Kinetic response of a <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> cell line to different medium formulations and culture conditions

In the past few years, Drosophila melanogaster cells have been employed for recombinant protein production purposes, and a comprehensive knowledge of their metabolism is essential for process optimization. In this work, the kinetic response of a Schneider S2 cell line, grown in shake flasks, in two different culture media, the serum-free SF900-II^® and the serum-supplemented TC-100, was evaluated. Cell growth, amino acids and glucose uptake, and lactate synthesis were measured allowing the calculation of kinetic parameters. The results show that S2 cells metabolism was able to adjust to different environmental situations, as determined by medium formulation, as well as by the particular situation resulting from the culture conditions. Cells attained a 163% higher final cell concentration (1.4 × 10⁷ cells mL⁻¹) in SF900 II^® medium, when compared to serum-supplemented TC-100 medium. Also, a maximum specific cell growth rate 52% higher in SF900 II^® medium, when compared to serum-supplemented TC-100 one, was observed. Glutamine was the growth limiting factor in SF900 II^® medium, while glucose, sometimes associated with glutamine, controlled growth in serum-supplemented TC-100 medium based formulation. The different pattern of lactate production is an example of the versatility of the metabolism of these cells. This by-product was produced only in glutamine limitation, but the amount synthesized depended not only on the excess glucose, but on other medium components. Therefore, in serum-supplemented TC-100 medium a much smaller lactate amount was generated. Besides, glucose was identified not only as a growth limiting factor, but also as a viability limiting factor, since its depletion accelerated cell death.     

Influence of temperature rise on kinetic parameters during batch propagation of Kluyveromyces fragilis in cheese whey under ambient conditions

Three 5 l working volume fermenters were used to investigate the growth of the yeast Kluyveromyces fragilis in acid cheese whey under ambient temperature in order to assess the specific growth rate and yield, the lactose and oxygen uptake rates during the various phases of batch culture, the effect of increasing temperature on the various kinetic parameters, and the need for a cooling unit for single cell production batch systems. The initial dissolved oxygen in the medium was 5.5 mg l^–1 and the pH was maintained at 4.5. The observed lag phase, specific growth rate and maximum cell number were 4 h, 0.2 h^–1 and 8.4 × 10⁸ cells ml^–1, respectively. About 99% of the lactose in cheese whey was utilized within 20 h, 85% during the exponential growth phase. The specific lactose utilization rates by K. fragilis were 0.20 × 10^–12, 1.457 × 10^–12, 0.286 × 10^–12 and 0.00 g lactose cell^–1 h^–1, for the lag, exponential, stationary and death phases, respectively. The dissolved oxygen concentration in the medium decreased as the cell number increased. The lowest oxygen concentration of 1.2 mg l^–1 was observed during the stationary phase. The volumetric oxygen transfer coefficient was 0.41 h^–1 and the specific oxygen uptake rates were 0.32 × 10^–12, 2.14 × 10^–12, 0.51 × 10^–12 and 0.003 × 10^–12 mg O₂ cell^–1 h^–1, for the lag, exponential, stationary and death phases, respectively. The maximum temperature recorded for the medium was 33 °C, indicating that a cooling unit for batch production of single cell protein at ambient temperature is not needed for this type of bioreactor. The increase in medium temperature affected the cell growth and the lactose and oxygen uptake rates.     

Cell immobilization in composite agar layer microporous membrane structures: growth kinetics of gel-entrapped cultures and cell leakage limitation by a microporous membrane

Agar discs containing different amounts of viable Escherichia coli cells (from 10 to 10⁶ organisms·g^–1 agar) were incubated in a nutrient medium and the growth of agar-entrapped bacteria and free (released) cells was monitored. The study was repeated with composite immobilized-cell structures obtained by placing a microporous membrane filter between the gel matrix and the incubation medium. In both cases, immobilized cells grew exponentially and reached a peak concentration an order of magnitude higher than that of free (suspended) cell cultures. The maximum specific growth rates of entrapped bacteria, ranging between 0.0115 min^–1 and 0.0145 min^–1, i.e., slightly higher than that of control free cultures (0.011 min^–1), showed no clear dependence on the initial cell loading (ICL). The microporous filter proved efficient in limiting cell leakage since it noticeably lengthened the leakage time at a given ICL. This efficiency, however, decreased at high ICL and high growth rate of immobilized organisms. Correspondence to: G.-A. Junter     

