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Kinetics study of hybridoma growth and antibody production Henry, Olivier


Monoclonal Antibodies (MAb) produced by hybridomas have an expanding market for use in diagnostic assays, affinity separations and therapeutic applications. The objectives of this thesis were to study the kinetics of growth and antibody production of a hybridoma cell line in five different modes of operation and establish to what extent these kinetics are comparable from one mode to another. Hybridoma cells producing monoclonal antibodies were grown in batch, fed-batch, semi-continuous, continuous and perfusion cultures. The kinetics were compared in terms of growth rate, nutrient consumption rate and antibody formation rate. Batch cultures reached a maximum cell concentration of around 3x106 cells/mL and antibody concentration of 100 |j,g/mL. Glutamine was likely the substrate limiting the batch cultures since its depletion coincided with the end of exponential growth phase. No other amino acid was consumed completely and the levels of toxic metabolites (23 mM lactate; 2.6 mM ammonium) were much lower than those reported to affect growth or MAb production. A simple unstructured model was developed based on the batch data during the exponential phase. Simulations were performed and compared with experimental results obtained in semicontinuous and continuous cultures at dilution rates varying from 0.016 to 0.036 h"1. Cell yields from glucose in batch exponential phase were approximately 30% less than in the continuous processes. The cell specific glucose and glutamine uptake rates increased at greater dilution rates in both continuous and daily fed semi-continuous cultures. Comparing the cultures on the basis of specific dilution rates, perfusion results were in good agreement with semi-continuous and continuous data in terms of glutamine and glucose uptake rates. Although the specific antibody production rate only varied within a narrow range (0.7 to 2 pg/cell.h), the data suggest that the formation of antibody was growth-associated. It is concluded that modeling and analysis of semi-continuous data can describe continuous processes sufficiently well to be a useful tool for perfusion system optimization, while providing the relative simplicity of batch operation. In fed-batch, through the daily addition of a concentrate feed, cultures yielded antibody concentrations two times greater (200 (Xg/mL) compared to batch. This increase in final antibody concentration was closely related to the increase in the viable cell index.

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