonia levels. However, the specific antibody production rate was unaffected. It is hoped that the results presented in this thesis will contribute significantly to a better understanding of cell physiology in bioreactor environments, and provide coherent design principles for the optimization of mammalian cell culture technology.