Effects of peptone on hybridoma growth and monoclonal antibody formation

Hybridoma WuT3 secreting a monoclonal antibody against T lymphocytes was grown in RPMI 1640 medium supplemented with 1% human serum. The effect of the concentration of peptone, as an additive, was investigated on cell growth, monoclonal antibody formation, and cell metabolism over 0–10 g l^–1 range. It was found that 1–5 g l^–1 peptone can significantly promote the growth of cells and increase the formation of monoclonal antibody, especially at 3–5 g l^–1, when both the accumulating level and secretion rate of monoclonal antibody are higher than that at other peptone concentrations. Based on glucose, lactate and ammonia analysis data, the efficiency of glycolysis was assessed and the utilization of amino acids was more efficient at 3–5 g l^–1 peptone. The cell growth and monoclonal antibody formation were inhibited at higher peptone concentrations, e.g. 10 g l^–1.     

Improvement of the culture stability of non-anchorage-dependent animal cells grown in serum-free media through immobilization

A murine hybridoma cell line producing a monoclonal antibody against penicillin-G-amidase and a murine transfectoma cell line secreting a monovalent chimeric human/mouse Fab-antibody fragment were cultivated in three different media (serum-containing, low protein serum-free, and iron-rich protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In static batch cultures in flasks both cell lines showed similar good growth in all three media.In suspension in a stirred reactor, the hybridoma cell line could be cultivated satisfactory only in serum-containing medium. In low protein serum-free medium, Pluronic F68 had to be added to protect the hybridoma cells against shear stress. But even with this supplement only batch, not chemostat mode was possible. In iron-rich protein-free medium the hybridoma cells grew also in continuous chemostat mode, but the stability of the culture was low. The transfectoma cell line did not grow in stirred reactors in any of the three media.Good results with both cell lines were obtained in fixed bed experiments, where the cells were immobilized in macroporous Siran^®-carriers. The media, which were optimized in flask cultures, could be used without any further adaptation in the fixed bed reactor. Immobilization improved the stability and reliability of cultures of non-adherent animal cells in serum-free media tremendously compared to suspension cultures in stirred reactors. The volume-specific glucose uptake rate, an, indicator of the activity of the immobilized cells, was similar in all three media. Deviations in the metabolism of immobilized and suspended cells seem to be mainly due to low oxygen concentrations within the macroporous carriers, where the cells are supplied with oxygen only by diffusion.List of symbols c substrate or product concentration mmol l^–1 - c₀ substrate or product concentration in the feed mmol l^–1 - c_Glc glucose concentration mmol l^–1 - c_Gln glutamine concentration mmol l^–1 - c_Amm ammonia concentration mmol l^–1 - c_Lac lactate concentration mmol l^–1 - c_FAB concentration of Fab# 10 antibody fragment g l^–1 - c_MAb monoclonal antibody concentration mg l^–1 - D dilution rate d^–1 - q cell-specific substrate uptake or metabolite production rate mmol cell^–1 h^–1 - q_Glc cell-specific glucose uptake rate mmol cell^–1 h^–1 - q_Gln cell-specific glutamine uptake rate mmol cell^–1 h^–1 - q_MAb cell-specific MAb production rate mg cell^–1 h^–1 - q^* volume-specific substrate uptake or metabolite production rate mmol l^–1 h^–1 - q^*FB volume-specific substrate uptake or metabolite production rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Glc volume-specific glucose uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Gln volume-specific glutamine uptake rate related to the fixed volume mmol l_FB ^–1 h^–1 - q^*FB,MAb volume-specific MAb production rate related to the fixed volume mg l_FB ^–1 h^–1 - q^*FB,02 volume-specific oxygen uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - t time h - U superficial flow velocity mm s^–1 - V medium volume in the conditioning vessel of the fixed bed reactor l - V_FB volume of the fixed bed l - x_v viable cell concentration cells ml^–1 - y_Amm,Gln yield of Ammonia from glutamine - y_Lac,Glc yield of lactate from glucose - specific growth rate h^–1 - _d specific death rate h^–1     

Propagation ofSpodoptera frugiperda cells (Sf9) and production of recombinant proteins with the baculovirus expression system using improved spinner flasks with membrane aeration

