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Gram Quantities of MAb Produced with Simple Bioreactor in Serum-Free Perfusion Culture Replacing Ascitic Fluid Production

By J. Doyle, L. Johnstone and L. Cottis
AGEN Biomedical Ltd. Queensland, Australia

The CelliGen Plus immobilized-bed perfusion was investigated as a means for producing large quantities of MAb from a hybridoma, as an alternative to in vivo production. This system best represented a production facility that was easy to operate, flexible in its use, small enough to enable autoclaving and yet large enough to produce the required quantities of MAb. Three cell culture trials were performed using serum-free medium. Each run proceeded in the same manner, with MAb yields of 39.3, 33.8 and 30.3 grams. The perfusion system enabled us to scale down the size of the reactor needed, as the yields were significantly higher than that obtainable in batch fermentation which would have necessitated a bioreactor of several hundred liters.

Materials and Methods
All runs were performed in a 7.5 Liter NBS CelliGen Plus bioreactor with the basket impeller (New Brunswick Scientific, Edison, NJ). Working volume was 5 L and the bed volume was 2.6 L. Cells were inoculated at a cell density of 2 x 105 cells/mL at > 80% viability. Temperature was maintained at 37C, pH 7.20 and top dissolved oxygen (DO) at 50% of air saturation. The pH was controlled via the interactive pH-DO controller with CO2 at the initial stage and then with an 8% NaHCO3 solution when perfusion reached 2 volumes/day. Agitation rate was initiated at 80 rpm and increased throughout the run to maintain the bottom DO concentration at > 35% air saturation. The maximal rate did not exceed 150 rpm. Gas supply was via the ring sparger at a rate not exceeding 0.13 LPM and this was only increased when the top DO setpoint could not be maintained.

Gibcos Hybridoma Serum-Free Medium was used, supplemented with between 2 - 4 mM L-glutamine and 100 U/mL penicillin and 100 Ug/mL streptomycin. A separate 5% glucose solution was fed to the cultures when the perfusion rate reached 2 volumes/day to maintain the residual glucose concentration at > 1 g/L. The hybridoma cell line used produces an IgG1 MAb. Perfusion at 1 volume/ day (based on the bed volume), was initiated 24 hours after the commencement of the run, with increases to 2 then 3 volumes/day also 24 hours apart. The perfusion rate was maintained at 3 volumes/day for the duration.

Results and Discussion
The MAb produced in these cultivations has been traditionally produced from ascitic fluid with an average yield after purification of 5 mg/mouse. Yields produced using a contract service in 10 & 250 L batch reactors produced ~35 mg/L before purification. Using the CelliGen Plus perfusion system, yields of between 4 - 5 times that of the batch fermentation were obtained, based on purified material from the perfusion system but unpurified material from the batch system ( see table ).

All AGEN staff found the CelliGen Plus bioreactor very user friendly, easily simplifying an otherwise complicated and laborious procedure. The bioreactors display and associated BioCommand software provided fermentation information at the touch of a fingertip and trend graphing of process controls could also be viewed via BioCommand software. These features made the data handling, interpreting and report writing much simpler, something much appreciated by all staff. For CelliGen Plus literature contact your local NBS sales representative or write us at



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