Stimulation of monoclonal antibody production by human-human hybridoma cells with an elevated concentration of potassium or sodium phosphate in serum-free medium

Potassium or sodium phosphate was found to stimulate the production of human monoclonal antibody by human-human hybridoma HB4C5. The addition of 15 mM Na-phosphate (pH 7.4) into serum-free culture medium increased the antibody production up to 4-fold, when seeded at cell density of 1×10⁵ cells/ml in dishes. At the higher cell density of 5×10⁵ cells/ml, K-phosphate was more effective than Na-phosphate, at the same concentration. In large-scale continuous culture, the addition of 10 mM Na-phosphate into serum-free culture medium stimulated antibody production by HB4C5 cells 6-fold.     

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     

Strategies to extend longevity of hybridomas in culture and promote yield of monoclonal antibodies

Summary Cell viability was improved by supplemental feeding of amino acids and vitamins in batch culture of hybridoma cells. Cells could be maintained over a 10 day period following exponential growth at a constant viable cell concentration of 2.1×10⁶ cells/ml. Concentrations of monoclonal antibody (MAb) reached 140 mg/l, a value of nearly four times that found in typical batch culture. Lactate formation appeared to occur only during active exponential growth and not during the stationary phase.     

Performance of an external loop air-lift bioreactor for the production of monoclonal antibodies by immobilized hybridoma cells

Summary The performance of an external loop air-lift bioreactor was investigated by assessing the inter-relationships between various hydrodynamic properties and mass transfer. The feasibility of using this bioreactor for the production of monoclonal antibodies by mouse hybridoma cells immobilized in calcium alginate gel beads and alginate/poly-l-lysine microcapsules was also examined. When the superficial gas velocity, V _g , in the 300 ml reactor was varied from 2 to 36 cm/min, the average liquid velocity increased from 3 to 14 cm/sec, the gas hold-up rose from 0.2 to 3.0%, and the oxygen mass transfer coefficient, k _L a, increased from 2.5 to 18.1 h^-1. A minimum liquid velocity of 4 cm/s was required to maintain alginate gel beads (1000 m diameter, occupying 3% of reactor volume) in suspension. Batch culture of hybridoma cells immobilized in alginate beads followed logarithmic growth, reaching a concentration of 4×10⁷ cells/ml beads after 11 days. Significant antibody production did not occur until day 9 into the culture, reaching a value of 100 g/ml of medium at day 11. On the other hand, bioreactor studies with encapsulated hybridoma cells gave monoclonal antibody concentrations of up to 800 g/ml capsules (the antibody being retained within the semipermeable capsule) and maximum cell densities of 2×10⁸ cells/ml capsule at day 11. The volumetric productivities of the alginate gel immobilized cell system and the encapsulated cell system were 9 and 3 g antibody per ml of reactor volume per day, respectively. The main advantage of the bioreactor system is its simple design, since no mechanical input is required to vary the hydrodynamic properties.     

Growth characterizations of a human monocytic leukemia cell line in a serum-free medium

A clone, AH-01S, derived from a human monocytic leukemia cell line, THP-1, grew rapidly in a serum-free medium containing insulin, transferrin, ethanolamine, and sodium selenite. In batch culture using the serum-free medium, the AH-01S cells proliferated at a specific growth rate (μ) of 0.30 to 0.50 (1/day) from a cell concentration of 1 × 10⁴ cells/ml to 1.6 × 10⁶ cells/ml, an increase of 160 times. A higher cell concentration of 0.45 × 10⁷ cells/ml (cell volume ratio was 0.5%) was obtained in spinner flask culture using the serum-free medium. A mean specific growth rate 0.50 (1/day) was also observed in a culture in a fully instrumented cell culture fermentor. However, μ decreased drastically after the cell concentration reached 1.5 × 10⁶ cells/ml. Analyses of medium composition during cultivation revealed that under lower cell concentration, l-glutamine was the main carbon source while glucose was converted to lactate almost stoichiometrically, and that the production of lactate from glucose decreased at higher cell concentrations. To obtain cultures of 1 × 10⁹ cells, 1,200 to 1,300 mg of a carbon source (glucose) and 400 to 500 of amino acids were consumed during high cell concentration cultivation of the AH-01S cells in the serum-free medium.     

