ENHANCED ANTIBODY-PRODUCTION ASSOCIATED WITH ALTERED AMINO-ACID-METABOLISM IN A HYBRIDOMA HIGH-DENSITY PERFUSION CULTURE ESTABLISHED BY GRAVITY SEPARATION

A high density hybridoma perfusion culture was established by separati ng and recycling cells from the product stream to the reactor using a simple external sedimentation-based separator - an inclined modified E rlenmeyer flask. After 3 weeks, when the optimal perfusion rate of 1.0 day-1 had been reached, viable cell density stabilized at around 10 X 10(6) cells ml-1, a level five times that obtained by simple batch cu lture. The efficiency of the separator was enhanced by cell flocculati on. Specific antibody productivity, which was initially 0.4 mug 1 X 10 (6) cells-1 h-1, decreased to half that value while cell density was i ncreasing, but recovered to the initial level when the culture finally stabilized at a high cell density. During the final phase, when viabl e cell density and specific antibody production were high, there was a marked shift in metabolism. Consumption of the two most important sub strates for energy generation, glucose and glutamine, caused their bro th concentrations to decrease to 1.5 mM and 1 mM, respectively, from i nput medium concentrations of 25 mM and 10 mM, respectively. At the sa me time there was an increase in the specific production of glycine an d aspartate, their broth concentrations reaching 1.5 mM and 0.02 mM, r espectively. We suggest that this shift in metabolism results in enhan ced production of ATP from glutamine. The specific glucose consumption and lactate production also indicate that there is a shift to more en ergy efficient metabolism. The mechanism whereby this leads to enhance d specific antibody production remains to be elucidated. Nevertheless, the combination of high cell density and enhanced productivity obtain ed with the present perfusion culture resulted in a high monoclonal an tibody production - 100 mg l-1 d-1.