Cell cycle model for growth rate and death rate in continuous suspension hybridoma cultures

As a result of recent advances in flow cytometry, renewed interest is shown in modeling the kinetic behavior of cells in culture on the basis of cell cycle parameters. An important but often overlooked kinetic variable in hybridoma cultures is the cell death rate. Not only the overall cell growth but also the kinetics of nutrient metabolism and monoclonal antibody production have been shown to depend on the cell death rate in continuous suspension hybridoma cultures. The present study shows that the death rate in hybridoma cultures is proportional to the fraction of cells arrested in the G(1) phase of the cell cycle. The steady-state cell age distributions in the various phases of the division cycle have been calculated analytically. A simple mathematical model has been used to produce the profiles of the cycling and arrested cell fractions with respect to the dilution rate. The calculated steady-state growth rate, death rate, and viability profiles are shown to be in agreement with recently published experimental data from continuous suspension hybridoma cultures. (c) 1992 John Wiley & Sons, Inc.     

Model simulation and analysis of perfusion culture of mammalian cells at high cell density.

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.     

Correlation between steady-state cell concentration and cell death of hybridoma cultures in chemostat

In the present study, the steady-state cell density (X) of chemostat cultures of murine hybridoma was varied by the concentration of glucose and glutamine in culture medium and the dissolved oxygen partial pressure. Except at low glutamine and low oxygen levels, the specific death rate (k(d)) of the cultures was found to decrease with increasing dilution rate (D). However, the plot of k(d) vs. X/D yielded linear relation, which suggests that cell death was due to a non-growth-linked inhibitory product of the cells. The k(d) value measured at low glutamine and low oxygen levels remained practically unchanged over a wide range of D between 0.020 and 0.029 h(-1). The k(d) for low oxygen cultures was always lower than the values obtained in low glucose and low glutamine cultures. A low-molecular-weight component of possibly less than 3000 MW was detected to be cell-death-inducing in the supernatant of exponentially growing cultures. It was neither lactate nor ammonium. The autoinhibitor was not cell-line specific. (c) 1995 John Wiley & Sons, Inc.     

Continuous hybridoma suspension cultures with and without cell retention: kinetics of growth, metabolism and product formation.

A laboratory scale bioreactor was constructed from glass and polycarbonate materials whereby a track-etch membrane (3 microns pore diameter) was integrated into its two-part bottom flange. The reactor performance was evaluated for continuous hybridoma suspension cultures under various conditions of cell retention. A total retention experiment demonstrated that this type of stirred tank reactor cannot be operated at near zero growth rate conditions. Instead, at steady viable cell concentrations of congruent to 3 x 10(6) cells per ml, specific growth and death rates were estimated at 0.60 +/- 0.06 d-1. Specific substrate (glucose, glutamine, O2, amino acids) consumption, by-product (ammonia, alanine, amino acids) and product (antibody) production rates as well as various apparent molar yield coefficients were obtained and are compared to metabolic quotients and yield coefficients previously calculated from standard continuous culture experiments, i.e., without cell retention, at specific growth rates of 0.63 and 1.24 d-1. Furthermore, steady-state data on viable cell and antibody concentrations, spec. mAb productivities, and space-time yields determined before and after a step change (2.5-fold increase) in dilution rate at identical specific growth rates mu are presented.     

Specific monoclonal antibody productivity and the cell cycle-comparisons of batch,continuous and perfusion cultures

A selection of mouse hybridoma cell lines showed a variation of approximately two orders of magnitude in intracellular monoclonal antibody contents. The different levels directly influenced apparent specific monoclonal antibody productivity during the death phase but not during the growth phase of a batch culture. The pattern of changes in specific productivity during culture remained basically similar even though at different levels for all cell lines tested. Arresting the cells in the G₁ phase using thymidine increased the specific productivity, cell volume and intracellular antibody content but at the same time led to decreased viability. In continuous culture DNA synthesis decreased with decreasing dilution rate though without an accompanying change in cell cycle and cell size distributions. The data shows both the decrease in viability and intracellular antibody content to be important factors which influence the negative association between specific antibody productivity and growth rate. In high cell density perfusion culture, when the cell cycle was prolonged by slow growth, viability was low and dead, but not lysed, cells were retained in the system, the specific antibody productivity was nearly two fold higher than that obtained in either batch or continuous cultures. The results imply that the prolongation of G₁ phase and the increase in death rate of cells storing a large amount of antibody together cause an apparent increase in specific antibody productivity.     