TNT biodegradation and production of dihydroxylamino-nitrotoluene by aerobic TNT degrader <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain TM15 in an anoxic environment

Anaerobic bacteria have been used to produce 2,4-dihydroxylamino-nitrotoluene (2,4DHANT), a reductive metabolite of 2,4,6-trinitrotoluene (TNT). Here, an aerobic TNT biodegrader Pseudomonas sp. strain TM15 produced 2,4DHANT as evidenced by the molecular ion with m/z of 199 identified from LC-TOFMS analyses. TNT biodegradation with a high cell concentration (10⁹ cells/ml) led to a significant accumulation of 2,4DHANT in the culture medium, as well as hydroxylamino-dinitrotoluenes (HADNTs), although these products were not accumulated when a low cell concentration was used; also, the accumulation of diamino-nitrotoluene and of an unidentified metabolite were observed in the culture medium with the high cell concentration (10¹⁰ cells/ml). 2,4DHANT overproduction was a function of the aeration speed since cultures with low aeration speeds (30 rpm) had a 19-fold higher DHANT productivity than those aerated with high speeds (180 rpm); this indicates that molecular oxygen was related to the formation of 2,4DHANT. The quantification of dissolved oxygen (DO) in the media demonstrated that the productivity of 2,4DHANT was increased at low DO values. Moreover, supplying oxygen to the culture media produced a remarkable decrease of 2,4DHANT accumulation; these results clearly indicate that high 2,4DHANT production was a consequence of the oxygen deficit in the culture medium. This finding is useful for understanding the TNT biodegradation (bioremediation technology) in an anoxic environment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Suspension culture of drosophila cells employing a gyratory shaker

Summary To develop a method for culturing a large number of small-scale suspension cultures ofDrosophila melanogaster cells simultaneously, basic conditions were studied using a cell line GM₂ and a gyratory shaker. Under gyration at more than 180 rpm, a majority (>80%) of the cells still remained as suspension and grew normally. Lower speed of gyration caused adhesion of the cells to a substratum. Furthermore, size of the culture vessels was found to affect the pattern of cell growth. Five- or 10-ml Erlenmeyer flasks gave satisfactory results, but the growth curves in 30-ml flasks differed from flask to flask and the saturation level was lower. Besides, the growth curves in the latter case were quite different depending on the volume of the medium. A preliminary experiment showed that the type of flask might affect the pattern of a growth curve. Initial cell densities has to be more than 6×10⁴ cells per ml. Lower densities resulted in the longer doubling time or no increase in the cell number. Therefore the following conditions are recommended as a standard for gyration culture ofD. melanogaster cell, GM₂: speed of gyration, 180 rpm; culture vessel, 5- or 10-ml Erlenmeyer flask of a certain type; initial cell density, 1 to 5×10⁵ per ml. Both D20 and modified Schneider’s medium could be utilized as the medium.     

High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge

The influence of centrifugal force on the growth of cells was examined by exposing the cells of the mouse-human hybridoma X87 line to centrifugal force (100–500 G) for ten minutes twice a day and comparing the static culture with that of unexposed cells. In this experiment, both cell proliferation and specific antibody productivity were independent of the centrifugal effect, and gave the same results as in the case of no exposure to centrifugal force. High density cultivation of the mouse-human hybridoma X87 line was obtained by a perfusion system where the cells were separated from the culture medium by continuous centrifugation. In the serum-free culture, the maximum viable cell density exceeded 10⁷ cells/ml, and monoclonal antibody was stably produced for 37 days. The results in this culture were equivalent to those obtained by intermittent centrifugal cell separation from the culture medium, and separation by gravitational settlement.     

