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Dev Biol Stand. 1985;60:185-97.

Production of monoclonal antibodies in culture.


Factors that affected the production of monoclonal antibodies by a mouse-mouse hybridoma cell line, propagated in vitro in stirred vessels, were investigated. The purpose of the research was to estimate the efficiency of this system for large scale production of monoclonal antibodies. The antibody produced by these hybridoma cells was an IgG2a, specific for a surface antigen on Rhizobium japonicum NR-7 cells. Antibody content in the culture supernatant was determined by a radial-immunodiffusion assay using rabbit anti-mouse IgG antibodies in the immobile phase and mouse IgG (the monoclonal antibody) as the antigen in the mobile phase. This method was found to be more reproducible and reliable compared with an ELISA method. Cells were adapted to grow in an inexpensive, low protein content medium based on Dulbecco's Modified Eagle Medium (DMEM) supplemented with 0.25% Primatone RL, 0.01% Pluronic polyol F-68 and fetal bovine serum as low as 1%. Doubling time for the cells averaged 24 hrs, and final yields reached 2 X 10(6) cells per ml. The hybridoma cells were grown in the newly developed medium in 3 liter fermentors. Monoclonal antibody was produced during the early growth phase (3 days), however, most of the antibody was produced during a later growth phase (3-10 days) when 30 to 90% of the cells were dead. Final antibody yields were estimated to be 100-200 micrograms/ml. A low level of dissolved oxygen (25% air saturation) in the culture was found to increase the amount of antibody produced as compared with cells propagated at 60% air saturation (the optimal level for cell propagation) since the cells were kept alive for longer periods at the lower dissolved oxygen concentration. Using a fed-batch propagation method we were able to keep cells alive for long periods (up to 1 month) at a concentration of about 1 X 10(6) cells per ml, and thus to increase further monoclonal antibody production. Yields of 300-400 micrograms/ml were obtained.

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