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1.
A photo-crosslinkable resin, BIX12, was selected from among various photo-crosslinkable resins for the immobilization of animal cells. BIX12 had no cytotoxic effect on the growth of hybridoma cells and the production of monoclonal antibody, although other photo-crosslinkable resins had significant inhibitory effects. Using BIX12-alginate hybrid gel particles, hybridoma cells could grow in the resins and produce monoclonal antibody. For the continuous production of monoclonal antibody, perfusion culture using a fluidized-bed bioreactor with direct air bubbling was carried out. By this cultivation, monoclonal antibody could be produced stably for more than 50 d. A high viable cell density of more than 107 cells/ml-gel was attained, and the antibody productivity was improved 8.5-fold compared with conventional suspension culture using a spinner flask. Anchorage-dependent cells were also immobilized in the resin particles by three immobilization procedures. Among these procedures, porous BIX12 formed by adding gelatin powder provided good support strength and allowed the cells to grow on the surface inside of the support.  相似文献   

2.
A composite gel system has been developed combining the chemical and physical properties of calcium alginate and agarose gels. The results of growing composite gel immobilized hybridoma SPO1 cells in a protein-free medium within a fluidized-bed perfusion bioreactor are presented in this paper. During the continuous operation of this system, the total cell density reached 3.9×107 cells per ml of beads (viability 79.6%). The specific productivity of monoclonal antibody of the immobilized hybridoma cells reached more than 1.5 g per 106 viable cells per hour, compared with 0.5 for non-immobilized viable cells grown in a one liter agitated bioreactor with the same medium. Significant increases in cell metabolic activities, including substrate utilization and byproduct formation, were also observed. Leaching of materials from the beads was evident and the major fraction of released materials was alginate.  相似文献   

3.
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.  相似文献   

4.
In order to test the feasibility of using calcium alginate-entrapped hybridoma cells for IgM production, HO-22-1 hybridoma cells entrapped into calcium alginate beads with varying alginate concentrations were cultivated in spinner flasks. It was observed that the IgM produced by the entrapped cells could diffuse out of the calcium alginate beads regardless of alginate concentrations tested (0.8–2.5%). Since the increase in alginate concentrations showed an adverse effect on cell growth and maximum cell concentration, the use of lower alginate concentration was desirable for higher volumetric monoclonal antibody (MAb) productivity. When the entrapped cells in 0.8% alginate beads were cultivated in repeated-fed batch mode, the reduction of serum concentration in the medium from 10% to 1% did not decrease the volumetric IgM production. Taken together, the data obtained here showed the feasibility of using calcium alginate-entrapped hybridoma cells for IgM production.Alginate was generously provided by the Kelco company. This work was supported by the Ministry of Science and Technology, Korea.  相似文献   

5.
Recombinant Escherichia coli whole cells harboring Bacillus licheniformis l-arabinose isomerase (BLAI) were immobilized with alginate. The operational conditions for immobilization were optimized with response surface methodology. Optimal alginate concentration, Ca2+ concentration, and cell mass loading were 1.8% (w/v), 0.1 M, and 44.5 g L−1, respectively. The interactions between Ca2+ concentration, alginate concentration, and initial cell mass were significant. After immobilization of BLAI, cross-linking with 0.1% glutaraldehyde significantly reduced cell leakage. The half-life of immobilized whole cells was 150 days, which was 50-fold longer than that of free cells. In seven repeated batches for l-ribulose production, the productivity was as high as 56.7 g L−1 h−1 at 400 g L−1 substrate concentration. The immobilized cells retained 89% of the initial yield after 33 days of reaction. Immobilization of whole cells harboring BLAI, therefore, makes a suitable biocatalyst for the production of l-ribulose, particularly because of its high stability and low cost.  相似文献   