Production of rat monoclonal antibody from rat x mouse hybridoma cell lines using microencapsulation technology

Summary The cell growth and monoclonal antibody production characteristics of two rat x mouse heterohybridoma cell lines, designated 187.1 and M1/9.3, were investigated using a biocompatible microencapsulation technology. Both cell lines, derived from the fusion of immunized rat spleen cells with either the NS1 or X63Ag8.653 myeloma cell lines, were found to reach a maximum intracapsular cell density of 1.3 to 1.5×10⁷ cells/ml during a 27-d culture period. During this period, rat monoclonal antibody accumulated in the intracapsular space of both cultures to a final concentration of 2.0 to 2.8 mg/ml. Comparison of the concentration of rat monoclonal antibody in the extracapsular vs. the intracapsular space of both cultures indicated that significantly less than 1% of the antibody produced by the encapsulated hybridoma cells was capable of transiting the microcapsule membrane during the culture period. Due to the partition of the rat monoclonal antibody within the intracapsular space, the initial purity of the antibody harvested from 21-d microcapsule cultures of 187.1 and M1/9.3 cells was approximately 48 and 75% by weight, respectively. Analysis of the intracapsular protein by sodium dodecyl sulfoxide gel electrophoresis at different times during the culture period demonstrated that the principal contaminant associated with the unpurified antibody was bovine serum albumin.     

Step-up/step-down perfusion approach for increased mAb 520C9 production by a hybridoma cell line

The hybridoma cell line, HB-8696, produces a monoclonal antibody, 520C9 (mouse IgG₁) that recognizes the breast cancer oncoprotein, c-erbB2. The effect of perfusion rate (volume of fresh feed/working volume of reactor/day) on cell growth and mAb production was investigated but perfusion at a constant rate and at an arbitrarily increased rate could not maintain exponential cell growth or a higher specific mAb production rate. An optimum step-up/step-down perfusion strategy is therefore proposed for maintaining a steady state production phase at high cell density for ten days. The optimum step-up perfusion could achieve fast cell growth by avoiding any nutrient limited condition and the following optimum step-down perfusion could potentially maintain high live cell density and reduced product dilution as well. The maximum viable cell achieved under optimum perfusion strategy was 2.3 × 10⁷ cells/ml which was 19-fold higher than in optimum batch culture. The mAb yield and volumetric productivity were significantly improved to 52 and 50 mg/l day compared to 25 and 3.8 mg/l day in optimum batch, respectively, and could be maintained for up to ten days.     

Use of Antibiotics in the Preparation of Canine Kidney Tissue Culture Vaccines to Eliminate Leptospiral Infection Hazards

The potential leptospiral infection hazard in the use of vaccines prepared from canine kidney monolayer cultures was studied. Cell cultures were prepared from kidneys of dogs experimentally infected with Leptospira serotype canicola. Viable leptospires were found in kidney cell suspensions at the time of seeding, surviving trypsinization either at room temperature for approximately 2 hr or overnight at 4 C, even in the presence of antibiotics. In tissue cultures maintained without antibiotics, leptospires were cultured up to the time of involution of cells at 25 to 34 days of incubation. Cytopathogenic effects of leptospires on cultured kidney cells were not noted; neither was growth of leptospires remarkable. Generally, the leptospire culture titer decreased to 10^-4 or 10^-5 at the 4th hr or 1st day of incubation to 10^-1 or negative by the 30th or 34th day of incubation. The addition of either a combination of penicillin (100 units per ml) plus streptomycin (100 μg/ml) or polymyxin B (50 units per ml) plus dihydrostreptomycin (100 μg/ml) to seeding cell suspensions resulted in the elimination of viable leptospires by the 4th hr of incubation. From cell cultures treated with neomycin (100 μg/ml) or chloramphenicol (100 μg/ml), leptospires were recovered, respectively, after 24 and 48 hr, but not thereafter. It was apparent that antibiotics, particularly the combination of polymyxin B and dihydrostreptomycin, could be effectively used to eliminate leptospires in tissue culture. Other antibiotics with known antileptospiral activities probably would be effective also. If antibiotics are not used in canine kidney tissue culture employed for viral vaccine preparations, rigid testing for the presence of leptospires in donor dogs and tissue-culture vaccine is indicated.     

