Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus-infected insect cells

The baculovirus infection process ofSpodoptera frugiperda (Sf9) insect cells in oxygen-controlled bioreactors in serum-free medium was investigated using a recombinantAutographa californica (AcNPV) virus expressing -galactosidase enzyme as a model system. A variety of monitoring techniques including trypan blue exclusion, fluorescent dye staining, oxygen uptake rate (OUR) measurements, and glucose consumption were applied to infected cells to determine the best way of evaluating cell integrity and assessing the course of baculovirus infection. The metabolism of newly-infected cells increased 90% during the first 24 hours, but as infection proceeded, and cells gradually succumbed to the baculovirus infection, the cytopathic effect of the baculovirus on the cells became evident. Oxygen and glucose uptake rate measurements appeared to more accurately assess the condition of infected cells than conventional trypan blue staining, which tended to overestimate cell viability in the mid stages of infection. The optimal harvest time varied, depending on which technique — SDS-PAGE, chromogenic (ONPG) or fluorometric (C₁₂FDG) — was used to monitor -galactosidase production. Specific -galactosidase production was found to be insensitive to a wide range of culture dissolved oxygen tensions, whereas resuspending cells in fresh medium prior to infection increased volumetric productivity approximately two-fold (800,000 units -galactosidase/ml) compared to cultures infected in batch mode and allowed successful infections to occur at higher cell densities.Abbreviations ONPG ortho-phenyl 2--D-galactopyranoside - OUR oxygen uptake rate (-mol O₂/liter/hour) - q_glucose specific glucose uptake rate (mg glucose/10⁶cell/hour) - q_glutamine specific glutamine uptake rate (mg glutamine/10⁶cell/hour) - qO₂ specific oxygen uptake rate (-mol O₂/10⁶cell/hour) - MOI virus multiplicity of infection (viral plaque forming units/cell)     

Insect cells respiratory activity in bioreactor

Specific respiration rate ( ) is a key parameter to understand cell metabolism and physiological state, providing useful information for process supervision and control. In this work, we cultivated different insect cells in a very controlled environment, being able to measure . Spodoptera frugiperda (Sf9) cells have been used through virus infection as host for foreign protein expression and bioinsecticide production. Transfected Drosophila melanogaster (S2) cells can be used to produce different proteins. The objective of this work is to investigate respiratory activity and oxygen transfer during the growth of different insect cells lines as Spodoptera frugiperda (Sf9), Drosophila melanogaster (S2) wild and transfected for the expression of GPV and EGFP. All experiments were performed in a well-controlled 1-L bioreactor, with SF900II serum free medium. Spodoptera frugiperda (Sf9) cells reached 10.7 × 10⁶ cells/mL and maximum specific respiration rate () of 7.3 × 10⁻¹⁷ molO₂/cell s. Drosophila melanogaster (S2) cells achieved 51.2 × 10⁶ cells/mL and of 3.1 × 10^–18 molO₂/cell s. S2AcGPV (expressing with rabies virus glycoprotein) reached 24.9 × 10⁶ cells/mL and of 1.7 × 10^–17 molO₂/cell s, while S2MtEGFP (expressing green fluorescent protein) achieved 15.5 × 10⁶ cells/mL and  = 1.9 × 10⁻¹⁷ molO₂/cell s. Relating to the Sf9, S2 cells reached higher maximum cell concentrations and lower specific respiration rate, which can be explained by its smaller size. These results presented useful information for scale-up and process control of insect cells.     