Growth and production modeling in hybridoma continuous cultures

Several experimental data on continuous cultures of hybridoma cells show that monoclonal antibody productivity is a decreasing function of dilution rate. It has been suggested that this unusual behavior may be due to the arrest of a fraction of cycling cells at a critical point of Phase G(1). Although this hypothesis has been recently investigated by using population balance models, mathematical analysis has been performed without accounting for the dynamics of the arrested cells properly. In this article, a more general and accurate approach is presented and new specific assumptions are introduced to characterize the arrest and the later progress through the cycle. Two different models (stochastic and deterministic) and two different critical points for the arrest (at the beginning and at the end of G(1)) are considered. The cell cycle parameters are estimated so that data predicted by the model fit those reported in the literature. In particular, the fraction of arrested cells, the cell arrest probability, and the mean cell generation time are computed as functions of the dilution rate. Results so far obtained predict that there is an optimal value of dilution rate for maximizing specific production rate of monoclonal antibody. (c) 1993 John Wiley & Sons, Inc.     

Sand administration as an instrument for biofilm control of Pseudomonas putida ATCC 11172 in chemostat cultures

Pseudomonas putida ATCC 11172 was grown in chemostat on L-asparagine or phenol as the sole, limiting carbon and energy source. The growth characteristics of a culture where a biofilm was present, were compared with one where the biofilm was strongly reduced by the grinding and shearing effect of sand suspended in the culture. In the presence of the intact biofilm, the curve of steady-state biomass versus dilution rate diverged greatly from the theoretical pattern predicted by conventional chemostat models. The sand strongly retarded the biofilm formation and to a high degree restored the shape of the biomass versus dilution rate curve to a more conventional pattern. The maximum specific growth rate (mu(max)) could not be calculated from the biofilm cultures. However using the sand cultures, mu(max) was determined to 0.64 h(-1) with L-asparagine as the carbon source and 0.49 h(-1) with phenol which compare favorably with the respective mu(max) values calculated from batch cultures.Incorporation of sand into strongly agitated cultures is recommended as an efficient and simple means of controlling biofilm formation in continuous cultures. The method may enable the gathering of basic kinetic data difficult to obtain in the presence of biofilm.     

Kinetic studies and unstructured models of lymphocyte metabolism in fed-batch culture

The growth of two lymphocyte cell lines, a hybridoma cell line and a human cutaneous T cell lymphoma (HuT78), was studied in fed-batch culture, and unstructured models of growth developed. A criteria was established to insure that the growth rate varied by less than a specified tolerance throughout the culture period. Glutamine and serum were growth-limiting nutrients for both cell lines with half-maximal growth rates at 0. 53 mM glutamine and 0. 55%(v/v) serum for the hybridoma cells and 0. 21 mM glutamine and 1. 5% serum for the HuT-78 cells. Over the range of glucose concentrations from 5. 5 mM to 28 mM, the specific growth rate of hybridoma cells was independent of glucose concentration, whereas glucose concentrations above 5. 5 mM inhibited HuT-78 growth. For both cell lines, the growth rate was significantly inhibited by the addition of ammonium, although the hybridoma cell line was more affected by ammonia than was the HuT-78 cell line. Growth of HuT-78 cells increased in the presence of interleukin-2. Unstructured models for the hybridoma cells were similar to other models presented in the literature. Applications of these models to adoptive immunotherapy are discussed.     

