Optimisation of Microbial 3-methylcatechol Production as Affected by Culture Conditions

Pseudomonas putida MC2 can be used to produce catechols. The accumulation, specific and volumetric production rates of 3-methylcatechol from toluene have been studied. Production rates were shown to depend on growth medium, pH and toluene concentration. Minimal glucose medium and rich LB medium were the best growth media for 3-methylcatechol production. A lower pH often resulted in slower growth and a higher specific production rate, but a lower volumetric production rate. Specific production rates also increased at higher initial toluene concentrations. The best process conditions in terms of substrate conversion and specific production rate were found in minimal glucose medium at an initial aqueous toluene concentration of 1.0 &#117 mM and an initial pH of 6. At pH 7 and 2.0 &#117 mM toluene, more product was accumulated at a lower specific rate, but at a higher volumetric production rate.     

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.     

Integrated bioproduction and extraction of 3-methylcatechol

Pseudomonas putida MC2 is a solvent-tolerant strain that accumulates 3-methylcatechol. In aqueous media, 10 mM of 3-methylcatechol was produced and production was limited by 3-methylcatechol toxicity to the biocatalyst. Production levels increased by introduction of a second, organic phase that provides the substrate toluene and extracts the product from the culture medium. Octanol was shown to be an appropriate second phase with respect to tolerance of the strain for this solvent and with respect to partitioning of both substrate and product. Per unit of overall reactor volume (octanol and water), best results were obtained with 50% (v/v) of octanol: an overall 3-methylcatechol concentration of 25 mM was reached with 96% of the product present in the octanol phase. These product concentrations are much higher than in aqueous media without organic solvent, indicating that biocatalysis in an organic/aqueous two-phase system is an improved set-up for high production levels of 3-methylcatechol.     

A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: effect of nutrient concentration, dilution rate, and pH

Hybridomas are finding increased use for the production of a wide variety of monoclonal antibodies. Understanding the roles of physiological and environmental factors on the growth and metabolism of mammalian cells is a prerequisite for the development of rational scale-up procedures. An SP2/0-derived mouse hybridoma has been employed in the present work as a model system for hybridoma suspension culture. In preliminary shake flask studies to determine the effect of glucose and glutamine, it was found that the specific growth rate, the glucose and glutamine metabolic quotients, and the cumulative specific antibody production rate were independent of glucose concentration over the range commonly employed in cell cultures. Only the specific rate of glutamine uptake was found to depend on glutamine concentration. The cells were grown in continuous culture at constant pH and oxygen concentration at a variety of dilution rates. Specific substrate consumption rates and product formation rates were determined from the steady state concentrations. The specific glucose uptake rate deviated from the maintenance energy model(1) at low specific growth rates, probably due to changes in the metabolic pathways of the cells. Antibody production was not growth-associated; and higher specific antibody production rates were obtained at lower specific growth rates. The effect of pH on the metabolic quotients was also determined. An optimum in viable cell concentration was obtained between pH 7.1 and 7.4. The viable cell number and viability decreased dramatically at pH 6.8. At pH 7.7 the viable cell concentration initially decreased, but then recovered to values typical of pH 7.1-7.4. Higher specific nutrient consumption rates were found at the extreme pH values; however, glucose consumption was inhibited at low pH. The pH history also influenced the behavior at a given pH. Higher antibody metabolic quotients were obtained at the extreme pH values. Together with the effect of specific growth rate, this suggests higher antibody production under environmental or nutritional stress.     

A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: effect of nutrient concentration, dilution rate, and pH. Reprinted from Biotechnology and Bioengineering, Vol. 32, Pp 947-965 (1988)