Agitation effect on growth and metabolic behavior of plant cell suspension cultures of Thevetia peruviana at bench scale reactor

Plant cell suspension cultures of Thevetia peruviana has been explored for pharmaceutical compounds production. The aim of this study was to evaluate the agitation rate effect on growth and metabolism behavior of T. peruviana cells grown in a 7 L stirred tank reactor. Increases in agitation rates favored cell growth, secondary metabolites production and metabolic activity. The highest biomass concentration 11.92?±?0.25 g DW/L was reached at 550 rpm. The oxidase-reductase activity was stimulated at 550 and 800 rpm. Guaiacol peroxidase activity showed an increase for 300 and 550 rpm after day 7. High levels of extracellular Reactive Oxygen Species (ROS) were observed at day 7 for 550 and 800 rpm. Intracellular phenolic compounds (PC) showed an upward trend until day 7 with a maximum phenolic production of 57.78?±?4.70 mg EGA/100 g FW for 550 rpm. These results indicated that cells responded to ROS stress in a non-enzymatic manner during the first 7 days of culture, increasing PC production with antioxidant capacity. After 7 days, cells responded enzymatically. Intracellullar cardiac glycoside showed a relative increase of 1.7 and 2.1 times for 550 and 800 rpm, respectively. The maximum extracellular production of cardiac glycoside for 550 and 800 rpm was 770.34?±?42.84 mg EP/L. Taken together this study established reactor culture conditions for production of cardiac glycosides and PC, especially taxifolin.

     

Production of human-mouse chimeric antibody by high cell density perfusion culture

Two mouse myeloma cell lines which were transfected with chimeric mouse variable-human constant immunoglobulin heavy and light chain genes have been cultured at high cell density in a settling perfusion culture vessel to produce chimeric antibody specific for human common acute lymphocytic leukemia antigen (cALLA).J558L transfectant proliferated well in a serum-free medium (ITES-eRDF) to a viable cell density of 3.7×10⁷ cells/ml and produced chimeric antibody to a maximum value of 60 g/ml in 120 ml scale vessel. X63Ag8.653 transfectant reached a density of 1.9×10⁷ cells/ml in 1.2 I scale vessel in serum supplemented medium (10% FCS-eRDF) and produced chimeric antibody which consisted of chimeric gamma and chimeric kappa chains to a maximum value of 5.8 g/ml.     

Effectiveness of vitamin A acetate for enhancing the production of lung cancer specific monoclonal antibodies

The antibody productivity of the human–human hybridoma cell line AE6, which produces the lung cancer specific human monoclonal antibody AE6F4, was enhanced fourfold upon stimulation with 1 μg/ml of vitamin A acetate for one day. The enhancement lasted for about two weeks, and could be repeated by another stimulation with vitamin A acetate. The enhancing effect of vitamin A acetate was influenced by the cell density. Enhancement was clearly observed when the cell density was under 10⁶ cells/ml. However, when the cell density was over 10⁷ cells/ml, enhancement was observed weakly or not at all. Although the enhancing effect of vitamin A acetate is not unique to AE6 cells, not all human–human hybridoma cell lines show increased productivity upon VA acetate stimulation. This study suggests that the response to vitamin A acetate may be related to the properties of a particular fusion partner which the hybridoma cell inherits. The efficacy of vitamin A acetate for production of human monoclonal antibodies using human–human hybridomas is discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Factors in the Fermentation-Inhibition Test for Measurement of Growth-Inhibiting Antibody to Mycoplasma pneumoniae

