Determinants and rate laws of growth and death of hybridoma cells in continuous culture

Abstract

Experimental data from six hybridoma cell lines grown under diverse experimental conditions in both normal continuous and perfusion cultures are analyzed with respect to the significance of nutrients and products in determining the growth and death rates of cells and with respect to their mathematical modeling. It is shown that neither nutrients (glucose and glutamine) nor the common products lactic acid, ammonia, and monoclonal antibody can be generally assumed to be the clear‐limiting or inhibiting factors for most of the cultures. Correspondingly, none of the unstructured models existing in the literature can be generally applied to describe the experimental data obtained over a relatively wide range of cultivation conditions as considered in this work. Surprisingly, for all cultures the specific growth rate (μ) almost linearly correlates with the ratio of the viable cell concentration (NV) to the dilution (perfusion) rate (D). Similarly, the specific death rate (kd) is a function of the ratio of the total cell concentration (Nt) to the dilution (perfusion) rate. These results strongly suggest the formation of not yet identified critical factors or autoinhibitors that determine both the growth and death rates of hybridoma cells. Based on these observations, simple kinetic models are developed for μ and kd which describe the experimental data satisfactorily. Analysis of the experimental data with the kinetic models reveals that under the current cultivation conditions the formation rate of the autoinhibitor(s) or the sensitivity of cell growth and death to the autoinhibitor(s) is mainly affected by the medium composition. Irrespective of the cell lines, cells grown on serum‐containing media have almost the same model parameters, which are distinctively different from those of cells grown on serum‐free media. Furthermore, in contrast to the prevailing view, kd is shown to positively correlate with μ if the effects of cell concentration and dilution (perfusion) rate are considered. Several important implications of these findings are discussed for the optimization and control of animal cell culture. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 642–654, 1998

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

“…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.…”

“…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).…”

Modelling of Mammalian Cells and Cell Culture Processes

“…Zeng et al (1998) proposed in a comparison of six hybridoma cell lines that the specific growth rate is limited by either an auto-inhibitor or an unknown limiting growth factor, which manifests itself through the strong dependence of on the ratio of cell number to dilution rate N v /D. The growth rate correlates negatively and almost linearly for all cultures, in both chemostat and perfusion modes, with the ratio of viable cell number to the dilution (perfusion) N v /D, until cessation of growth is reached.…”

“…The work of Lee et al (1995) proposed the dependence of specific cell death rate on a non-growth linked auto-inhibitor and suggested a positive linear correlation between K d and N v /D. Zeng et al (1998) tested this relation on a number of hybridoma cell lines and found it to hold only for some chemostat cultures and only at relatively low values of N v /D. For higher values, the relation becomes severely non-linear.…”

“…For higher values, the relation becomes severely non-linear. Instead, based on evidence of a dependence of K d on , Zeng et al (1998) modified the relation of Lee et al (1995) to incorporate this so that the same inhibitor that affects growth through also affects death. They also make a correlation with N t not N v thus finding a more robust linear relation between K d / and N t /D that is cell line non-specific, yet strongly media dependent.…”