Hybridomas are finding increased use for the production of a wide variety of monoclonal antibodies. Understanding the roles of physiological and environmental factors on the growth and metabolism of mammalian cells is a prerequisite for the development of rational scale-up procedures. An SP2/0-derived mouse hybridoma has been employed in the present work as a model system for hybridoma suspension culture. In preliminary shake flask studies to determine the effect of glucose and glutaminE, it was found that the specific growth rate, the glucose and glutamine metabolic quotients, and the cumulative specific antibody production rate were independent of glucose concentration over the range commonly employed in cell cultures. Only the specific rate of glutamine uptake was found to depend on glutamine concentration. The cells were grown in continuous culture at constant pH and oxygen concentration at a variety of dilution rates. Specific substrate consumption rates and product formation rates were determined from the steady state concentrations. The specific glucose uptake rate deviated from the maintenance energy model(1) at low specific growth rates, probably due to changes in the metabolic pathways of the cells. Antibody production was not growth-associated; and higher specific antibody production rates were obtained at lower specific growth rates. The effect of pH on the metabolic quotients was also determined. An optimum in viable cell concentration was obtained between pH 7.1 and 7.4. The viable cell number and viability decreased dramatically at pH 6.8. At pH 7.7 the viable cell concentration initially decreased, but then recovered to values typical of pH 7.1-7.4. Higher specific nutrient consumption rates were found at the extreme pH values; however, glucose consumption was inhibited at low pH. The pH history also influenced the behavior at a given pH. Higher antibody metabolic quotients were obtained at the extreme pH values. Together with the effect of specific growth rate, this suggests higher antibody production under environmental or nutritional stress.     

High-rate 3-methylcatechol production in Pseudomonas putida strains by means of a novel expression system

The bioconversion of toluene into 3-methylcatechol was studied as a model system for the production of valuable 3-substituted catechols in general. For this purpose, an improved microbial system for the production of 3-methylcatechol was obtained. Pseudomonas putida strains containing the todC1C2BAD genes involved in the conversion of toluene into 3-methylcatechol were used as hosts for introducing extra copies of these genes by means of a novel integrative expression system. A construct was made containing an expression cassette with the todC1C2BAD genes cloned under the control of the inducible regulatory control region for naphthalene and phenanthrene degradation, nagR. Introducing this construct into wild-type P. putida F1, which degrades toluene via 3-methylcatechol, or into mutant P. putida F107, which accumulates 3-methylcatechol, yielded biocatalysts carrying multiple copies of the expression cassette. As a result, up to 14 mM (1.74 g l(-1)) of 3-methylcatechol was accumulated and the specific production rate reached a level of 105 micromol min(-1) g(-1) cell dry weight, which is four times higher than other catechol production systems. It was shown that these properties were kept stable in the biocatalysts without the need for antibiotics in the production process. This is an important step for obtaining designer biocatalysts.     

A pH Control System Based on Malate Decarboxylation for the Cultivation of Lactic Acid Bacteria

Most species of lactic acid bacteria decarboxylate l-malate to lactate and CO(2) if an energy source such as glucose is present. A proton is taken up in the reaction, which prevents pH decreases in the growth medium caused by lactic acid production from glucose fermentation. MRS broth (pH 7.0) (Difco Laboratories) containing 10 mM glucose and various concentrations of l-malate (0, 25, 50, 75, and 100 mM) was used to cultivate Lactobacillus plantarum. After 72 h at 37 degrees C, all malate was decarboxylated and all glucose was fermented, with resultant final pH values of 4.5, 6.3, 6.9, 7.3, and 7.5, respectively. When d-malate (which cannot be decarboxylated) was substituted for l-malate, the final pH values were 4.5, 5.2, 5.6, 5.8, and 5.9. By varying the ratios of glucose to l-malate in the growth medium, it was possible to obtain pH values which were lower, the same, or higher than the initial pH values. In contrast, buffers such as phosphate only retard decreases in pH. l-Malate, when compared with K(2)PO(4) on an equal molar basis, provided greater resistance to decreases in pH. Higher specific growth rates were observed for L. plantarum and Leuconostoc mesenteroides when l-malate rather than K(2)PO(4) was incorporated into the growth medium.     

Influence of Environmental Factors and Medium Composition on Vibrio gazogenes Growth and Prodigiosin Production