Factors in the fermentation-inhibition test for the measurement of growth-inhibiting antibody in serum to Mycoplasma pneumoniae were studied. The fermentation-inhibiting antibody titer, as read on the day when the color of the control cup without serum had just changed to yellow, was constant among inoculum dilutions of 10^–2 to 10^–5 (10⁶ to 10³ CFU/ml) of a M. pneumoniae broth culture. The use of 10^–3 to 10^–5 dilutions (10⁵ to 10³ CFU/ml) was adequate for inoculation, inasmuch as one day delay in reading did not result in a significant decrease in the test. Heat inactivation of the serum gave no significant effect on the titer. The test was simple and reliable. The growth-inhibiting antibody was shown to be detectable in the test, when the growth of M. pneumoniae was suppressed at least to 1/100 of the growth of the control without serum. The growth-inhibiting antibody titer rose later than the complement-fixing antibody titer in some cases after M. pneumoniae infection. It is suggested that, when an erythromycin-sensitive strain of M. pneumoniae is used, the titer transiently rises and does not show a real growth-inhibiting antibody titer in sera from patients under erythromycin administration.     

Increase of hybridoma productivity using an original dialysis culture system

Hybridoma cell growth and monoclonal antibody production were investigated with a laboratory-made system in which cells were grown in dialysis tubing (MW cut-off 25 kD). The dialysis system contained 10 ml of cell suspension and was immersed in 200 ml of culture medium which when replaced or was at 4-day intervals. With this system, monoclonal antibody concentrations similar to those observed in ascites (concentrations in the order of one gramme per liter) were obtained. With no medium replacement, the antibody production was 3.3 g/l and the cell productivity 3.2×10^–8 g of IgM produced per cell in one minute. With medium replacement the antibody production was higher, 4.4 g/l but the cell productivity was lower, 1.49×10^–8 g per cell in one minute. Cells cultivated in non-optimized conditions were better producers than cells growing in a good environment.Abbreviations FCS fetal calf serum - Ig immunoglobulin - MAb monoclonal antibody - MW molecular weight - MWCO molecular weight cut off - RM replaced medium - NRM non replaced medium     

High cell density suspension culture of mammalian anchorage independent cells: Oxygen transfer by gas sparging and defoaming with a hydrophobic net

Gas sparging directly into the culture-broth is not done in cell culture, except when the gas flow rate is very small, because much foaming occurs.During screening of defoaming methods, foam was observed to be broken up effectively when it made contact with a net fabricated from hydrophobic materials. Providing a highly efficient oxygen supply to suspension culture was tried using the new defoaming method. In a 5 1 reactor equipped with the foam-eliminating net fabricated with polysiloxane, oxygen was transferred at 21 mmole/l·h equivalent to an about forty-fold higher rate than in conventional surface aeration. This was equivalent to a consumption rate of 1×10⁸ cells/ml, even at a low oxygen gas flow rate of 0.1 cm/s corresponding to a fourth of the gas flow rate when foam leaked through the net.Perfusion culture of rat ascites hepatoma cell JTC-1 was successfully carried out in the 51 scale culture system with the net and a hydrophobic membrane for cell filtration. The viable cell concentration reached 2.7×10⁷ cells/ml after twenty-seven days, in spite of the nutrient-deficient condition of the lower medium exchange rate, that is, a working volume a day, and viability was maintained at more than 90%. In a 1.21 scale culture of mouse-mouse hybridoma cell STK-1, viable cell concentration reached 4×10⁷ cells/ml. These results showed that oxygen transfer by gas sparging with defoaming was useful for high density suspension culture. A foam-breaking mechanism was proposed.Abbreviations Eagle's MEM Eagle's minimal essential medium - Dulbecco's modified Eagle MEM Dulbecco's modified Eagle minimal essential medium     

Osteosarcomagenic doses of radium (224Ra) and infectious endogenous retroviruses enhance proliferation and osteogenic differentiation of skeletal tissue differentiating in vitro

Cartilage tissue from embryonic mice which undergoes osteogenic differentiation during in vitro cultivation was used to study the effect of osteosarcomagenic doses of -irradiation and bone-tumor-inducing retroviruses on proliferation and phenotypic differentiation of skeletal cells in a defined tissue culture model. Irradiated mandibular condyles showed dose-dependent enhancement of cell proliferation at day 7 of the culture and increased osteogenic differentiation at day 14. Maximal effects were found with 7.4 Bq/ml of²²⁴Ra-labeled medium. Doses of 740 and 7400 Bq/ml of²²⁴Ra-labeled medium induced increasing cell death. Retrovirus infection enhanced osteogenic differentiation and extended the viability of irradiated cells. After transplantation none of the treated tissues developed tumors in syngeneic mice.     