Relationships Between Slc1a5 and Osteoclastogenesis

Slc1a5 (ASCT2) encodes a small neutral amino-acid exchanger and is the most well-studied glutamine transporter in cancer cells. To investigate the role of Slc1a5 in osteoclastogenesis, we developed Slc1a5-deficient mice by using a conventional gene-targeting approach. The Slc1a5^−/− mice showed no obvious abnormalities in growth. Glutamine uptake was assessed in Slc1a5^+/+ and Slc1a5^−/− bone marrow cells stimulated with RANKL. The rate of glutamine uptake in Slc1a5^−/− bone marrow cells was reduced to 70% of that of cells from Slc1a5^+/+ bone marrow. To confirm the involvement of Slc1a5 in osteoclast formation, bone marrow cells derived from Slc1a5^+/+ or Slc1a5^−/− mice were stimulated with RANKL and macrophage colony-stimulating factor and stained with tartrate-resistant acid phosphatase. The bone resorption activity and actin ring formation of stimulated cells were measured. The formation of multinucleated osteoclasts in bone marrow cells isolated from Slc1a5^−/− mice was severely impaired compared with those from Slc1a5^+/+ mice. RANKL-induced expression of ERK, NFκB, p70S6K, and NFATc1 was suppressed in Slc1a5^−/− osteoclasts. These results show that Slc1a5 plays an important role in osteoclast formation.

Osteoclasts are giant multinucleated cells of hematopoietic origin that are responsible for bone resorption. The differentiation of osteoclasts can be induced by treating bone marrow macrophages with RANKL.² After stimulation, bone marrow macrophages mature and then fuse to become multinucleated osteoclasts. The processes of osteoclastogenesis and bone resorption are known to be energy-demanding,⁸ but little is known about the amino acid requirements of osteoclasts. In this study, we investigated the role of glutamine in osteoclastogenesis. Glutamine was selected for this work because it provides an excellent example of amino acid metabolism.Although glutamine acts as an essential amino acid in some specific physiologic situations, it is classified as a nonessential amino acid.⁵ The need for the biosynthesis and metabolism of amino acids is significantly increased in cells with high rates of proliferation, such as functionally active cells and cancer cells. The activity of amino acid synthetases such as glutamine synthetase is increased in these cells. In addition, glutamine transporters on the plasma membrane are important, because they mediate glutamine uptake to meet the intracellular glutamine demand. The transporter Slc1a5, also known as ASCT2, is particularly important for glutaminolysis and mTOR signaling.^14,16Glutamine concentrations in tissue and blood are regulated by the activities of glutamine synthetase and glutaminase. ­Endogenous synthesis cannot meet the cell’s demands for glutamine in conditions including cancer, infections, and intense physical exercise. Glutamine is released into the blood from the lungs, adipocytes, and skeletal muscles and is transported into the cytoplasm via glutamine acid transporter molecules on the cell membrane. Glutamine is required for the growth of cancer cells; upregulation of the expression of the proteins involved in glutamine transport has been observed in tumor cells.⁴ Slc1a5 (ASCT2) is a small neutral amino acid exchanger that is overexpressed in many cancers and is the most well-described glutamine transporter in cancer cells.⁹ However, previous studies^1,10,22,23 have reported that silencing, deletion, and amino-acid analog substitution of Slc1a5 in cancer cells generated different results for mTORC1 signaling, proliferation, and cell migration.^{1,3,4,10,22,23} Additional work^3,4 has shown that Slc1a5 is indispensable for tumor growth and mTORC1 signaling. Slc1a5 is important in accumulating nonessential amino acids to quickly restore amino acid composition during imbalanced amino acid usage,⁴ whereas Slc38a1 (SNAT1) and Slc38a2 (SNAT2) mediate the net import of glutamine.In bone homeostasis, glutamine is a critical regulator of energy for protein and nucleic acid synthesis via the tricarboxylic acid cycle. Active glutamine metabolism stimulates the proliferation and differentiation of osteoblasts, chondrocytes, and osteoclasts. The enzyme glutaminase deaminates glutamine to form glutamate. Glutaminase deficiency in osteoblasts and chondrocytes leads to reduced osteoblast formation and decreased bone mass, resulting in potentially dangerous conditions, such as osteoporosis.²⁴ In osteoclasts, glutamine is an important source of fuel for protein and nucleic acid biosynthesis. Therefore, Slc1a5 deficiency in mice may influence bone homeostasis, including osteoclastogenesis. We therefore created Slc1a5-deficient mice to investigate the contribution of Slc1a5 to the development and functional properties of osteoclasts.     

