FILTRATION-BASED PERFUSION OF HYBRIDOMA CULTURES IN PROTEIN-FREE MEDIUM - REDUCTION OF MEMBRANE FOULING BY MEDIUM SUPPLEMENTATION WITH DNASE-I

In this study, a filtration-based perfusion process was developed for the production of monoclonal antibodies (IgM) by suspended hybridoma c ells,grown in protein-free medium. It was found that the use of protei n-free medium for perfusion culture generated the formation of numerou s visible suspended particles consisting of dead cells and cellular de bris aggregated into fibrous material. Surprisingly high apparent viab ilities were observed in such protein-free cultures. In addition, memb rane fouling occurred more rapidly in protein-free medium than in conv entional serum-supplemented medium. By the addition of deoxyribonuclea se I (DNase I) to the protein-free medium, it was possible to prevent the formation of aggregates and to follow the evolution of the total c ell population more accurately. Moreover, DNase I significantly reduce d the fouling of filtration membranes, and that, for two different typ es of separation systems (cross-flow and vortex-flow filtration) and t wo different types of membranes (polycarbonate and hydrophilized polys ultone). From these results, it is clear that the presence of DNA frag ments liberated following cellular death is playing an important role in membrane fouling. Longevity of filtration membranes was found to be considerably greater using a vortex-flow filtration module than with a static plate-and-frame cross-flow filtration module. The use of vort ex-flow filtration in conjunction with DNase I allowed maintenance of perfusion cultures for more than 1 month without membrane fouling or a ntibody retention and with a constant permeate IgM concentration of 25 0 mg/L. Hybridoma cells appeared to gradually adapt to increasing rota tional speed in the vortex-flow filtration module. (C) 1994 John Wiley and Sons, Inc.