Vibrio gazogenes ATCC 29988 growth and prodigiosin synthesis were studied in batch culture on complex and defined media and in chemostat cultures on defined medium. In batch culture on complex medium, a maximum growth rate of 0.75 h⁻¹ and a maximum prodigiosin concentration of 80 ng of prodigiosin · mg of cell protein⁻¹ were observed. In batch culture on defined medium, maximum growth rates were lower (maximum growth rate, 0.40 h⁻¹), and maximum prodigiosin concentrations were higher (1,500 ng · mg of protein⁻¹). In batch culture on either complex or defined medium, growth was characterized by a period of logarithmic growth followed by a period of linear growth; on either medium, prodigiosin biosynthesis was maximum during linear growth. In batch culture on defined medium, the initial concentration of glucose optimal for growth and pigment production was 3.0%; higher levels of glucose suppressed synthesis of the pigment. V. gazogenes had an absolute requirement for Na⁺; optimal growth occurred in the presence of 100 mM NaCl. Increases in the concentration of Na⁺ up to 600 mM resulted in further increases in the concentration of pigment in the broth. Prodigiosin was synthesized at a maximum level in the presence of inorganic phosphate concentrations suboptimal for growth. Concentrations of KH₂PO₄ above 0.4 mM caused decreased pigment synthesis, whereas maximum cell growth occurred at 1.0 mM. Optimal growth and pigment production occurred in the presence of 8 to 16 mg of ferric ion · liter⁻¹, with higher concentrations proving inhibitory to both growth and pigment production. Both growth and pigment production were found to decrease with increased concentrations of p-aminobenzoic acid. The highest specific concentration of prodigiosin (3,480 ng · mg protein⁻¹) was observed in chemostat cultures at a dilution rate of 0.057 h⁻¹. The specific rate of prodigiosin production at this dilution rate was approximately 80% greater than that observed in batch culture on defined medium. At dilution rates greater than 0.057 h⁻¹, the concentration of cells decreased with increasing dilution rate, resulting in a profile comparable to that expected for linear growth kinetics. No explanation could be found for the linear growth profiles obtained for both batch and chemostat cultures.     

Optimized production of active alpha-glucosidase by recombinant Escherichia coli. evaluation of processes using in vivo reactivation from inclusion bodies

During fast production in recombinant Escherichia coli, the enzyme alpha-glucosidase from Saccharomyces cerevisiae accumulates partially as inclusion bodies. The inclusion bodies are reactivated inside the cell upon temperature downshift. This in vivo reactivation was most efficient on complex medium with inclusion body production at 42 degrees C and reactivation at 30 degrees C, yielding volumetric activities 85% higher than those of extended isothermal production at low temperature. On defined medium, however, in vivo reactivation was slow. In fed-batch cultivations, feeding controls the specific growth rate independent of the temperature. Here, high growth rates fostered inclusion body formation even at low temperature. Intermediate growth rates permitted accumulation of active alpha-glucosidase without affecting the total amount of alpha-glucosidase. Low growth rates yielded similar activities and additionally prevented inclusion body formation. Moreover, high growth rates during production forestalled subsequent in vivo reactivation. Accumulation of activity after temperature reduction was possible with intermediate growth rates. The best time for temperature shift was concomitant to induction. Thus, in fed-batch culture, isothermal production at 30 degrees C and with a set growth rate of 0.12 h(-)(1) controlled by feeding was most efficient for production of active alpha-glucosidase. Compared to production under optimal conditions on complex medium, the specific and volumetric activities obtained were 3 and 45 times higher, respectively.     

Glucose-limited chemostat culture of Chinese hamster ovary cells producing recombinant human interferon-gamma

A Chinese hamster ovary (CHO) cell line expressing recombinant human interferon-gamma (IFN-gamma) was grown under glucose limitation in a chemostate at a constant dilution rate of 0.015 h(-1) with glucose feed concentrations of 2.75 mM and 4.25 mM. The changes in cell concentration that accompanied changes in the glucose feed concentration indicated that the cells were glucose-limited. The cell yield on glucose remained constant, but there was a decline in residual glucose concentration and a reduced lactate yield from glucose in the latter stages of the culture. The consumption rates for many of the essential amino acids were increased later in the culture. The volumetric rate of interferon-gamma production was maintained throughout the course of this culture, indicating that IFN-gamma expression was stable under these conditions. However, the specific rate of IFN-gamma production was significantly lower at the higher glucose feed concentration. Under glucose limitation, the proportion of fully glycosylated IFN-gamma produced by these cells was less than that produced in the early stages of batch cultures. The proportion of fully glycosylated IFN-gamma increased during transient periods of glucose excess, suggesting that the culture environment influences the glycosylation of IFN-gamma.     

Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 2. Effects of serum concentration, dissolved oxygen concentration, and medium pH in a batch reactor.