REGULATION OF INITIATION OF DNA SYNTHESIS IN CHINESE HAMSTER CELLS : II. Induction of DNA Synthesis and Cell Division by Isoleucine and Glutamine in G1-Arrested Cells in Suspension Culture

Suspension cultures of Chinese hamster cells (line CHO), which had stopped dividing and were arrested in G₁ following growth to high cell concentrations in F-10 medium, could be induced to reinitiate DNA synthesis and to divide in synchrony upon addition of the appropriate amounts of isoleucine and glutamine. Both amino acids were required to initiate resumption of cell-cycle traverse. Deficiencies in other amino acids contained in F-10 medium did not result in accumulation of cells in G₁, indicating a specific action produced by limiting quantities of isoleucine and glutamine. In the presence of sufficient glutamine, approximately 2 x 10^-6 M isoleucine was required for all cells to initiate DNA synthesis in a population initially containing 1.5 x 10⁵ cells/ml. Under similar conditions, about 4 x 10^-6 M isoleucine was required for all G₁-arrested cells to progress through cell division. In contrast, 1 x 10^-4 M glutamine was necessary for maximum initiation of DNA synthesis in G₁ cells, along with sufficient isoleucine. A technique for rapid production of G₁-arrested cells is described in which cells from an exponentially growing population placed in F-10 medium deficient in both isoleucine and glutamine or isoleucine alone accumulated in G₁ after 30 hr.     

Functional intracellular glutaminase activity in intact astrocytes

Numerous cellular metabolites such as glutamine, glutamate, phosphate, calcium, ammonia and acetyl derivatives are known to affect the phosphate-activated glutaminase activity in whole cell homogenates or extracts. Since measurements in extracts under non-physiological conditions may obscure the actual intracellular metabolic flux, the functional intracellular phosphate-activated glutaminase activity was measured by the formation of³H₂O froml-[2-³H]glutamine (Anal. Biochem. 127:134–142, 1982) in cultures of intact astrocytes, untreated and treated with dibutyryl c-AMP (DiBcAMP), in the presence of several potential effectors. These values were compared with enzyme levels determined in extracts from identical cells. The rate of¹⁴CO₂ release froml-[1-¹⁴C]glutamine was also measured in both untreated and DiBcAMP treated astrocytes. The intracellular activity of glutaminase for untreated cells assayed in MEM medium with 1mM radioactive glutamine was 88 nmol/mg protein/h and in DiBcAMP treated cells the rate was 153 nmol/mg protein/h. However, the enzymatic activity measured under optimal conditions in extracts from both untreated and treated cells was much higher, but essentially the same, about 1,750 nmol/mg protein/h. The rate of¹⁴CO₂ release froml-[1-¹⁴C]glutamine was 74 and 133 nmol/mg protein/h in untreated and DiBcAMP treated cells, respectively. This represents approximately 85% of the intracellular glutaminase activity. Furthermore, increasing the concentration of glutamine in the medium from 1 to 6.4 mM increased glutaminase intracellular activity about 3 fold in both untreated and treated cells. Addition of 250 M glutamate to the medium inhibited intracellular glutaminase activity by 70% under both treatment conditions. Deletion of glucose stimulated glutaminase activity. In contrast the removal of fetal bovine serum decreased activity by 35%. The addition of 10 mM phosphate and the alpha keto acids of isoleucine and valine marginally increased intracellular glutaminase activity. The addition of 0.4 mM ammonium chloride to the medium had no effect. An increase in media pH from 6.8 to 7.7 increased intracellular glutaminase activity almost 2 fold. These results provide evidence that phosphate-activated glutaminase activity in vivo is regulated by cellular metabolites, that its functional activity is 5–9% of the rate obtained using extracts, and this functional activity is sufficient to account for the rate of glutamine oxidation.Special issue dedicated to Dr. Elling Kvamme     