6.
Summary We have cultivated a hybridoma cell-line H505AC in suspension and alginate immobilized cell bioreactors to produce anti-Hepatitis B surface Antigen (anti-HBsAg) monoclonal antibody IgM. The specific IgM production rates correlate linearly with the specific glucose consumption rate in suspension culture with a maximum production rate of 300 g/106 cells/day. In alginate-cell immobilized airlift bioreactor, a total of 1143 milligrams IgM was produced in 9 days operation with a volumetric productivity 44.1 mg/day.L  相似文献   

7.
Oxygen consumption by hybridoma cells immobilized in 1- and 3.9-mm-diameter calcium alginate beads was measured. The entrapped cells consumed oxygen at about 10 mumol/min per 10(9) cells, regardless of the bead size and cell loading. In contrast, the same cells in suspension culture respire at specific rates of 3-8 mumol/min per 10(9) cells (depending on the cell density). The growth rate of the immobilized cells was significantly reduced, while specific antibody production was comparable to that of free cells.  相似文献   

8.
Animal cell perfusion high density culture is often adopted for the production of biologicals in industry. In high density culture sometimes the productivity of biologicals has been found to be enhanced. Especially in immobilized animal cell culture, significant increase in the productivity has been reported. We have found that the specific monoclonal antibody (MAb) productivity of an immobilized hybridoma cell is enhanced more than double. Several examples of enhancing productivities have been also shown by collagen immobilized cells. Immobilized cells involve some different points from non-immobilized cells in high density culture: In immobilized culture, some cells are contacted together, resulting in locally much higher cell concentration more than 108 cells/ml. Information originating from a cell can be easily transduced to the others in immobilized culture because the distance between cells is much nearer. Here we have performed collagen gel immobilized culture of recombinant BHK cells which produce a human IgG monoclonal antibody in a protein-free medium for more than three months. In this high density culture a stabilized monoclonal antibody production was found with around 8 times higher specific monoclonal antibody productivity compared with that in a batch serum containing culture. No higher MAb productivity was observed using a conditioned medium which was obtained from the high density culture, indicating that no components secreted from the immobilized cells work for enhancing monoclonal antibody production. The MAb productivity by the non-immobilized cells obtained by dissolving collagen using a collagenase gradually decreased and returned to the original level in the batch culture using a fresh medium. This suggests that the direct contact of the cells or a very close distance between the cells has something to do with the enhancement of the MAb productivity, and the higher productivity is kept for a while in each cell after they are drawn apart.  相似文献   

9.
Summary The effect of the inlet gas oxygen concentration on hybridoma growth, viability and monoclonal antibody production was studied. Maximum viable cell cencentrations of 1.2×106 cells/mL were obtained in split-flow bioreactors mixed with a gas containing 10% oxygen. However, maximum antibody concentration was attained at a sub-optimal level of growth with an oxygen concentration of 2.5%.  相似文献   

10.
Ethanol fermentation by cells of Zymomonas mobilis immobilized in calcium alginate gel has been studied using 5 to 30 wt% initial d-glucose in the medium. Up to 27% d-glucose was completely fermented and the maximum ethanol concentration of 12.6% (w/v) was obtained using an immobilized cell concentration of 58 g dry wt l?1 of bead volume. The ethanol yield coefficient was almost unaffected by initial d-glucose concentration and its value was >95% of theoretical. The rates of ethanol production and d-glucose utilization first increased, with an increase in initial d-glucose concentration up to 13.6%, and then started to decrease upon a further increase in initial d-glucose concentration. Cell leakage from the calcium alginate beads was very low.  相似文献   

11.
In the present study, the optimum conditions for the production of xylanase by immobilized spores of Trichoderma reesei SAF3 in calcium alginate beads were determined. The operational stability of the beads during xylanase production under semi-continuous fermentation was also studied. The influence of alginate concentration (1, 2, 3, and 4%) and initial cell loading (100, 200, 300, 400, and 500 beads per flask) on xylanase production was considered. The production of xylanase was found to increase significantly with increasing concentration of alginate and reached a maximum yield of 3.12 ± 0.18 U ml−1 at 2% (w/v). The immobilized cells produced xylanase consistently up to 10 cycles and reached a maximum level at the forth cycle (3.36 ± 0.2 U ml−1).  相似文献   