Variation and modeling of the probability of plasmid loss as a function of growth rate of plasmid-bearing cells of Escherichia coli during continuous cultures

A large number of models concerning cultures of genetically engineered bacteria have been described. Among them, some are specifically adapted to continuous cultures and lead to the determination of two variables: (i) the difference in the specific growth rates between plasmid-carrying cell and plasmid-free cells (deltamu) and (ii) the frequency of plasmid loss by plasmid-containing cells (p(r)mu(+)). Until now, studies have been performed on the global expression p(r)mu(+) and deltamu, whose value during continuous assays have been supposed approximately constant (mean value) and not on separate values of both terms p(r) and mu(+), respectively, probability of plasmid loss and specific growth rate of the plasmid-carrying cells. So far these studies do not allow examination of the relationship between these two last parameters. Experimental results were obtained with Escherichia coli C600 galk (GAPDH), a genetically engineered strain that synthetizes an elevated quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). From data obtained during continuous cultures, it is shown that during an assay, deltamu, and p(r)mu(+) do not remain constant. An appropriate mathematical analysis of the expression of mu(-) (specific growth rate of the plasmid-free cells) and mu(+) has been built up. This allows the evaluation of the values of mu(+) and mu(-) during the continuous cultures carried out at different dilution rates. Values of p(r) have been calculated from these data. Indeed our results show that p(r) increases with mu(+). A modeling approach which allows correct simulation of this variation is also proposed. This model is derived from the Hill equation regarding cooperative binding of enzymic type reaction. (c) 1993 John Wiley & Sons, Inc.     

High density culture of HeLa cells in a CelliGen perfusion system

A hollow fiber cartridge may be used in an extraneous recycle loop to facilitate perfusion operation of a stirred tank bioreactor. Retention of cells while removing waste products and replenishment with fresh nutrients allows higher than normal cell densities obtained in batch or continuous culture systems. This system successfully propagated HeLa cells to over 11 million viable cells per milliliter. Much higher perfusion rates (up to 4 vessel volumes per day) were necessary for high density culture of HeLa cells compared to BHK or a hybridoma cell line because of a much higher specific cellular metabolic rate. Cell specific glucose consumption rate, lactate production and ammonia production rates are several times higher for HeLa cells. Reproducible high cell densities and viabilities can be repeatedly obtained after harvest and dilution of a HeLa cell culture by partial drainage and reconstitution in the bioreactor.     

Cultivation of hybridoma cells in continuous cultures: kinetics of growth and product formation

Mouse hybridoma cells were grown in suspension in continuous stirred bioreactors. Cell growth, substrate utilization, and monoclonal antibody (MAb) production were studied using serum-free medium. Steady-state data were obtained at different dilution rates, between 0.012 and 0.039 h(-1) Viability was profoundly affected by dilution rate, particularly near the lower end of the dilution-rate range investigated. MAb concentration and productivity went through a maximum with respect to dilution rate. Lactate yield on glucose declined with in creasing dilution rate. Experiments were carried out to study the effects of medium glucose concentration on cell growth, product formation, and lactate yield on glucose. Reduction of glucose concentration in the feed medium did not considerably affect cell density and MAb concentration in the culture, but lactate levels dropped sharply; lactate yield on glucose declined substantially, indicating alterations in cell metabolic path ways for energy metabolism. Optimization strategy for continuous cell culture is discussed.     

Biological responses of hybridoma cells to hydrodynamic shear in an agitated bioreactor.

Effects of long-term hydrodynamic shear on hybridoma cells were investigated in a 250-ml continuous stirred-tank reactor (CSTR). Cells grown at steady state were subjected to step changes in agitation rates. Cell viability, glucose consumption, and monoclonal antibody (MAb) production were determined at high agitation rates and compared with the control (100 rev min-1). Impeller tip speeds higher than 40 cm s-1 caused a significant drop in cell concentration and respiration activity, and increased lactate dehydrogenase (LDH) release to the culture medium. Also, high agitation speeds caused a decrease in MAb concentration and an increase in specific glucose consumption rate. The effects of dilution rate and serum concentration on the sensitivity of hybridoma cells to hydrodynamic shear were determined. Serum was found to protect the cells against shear damage and had a significant positive effect on hybridoma growth and MAb production. Shear damage on cells in CSTR was approximated to first-order kinetics. The death rate constant increased sharply at impeller tip speeds above 40 cm s-1.     