The use of serum-free medium for the production of functionally active humanised monoclonal antibody from NS0 mouse myeloma cells engineered using glutamine synthetase as a selectable marker

A protein-free growth medium (W38 medium) had previously been developed for the NS0 mouse myeloma cell line which is cholesterol-auxotrophic. This paper describes the development of a protein-free growth medium for NS0 cells expressing humanised monoclonal antibody using GS (glutamine synthetase) as a selectable marker. Several GS-engineered NS0 cell lines expressing humanised monoclonal antibody grew in a modification of W38 medium which maintained GS-selection, supplemented with cholesterol, phosphatidylcholine and -cyclodextrin. Further studies showed that additional glutamic acid, asparagine, ribonucleosides and choline chloride improved cell growth. Amino acid analysis identified a number of amino acids that were being depleted from the culture medium. NS0 cell lines 9D4 and 2H5 expressing CAMPATH-1H^* were adapted to enable them to grow serum-free in the absence of cholesterol and -cyclodextrin. Cholesterol-independent 9D4 (9D4.CF) cells grown in shake flask culture using an enriched protein-free medium (WNSD medium), supplemented with human recombinant insulin (Nucellin), reached a maximum cell density to 1.86×10⁶ cells ml^–1 producing 76.6 mg l^–1 of antibody. CAMPATH-1H antibody produced using serum-free medium was found to be functionally activein vitro in the Antibody Dependant Cellular Cytotoxicity (ADCC) assay.Abbreviations C cholesterol - CD cyclodextrin - dhfr dihydrofolate reductase - F68 Pluronic F68 - GS glutamine synthetase - MSX methionine sulphoximine - P phosphatidylcholine - PC-FBS phosphatidylcholine, cholesterol and foetal bovine serum - RPMI RPMI 1640 medium - ADCC Antibody-dependant cellular cytotoxicity     

Glutamine metabolism in AS-30D hepatoma cells. Evidence for its conversion into lipids via reductive carboxylation

A study was undertaken to assess the role of a physiological concentration of glutamine in AS-30D cell metabolism. Flux of¹⁴C-glutamine to¹⁴CO₂ and of¹⁴C-acetate to glutamate was detected indicating reversible flux between glutamate and TCA cycle -ketoglutarate. These fluxes were transaminase dependent. A flux analysis was compared using data from three tracers that label -ketoglutarate carbon 5, [2-¹⁴C]glucose, [1-¹⁴C]acetate and [5-¹⁴C]glutamine. The analysis indicated that the probability of flux of TCA cycle -ketoglutarate to glutamate was, at minimum, only slightly less than the probability of flux of -ketoglutarate through -ketoglutarate dehydrogenase. The apparent Km for oxidative flux of [¹⁴C]glutamine to¹⁴CO₂, 0.07 mM, indicated that this flux was at a maximal rate at physiological, 0.75 mM, glutamine. Although oxidative flux through -ketoglutarate dehydrogenase was the major fate of glutamine, flux of glutamine to lipid via reductive carboxylation of -ketoglutarate was demonstrated by measuring incorporation of [5-¹⁴C]glutamine into¹⁴C-lipid. In media containing glucose (6 mM), and glutamine (0.75 mM) 47 per cent of the lipid synthesized from substrates in the media was derived from glutamine via reductive carboxylation and 49 per cent from glucose. These findings of nearly equal fluxes suggest that lipogenesis via reductive carboxylation may be an important role of glutamine in hepatoma cells.     

