Fed-batch cultivation of animal cells using different medium design concepts and feeding strategies

In animal cell cultivation, cell density and product concentration are often low due to the accumulation of toxic end-products such as ammonia and lactate and/or the depletion of essential nutrients. A hybridoma cell line (CRL-1606) was cultivated in T-flasks using a newly devised medium feeding strategy. The goals were to decrease ammonia and lactate formation by the design of an initial medium which would provide a starting environment to achieve optimal cell growth. This was followed by using a stoichiometric equation governing animal cell growth and then designing a supplemental medium for feeding strategy used to control the nutritional environment. The relationship between the stoichiometric demands for glutamine and nonessential amino acids was also studied. Through stoichiometric feeding, nutrient concentrations were controlled reasonably well. Consequently, the specific production rate of lactate was decreased by fourfold compared with conventional fed-batch culture and by 26-fold compared with conventional batch culture. The specific production rate of ammonia was decreased by tenfold compared with conventional fed-batch culture and by 50-fold compared with conventional batch culture. Most importantly, total cell density and monoclonal antibody concentration were increased by five- and tenfold respectively, compared with conventional batch culture. (c) 1994 John Wiley & Sons, Inc.     

Effects of ammonia and lactate on hybridoma growth, metabolism, and antibody production

The influence of ammonia and lactate on cell growth, metabolic, and antibody production rates was investigated for murine hybridoma cell line 163.4G5.3 during batch culture. The specific growth rate was reduced by one-half in the presence of an initial ammonia concentration of 4 mM. Increasing ammonia levels accelerated glucose and glutamine consumption, decreased ammonia yield from glutamine, and increased alanine yield from glutamine. Although the amount of antibody produced decreased with increasing ammonia concentration, the specific antibody productivity remained relatively constant around a value of 0.22 pg/cell-h. The specific growth rate was reduced by one-half at an initial lactate concentration of 55 mM. Although specific glucose and glutamine uptake rates were increased at high lacatate concentration, they showed a decrease after making corrections for medium osmolarity. The yield coefficient of lactate from glucose decreased at high lactate concentrations. A similar decrease was observed for the ammonia yield coefficient from glutamine. At elevated lactate concentrations, specific antibody productivities increased, possibly due to the increase in medium osmolarity. The specific oxygen uptake rate was insensitive to ammonia and lactate concentrations. Addition of ammonia and lactate increased the calculated metabolic energy production of the cells. At high ammonia and lactate, the contribution of glycolysis to total energy production increased. Decreasing external pH and increasing ammonia concentrations caused cytoplasmic acidification. Effect of lactate on intracellular pH was insignificant, whereas increasing osmolarity caused cytoplasmic alkalinization.     

Comparison of specific rates of hybridoma growth and metabolism in batch and continuous cultures

For the mouse hybridoma cell line VO 208, kinetics of growth, consumption of glucose and glutamine, and production of lactate, ammonia and antibodies were compared in batch and continuous cultures. At a given specific growth rate, different metabolic activities were observed: a 40% lower glucose and glutamine consumption rate, but a 70% higher antibody production rate in continuous than in batch culture. Much higher metabolic rates were also measured during the initial lag phase of the batch culture. When representing the variation of the specific antibody production rate as a function of the specific growth rate, there was a positive association between growth and antibody production in the batch culture, but a negative association during the transient phase of the continuous culture. The kinetic differences between cellular metabolism in batch and continuous cultures may be result of modifications in the physiology and metabolism of cells which, in continuous cultures, were extensively exposed to glucose limitations.Institut National Polytechnique de Lorraine, ENSAIA BP 172, 2 avenue de la forêt de Haye, 54505, Vandoeuvre Cedex France     

Ammonia inhibition of hybridomas propagated in batch, fed-batch, and continuous culture

The nature and temporal development of ammonia inhbition were investigated in batch, fed-batch, and continuous cultures. Significant inhibition was observed when cells were inoculated in serum-containing or chemically defined medium containing more than 2 mM of ammonia. In contrast, no inhibition was observed at greater than 10 mM when the ammonia concentration was gradually increased over the span of a batch culture by feeding ammonium chloride. Strong growth inhibition was observed after each of five step changes (2.8 --> 3.7 --> 4.0 --> 4.9 --> 7.7 --> 13.5 mM) in continuous culture. Following a period of adaptation at each higher value, the viable cell density stabilized at a new lower value. The lowering in viable cell density was caused by an increase in specific death rate and a decreased cell yield on glucose, glutamine, and oxygen. Increased ammonia concentration had little or no effect on the steady-state specific growth kinetics or specific antibody productivity. (c) 1994 John Wiley & Sons, Inc.     

Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture

Summary Recombinant human interferon- production by Chinese hamster ovary cells was restricted to the growth phase of batch cultures in serum-free medium. The specific interferon production rate was highest during the initial period of exponential growth but declined subsequently in parallel with specific growth rate. This decline in specific growth rate and interferon productivity was associated with a decline in specific metabolic activity as determined by the rate of glucose uptake and the rates of lactate and ammonia production. The ammonia and lactate concentrations that had accumulated by the end of the batch culture were not inhibitory to growth. Glucose was exhausted by the end of the growth phase but increased glucose concentrations did not improve the cell yield or interferon production kinetics. Analysis of amino acid metabolism showed that glutamine and asparagine were exhausted by the end of the growth phase, but supplementation of these amino acids did not improve either cell or product yields. When glutamine was omitted from the growth medium there was no cell proliferation but interferon production occurred, suggesting that recombinant protein production can be uncoupled from cell proliferation. Offprint requests to: P. M. Hayter     

Transient responses of hybridoma metabolism to changes in the oxygen supply rate in continuous culture

Oxygen is an important nutrient that may limit the productivity of commercial cell culture reactors. The transient responses of hybridoma growth and metabolism to step changes in the oxygen supply rate have been examined for dissolved oxygen concentrations (DO) ranging from 0.1% to 10% of air saturation in continuous culture. Metabolic quotients are reported for glucose, lactate, ammonia, oxygen, glutamine, alanine and other amino acids. A majority of the estimated ATP production was due to oxidative phosphorylation under all conditions tested. Decreases in the oxygen supply rate below the value required to maintain 0.5% DO caused the viable cell concentration to decrease. Glycolysis was enhanced at the lower oxygen concentrations, and after an initial decrease, the specific glutamine consumption rate was also higher. High residual glutamine concentrations occurred below 0.5% DO. Oxidation of other amino acids and production of serine were also inhibited. The cells subsequently adapted to low oxygen concentrations. The increase in cell concentration following the return to 10% DO was preceded by increased biosynthetic activity, as evidenced by transiently reduced yields of lactate from glucose, and alanine and ammonia from glutamine.     

Influence of inoculum age on hybridoma culture kinetics

To determine the influence of the inoculum age on the kinetics of hybridoma growth and metabolism, spinner flasks have been inoculated with cells previously propagated in T flasks for 43, 52, 62 and 71 hr respectively. Increasing the age of the inoculum is found to result in a longer lag phase, in a lower maximum specific growth rate and in a reduced maximal cell density. During the growth phase specific rates of glucose and glutamine uptake and of ammonia and lactate production are similar. However, with the older inoculum, much higher metabolic activities are observed during the lag phase. The production of antibodies is delayed with increasing inoculum age, but the final antibody concentrations are similar, which indicates a higher specific antibody production rate when inoculating with older cells.     

重组CHO细胞培养过程中氨对细胞代谢的影响

研究了重组CHO细胞批培养过程中,氨浓度对细胞的葡萄糖、谷氨酰胺及其它氨基酸代谢的影响。表明,细胞对葡萄糖和谷氨酰胺的得率系数随着氨浓度的增加而降低,起始氨浓度为566mmol/L的批培养过程与起始氨浓度为021mmol/L的批培养过程相比,细胞对葡萄糖和谷氨酰胺的得率系数分别下降了78%和74%,细胞对其它氨基酸的得率系数也分别下降了50%～70%。氨浓度的增加明显地改变了细胞的代谢途径,葡萄糖代谢更倾向于厌氧的乳酸生成。在谷氨酰胺的代谢过程中,谷氨酸经谷氨酸脱氢酶进一步生成α酮戊二酸的过程受到了氨的抑制,而氨对谷氨酸经谷氨酸转氨酶反应生成α酮戊二酸的过程有促进作用,但总体上谷氨酸进一步脱氨生成α酮戊二酸的反应受到了氨的限制。     

Fed‐batch CHO cell t‐PA production and feed glutamine replacement to reduce ammonia production