Cell metabolism measurements in culture via microelectronic biosensors

Serum free fermentation procedures of cell cultures have got a wide application in production of biochemicals. But, cells cultured in serum free media in general are more sensitive to changes in culture condition, especially to nutrient limitation. There are no substances from serum which can support the cells when conditions are changing. In this study special attention is directed to amino acid utilization of mouse hybridoma in batch, chemostat and perfusion fermentations. Detailed data are presented which show the considerable difference of amino acid consumption rates in different fermentation modes. Already, in batch mode there are differences of the two investigated mouse hybridoma cell lines, although they are derived from the same myeloma line. In chemostat running at a dilution rate representing maximal growth rate most of the consumption rates are significant higher than in batch. On the other hand, in perfusion mode the rates are lower than in batch. This indicates clearly the different conditions of the fermentation modes. Therefore, it is necessary to develop serum free processes under the desired production conditions. An accurate analysis of the process is strongly recommended.     

Unveiling steady-state multiplicity in hybridoma cultures: the cybernetic approach

Mammalian cells grown in suspension produce waste metabolites such as lactate, alanine, and ammonia, which reduce the yield of cell mass and the desired product on the nutrients supplied. Previous studies (Cruz et al., 1999; Europa et al., 2000; Follstad et al., 1999) have shown that the cells can be made to alter their metabolism by starving them on their nutrients in continuous cultures at low dilution rates or starting the culture as a fed-batch. This leads to multiple steady states in continuous reactors, with some states being more favorable than others. Mathematical models that take into account the metabolic regulation that leads to these multiple steady states are invaluable tools for bioreactor control. In this article we present a cybernetic modeling strategy in which Metabolic Flux Analysis (MFA) is used to guide the cybernetic formulation. The hybridoma model presented as a result of this strategy considers the partially substitutable, partially complementary nature of glucose and glutamine. The choice of competitions within the network is guided by MFA and the model is successful in explaining the three multiple steady states observed. The cybernetic model though identified for the hybridoma experiments of Hu and others (Europa et al., 2000) seem generally applicable to mammalian systems as it captures the pathways that are common to mammalian cells grown in suspension. The model presented here could be used for start-up strategies for continuous reactors and model-based feedback control for maintaining high productivity of the reactor.     

Influence of dissolved oxygen concentration on growth,mitochondrial function and antibody production of hybridoma cells in batch culture

The effects of dissolved oxygen concentration on hybridoma cell growth, metabolism, and antibody production were studied. A mouse hybridoma cell line producing an IgG1 directed at a consensus -interferon was grown in batch cultures in a 5 dm³ stirred bioreactor at dissolved oxygen (DO) concentrations of 5, 30, 90 and 95% or air saturation. High oxygen tension (95% of air saturation) reduced specific growth rate without affecting cell viability. At lower dissolved oxygen levels, specific growth rates were approximately independent of DO, although changes in mitochondrial function and antibody production were observed. Flow cytometry assays of a fluorescent mitochondria-specific marker (Rhodamine 123) show significant single-cell heterogeneities during late exponential growth and greater average fluorescence in cultures grown at 95% DO. The quantity of cell-surface immunoglobulin, measured by an immunofluorescent flow cytometric technique, was the same at high (95%) and low (5%) dissolved oxygen concentrations. Myeloma cells of the type used in constructing the above hybridoma line were much less sensitive to dissolved oxygen level. Specific respiration rate, pyruvate utilization rate, cytochrome oxidase activity, and succinate-cytochrome c oxidoreductase activity were significantly greater (62–116%) for the hybridoma cells than for the myeloma cells in T-flask cultures.     