The effects of serum, dissolved oxygen (DO) concentration, and medium pH on hybridoma cell physiology were examined in a controlled batch bioreactor using a murine hybridoma cell line (167.4G5.3). The effect of serum was also studied for a second murine hybridoma cell line (S3H5/gamma 2bA). Cell growth, viability, cell density, carbohydrate and amino acid metabolism, respiration and energy production rates, and antibody production rates were studied. Cell growth was enhanced and cell death was decreased by increasing the serum level. The growth rates followed a Monod-type model with serum being the limiting component. Specific glucose, glutamine, and oxygen uptake rates and specific lactate and ammonia production rates did not change with serum concentrations. Amino acid metabolism was slightly influenced by the serum level. Cell growth rates were not influenced by DO between 20% and 80% air saturation, while the specific death rates were lowest at 20-50% air saturation. Glucose and glutamine uptake rates increased at DO above 10% and below 5% air saturation. Cell growth rate was optimal at pH 7.2. Glucose and glutamine uptake rates, as well as lactate and ammonia production rates, increased above pH 7.2. Metabolic rates for glutamine and ammonia were also higher below pH 7.2. The consumption or production rates of amino acids followed the glutamine consumption very closely. Cell-specific oxygen uptake rate was insensitive to the levels of serum, DO, and pH. Theoretical calculations based on experimentally determined uptake rates indicated that the ATP production rates did not change significantly with serum and DO while it increased continually with increasing pH. The oxidative phosphorylation accounted for about 60% of total energy production. This contribution, however, increased at low pH values to 76%. The specific antibody production rate was not growth associated and was independent of serum and DO concentrations and medium pH above 7.20. A 2-fold increase in specific antibody production rates was observed at pH values below 7.2. Higher concentrations of antibody were obtained at high serum levels, between 20% and 40% DO, and at pH 7.20 due to higher viable cell numbers obtained.     

Enhancement of erythropoietin production in recombinant Chinese hamster ovary cells by sodium lactate addition

The stabilization of optimum pH for cells can cause a higher erythropoietin (EPO) production rate and a good growth rate with the prolonged culture span in recombinant Chinese hamster ovary (r-CHO) cells. Our strategy for stabilizing the optimum pH in this study is to reduce the lactate production by adding sodium lactate to a culture medium. When 40 mM sodium lactate was added, a specific growth rate was decreased by approximately 22% as compared with the control culture. However the culture longevity was extended to 187 h, and more than a 2.7-fold increase in a final accumulated EPO concentration was obtained at 40 mM of sodium lactate. On the condition that caused the high production of EPO, a specific glucose consumption rate and lactate production rate decreased by 23.3 and 52%, respectively. Activity of lactate dehydrogenase (LDH) in r-CHO cells increased and catalyzed the oxidation of lactate to pyruvate, together with the reverse reaction, at the addition of 40 mM sodium lactate. The addition of 40 mM sodium lactate caused the positive effects on a cell growth and an EPO production in the absence of carbon dioxide gas as well as in the presence of carbon dioxide gas by reducing the accumulation of lactate.     

Optimized nitrate reductase assay predicts the rate of nitrate utilization in the halotolerant microalga Dunaliella viridis

An in situ method for measuring nitrate reductase (NR) activity in Dunaliella viridis was optimized in terms of incubation time, concentration of KNO₃, permeabilisers (1-propanol and toluene), pH, salinity, and reducing power (glucose and NADH). NR activity was measured by following nitrite production and was best assayed with 50 mM KNO₃, 1.2 mM NADH, 5% 1-propanol (v/v), at pH 8.5. The estimated half-saturation constant (K_s) for KNO₃ was 5 mM. Glucose had no effect as external reducing power source, and NADH concentrations >1.2 mM inhibited NR activity. Nitrite production was linear up to 20 min; longer incubation did not lead to higher nitrate reduction. The use of the optimized assay predicted the rate of NO ₃ ⁻ removal from the external medium by D. viridis with high degree of precision. This revised version was published online in September 2006 with corrections to the Cover Date.     

H2 production from chemostat fermentation of glucose byClostridium butyricum andClostridium pasteurianum in ammonium- and phosphate limitation

Summary In ammonium-limitation (4.55 mM NH₄ ⁺) at a dilution rate (D)=0.081 h^–1,Clostridium butyricum produced 2 mol H₂ per mol glucose consumed at pH 5.0, but at a low fermentation rate. At higher pH, important amounts of extracellular protein were produced. Phosphatelimitation (0.5 mM PO₄ ^–3) at D=0.061 h^–1 and pH 7.0 were the best conditions tested for hydrogen gas production (2.22 mol H₂ per mol glucose consumed) at a high fermentation rate. Steady-state growth at lower pH and with 0.1 mM PO₄ ^–3 resulted in proportional higher glucose incorporation into biomass and lower H₂ production. C. pasteurianum in NH₄ ⁺ limitation showed higher fermentation rates thanC. butyricum and a stabilized H₂ production around 2.08 (±0.06) mol per mol glucose consumed at various defined pH conditions, although the acetate/butyrate ratio increased to 1 at pH 7.0. The latter was also observed in phosphate-limitation, but here H₂ production was maximal (1.90 mol. per mol glucose consumed) at the lowest pH (5.5) tested.     