Adaptation of cholesterol-requiring NS0 mouse myeloma cells to high density growth in a fully defined protein-free and cholesterol-free culture medium

NS0 has been used as a fusion partner for the production of hybridomas and has more recently been engineered to produce recombinant protein. A protein-free culture medium, designated W38 medium, has previously been developed which supported high density growth of rat myeloma and hybridoma cell lines. NS0 cells failed to grow in W38 medium and in a number of protein-free culture media which support the growth of other myeloma cell lines. NS0 cells are derived from the NS-1 cell line, which is known to require exogencus cholesterol. It was found that NS0 cells grew in W38 medium supplemented with phosphatidylcholine, cholesterol, and albumin and that NS0 were auxotrophic for cholesterol. Protein-free growth of NS0 cells was achieved by using -cyclodextrin to replace albumin as a lipid carrier. The maximal cell density reached in this protein-free medium was in excess of 1.5×10⁶ cell ml^–1. The lipid supplements in the medium precipitated after a few days storage at +4°C. In order to overcome this problem a protocol was developed which allowed NS0 cells to be adapted to cholesterol-independent growth in W38 medium. NS0.CF (cholesterol-independent NS0 cells) were cultured continuously in W38 medium for several months. In shake flask culture a cell density of 2.4×10⁶ cells ml^–1 was achieved in W38 medium compared with 1.41×10⁶ cells ml^–1 in RPMI 1640 medium containing 10% foetal bovine serum. NS0.CF cells readily grew in a 1 litre stirred bioreactor using W38 medium supplemented with Pluronic F68 reaching a density of 3.24×10⁶ cells ml^–1. NS0.CF were cloned protein-free by limiting dilution in W38 medium, giving colonies in wells that were seeded at an average density of 0.32 cells per 200 l. This study has demonstrated for the first time the growth of a cholesterol-requiring mouse myeloma cell line in a completely defined protein-free medium and its subsequent adaptation to cholesterol-independence.Abbreviations BSA bovine serum albumin - C cholesterol - CD cyclodextrin - F68 Pluronic F68 - GS glutamine synthetase - P phosphatidylcholine - PC-FBS phosphatidylcholine, cholesterol and foetal bovine serum - RPMI RPMI 1640 medium - MSX methionine sulphoximine     

Scale up cultivation of primary human umbilical vein endothelial cells on microcarriers from spinner vessels to bioreactor fermentation

Five types of dextran-based microcarriers (Dormacell, Pfeifer and Langen) with different concentrations of dimeric DEAE anion-exchange groups (nitrogen contents from 1.2 up to 2.9%) were tested as growth substrates for the cultivation of human umbilical vein endothelial cells (HUVECs). All microcarriers were gelatinized before use to improve cell adhesion. The one with the highest DEAE-group density was found to be most suitable for HUVEC propagation reaching final cell densities of 8×10⁵ viable cells ml^-1 (95% viability) using microcarrier concentrations of 3 g l^–1. Furthermore, metabolic data of glucose/lactate and amino acid metabolism are presented in this study. The concentrations of 18 amino acids were monitored throughout cultivation. A considerable decrease of glutamine and inverse increase of glutamate was observed. Cultivation with initial glucose concentration of 16.5 mmol l^–1 resulted in high glutamine consumption rates, whereas high glucose-supplemented starting culture medium (30 mmol l^-1) gave considerably lowered rates, indicating altered glutamine metabolism due to different glucose feeding. The glucose consumption and lactate production rates increased 2.6 fold and 3.5 fold, respectively, due to switch over from low to high glucose supplemented cultures. The rate of glucose metabolism was found not to be directly related to cell growth, because almost identical growth rates and doubling times were obtained. Considering the remaining 16 amino acids measured, serine concentrations considerably declined and glycine as well as alanine concentrations raised strongly. Most amino acid values were found insignificantly altered during 14 days of cultivation. Spinner vessel cultures served as inoculum for up scale propagation of HUVECs in membrane stirred 2 liter bioreactors. About 5×10⁹ HUVECs were produced, which were used for the isolation and structural characterization of glycosphingolipids, cell membrane compounds, which are suggested to be involved in e.g. selectin-carbohydrate interaction (cell-cell adhesion), carcinogenesis and atherogenesis.Abbreviations HUVECs human umbilical vein endothelial cells - PBS phosphate buffered saline     

A new culture system for the cultivation of mammalian cells for the production of several biologically active substances

A maximum cell density of 8×10⁶ viable human fibroblast cells/ml was obtained on microcarriers at a perfusion rate of 0.6 mL/min — a rate which maintained a quasi-steady state. The maximum tPA production was approximately 1.2 g/ml and obtained when the cell density was relatively constant during the later periods of cultivation. Specific UTP and tPA production rates had a correlation factor of 0.85, and cell growth to oxygen consumption had an 0.92 correlation factor. ATP generation was strongly correlated to glutamine consumption, with a lower correlation to oxygen utilization.     