Industrial therapeutic protein production has been greatly improved through fed‐batch development. In this study, improvement to the productivity of a tissue‐plasminogen activator (t‐PA) expressing Chinese hamster ovary (CHO) cell line was investigated in shake flask culture through the optimization of the fed‐batch feed and the reduction of ammonia generation by glutamine replacement. The t‐PA titer was increased from 33 mg/L under batch conditions to 250 mg/L with daily feeding starting after three days of culture. A commercially available fed‐batch feed was supplemented with cotton seed hydrolysate and the four depleted amino acids, aspartic acid, asparagine, cysteine, and tyrosine. The fed‐batch operation increased the generation of by‐products such as lactate and ammonia that can adversely affect the fed‐batch performance. To reduce the ammonia production, a glutamine‐containing dipeptide, pyruvate, glutamate, and wheat gluten hydrolysate, were investigated as glutamine substitutes. To minimize the lag phase as the cells adjusted to the new energy source, a feed glutamine replacement process was developed where the cells were initially cultured with a glutamine containing basal medium to establish cell growth followed by feeding with a feed containing the glutamine substitutes. This two‐step feed glutamine replacement process not only reduced the ammonia levels by over 45% but, in the case of using wheat gluten hydrolysate, almost doubled the t‐PA titer to over 420 mg/L without compromising the t‐PA product quality or glycosylation pattern. The feed glutamine replacement process combined with optimizing other feed medium components provided a simple, practical, and effective fed‐batch strategy that could be applied to the production of other recombinant therapeutic proteins. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Transient responses of hybridoma cells to lactate and ammonia pulse and step changes in continuous culture

Ammonia and lactate are the major byproducts from mammalian cells grown in medium containing glutamine and glucose. Both can be toxic to cells, and may limit the productivity of commercial bioreactors. The transient and steady-state responses of hybridoma growth and metabolism to lactate and ammonia pulse and step changes in continuous suspension culture have been examined. No inhibition was observed at 40 mM lactate. Cell growth was inhibited by 5 mM ammonia, but the cells were able to adapt to ammonia concentrations as high as 8.2 mM. Ammonia production decreased and alanine production increased in response to higher ammonia concentrations. Increased ammonia concentrations also inhibited glutamine and oxygen consumption. The specific oxygen consumption rate decreased by an order of magnitude after an ammonia pulse to 18 mM. Under these conditions, over 90% of the estimated ATP production was due to glycolysis and a large fraction of glutamine was converted to lactate.     

Optimization of reovirus production from mouse L-929 cells in suspension culture

Reovirus serotype 3 Dearing (T3D) has shown potential as a novel cancer therapy. To support the increasing demand for reovirus, a two-stage perfusion mode scheme is proposed for cell growth and reovirus production. Mouse L-929 cells were used as the host for reovirus infection due to their ability to grow well in suspension culture. Several L-929 cell growth and reovirus infection characteristics were investigated and optimized in spinner flask batch cultures. For the growth of L-929 cells, a balanced nutrient-fortification of SMEM medium increased the maximum cell density by 30%, compared to normal SMEM; however, ammonia and lactate accumulations were found to inhibit further cell growth. For the production of reovirus, approximately 90% increase in viral yield resulted when the infection temperature was reduced from 37 to 33 degrees C. Infectious reovirus particles were shown to be stable in conditioned medium at 37 and 33 degrees C. The final virus titer was dependent on the multiplicity of infection (MOI) and the host cell density at the time of infection. A combination of an MOI of 0.1 pfu/cell and an initial host cell density of 1.0 x 10(6) cells/mL in fortified medium resulted in a maximum virus titer of (4.59 +/- 0.16) x 10(9) pfu/mL and a specific yield of (2.34 +/- 0.08) x 10(3) pfu/cell. At an optimal harvest time of the infection process, 99% of the virus was associated with the cellular debris. Finally, the presence of 5.0 mM ammonia in the culture medium was shown to seriously inhibit the reovirus yield, whereas lactate concentrations up to 20 mM had no effect.     

Software sensors for the monitoring of perfusion cultures: Evaluation of the hybridoma density and the medium composition from glucose concentration measurements

New software sensors based on the Extended Kalman Filter technique have been developed for the monitoring of animal cell perfusion cultures. They use a kinetic model describing the growth, death and metabolism of hybridoma cells as a function of the medium composition. The model was initially validated on a batch culture and found to correctly predict the continuous perfusion culture kinetics, except for the production of ammonia and lactate. Using the measurement of a single component in the culture medium, in this case glucose, the Extended Kalman Filter provides an excellent evaluation of the time variation of the concentrations of living and dead cells, of glutamine and antibodies, during the whole perfusion culture for a retained cell density rising from 1 to 11×10⁶ cells.ml^–1 inside the reactor.     

The influence of high substrate concentrations on microbial kinetics

High substrate concentrations inhibit growth and may distort the metabolism of microorganisms. Mechanisms causing substrate inhibition are discussed and used to derive several mathematical models representative of the entire concentration range, including stimulation of growth by low substrate concentrations. These kinetic models are tested with a variety of batch culture measurements of specific growth rate and respiration rate at widely-ranging substrate concentrations. Using one of the kinetic models, equations are developed for batch, continuous, and exponential-feed reactors. Comparison of results obtained in continuous culture with results from exponential-feed culture systems is shown to offer a novel experimental method for evaluating the effect of the cell age distribution on the properties and metabolic activity of a culture.     