Glucose utilization, pH reduction and density dependent inhibition in cultures of chick embryo fibroblasts.

The multiplication rate of sparse cultures of chick embryo cells is only slightly lower at pH 6.9 than at pH 7.4. There is, however, a marked reduction in the multiplication rate of the pH 6.9 cultures before they reach confluency. Cultures at pH 7.4 continue to multiply beyond confluency with only a slight decrease in the multiplication rate. Eighty to ninety percent of the glucose taken up by the cells growing at each pH is converted to lactic acid which is released into the medium. Metabolic reduction in pH of the medium is almost entirely accounted for by the amount of lactic acid produced by the cells. Neither the intracellular nor extracellular accumulation of lactic acid nor the accompanying reduction in pH is sufficient to explain density dependent inhibition of the rate of multiplication of chick cells. The rate of lactic acid production and the multiplication rate of chick cells are independent of glucose concentration in the range of 2--16 mM. In view of the kinetic parameters for the uptake of glucose, this shows that glycolysis is not limited by the rate of glucose uptake and that depletion of glucose from the medium cannot account for the onset of density dependent inhibition of multiplication. However, when cells reach very high population densities, conventional glucose concentrations of 5 mM can be depleted overnight by chick cells. Since the multiplication rate of cells is dependent on glucose concentration when it falls below 2 mM, depletion of glucose may cause some growth inhibition in crowded cultures supplied with conventional medium.     

Optimization of fusion proinsulin production by high cell-density fermentation of recombinantE. coli

The optimum conditions for mass production of fusion proinsulin were studied in recombinantEscherichia coli strain BL21 (DE3) [pT7-PI] using fed-batch culture employing pH-stat method. Yeast extract was found to enhance both the growth rate of recombinantE. coli strain BL21 (DE3) [pT7-PI] and its cell mass yield. When the glucose concentration was 10 g/L in the initial medium, 10 g/L concentration of yeast extract was found to be optimal to control the acetate production and to augment both the cell mass yield and the growth rate. Optimum ratio of glucose to yeast extract to minimize the cost of the feeding medium in the fed-batch culture was calculated to be 1.225 and verified by the subsequent experiments. The appropriate inducer concentration and induction time were examined with isopropyl-β-D-thiogalactopyranoside (IPTG). Irrespective of the induction time, IPTG induction resulted in the reduction of growth rate, but the expression level of the fusion protein was maintained at the level of about 20% of the total proteins. Since the volumetric productivity was well maintained in the range between 0.15 and 0.18 g/L.hr at the inducer concentration of above 0.025 mM, the appropriate inducer concentration, in relation to the inducer cost, is considered to be about 0.025 mM.     

Effect of ammonia production on intracellular pH: Consequent effect on adenovirus vector production

Recombinant adenoviral vectors (AdV) have proven to be highly efficient for the delivery and expression of foreign genes in a broad spectrum of cell types and species both for vaccination and gene therapy in a number of specific applications. In this study, the effect of ammonia production on intracellular pH (pH(i)) and consequently inhibition of AdV production at high cell densities is assessed. Different specific ammonia production rates were obtained for 293 cells adapted to grow in glutamate supplemented medium (non-ammoniagenic medium) as compared with 293 cells growing in glutamine supplemented medium (ammoniagenic medium); pH(i) was observed to be lower during cell growth and AdV production at both high and low CCI in the ammoniagenic medium, where the specific ammonia production rate is higher. In addition, after infection at CCI of 3x10(6)cell/ml, the cell viability decreased significantly in the ammoniagenic medium, attributed to the activation of an acidic pathway of apoptosis. Furthermore, AdV DNA was observed to be degraded at the observed pH(i) in the ammoniagenic medium, decreasing significantly the amount of AdV DNA available for encapsulation. To elucidate the pH(i) effect upon AdV production, 293 cells were infected at a CCI of 1 x 10(6)cell/ml in the non-ammoniagenic medium with a manipulated pH(i) as observed at the time of infection at CCI of 3 x 10(6)cell/ml in the ammoniagenic (pH(i) 7.0) and non-ammoniagenic (pH(i) 7.3) media; AdV volumetric productivities were observed to be lower when the cells were exposed to the lower pH(i). Thus, the importance of controlling all the factors contributing to pH(i) on AdV production, such as ammonia production, has been established.     