Summary It has been shown that the growth of Spodoptera frugiperda cells is significantly reduced or ceased under oxygen limiting culture conditions. This paper describes the use of a new membrane-aerated spinner flask which was compared to conventional surface-aerated spinner flasks with regard to growth of the insect cell line Sf9 and recombinant protein production after infection with baculovirus. Using a commercially available serum-free culture medium Sf9 cells reached highest cell densities (3×10⁶ ml^–1) in the membrane-aerated spinner flask. Production of recombinant protein was also influenced by the oxygen supply. In the membrane-aerated spinner flask and in a surface-aerated spinner flask with reduced filling volume more than 20000 U ml^–1 of a recombinant interleukin-2 variant were accumulated whereas only 100 U ml^–1 were produced in a surface-aerated spinner flask with insufficient oxygen supply. Sufficient oxygenation appears to be essential for proliferation of Sf9 cells as well as recombinant protein production after infection with baculovirus. Membrane oxygenation allows sufficient oxygen supply at high cell density and an at least 2.5 fold higher filling volume per spinner unit.     

Effect of mechanical agitation on hybridoma cell growth

Hybridoma cells (S3H5/2bA2) were grown in spinner flasks at different agitation speeds. It was found that cells in stationary and decline phases of growth were sensitive to shear force caused by agitation but cells in growth phase seemed less sensitive to the shear forces introduced. The death rate was found to be. 0.007 hr^–1 in T flasks but 0.018 hr^–1 and 0.028 hr^–1 at 100 and 200 rpm, respectively, while the growth rate was about 0.05 hr^–1 for all cases.     

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.     

Invertase and phosphatase of yeast in a phosphate-limited continuous culture

Summary Deficiency of inorganic phosphate caused the hyper production of invertase and the derepression of acid phosphatase in a continuous culture ofSaccharomyces carlsbergensis. The specific invertase activity was 40,000 enzyme units per g dry cell weight at a dilution rate lower than 0.05 h^–1 with a synthetic glucose medium of which the molecular ratio of KH₂PO₄ to glucose was less than 0.006. This activity is eight fold higher than in a batch growth and 1.5 fold as much as the highest enzyme activity observed so far in a glucose-limited continuous culture.For the hyper production of invertase, it is necessary to culture the yeast continuously by keeping the Nyholm's conservative inorganic phosphate concentration at less than 0.2 m mole per g dry weight cell. The derepression of acid phosphatase brought about by phosphate deficiency, was similar in both batch and continuous cultures.Nomenclature D dilution rate of continuous culture (h^–1) - E_i invertase concentration in culture (enzyme unit l^–1) - E_p acid phosphatase concentration in culture (enzyme unit l^–1) - P inorganic phosphate concentration in culture (mM) - S glucose concentration in culture (mM) - X cell concentration in culture (g dry weight cell l^–1) Greek Letter specific rate of growth (h^–1) Suffix f feed - 0 initial value     

Determination of cell lysis and death kinetics in continuous hybridoma cultures from the measurement of lactate dehydrogenase release

The death of the hybridoma VO 208 in a continuous culture at pH 7 and 6.8 was investigated by measuring both the appearance of visible dead cells which do not exclude the trypan blue dye and the release of lactate dehydrogenase (LDH) in the culture medium. The intracellular LDH was found to be completely released either when live cells lysed or when they were transformed into visible dead cells. No significant lysis of blue dead cells could be observed at the two different pH. Using a LDH balance over the culture system, cell lysis was found negligible at pH 7, but accounted for 20% of the total cell death at pH 6.8. A methodology is proposed to evaluate the rate constants of hybridoma lysis and total death. For the investigated cell line in continuous culture, the calculated total cell death rate constant was found to increase from 0.002 h^–1 to 0.01 h^–1 when decreasing the pH from 7 to 6.8.Abbreviations D dilution rate (h^–1) - k_b specific trypan-blue dead cells appearance rate (h^–1) - k_L specific lysis rate of viable cells (h^–1) - k_d specific death rate (h-1) - LDH₀ lactate dehydrogenase activity in the feed culture medium (IU.l^–1) - LDH lactate dehydrogenase activity in the outlet culture medium (IU.l^–1) - LDH_i intracellular lactate dehydrogenase activity of viable cells (IU.10^–9 cells) - r_LDH total rate of LDH release (IU.h^–1.L^–1) - r_b transformation rate of viable cells into blue dead cells (10⁹ cells.h^–1.L^–1) - x_v viable cell concentration (10⁹ cells.l^–1) - x_b trypan-blue dead cell concentration (10⁹ cells.l^–1)     

