Glutamine replenishment and ammonia removal in hybridoma cell cultures via immobilized glutamine synthetase
AuthorOkeson, Carl D.
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PublisherThe University of Arizona.
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AbstractHybridoma cells utilize glutamine as their primary nitrogen source and excrete ammonia as a metabolic waste product. This ammonia can quickly accumulate to toxic levels in hybridoma culture media, and can severely reduce monoclonal antibody production (Ozturk et al., 1991). The enzyme glutamine synthetase (E.C. 188.8.131.52), which catalyzes the reaction UNFORMATTED EQUATION FOLLOWS: NH₄⁺ + L-glutamate + ATP Mg²⁺ → L-glutamine + ADP +Pᵢ +H⁺, UNFORMATTED EQUATION ENDS was evaluated as a means of reducing ammonia and replenishing glutamine in hybridoma culture medium. The effect of each enzyme reactant on soluble glutamine synthetase activity was quantified, and enzymatic reaction equilibrium evaluated. Enzyme reaction rates in two culture media, both with and without serum, were compared. Glutamine synthetase was immobilized via three different methods, and their effects compared. Cell sensitivity to each enzymatic reactant was studied. Finally, immobilized glutamine synthetase was incorporated in a hybridoma cultivation, and its effect on culture characteristics evaluated.
Degree ProgramGraduate College
Agriculture and Biosystems Engineering