An unstructured kinetic model of macromolecular metabolism in batch and fed-batch cultures of hybridoma cells producing monoclonal antibody

Growth profiles of the batch and fed-batch culture of hybridoma cells producing monoclonal antibody were simulated using an unstructured model. The model describes the production of cellular macromolecules and monoclonal antibody, the metabolism of glucose and glutamine with the production of lactate and ammonia, and the profiles of cell growth in batch and fed-batch culture. Equations describing the cells arrested in G1 phase [T.I. Linardos, N. Kalogerakis, L.A. Behie, Biotechnol. Bioeng. 40 (1992) 359–368; E. Suzuki, D.F. Ollis, Biotechnol. Bioeng. 34 (1989) 1398–1402] were included in this model to describe the increase of the specific antibody productivity in the near-zero specific growth rate, which was observed in the recent experiments in fed-batch cultures of this study and the semi-continuous culture of hybridoma cells [S. Reuveny, D. Velez, L. Miller, J.D. Macmillan, J. Immnol. Methods 86 (1986) 61–69]. This model predicted the increase of specific antibody production rate and the decline of the specific production rate of cellular macromolecules such as DNA, RNA, protein, and polysaccharide in the late exponential and decline phase of batch culture and at lower specific growth rates in the fed-batch culture.     

Substitution of glutamine by pyruvate to reduce ammonia formation and growth inhibition of mammalian cells

In mammalian cell culture technology glutamine is required for biomass synthesis and as a major energy source together with glucose. Different pathways for glutamine metabolism are possible, resulting in different energy output and ammonia release. The accumulation of ammonia in the medium can limit cell growth and product formation. Therefore, numerous ideas to reduce ammonia concentration in cultivation broths have been developed. Here we present new aspects on the energy metabolism of mammalian cells. The replacement of glutamine (2 mM) by pyruvate (10 mM) supported cell growth without adaptation for at least 19 passages without reduction in growth rate of different adherent commercial cell lines (MDCK, BHK21, CHO-K1) in serum-containing and serum-free media. The changes in metabolism of MDCK cells due to pyruvate uptake instead of glutamine were investigated in detail (on the amino acid level) for an influenza vaccine production process in large-scale microcarrier culture. In addition, metabolite profiles from variations of this new medium formulation (1-10 mM pyruvate) were compared for MDCK cell growth in roller bottles. Even at very low levels of pyruvate (1 mM) MDCK cells grew to confluency without glutamine and accumulation of ammonia. Also glucose uptake was reduced, which resulted in lower lactate production. However, pyruvate and glutamine were both metabolized when present together. Amino acid profiles from the cell growth phase for pyruvate medium showed a reduced uptake of serine, cysteine, and methionine, an increased uptake of leucine and isoleucine and a higher release of glycine compared to glutamine medium. After virus infection completely different profiles were found for essential and nonessential amino acids.     

Influence of culture modes on the microbial production of hyaluronic acid by <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis>

The principal objective of this study was to assess the effects of culture modes including batch culture, pulse fed-batch culture, constant feeding rate fed-batch culture, and exponential fed-batch culture on the production of hyaluronic acid (HA) by Streptococcus zooepidemicus. Batch cultures had the highest levels of HA productivity, whereas fed-batch cultures were more favorable with regard to cell growth, and exponential fed-batch cultures evidenced the highest cell concentrations. A two-step culture model was proposed to enhance HA production: an exponential fed-batch culture was conducted prior to 8 h and then sucrose supplementation was applied for 8 h to start the batch fermentation of S. zooepidemicus. HA production and productivity were increased by 36 and 37% in the proposed two-step culture process as compared with that observed in the batch culture, respectively. The proposed two-step culture model can be applied in the production of secondary metabolites, and particularly of the exopolysaccharides.     