Methods for animal cell immobilization using electrostatic droplet generation

Summary A technique for the production of small polymer microbeads (less than 150 m diam.) utilizing electrostatic spraying is presented. Video and image analysis were performed to reveal details of the mechanism of droplet formation. Microbeads coated with poly-L-lysine were prepared and found to be effective as microcarriers for culturing surface immobilized insect cells to a high cell density (1.55 × 10⁷ cells/ml microbeads).     

Enhanced antibody production associated with altered amino acid metabolism in a hybridoma high-density perfusion culture established by gravity separation

A high density hybridoma perfusion culture was established by separating and recycling cells from the product stream to the reactor using a simple external sedimentation-based separator — an inclined modified Erlenmeyer flask. After 3 weeks, when the optimal perfusion rate of 1.0 day^–1 had been reached, viable cell density stabilized at around 10×10⁶ cells ml^–1, a level five times that obtained by simple batch culture. The efficiency of the separator was enhanced by cell flocculation. Specific antibody productivity, which was initially 0.4 g 1×10⁶ cells^–1 h^–1, decreased to half that value while cell density was increasing, but recovered to the initial level when the culture finally stabilized at a high cell density. During the final phase, when viable cell density and specific antibody production were high, there was a marked shift in metabolism. Consumption of the two most important substrates for energy generation, glucose and glutamine, caused their broth concentrations to decrease to 1.5 mM and 1 mM, respectively, from input medium concentrations of 25 mM and 10 mM, respectively. At the same time there was an increase in the specific production of glycine and aspartate, their broth concentrations reaching 1.5 mM and 0.02 mM, respectively. We suggest that this shift in metabolism results in enhanced production of ATP from glutamine. The specific glucose consumption and lactate production also indicate that there is a shift to more energy efficient metabolism. The mechanism whereby this leads to enhanced specific antibody production remains to be elucidated. Nevertheless, the combination of high cell density and enhanced productivity obtained with the present perfusion culture resulted in a high monoclonal antibody production –100 mg l^–1 d^–1.     

The effect of dissolved oxygen tension and the utility of oxygen uptake rate in insect cell culture

Dissolved oxygen tension and oxygen uptake rate are critical parameters in animal cell culture. However, only scarce information of such variables is available for insect cell culture. In this work, the effect of dissolved oxygen tension (DOT) and the utility of on-line oxygen uptake rate (OUR) measurements in monitoring Spodoptera frugiperda (Sf9) cultures were determined. Sf9 cells were grown at constant dissolved oxygen tensions in the range of 0 to 30%. Sf9 metabolism was affected only at DOT below 10%, as no significant differences on specific growth rate, cell concentration, amino acid consumption/production nor carbohydrates consumption rates were found at DOT between 10 and 30%. The specific growth rate and specific oxygen uptake rate followed typical Monod kinetics with respect to DOT. The calculated _max and max were 0.033 h^-1 and 3.82×10^-10 mole cell^-1h^-1, respectively, and the corresponding saturation constants were 1.91 and 1.57%, respectively. In all aerated cultures, lactate was consumed only after glucose and fructose had been exhausted. The yield of lactate increased with decreasing DOT. It is proposed, that an apparent DOT in non-instrumented cultures can be inferred from the lactate yield of bioreactors as a function of DOT. Such a concept, can be a useful and important tool for determining the average dissolved oxygen tension in non-instrumented cultures. It was shown that the dynamic behavior of OUR can be correlated with monosaccharide (fructose and glucose) depletion and viable cell concentration. Accordingly, OUR can have two important applications in insect cell culture: for on-line estimation of viable cells, and as a possible feed-back control variable in automatic strategies of nutrient addition.Abbreviations DOT Dissolved oxygen tension - OUR Oxygen uptake rate - specific oxygen uptake rate - specific growth rate - X_v viable cell concentration - C_L, C^*, and oxygen concentrations in liquid phase, in equilibrium with gas phase, and medium molar concentration, respectively - H Henry's constant - K_La volumetric oxygen transfer coefficient - P_T total pressure - oxygen partial pressure - oxygen molar fraction - i discrete element     