Effect of coexisting cells on the NGF-inducing neurite growth from PC12h-R

Possible roles of coexisting cells in inducing neurite growth from a nerve cell were studied. Nerve growth factor (NGF)-inducing neurite growth from PC12h-R (a cell line derived from cultured nerve cells) was investigated at various cell densities. At the cell density 10²10⁴ cells/ml neurites appeared even without NGF. In contrast, no neurite appeared without NGF in single cell culture. The neurite growth observed in plural cell culture without NGF was only partially inhibited by antibody to NGF receptor (Ab-NGFR). However, the effect of the used medium alone was mostly inhibited by Ab-NGFR. These results suggest that the neurite inducing potency of coexisting cells is via different sites than the NGF receptor.Abbreviations Ab-IgG-FITC anti-mouse-IgG labeled with fluorescein isothiocyanate - Ab-NF monoclonal antibody to neurofilament 160 kD - Ab-NGFR monoclonal antibody to NGF receptor - BDNF brain-derived neurotrophic factor - D-medium medium for differentiation culture - DMEM Dulbecco's modified Eagle's medium - M-medium medium for multiplication culture - NGF nerve growth factor - NGFR NGF receptor - NT-3 neurotrophin-3 - PC12 pheochromocytoma cell line - PC12h-R subclone of PC12 - Sup-D supernatant of D-medium     

An investigation of cell density effects on hybridoma metabolism in a homogeneous perfusion reactor

In this work, metabolite and antibody production kinetics of hybridoma cultures were investigated as a function of cell density and growth rate in a homogeneous perfusion reactor. Hydrophilized hollow fiber polypropylene membranes with a pore size of 0.2 m were used for medium perfusion. Oxygen was supplied to the cells through thin walled silicone tubing. The mouse-mouse hybridoma cells were grown in three identical bioreactors at perfusion rates of 1.1, 2.0, and 3.2/day for a period of eight days during which the viable cell concentrations reached stable values of 2.6×10⁶, 3.5×10⁶, and 5.2×10⁶ cells/ml, respectively. Total cell densities reached values ranging from 8×10⁶ to 1×10⁶ cells/ml. Specific substrate consumption and product formation rates responded differently to changes in cell density and apparent specific growth rate, which were not varied independently. Using multiple regression analysis, the specific glucose consumption rate was found to vary with viable cell density while the specific glutamine uptake and lactate production rates varied with both viable cell density and apparent specific growth rate. These results suggest that cell density dictates the rate of glucose consumption while the cell growth rate influences how glucose is metabolized, i.e., through glycolysis or the TCA cycle. The specific antibody production rate was found to be a strong function of cell density, increasing as cell density increased, but was essentially independent of the specific growth rate for the cell line under study.List of Symbols MAb monoclonal antibody - X _v viable cell density (cells/ml) - X _d nonviable cell density (cells/ml) - specific growth rate (1/day) - k _d specific death rate (1/day) - D dilution rate (1/day) - S _f substrate concentration in feed (g/l or mM) - S substrate concentration (g/l or mM) - P _f product concentration in feed (g/l or g/ml) - P product concentration (g/l or ug/ml) - q _s specific consumption rate of substrate (g/hr/cell or mmol/hr/cell) - q _p specific production rate of product (g/hr/cell) - q _MAb specific production rate of monoclonal antibody (g/hr/cell) This work was supported in part by a grant for the National Science Foundation (BCS-9157851) and by matching funds from Merck and Monsanto. We sincerely thank Mr. Roland Buchele of Akzo Inc. (Germany) for donation of the polypropylene membranes, Dr. Michael Fanger (Dartmouth Medical School) for the hybridoma cell line, Dr. Sadettin Ozturk (Verax Corp., Lebanon, NH) for technical discussions regarding reactor design, and Dr. Derrick Rollins (Iowa State University) for advice on statistical methods.