12.
The role of functionalized alginate gels as immobilized matrices in production of l (+) lactic acid by Lactobacillus delbrueckii was studied. L. delbrueckii cells immobilized in functionalized alginate beads showed enhanced bead stability and selectivity towards production of optically pure l (+) lactic acid in higher yields (1.74Yp/s) compared to natural alginate. Palmitoylated alginate beads revealed 99% enantiomeric selectivity (ee) in production of l (+) lactic acid. Metabolite analysis during fermentation indicated low by-product (acetic acid, propionic acid and ethanol) formation on repeated batch fermentation with functionalized immobilized microbial cells. The scanning electron microscopic studies showed dense entrapped microbial cell biomass in modified immobilized beads compared to native alginate. Thus the methodology has great importance in large-scale production of optically pure lactic acid.  相似文献   

13.
Summary Auxotrophic mutant cells of Corynebacterium glycinophilum with high l-serine production activity were immobilized by entrapment with various gel materials, such as synthetic prepolymers and natural polysaccharides. The entrapped cells were used for estimation of l-serine productivity in a medium supplemented with glycine as a precursor. Based on the above criteria, including cell growth in gels and cell leakage from gels, calcium alginate was the most suitable gel material. Continuous l-serine fermentation with calcium alginate-entrapped growing cells was successfully achieved in an air-bubbled reactor for at least 13 days.  相似文献   

14.
Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for use in the continuous production of ethanol. Yeasts were grown in medium supplemented with ethanol to selectively screen for a culture which showed the greatest tolerance to ethanol inhibition. Yeast beads were produced from a yeast slurry containing 1.5% alginate (w/v) which was added as drops to 0.05M CaCl2 solution. To determine their optimum fermentation parameters, ethanol production using glucose as a substrate was monitored in batch systems at varying physiological conditions (temperature, pH, ethanol concentration), cell densities, and gel concentration. The data obtained were compared to optimum free cell ethanol fermentation parameters. The immobilized yeast cells examined in a packed-bed reactor system operated under optimized parameters derived from batch-immobilized yeast cell experiments. Ethanol production rates, as well as residual sugar concentration were monitored at different feedstock flow rates.  相似文献   

15.
The aim of this study was to produce two isozymes of α-amylase by immobilization of a newly isolated soil bacterium. The bacterium was identified as Bacillus thuringiensis CKB19 on the basis of its 16S rRNA profile. Enzyme production by free cells increased linearly with cell growth up to 34 h in starch containing enriched liquid media. The active bacterial cells were immobilized in Caalginate beads, and operational stability of the entrapped cell was optimized for amylase production. Enzyme production was optimal at an alginate concentration of 2 g% (w/v), calcium chloride concentration of 1 M, and with 300 beads (each bead contained 2 × 107 cells)/250 mL flask. Amylase production by the immobilized cells was about 3 times higher than free cell fermentation after 34 h of incubation. It was observed that the immobilized bacterium secreted two different amylases (Am-I and Am-II) into the culture fluid. The molecular masses of Am-I and Am-II were 59.6 and 44.7 kd, respectively, and showed optimum activity at pH 5.0 and 9.0. Both amylases showed optimum activity at 40°C and were stable at the same temperature, with losses of only 10 and 20% (for Am I and Am II, respectively) of their original activities after 24 h of incubation. Further, both amylases were salt tolerant (up to 4 M NaCl) and hydrolyzed raw starchy foods into glucose. All these characteristics make this enzyme mixture suitable for use as a digestive aid and for the improvement of digestibility of animal feed ingredients.  相似文献   

16.
Summary Continuous production of monoclonal antibody was achieved in serum-free medium by hybridoma cells immobilized by calcium alginate. The cells were cultivated in an expanded bed fermentor under mild flow conditions which reduced destruction of the immobilized gel particles. Monoclonal antibody was produced continuously for more than 40 days.  相似文献   

