Use of the Centritech Lab Centrifuge for Perfusion Culture of Hybridoma Cells in Protein-Free Medium Mark Johnson, †,‡,§ Ste ´ phane Lanthier, †,‡ Bernard Massie, ‡ Gilles Lefebvre, ‡ and Amine A. Kamen* ,‡ Biomira Inc., Edmonton Research Park, 9411 20th Street, Edmonton, Alberta, Canada T6N 1E5, and Groupe d’Inge ´nierie des cellules animales, Institut de recherche en biotechnologie, Conseil National de Recherches du Canada, 6100 Royalmount Avenue, Montre ´al, Que ´bec, Canada H4P 2R2 As part of an effort to develop a suspension-culture perfusion-based process with high flow rate without the fouling and antibody retention inherent to filter-based cell- separation devices, we have evaluated and contributed to the development of the Centritech Lab centrifuge for the perfusion culture of hybridoma cells in protein-free medium. Culture start-ups showed that cell growth and monoclonal-antibody (MAb) production rates were similar in both a spinner flask and continuous centrifugation coupled to a bioreactor. The centrifuge efficiently separated viable cells from dead ones. Viable-cell recoveries were never below 98%, whereas dead-cell recoveries were usually around 80%. The cell content of the centrifuge supernatant and concentrate was strongly determined by the total amount of cells, viable and dead, in the culture broth, but an influence of the centrifugation parameters (feed rate, times of separation and discharge, and rotor speed) was observed. This understanding of the separation process inside the centrifuge is important and may apply to other similar devices. Monoclonal antibodies were not retained in the bioreactor during centrifugation perfusion. However, whereas similar growth rates were obtained in perfusion cultures using either continuous centrifugation or filtration, MAb concentrations were 35% lower in the former case. Utilization of the centrifuge in an intermittent fashion decreased the daily cell residence time outside the bioreactor, the daily pelleted-cell residence time in the centrifuge, and the frequency of cell passage to the centrifuge. This led to higher viable-cell numbers in the bioreactor and an accompanying increase in MAb concentrations, 225-250 mg of IgM L -1 , equal to the performance of filter- based perfusion systems with the same cell line. It was hypothesized that having cells periodically packed at the bottom of the centrifuge insert (up to 800 × 10 6 cells mL -1 ) is deleterious to the culture by exposing the pelleted cells to prolonged nutrient limitations. Introduction As the commercial potential of animal cell products increases (Spier, 1993), interest in perfusion processes grows (see 1994 and 1995 Meeting Proceedings of the European Society of Animal Cell Technology and the Cell Culture Engineering). The recent approvals of recombi- nant Factor VIII from transfected BHK cells (Kogenate) in the U.S. and the monoclonal antibody Centoxin in Europe mark the growing acceptance by the international regulatory agencies for continuous-perfusion culture with batchwise purification for the production of biopharma- ceuticals from mammalian cell lines (Bo ¨deker et al., 1994a,b). Perfusion processes can eliminate the nutrient-limita- tion and inhibitor-accumulation problems inherent to animal cell-suspension cultures. High viable-cell densi- ties and product yields have been observed using perfu- sion for the production of monoclonal antibodies (MAbs) (Banik and Heath, 1994; de la Broise et al., 1991; Flickinger et al., 1990; Hiller et al., 1993; Mercille et al., 1994; Mohan et al., 1993; Munster et al., 1991; Smith et al., 1991), and perfusion offers the added advantage of rapid removal from cultures of easily inactivated products (Prior et al., 1989). Procedures for cell-spent medium separation in per- fusion systems include cell encapsulation or immobiliza- tion, filtration, and gravitational sedimentation, but few of these methods enable easy scale-up and long-term trouble-free operation (de la Broise et al., 1992; Mercille et al., 1994; Tokashiki et al., 1990; Tokashiki and Takamatsu, 1993). In the case of immobilization, bio- mass sampling is either not possible or limited, leaving the monitoring of culture state and productivity to indirect measures. Mass-transfer resistances are also a major concern for the scale-up of cell immobilization and related techniques. These problems can be overcome by using filtration to separate the cells from the spent medium in suspension cultures, but membrane fouling is a major impediment to the utility of filtration. The use of the vortex flow filter (VFF), designed to reduce clogging by the action of Taylor vortexes on the surface of the membranes (Hildebrandt and Saxton, 1988), can partly solve the problem. Filter fouling is caused in large part by the release of high-molecular-weight DNA from dying cells. The addition of deoxyribonuclease I to † Biomira Inc. ‡ Conseil National de Recherches du Canada. § Present address: Biomira Inc., 6100 Royalmount Avenue, Montre ´al, Que ´bec, Canada H4P 2R2. * To whom all correspondence should be addressed: telephone, (514) 496-2264; FAX, (514) 496-5143; e-mail, kamen@biotech. lan.nrc.ca. 855 Biotechnol. Prog. 1996, 12, 855-864 S8756-7938(96)00072-0 CCC: $12.00 © 1996 American Chemical Society and American Institute of Chemical Engineers