Induction of apoptosis in oxygen-deprived cultures of hybridoma cells

It is now well documented that apoptosis represents the prevalent mode of cell death in hybridoma cultures. Apoptotic or programmed cell death occurs spontaneously in late exponential phase of batch cultures. Until lately, no specific triggering factors had been identified. Recently, we observed that glutamine, cystine or glucose deprivation induced apoptosis in both hybridoma and myeloma cell lines whereas accumulation of toxic metabolites induced necrotic cell death in these cells. Other triggering factors such as oxygen deprivation might also be responsible for induction of apoptosis. In the present study, induction of cell death by exposure to anoxia was examined in batch culture of the SP2/0-derived hybridoma D5 clone. The mode of cell death was studied by morphological examination of acridine orange-ethidium bromide stained cells in a 1.5 L bioreactor culture grown under anoxic conditions for 75 hours. Under such conditions, viable cell density levelled off rapidly and remained constant for 25 hours. After 45 hours of anoxia, cell viability had decreased to 30% and the dead cell population was found to be 90% apoptotic. In terms of cellular metabolism, anoxia resulted in an increase in the utilization rates of glucose and arginine, and in a decrease in the utilization rate of glutamine. The lactate production rate and the yield of lactate on glucose increased significantly while the MAb production rate decreased. These results demonstrate that glycolysis becomes the main source of energy under anoxic conditions.Cells incubated for 10 hours or less under anoxic conditions were able to recuperate almost immediately and displayed normal growth rates when reincubated in oxic conditions whereas cells incubated for 22 hours or more displayed reduced growth rates. Nonetheless, even after 22 h or 29 h of anoxia, cells reincubated in oxic conditions showed no further progression into apoptosis. Therefore, upon removal of the triggering signal, induction of apoptosis ceased.Abbreviations VNA Viable non-apoptotic cells - VA Viable apoptotic cells - NVNA Nonviable non-apoptotic or necrotic cells - NVA Nonviable apoptotic cells - CF Chromatin-free cells (late nonviable apoptotic cells) - AO Acridine orange - EB Ethidium Bromide - MAb Monoclocnal antibody - D.O. Dissolved oxygen - qMAb Specific MAb production rate (mg. (10⁹ cells)^–1.day^–1) - Specific growth rate (h^–1) - X_v Viable cell number (10⁵ cells.mL^–1) - X_t Total cell number (10⁵ cells.mL^–1) - Y_lac/glc Yield coefficient of lactate on glucose (mM lactate produced/mM glucose consumed)     

Production of glutamine by micro-gel bead-immobilized Corynebacterium glutamicum 9703-T cells in a stirred tank reactor

