Model Simulation and Analysis of Perfusion Culture of Mammalian Cells at High Cell Density

Abstract

Rate equations recently proposed by the authors for growth, death, consumption of nutrients, and formation of lactic acid, ammonium, and monoclonal antibody of hybridoma cells are used to simulate and analyze the behavior of perfusion cultures. Model simulations are in good agreement with experimental results from three different cell lines under varied perfusion and cell bleed rates except for cultures with very low viability. Analysis of simulations and experimental results indicates that in perfusion cultures with a complete cell separation cell bleed rate is a key parameter that strongly affects all the process variables, whereas the perfusion rate mainly affects the total and viable cell concentrations and the volumetric productivity of monoclonal antibody. Growth rate, viability, and specific perfusion rate of cells are only a function of the cell bleed rate. This also applies to cultures with partial cell separation in the permeate if the effective cell bleed rate is considered. It is suggested that the (effective) cell bleed rate of a perfusion culture should be carefully chosen and controlled separately from the perfusion rate. In general, a low cell bleed rate that warrants a reasonable cell viability appears to be desirable for the production of antibodies. Furthermore, model simulations indicate the existence of an optimum initial glucose concentration in the feed. For the cell lines considered, the initial glucose concentration used in normal cell culture media is obviously too high. The initial glutamine concentration can also be reduced to a certain extent without significantly impairing the growth and antibody production but considerably reducing the ammonia concentration. The mathematical model can be used to predict these optimum conditions and may also be used for process design.

Proliferation control strategies to improve productivity and survival during CHO based production culture

“…Inefficient use (higher uptake than required for normal cell growth) of glucose and glutamine leads to rapid biomass generation, nutrient depletion and by-product accumulation (lactate and ammonium ions, etc.) which often have inhibitory effects on culture longevity and productivity (Ozturk et al 1992;Zeng et al 1998;Zeng and Deckwer 1999). Finding alternative compounds that can be used efficiently as carbon and energy sources with lower uptake rates is important because slow consumption of nutrients can lead to reduced/ arrested cell growth (Altamirano et al 2001b).…”

“…Long lag phases are undesirable, therefore high growth rates are essential at the beginning of a process to achieve sufficient cells for maximal production. Uncontrolled proliferation beyond a certain desired cell density is also undesirable due to nutrient and oxygen depletion, toxic metabolite accumulation, cell death, and degradation of the products (Al-Rubeai and Singh 1998;Zeng et al 1998, Zeng andDeckwer 1999). Proliferation control strategies are typically implemented in the mid-or late-phases of exponential growth and have been shown to improve both the productivity and duration of production processes.…”

Model‐based identification of cell‐cycle‐dependent metabolism and putative autocrine effects in antibody producing CHO cell culture

“…The understanding of the interplay of cell-cycle-dependent population dynamics and a putative autocrine factor (also known as autoinhibitor or signal factor) in antibody producing CHO cells is still unknown. In the past, model-based approaches were used to identify a putative autocrine factor that influences the growth and death rates of various hybridoma cell lines in continuous and perfusion culture (Zeng, 1996;Zeng & Deckwer, 1999). Recent studies mainly focused on the effects of lowmolecular weight peptides (1-10 kDa) from hydrolysates on the growth and productivity of mammalian cell culture (Chabanon et al, 2008;Franěk & Fussenegger, 2005;Michiels et al, 2011;Spearman, Lodewyks, Richmond, & Butler, 2014).…”

Uncoupling of cell growth and proliferation results in enhancement of productivity in p21CIP1‐arrested CHO cells

“…To obtain sufficient cells for production, initial high growth rates are essential. However, in certain cell culture processes uncontrolled proliferation beyond a certain desired cell density causes nutrient and oxygen depletion, accumulation of toxic metabolites, cell death, and degradation of the product Mitchell et al, 1991;Simpson et al, 1998;Zeng et al, 1998, Zeng andDeckwer, 1999). The production processes can be improved by regulation of proliferation to allow initial rapid cell growth to achieve a sufficiently high cell density for production, followed by a production period at reduced proliferation rates to prevent excess cell densities (Al-Rubeai et al, 1992;Kaufmann et al, 2001;Mueller et al, 1999;Simpson et al, 1999;Suzuki and Ollis, 1990).…”