Growth of the toxic dinoflagellate Alexandrium minutum (Dinophyceae) using high biomass culture systems

Toxic dinoflagellates are important in natural ecosystems and are ofglobal economic significance because of the impact of toxic blooms onaquaculture and human health. Both the organisms and the toxins they producehave potential for biotechnology applications. We investigated autotrophicgrowth of a toxic dinoflagellate, Alexandrium minutum, inthree different high biomass culture systems, assessing growth, productivityandtoxin production. The systems used were: aerated and non-aerated2-L Erlenmeyer flasks; 0.5-L glass aerated tubes; anda 4-L laboratory scale alveolar panel photobioreactor. A range ofindicators was used to assess growth in these systems. Alexandriumminutum grew well in all culture conditions investigated, with amarked increase in both biomass and productivity in response to aeration. Thehighest cell concentration (4.9 × 10⁵ cellsmL^–1) and productivity (2.6 ×10⁴cells mL^–1d^–1) was achieved inthe aerated glass culture tubes. Stable growth of A.minutum in the laboratory scale alveolar panel photobioreactor wasmaintained over a period of five months, with a maximum cell concentration of3.3 × 10⁵ cells mL^–1, a meanproductivity of 1.4 × 10⁴ cells mL^–1d^–1, and toxin production of approximately 20g L^–1 d^–1 with weeklyharvesting.     

Influence of acetate on the growth of recombinant Escherichia coli JM103 and product formation

The influence of the acetate addeed to the M9 minimal medium and to the Luria-Bertani medium without and with glucose supplement on the growth of recombinant Escherichia coli J103 with three different types of multicopy plasmids and on the production of the fusion protein SpA :: EcoRI were investigated in shake flasks without and with induction of the gene expression by a temperature shift from 30 °C to 42 °C. At the beginning of the induction of gene expression concentrated LB-medium was added to the shake flask. Without this supplement of M9 medium no gene expression occurred.List of Symbols LB Luria Bertani cultivation medium (Table 2) - M9 cultivation medium (Table 1) - P enzym activity [U ml^–1] - te]TCC total cell count [10⁶ cells ml^–1] - specific growth rate [h-1]     

Studies on the production of useful chemicals,especially fatty acids in the marine diatom Nitzschia conspicua Grunow

A local marine diatom, Nitzschia conspicua Grunow, was cultured in enriched synthetic seawater using flasks (agitated by magnetic stirring) and a 1.2 l fermenter. Lipids, fatty acids, proteins, carbohydrates and ash of the flask cultures were determined at various stages of growth (day 3, 5, 7, 10, 13, 15 and 17). The fermenter culture was harvested during the stationary phase for similar chemical analyses. N. conspicua attained a higher biomass concentration during the stationary phase when cultured in the fermenter (188 mg dry weight l^–1) than in flasks (140–151 mg dry weight l^–1). However, both systems showed similar specific growth rates based on chlorophyll-a concentration. Appreciable amounts of the essential fatty acids 20:4 (0.6–4.7% total fatty acids) and 20:5 (1.9–4.7% total fatty acids) are present in this diatom. Maximal amounts of these fatty acids were produced after 7 days' growth (i.e. 2 days after the end of the exponential phase). Lipids, fatty acids, proteins, carbohydrates and ash varied with culture age in N. conspicua.author for correspondence     

Feasibility study on the use of hyperosmolar medium for improved antibody production of hybridoma cells in a long-term,repeated-fed batch culture

