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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
http://www.agen.com.au


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
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