Effects of medium glucose concentration and pH on docosahexaenoic acid content of heterotrophic Crypthecodinium cohnii

The effects of medium glucose concentration and pH on growth and docosahexaenoic acid (DHA, C22:6 ω-3) content of Crypthecodinium cohnii were investigated. Over a range of glucose concentrations (5–40 g l⁻¹) investigated, the highest specific growth rate (0.12 h⁻¹), highest cell dry weight concentration (3.13 g l⁻¹) and highest growth yield on glucose (0.6 g g⁻¹) were obtained at 20 g l⁻¹ glucose. However, the highest degree of fatty acid unsaturation (3.2) and highest DHA proportion (53.4% of total fatty acids) were achieved at 5 g l⁻¹ glucose. Low glucose concentrations enhanced the degree of fatty acid unsaturation and DHA formation. Medium pH also affected cell growth, fatty acid unsaturation and DHA proportion. When medium pH was 7.2, the highest specific growth rate (0.089 h⁻¹), highest cell dry weight concentration (2.73 g l⁻¹), highest growth yield on glucose (0.564 g g⁻¹), highest degree of fatty acid unsaturation (3.4) and highest DHA proportion (56.8% of total fatty acids) were obtained. Results suggest that glucose concentration and pH value could be effectively manipulated to achieve maximum DHA production by C. cohnii.     

Enhanced production of human monoclonal antibodies by the use of fructose in serum-free hybridoma culture media

It was found that the production of human monoclonal antibodies (MoAbs) by human-human hybridomas can be significantly enhanced by replacing glucose with fructose in the dish culture medium. Optimization of initial concentrations of fructose and glutamine, another influencing factor for MoAb production, enabled an enhanced production of human MoAb 2.1 times higher than that obtained using the conventional culture media employing glucose. It was shown by kinetic analysis that enhanced MoAb production at the optimum fructose concentration can be attributed to the retention of high specific antibody production rates and diminished time lag during the course of culture.These dish culture results with fructose-containing medium were successfully applied to the continuous perfusion culture with a slight modification, where 2.9- and 1.9-fold enhancements in specific antibody production rate and MoAb concentration, respectively, were attained as compared with the conventional glucose-containing medium.An inverse relationship was observed between the secreted concentrations of lactic acid and MoAb when the hybridoma was cultured in the media containing varying concentrations of fructose, i.e., the lower the lactic acid concentration, the higher the MoAb production andvice versa, suggesting that fructose at appropriate concentrations in the medium can serve as an alternative sugar for the efficient production of human MoAbs, with reduced pH shifts, for the serum-free culture of human-human hybridomas.     

Characteristics of CHO-K1 cell culture producing two types of recombinant soluble thrombomodulin

Recombinant CHO-K1 cells, expressing human soluble thrombomodulin, were cultured in a serum-free medium and characteristics of the culture associated with glucose and lactate were investigated. In 3 L fermentor (3LFM) cultures, the cell density was found to have a proportional relationship with the volumetric glucose consumption rate, and the specific glucose consumption rates were constant at about 0.2 mg/(10⁶ cells·d) despite many differences in the culture conditions. Thus, it was concluded that the glucose consumption rate is little influenced by the condition of the cells or the culture conditions, and that the cell density can be estimated by the glucose consumption rate calculated from glucose measurement. Two types of thrombomodulin (rsTMα and rsTMβ) were produced, in which rsTMβ possesses chondroitin-4-sulfate and has greater anticoagulant activities than rsTMα. Therefore, it is important to investigate the rsTMα and rsTMβ production properties, and to determine the optimal culture conditions for high rsTMβ production. The most important factor to increase the production of rsTMβ relative to rsTMα (the β/α ratio) was effective aeration. Moreover, a lower ratio of lactate production/glucose consumption (the L/G ratio) with sufficient oxygen, high glucose concentration, and a longer medium exchange interval contributed to a higher specific rsTMβ production rate. Since there was a linear relationship between the production rate of each type of rsTM and the overall rsTM production rate per liter, it is expected that the rsTMα and rsTMβ production rates may be able to be estimated from the overall rate and the rsTMβ production increased by increasing the overall rsTM production with a lower L/G ratio.