17.
Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l−1, the optimal production medium had the following initial concentrations of nutrients (g l−1): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l−1 h−1. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l−1 h−1. The packed bed could be used in repeated batch runs to produce lactic acid.  相似文献   

18.
利用经海藻酸钙包埋的重组大肠杆菌细胞催化D-半乳糖生产D-塔格糖,考察了细胞包埋量、反应条件对固定化细胞催化效率以及对D-塔格糖生产稳定性的影响。确定的最优转化条件为:温度65℃,pH 6.5,添加终浓度为1 mmol/L Mn2+,底物(D-半乳糖)浓度100 g/L,重组大肠杆菌细胞用量40 g/L。固定化小球在0.3%戊二醛溶液中交联30 min可以显著提高其在高温下的机械强度。考察了异构化反应体系中硼酸与底物间的摩尔比对产率的影响。研究结果表明,添加适量的硼酸可以改变原有的化学反应平衡,实现D-塔格糖的高产。利用D-半乳糖为底物在最优的反应条件下催化24 h,固定化细胞对D-半乳糖的转化率最高,可达65.8%,连续转化8批次的平均转化率为60.6%,为工业化生产D-塔格糖奠定了基础。  相似文献   

19.
Summary Recycled immobilized cells of Kluyveromyces fragilis ATCC 28244 were used for repeated batch production of ethanol from the inulin sugars derived from Jerusalem artichoke tubers. Using 10% initial sugar concentration, a maximum ethanol concentration of 48 g/l was achieved in 7 h when the immobilized cell concentration in the Ca alginate beads was 72 g dry wt. immobilized cell/l bead volume. The maximum ethanol production rate was 13.5 g ethanol/l bioreactor volume/h. The same Ca alginate beads containing the cells were used repeatedly for 11 batch runs starting with fresh medium at the beginning of each run. The ethanol yield was found to be almost constant at 96% of the theoretical for all 11 batch runs, while the maximum ethanol production rate during the last batch run was found to be 70% of the original ethanol rate obtained in the first batch run.  相似文献   

20.
The kinetics and long-term stability of continuous production of monoclonal antibody IgG2b by hybridoma HD-24 cells immobilized in a fibrous-bed bioreactor (FBB) were studied for a period of ~8 months. The cells were immobilized in the fibrous bed by surface attachment of cells and entrapment of large cell clumps in the void space of the fibrous matrix. A high viable cell density of 1.01 × 108/ml was attained in the bioreactor, which was about 63 times higher than those in conventional T-flask and spinner flask cultures. The continuous FBB produced IgG at a concentration of ~0.5 g/l, with reactor productivity of ~7 mg/h·l, which was about 23 times higher than those from conventional T-flask and spinner flask cultures. The IgG concentration can be further increased to ~0.67 g/l by using higher feed (glucose and glutamine) concentrations and running the reactor at a recycle batch or fed-batch mode. The long-term performance of this bioreactor was also evaluated. For a period of 36 days monitored, the MAb produced in the continuous well-mixed bioreactor at 50 h retention time (0.02/h dilution rate) was maintained at a steady concentration level of ~0.3 g/l with less than 8% drift. At the end of the study, it was found that ~25% of the cells were strongly attached to the fiber surfaces and the other ~75% entrapped or weakly immobilized in the fibrous matrix. The strongly attached cells had a high viability of ~90%, compared to ~75% for cells weakly immobilized and only ~1.4% for freely suspended cells, suggesting that the fibrous matrix preferentially retained and protected the viable (productive) cells. The FBB thus was able to maintain its long-term productivity because nonviable and dead cells were continuously washed off from the fibrous matrix. The high MAb concentration and production rate and excellent stability for continuous long-term production obtained in this study compare favorably to other bioreactor studies reported in the literature. The reactor performance can be further improved by providing better pH and aeration controls at higher feed concentrations. The FBB is easy to operate and scale-up, and thus can be used economically for industrial production of MAb.  相似文献   

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