The effectiveness of using micro-gel bead-immobilized cells for aerobic processes was investigated. Glutamine production by Corynebacterium glutamicum, 9703-T, cells was used as an example. The cells were immobilized in Sr-alginate micro-gel beads 500 m in diameter and used for fermentation processes in a stirred tank reactor with a modified impeller at 400 min^–1. Continuous production of glutamine was carried out for more than 220 h in this reactor and no gel breakage was observed. As a result of the high oxygen transfer capacity of this system, the glutamine yield from glucose was more than three times higher, while the organic acid accumulation was more than 24 times lower than those obtained with 3.0 mm-gel bead-immobilized cells in an airlift fermentor under similar experimental conditions. During the continuous fermentations there was evolution and proliferation of non-glutamine producing strains which led to a gradual decrease in the productivity of the systems. Although a modified production medium which suppresses cell growth during the production phase was effective in maintaining the productivity, the stability of the whole system was shortened due to high cell deactivation rate in such a medium.List of Symbols C kg/m³ glutamine concentration - C _A mol/m ³ local oxygen concentration inside the gel beads - C _AS mol/m ³ oxygen concentration at the surface of the gel beads - De m²/h effective diffusion coefficient of oxygen in the gel bead - DO mol/m³ dissolved oxygen concentration - F dm³/h medium flow rate - K h^–1 glutamine decomposition rate constant - Km mol/m³ Michaelis Menten constant - QO _2max mol/(kg · h) maximum specific respiration rate - R m radius of the gel beads - r m radial distance - t h time - V _C dm ³ volume of the gel beads - V _L dm ³ liquid volume in the reactor - Vm mol/(m³ · h) maximum respiration rate - X kg/m³ cell concentration - x r/R - y C _A /C_AS - h^–1 cell deactivation rate constant - Thiele modulus defined by R(Vm/De Km) ^1/2 - C _AS /Km - _C kg/(m³-gel · h) specific glutamine formation rate - _c dm³-gel/dm³ V _C /V _L      

Protein production (β-galactosidase) from a baculovirus vector inSpodoptera frugiperda andTrichpolusia ni cells in suspension culture

Protein production capabilities ofTrichpolusia ni (TN 368) cells andSpodoptera frugiperda (Sf9) cells were compared in GTC100 medium in suspension culture using as a vector a genetically engineeredAutographa californica nuclear polyhedrosis virus. TN 368 produces more -galactosidase than Sf9, on a per cell basis (2.2×10⁵ and 1.7×10⁵ units/ 10⁶ cells¹ respectively). In growth experiments serum-free medium supported a higher maximum Sf9 cell density (4±1×10⁶ cells/ml) than the serum- based media (1.5±5×10⁶ cells/ml in GTC100 and 2±1×10⁶ cells/ml in TNM-FH). However, using a cell density of 5×0⁵ cells/ml, the productivity per cell varied, from a low of 4.5×10⁴ units in EX-CELL-400 medium to a high of 7.6×10⁴ units in TNM-FH. The TN 368 cells were twice a large as Sf9 cells and appeared to be more shear sensitive than Sf9 cells.     

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     

Nitrogen metabolism inRhodopseudomonas globiformis

Rhodopseudomonas globiformis strain 7950 grew with a variety of amino acids, urea, or N₂ as sole nitrogen sources. Cultures grown on N₂ reduced acetylene to ethylene; this activity was absent from cells grown on nonlimiting NH ₄ ⁺ . Glutamate dehydrogenase could not be detected in extracts of cells of strain 7950, although low levels of an alanine dehydrogenase were present. Growth ofR. globiformis on NH ₄ ⁺ was severely inhibited by the glutamate analogue and glutamine synthetase inhibitor, methionine sulfoximine. High levels of glutamine synthetase (as measured in the -glutamyl transferase assay) were observed in cell extracts of strain 7950 regardless of the nitrogen source, although N₂ and amino acid grown cells contained somewhat higher glutamine synthetase contents than cells grown on excess NH ₄ ⁺ . Levels of glutamate synthase inR. globiformis were consistent with that reported from other phototrophic bacteria. Both glutamate synthase and alanine dehydrogenase were linked to NADH as coenzyme. We conclude thatR. globiformis is capable of fixing N₂, and assimilates NH ₄ ⁺ primarily via the glutamine synthetase/glutamate synthase pathway.Abbreviations GS glutamine synthetase - GOGAT Glutamineoxoglutarate aminotransferase - GDH Glutamate dehydrogenase - ADH Alanine dehydrogenase - MSO Methionine sulfoximine