To test the feasibility of using hyperosmolar medium for improved antibody production in a long-term, repeated fed-batch culture, the influence of various culture conditions (serum concentration and cultivation method) on the hybridoma cells' response to hyperosmotic stress resulting from sodium chloride addition was first investigated in a batch culture. The degree of cell growth depression resulting from hyperosmotic stress was dependent on serum concentrations and cultivation methods (static and agitated cultures). Depression of cell growth was most significant in agitated cultures with low serum concentration. However, regardless of serum concentrations and cultivation methods used, the hyperosmotic stress significantly increased specific antibody productivity (q _MAb). Increasing osmolality from 284 to 396 mOsm kg^–1 enhanced the q_MAb in agitated cultures with 1% serum by approximately 124% while the similar osmotic stress enhanced the q _MAb in static cultures with 10% serum by approximately 153%. Next, to determine whether this enhanced q_MAb resulting from hyperosmotic stress can be maintained after adaptation, long-term, repeated-fed batch cultures with hyperosmolar media were carried out. The cells appeared to adapt to hyperosmotic stress. When a hyperosmolar medium (10% serum, 403 mOsmkg^–1) was used, the specific growth rate improved gradually for the first four batches and thereafter, remained constant at 0.040±0.003 (average ± standard deviation) hr^–1 which is close to the value obtained from a standard medium (10% serum, 284 mOsmkg^–1) in the batch culture. While the cells were adpating to hyperosmotic stress, the q_MAb was gradually decreased from 0.388×10^–6 to 0.265×10^–6 g cell hr^–1 and thereafter, remained almost constant at 0.272±0.014× 10^–6 g cell^–1 hr^–1. However, this reduced q _MAb after adaptation is still approximately 98% higher than the q_MAb obtained from a standard medium in the batch culture.The authors would like to thank Dr.M. Kaminski for providing the hybridoma cell line used in this study. This work was supported by the Korea Science and Engineering Foundation.     

Effect of sugar concentration on ginsenoside biosynthesis in hairy root cultures of Panax quinquefolium cultivated in shake flasks and nutrient sprinkle bioreactor

The study assessed the influence of sugar concentration (10, 20, 30, 50, 70, 100, 120 g l^?1) on growth and ginsenoside biosynthesis in Panax quinquefolium hairy roots cultivated in shake flasks and a nutrient sprinkle bioreactor. The highest growth rate was achieved in medium containing 3–5 % sucrose. More than 70 g l^?1 or less than 20 g l^?1 sugar content in the medium induces significant inhibition of root growth when cultivated in shake flasks. The saponin content was determined using HPLC. The maximum yield (above 9 mg g^?1 d.w.) of the sum of six examined ginsenosides (Rb₁, Rb₂, Rc, Rd, Re and Rg₁) in hairy roots cultivated in shake flasks was obtained with 30 g l^?1 sucrose in the medium. The sucrose concentration in the medium was found to correlate with saponin content in bioreactor-cultured specimens. A higher level of protopanaxadiol derivatives was found for lower (20 and 30 g l^?1) sucrose concentrations; higher sucrose concentrations (50 and 70 g l^?1) in the medium stimulated a higher level of Rg group saponins.     

Improved methods for investigating the external redox potential in hybridoma cell culture

Because of the interest in understanding and optimizing secretion of proteins from mammalian cells, reliable and more reproducible methods are needed to monitor the external redox potential of animal cells in suspension culture. An improved off-line method was established that greatly reduces the typically long response time of redox electrodes in cell culture media and improves the standardization of redox probes. In addition, the dependence of medium redox potential on dissolved oxygen concentrations and pH was investigated using cell-free medium. Off-line as well as on-line redox potential measurements were then applied to spinner or bioreactor cultures of murine hybridoma cells. Serum containing or protein-free medium were used. The time dependence of the experimentally determined external redox potential was found to be affected not only by oxygen, pH, and medium composition. but to a significant extent by the rate of generation of reductants by hybridoma cells. The observed specific rate of medium reduction by generation of reductants (mV h^–1 viable cell^–1) decreased during exponential growth while cell number increased from 2×10⁵ viable cells ml^–1 to 3.5×10⁶ viable cells ml^–1. This rate, however, was essentially constant at –7.3 mV h^–1±3.7 mV h^–1 per 10¹⁰ viable cells during growth under conditions of constant dissolved oxygen tension and constant pH. Using these observations, the quantity of reductants synthesized and secreted into the medium by viable hybridoma cells was estimated to be approximately 1.3 mole h^–1 per 10¹⁰ viable hybridoma cells. The time course of specific monoclonal antibody secretion rate did not correlate with changes in the external oxidation/reduction potential in either serum containing or protein-free medium.     

