Comparison of cell propagation methods for their effect on monoclonal antibody yield in fermentors

Abstract

Four different systems for the propagation and maintenance of hybridoma cells in fermentors were compared to batch cultures for their effect on cell viability and monoclonal antibody production. For these studies cells were grown as either batch, fed-batch, semi-continuous, 2-stage, or perfusion types of cultures. The hybridoma line employed produced a monoclonal antibody reactive with a somatic antigen on Rhizobium japonicum cells. It was previously shown that final yields in batch cultures ranged between 100–200 μg/ml with an average production rate of 15 mg/l culture per day. Daily addition of fresh medium to batch cultures (fed-batch propagation) raised the titer to over 200 μg/ml and increased the productivity to about 27 mg/l per day. In the semi-continuous system, medium was periodically (twice daily) added to the culture and an equal amount of the culture was removed. With a replacement rate of 200 ml/l per day, cells were maintained at a level of 2.4 × 10 6/ml for over 2 months. The antibody titers averaged 170 μg/ml and the productivity was 34 mg/l per day. Further increase in productivity was achieved by applying a second stage to the semi-continuous fermentation where cells were periodically fed with glucose and glutamine. With this additional stage, an antibody titer of 310 μg/ml and a productivity of 62 mg/l per day, was obtained. A perfusion culturing method was found to be the most effective for production of McAb. A cylindrical filter with 5 μm openings was mounted around the stirring shaft of a 1 liter fermentor. Cells were grown on the outside of the filter with continuous feeding of growth medium while, at the same rate, cell-free supernatant liquid containing antibody was removed from the inner part of the filter. Cells were propagated in this system on a continuous basis for about a month. After 12 days the system reached steady-state conditions in which concentration of live cells, dead cells, glucose, lactic acid, ammonium and McAb were relatively constant. Average concentrations of 2.2 × 10 7 live cells/ml and 390 μg of antibody/ml, corresponding to a productivity of 660 mg/l per day, were achieved.