Influence of bcl-2 on antibody productivity in high cell density perfusion cultures of hybridoma

Apoptosis is an active, genetically determined death mechanism which can be induced by a wide range of physiological factors and by mild stress. It is the predominant form of cell death during the production of antibodies from murine hybridoma cell lines. A number of studies have now demonstrated that the suppression of this death pathway, by means of over-expression of survival genes such as bcl-2, results in improved cellular robustness and antibody productivity during batch culture. In the present study, the influence of bcl-2 expression on hybridoma productivity in two high density perfusion bioreactor systems was investigated. In the first system, a fixed-bed reactor, the DNA content in the spent medium was 25% higher in the control (TB/C3-pEF) culture than that found in the bcl-2 transfected (TB/C3-bcl2) cultures at all perfusion rates. This is indicative of a higher level of cell death in the control cell line. The average antibody concentration for the TB/C3-pEF cell line was 14.9 mg L-1 at perfusion rates of 2.6 and 5.2 d-1. However, for the TB/C3-bcl2 cell line it was 33 mg L-1 at dilution rates of 2 and 4 d-1. A substantial increase in antibody concentration was also found in the Integra Tecnomouse hollow fibre reactor. The antibody titre in the TB/C3-bcl2 cassette was nearly 100% higher than that in the TB/C3-pEF cassette during the cultivation period which lasted 6 weeks. Clearly, these results demonstrate the positive impact of bcl-2 over-expression on production of antibody in hybridoma perfusion cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of dilution rate on growth, productivity, cell cycle and size, and shear sensitivity of a hybridoma cell in a continuous culture

To study the effects of the growth rate of the hybridoma cell Mn12 on productivity, cell cycle, cell size, and shear sensitivity, six continuous cultures were run at dilution rate of 0.011, 0.021, 0.023, 0.030, 0.042, and 0.058 h(-1). This particular hybridoma cell appeared to be unstable in continuous culture with respect to specific productivity, as a sudden drop occurred after about 30 generations in continuous culture, accompanied by the appearance of two populations with respect to the cytoplasmic lgG content. The specific productivity increased with increasing growth rate. The shear sensitivity of the cell, as measured in a small air-lift loop reactor, increased with increasing growth rate. The mean relative cell size, as determined with a flow cytometer, increased with increasing growth rates. Furthermore, the fraction of cells in the S phase increased, and the fraction of cells in the G1/G0 phase decreased with increasing growth rates. (c) 1993 John Wiley & Sons, Inc.     

Mechanisms and kinetics of monoclonal antibody synthesis and secretion in synchronous and asynchronous hybridoma cell cultures.

The kinetics of monoclonal antibody synthesis and secretion have been studied in synchronous and asynchronous mouse hybridoma cell cultures. Pulse-labelling of IgG followed by immunoprecipitation and quantitation of synthesized and secreted IgG in synchronous cultures show maximum production during G1/S phases. Secretion takes place through exocytotic release of vesicle contents. Pulse-chase experiments show that 71% of the synthesized IgG is secreted within 8 h of the pulsing period and only a further 4% is secreted by 22 h. Higher specific antibody production (QA) is obtained if (a) cells are arrested and then maintained in G1/S phases, (b) viability is decreased during the death phase of batch culture, (c) the dilution rate is decreased in continuous culture or (d) cells are subjected to hydrodynamically induced stress. The increase in QA in all these cases is mainly due to the passive release of the accumulated intracellular antibody. DNA and protein synthetic activity peak during the early exponential phase and decline rapidly during mid and late exponential and death phases. Metabolic activity however peaks up to 20 h after the peak in DNA synthesis, and declines similarly during the death phase. The data are consistent with the idea that slow growth and higher death rates increase QA and that Ig secretion is probably subject to complex intracellular control.     

Effects of growth factors on cell proliferation and monoclonal antibody production of batch hybridoma cultures

Summary Effects of growth factors such as EGF, FGF and IL-2 on cell proliferation and monoclonal antibody production in a hybridoma cell line adapted to a completely defined serum-free medium were determined in batch cultures. The results indicate that the presence of growth factors in the medium enhances the antibody secretion without significantly affecting the growth rate. The specific antibody secretion rate of cells grown in serum-free medium supplemented with growth factors was 35% higher than those grown in serum-free medium alone.