Hybridoma growth and antibody production as a function of cell density and specific growth rate in perfusion culture

Abstract

Steady state metabolic parameters for hybridoma cell line H22 were determined over a wide range of cell densities and specific growth rates in a filtration based homogeneous perfusion reactor. Operating the reactor at perfusion rates of 0.75, 2.0, and 2.9 day(-1)(each at four different specific growth rates), viable cell densities as high as 2 x 10(7) cells/mL were obtained. For the cell line under investigation, the specific monoclonal antibody production rate was found to be a strong function of the viable cell density, increasing with increasing cell density. In contrast, most of the substrate consumption and product formation rates were strong functions of the specific growth rate. Substrate metabolism became more efficient at high cell densities and low specific growth rates. The Specific rates of metabolite formation and the apparent yields of lactate from glucose and ammonia from glutamine decreased at low specific growth rates and high cell densities. While the specific oxygen consumption rate was independent of the specific growth rate and cell density, ATP production was more oxidative at lower specific growth rate and higher cell density. These observed shifts are strong indications of the production potential of high-density perfusion culture.

Dynamic Modeling of Apoptosis and its Interaction with Cell Growth in Mammalian Cell Culture

“…higher in the higher specific growth rate culture and lower in lower specific growth rate culture. Similar observations have been reported for hybridoma in perfusion culture where it was found that specific glucose consumption and specific lactate production rates are highly correlated to the specific growth rate of the culture (Banik and Heath, 1995). In general, Figures 4.9 to 4.11 shows that caspase activation is correlated to initial cell density, i.e.…”

“…The perfusion rate depends on the type and culture behavior of the cell line, the concentration of nutrients in the feed and the level of toxicity. This mode of operation has been used for the production of monoclonal antibodies (Banik and Heath, 1995;Tang et al, 2007; and recombinant proteins (AlRubeai et al, 1999;Avgerinos et al, 1990). An example of a 500 L industrial perfusion system for monoclonal antibody production by hyridoma is described by (Deo et al, 1996) where the operation continued for 15 to 35 days to achieve commercial production of antibodies with a cell density of >10 x 10 6 cell/mL.…”

Apoptosis-resistant E1B-19K-expressing NS/0 myeloma cells exhibit increased viability and chimeric antibody productivity under perfusion culture conditions

“…This has been observed for cells cultivated in batch (Ramirez and Mutharasan, 1990), fed-batch (de Tremblay et al, 1993;Robinson et al, 1994), chemostat (Boraston et al, 1984), and perfusion modes (Al-Rubeai et al, 1992;Banik and Heath, 1995;Batt et al, 1990;de la Broise et al, 1991;Johnson et al, 1996;Hansen et al, 1993;Park and Ryu, 1994;Tokashiki and Takamatsu, 1993;Trampler et al, 1994), as well as in cells treated with cell-cycle blocking agents (Al-Rubeai and Emery, 1990;Jayme, 1991;Mercille and Massie, 1998;Oh et al, 1995;Øyaas et al, 1994a,b;Reddy and Miller, 1994;Suzuki and Ollis, 1990;Takahachi et al, 1994). Indeed, specific antibody production has been shown to be cell cycle dependent and optimum in the G 1 and/or G 2 /M phase (Cazzador and Mariani, 1993;Kromenaker and Srienc, 1991;Linardos et al, 1992;Richieri et al, 1991;Suzuki and Ollis, 1989).…”

“…1) is a typical feature of lymphoid cells in perfusion culture. This phenomenon has been observed with a variety of hybridoma cell lines in a number of different perfusion systems, such as those based on cross-flow filtration (de la Broise et al, 1991;Banik and Heath, 1995), vortex-flow filtration (Avgerinos et al, 1990;Mercille et al, 1994c), sedimentation (Kitano et al, 1986;Hansen et al, 1993), centrifugation (Johnson et al, 1996;Tokashiki and Tokamatsu, 1993), hollow-fiber cartridges (de la Broise et al, 1992;Park and Ryu, 1994), and more recently, acoustic filtration (Trampler et al, 1994). While it could be argued that these systems were not optimised and that the accumulation of dead cells was linked to hydrodynamic shear stress in the separation or bioreactor systems (Schmid et al, 1992), the results presented here propose a different explanation.…”

“…Therefore, the elevated death and renewal rates observed in perfusion cultures of NS/0 myeloma and D5 hybridoma cells were clearly not due to necrotic death but were the result of apoptosis most likely induced by a reduction in the availability of essential nutrients. Cells in the plateau phase of a perfusion culture are continuously under nutrient limitation (Banik and Heath, 1995;de la Broise et al, 1991;Mercille et al, 1994a,b;Park and Ryu, 1994;Schmid et al, 1992), explaining the typical occurrence of a steady-state viable cell plateau determined by the availability of one or more key nutrients. As demonstrated in previous studies, deprivation of glucose or amino acids such as glutamine or cystine leads to a rapid induction of apoptosis in lymphoid cells such as myelomas and hybridomas Perreault and Lemieux, 1993;Simpson et al, 1998;Singh et al, 1994a,b).…”