Analysis of the ammonia metabolism of rat primary hepatocytes and a human hepatocyte cell line Huh 7

Ammonia metabolism of ratprimary hepatocytes and a human hepatocyte cell line,Huh 7, at different concentrations of glutamine,glucose and ammonia was examined. During theincubation of the primary hepatocyte cells, glutamineand ammonia concentrations decreased, that of ureaincreased, and that of glucose remained the same. Inthe case of Huh 7 cells, glucose was consumed rapidly,the concentration of ammonia increased and that of urearemained the same. The major energy sources amongmedium components were glutamine for the primary cellsand glucose for Huh 7 cells, although the primaryhepatocytes may utilize intracellular glycogen asenergy source. As the glutamine concentration in theincubation medium increased, the specific rates of notonly glutamine consumption, but also ammonia productionby the primary cells and Huh 7 cells increased. Besides, specific urea production rate by the primarycells increased then. Increase of glucoseconcentration had no effect on glutamine and ammoniametabolism by both cells, although it increased glucoseconsumption by Huh 7 cells. The incubation of theprimary cells with higher ammonia concentrationincreased all specific rates of glutamine consumption,ammonia consumption and urea production. An increasein the ammonia concentration to 5 mM changed theammonia metabolism from production to consumption andincreased the specific glucose consumption rate. Consequently, increases in the glutamine and ammoniaconcentrations were revealed to have negative andpositive effects, respectively, on decreasing ammoniaconcentration by both of rat primary hepatocytes andHuh 7 cells.     

Acetate addition to an immobilized yeast column of ethanol production

Acetate, a by-product of ethanol fermentation by Saccharomyces cerevisiae, has been shown to inhibit cell growth if present in high concentrations. Consequently, acetate has been considered undesirable in systems where the production rate depends upon steady-state growth. Acetate, however, may be desirable in some systems since it increases the specific rate of ethanol production by increasing the maintenance requirements of yeast. In immobilized cell reactors using the crosslinking method, steady state is not achieved and cell overgrowth is a problem. This article presents the results of a study aimed at taking advantage of the use of acetate, both to reduce cell overgrowth and to increase productivity. Various concentrations of acetate were added to batch and plug flow systems, while monitoring the effects on cell growth and ethanol production. The productivity was increased by as much as 50% in an immobilized cell reactor (ICR), while cell growth was greatly reduced.     

Multiple steady states with distinct cellular metabolism in continuous culture of mammalian cells

Mammalian cells have the ability to proliferate under different nutrient environments by utilizing different combinations of the nutrients, especially glucose and the amino acids. Under the conditions often used in in vitro cultivation, the cells consume glucose and amino acids in great excess of what is needed for making up biomass and products. They also produce large amounts of metabolites with lactate, ammonia, and some non-essential amino acids such as alanine as the most dominant ones. By controlling glucose and glutamine at low levels, cellular metabolism can be altered and can result in reduced glucose and glutamine consumption as well as in reduced metabolite formation. Using a fed-batch reactor to manipulate glucose at a low level (as compared to a typical batch culture), cell metabolism was altered to a state with substantially reduced lactate production. The culture was then switched to a continuous mode and allowed to reach a steady-state. At this steady-state, the concentrations of cells and antibody were substantially higher than a control culture that was initiated from a batch culture without first altering cellular metabolism. The lactate and other metabolite concentrations were also substantially reduced as compared to the control culture. This newly observed steady-state was achieved at the same dilution rate and feed medium as the control culture. The paths leading to the two steady-states, however, were different. These results demonstrate steady-state multiplicity. At this new steady-state, not only was glucose metabolism altered, but the metabolism of amino acids was altered as well. The amino acid metabolism in the new steady-state was more balanced, and the excretion of non-essential amino acids and ammonia was substantially lower. This approach of reaching a more desirable steady-state with higher concentrations of cells and product opens a new avenue for high-density- and high-productivity-cell culture.     

Elevated pCO2 affects the lactate metabolic shift in CHO cell culture processes

The shift from lactate production to consumption in CHO cell metabolism is a key event during cell culture cultivations and is connected to increased culture longevity and final product titers. However, the mechanisms controlling this metabolic shift are not yet fully understood. Variations in lactate metabolism have been mainly reported to be induced by process pH and availability of substrates like glucose and glutamine. The aim of this study was to investigate the effects of elevated pCO₂ concentrations on the lactate metabolic shift phenomena in CHO cell culture processes. In this publication, we show that at elevated pCO₂ in batch and fed‐batch cultures, the lactate metabolic shift was absent in comparison to control cultures at lower pCO₂ values. Furthermore, through metabolic flux analysis we found a link between the lactate metabolic shift and the ratio of NADH producing and regenerating intracellular pathways. This ratio was mainly affected by a reduced oxidative capacity of cultures at elevated pCO₂. The presented results are especially interesting for large‐scale and perfusion processes where increased pCO₂ concentrations are likely to occur. Our results suggest, that so far unexplained metabolic changes may be connected to increased pCO₂ accumulation in larger scale fermentations. Finally, we propose several mechanisms through which increased pCO₂ might affect the cell metabolism and briefly discuss methods to enable the lactate metabolic shift during cell cultivations.