Optimisation of the cell release from immobilised cells of Bacillus simplex cultivated in culture media enriched with Cd2+: influence of Cd2+, inoculum size, culture medium and alginate beads characteristics

Calcium alginate beads were used to entrap a Bacillus sp. that has the ability to biosorb cadmium. During the batch incubation of alginate beads in a `rich' or a `poor' liquid medium, cell release out of the beads was noticed with a lag phase which was inversely proportional to the inoculum size (2×10⁷ or 2×10⁸ cells ml^–1 alginate), to the medium content, and proportional to the alginate concentration (10 or 15 g l^–1) and to the cadmium concentration (1, 5 or 10 mg l^–1). In addition, the cell release occurred more quickly when the medium was renewed. When the concentration was below 5 mg l^–1, the alginate matrix seemed to protect the bacteria against Cd²⁺ toxicity.     

Optimization of protein-production by the baculovirus expression vector system in shake flasks

Summary Shake flasks were successfully employed for the cultivation of Spodoptera frugiperda (Sf-9) insect cells and for the production of \-galactosidase, a recombinant model protein, utilizing the baculovirus expression vector system. The culture doubling time and maximal cell density were 20 h and 5 × 10⁶ cells/ml respectively. The optimal liquid volumes for flasks rotating at 100 rpm were 25–40% of the flask total volume. Enzyme production (about 600 mg/l) was best at a multiplicity of infection of between 1 and 20 and at a cell density at time of infection of 0.7 × 10⁶ cells/ml. At a rotation speed of 100 rpm, Pluronic F-68 had no effect on growth and enzyme production. Offprint requests to: Y. Shoham     

Interleukin-6 is antiproliferative to a mouse hybridoma cell line and promotive for its antibody productivity

Monoclonal antibody production by hybridoma cells at moderately slowed growth states would be favorable for commercial scale production since cells can devote their resources to performing the differentiated function, immunoglobulin production. We found that a purified recombinant human interleukin-6, which had been reported to support or stimulate proliferation of B cell hybridoma/plasmacytoma cells, suppressed growth of a hybridoma cell line in serum-free medium. In the presence of the interleukin, the growth-suppressed cells were viable for remarkably long periods in batch culture, and after removal of the interleukin from the culture medium, they started to proliferate at their normal growth rate. As the concentration of the interleukin increased in the culture, the growth rate decreased and the specific antibody productivity (antibody production rate per cell) increased to 5-fold of control at 10 U ml^–1 (2 ng ml^–1) of the interleukin.Abbreviations IL-1,2, and 6 interleukin-1, 2 and 6 - rhIL-6 recombinant human interleukin-6 - MCAb monoclonal antibody - TNP trinitrophenyl - unit (U) of interleukin-6 A unit (U) is equivalent to the amounts of IL-6 which gives one-half maximal IgM secretion by SKW6-CL4 cells (1U ml^–1=200 pg ml^–1)     

Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line

A serum-free medium, WCM5, has been developed for the large scale propagation of CHO (Chinese hamster ovary) cells which express recombinant protein using dihydrofolate reductase as a selectable marker. WCM5 was prepared by supplementing Iscoves medium without lecithin, albumin or transferrin with a number of components which were shown to benefit growth. WCM5 medium contained 5 mg l^–1 human recombinant insulin (Nucellin) but was otherwise protein-free. CHO 3D11^* cells which had been engineered to express a humanised antibody, CAMPATH^*-1H, were routinely grown using serum-containing medium. From a seeding density of 10⁵ cells ml^–1, cells grown in static culture with serum reached a maximal cell density of 6.5×10⁵ cells ml^–1 after 6 days in culture and produced a maximal antibody concentration of 69 mg l^–1 after 11 days in culture. CHO 3D11^* cells grown with serum were washed in serum-free medium then cultured in WCM5 medium. Following a period of adaptation the cell growth and product yield was superior to that achieved with serum-containing medium. CHO cells producing CAMPATH-1H grown in an 8000 l stirred bioreactor seeded with 2×10⁵ cells ml^–1 reached a maximal viable cell density of 2.16×10⁶ cells ml^–1 after 108 h in culture and a maximal antibody concentration of 131.1 mg l^–1 after 122 h in culture.Abbreviations CHO Chinese hamster ovary - dhfr dihydrofolate reductase - dhfr dihydrofolate reductase deficient - MTX methotrexate - H hypoxanthine - T thymidine - T/V trypsin versene - F12 Hams F12 medium - NEAA non essential amino acids