共查询到20条相似文献,搜索用时 15 毫秒
1.
Guozheng Wang Wenying Zhang Corey Jacklin David Freedman Lee Eppstein Avinoam Kadouri 《Cytotechnology》1992,9(1-3):41-49
This study describes two packed bed bioreactor configurations which were used to culture a mouse-mouse hybridoma cell line
(ATCC HB-57) which produces an IgG1 monoclonal antibody. The first configuration consists of a packed column which is continuously perfused by recirculating
oxygenated media through the column. In the second configuration, the packed bed is contained within a stationary basket which
is suspended in the vessel of a CelliGen™ bioreactor. In this configuration, recirculation of the oxygenated media is provided by the CelliGen Cell Lift impeller.
Both configurations are packed with disk carriers made from a non-woven polyester fabric. During the steady-state phase of
continuous operation, a cell density of 108 cells per cm3 of bed volume was obtained in both bioreactor configurations. The high levels of productivity (0.5 gram MAb per 1 of packed
bed per day) obtained in these systems demonstrates that the culture conditions achieved in these packed bed bioreactors are
excellent for the continuous propagation of hybridomas using media which contains low levels (1 %) of serum as well as serum-free
media.
These packed bed bioreactors allow good control of pH, dissolved oxygen and temperature. The media flows evenly over the cells
and produces very low shear forces. These systems are easy to set up and operate for prolonged periods of time.
The potential for scale-up using Fibra-cel carriers is enhanced due to the low pressure drop and low mass transfer resistance,
which creates high void fraction approaching 90% in the packed bed. 相似文献
2.
Cell growth and enzyme (alpha-amylase) production characteristics of Bacillus subtilis TN106 containing the recombinant plasmid pAT5 are investigated in batch and continuous cultures using a defined medium with glucose as the limiting nutrient. The batch culture studies demonstrate that the recombinant plasmid, reported earlier(1) to be stably maintained in the host, suffers from segregational and structural instabilities. The structural instability of this strain occurred during culture storage and can be eliminated in bioreactor experiments by using a modified inoculum preparation procedure. Such elimination allows an unbiased investigation of segregational instability via continuous culture studies. Such studies conducted with this fast growing microorganism, in the absence of antibiotic selection pressure, indicate a very efficient glucose utilization (very low residual glucose concentrations) over a wide range of dilution rates (0.16 h(-1) - 0.94 h(-1)). The nearly time-invariant and low residual glucose concentrations at each such dilution rate enable convenient estimation of growth parameters of the host and recombinant cells and frequency of segregational instability from transients in the resulting mixed cultures. The specific alpha-amylase activity exhibits an inverse relationship to the specific growth rate of recombinant cells. The growth of recombinant cells is not affected by the presence of antibiotic (kanamycin). The growth advantage of host cells over recombinant cells diminishes with increasing dilution rate. 相似文献
3.
Influence of plasmid origin and promoter strength in fermentations of recombinant yeast 总被引:1,自引:0,他引:1
The effects of plasmid promoter strength and origin of replication on cloned gene expression in recombinant Saccharomyces cerevisiae have been studied in batch and continuous culture. The plasmids employed contain the Escherichia coli lacZ gene under the control of yeast promoters regulated by the galactose regulatory circuit. The synthesis of beta-galactosidase was therefore induced by the addition of galactose. The initial induction transients in batch culture were compared for strains containing plasmids with 2mu and ARS1 origins. As expected, cloned gene product synthesis was much lower with the ARS1 plasmid: average beta-galactosidase specific activity was an order of magnitude below that with the 2mu-based plasmid. This was primarily due to the low plasmid stability of 7.5% when the plasmid origin of replication was the ARS1 element. The influence of plasmid promoter strength was studied using the yeast GAL1, GAL10, and hybrid GAL10-CYC1 promoters. The rate of increase in beta-galactosidase specific activity after induction in batch culture was 3-5 times higher with the GAL1 promoter. Growth rate under induced conditions, however, was 15% lower than in the absence of lacZ expression for this promoter system. The influence of plasmid promoter strength on induction behavior and cloned gene expression was also studied in continuous fermentations. Higher beta-galactosidase production and lower biomass concentration and plasmid stability were observed for the strain bearing the plasmid with the stronger GAL1 promoter. Despite the decrease in biomass concentration and plasmid stability, overall productivity in continuous culture using the GAL1 promoter was three times that obtained with the GAL10-CYC1 promoter. 相似文献
4.
Heterologous protein production by the yeast Saccharomyces kluyveri was investigated under aerobic glucose-limited conditions. Alpha-amylase from Aspergillus oryzae was used as model protein and the gene was expressed from a S. cerevisiae 2 micro plasmid. For comparison, strains of both S. kluyveri and S. cerevisiae were transformed with the same plasmid, which led to secretion of active alpha-amylase in both cases. The S. cerevisiae 2 micro plasmid was found to be stable in S. kluyveri as evaluated by a constant alpha-amylase productivity in a continuous cultivation for more than 40 generations. S. kluyveri and S. cerevisiae secreted alpha-amylase with similar yields during continuous cultivations at dilution rates of 0.1 and 0.2 h(-1) (4.8-5.7 mg (g dry weight)(-1)). At a dilution rate of 0.3 h(-1) the metabolism of S. kluyveri was fully respiratory, whereas S. cerevisiae produced significant amounts of ethanol. A fed-batch cultivation was carried out with S. kluyveri where the biomass concentration reached 85 g l(-1) and the alpha-amylase concentration reached 320 mg l(-1). Even though S. kluyveri could be grown to high cell density, it was also observed that it has a high maintenance coefficient, which resulted in low biomass yields at the low specific growth rates prevailing towards the end of the fed-batch cultivation. 相似文献
5.
Fikret Kargi 《Biotechnology letters》1988,10(3):181-186
Summary
Catharanthus
roseus cells producing indole alkaloids were grown on surfaces of Ca-alginate beads within the interspacial volume of a packed column. Production media was circulated through the packed column in an upflow mode. Growth and indole alkaloid formation were quantified and compared with suspension culture of cells. Final alkaloid concentration and alkaloid yield obtained in the packed bed was superior to those obtained in suspension culture. This is thought to be due to improved cell-cell contact and interaction in the packed column. 相似文献
6.
S Sayadi M Nasri F Berry J N Barbotin D Thomas 《Journal of general microbiology》1987,133(7):1901-1908
7.
In order to better understand the high plasmid stability in immobilized recombinant E. coli cells, the effects of dilution rate on the pTG201 plasmid stability, the copy number, and the catechol 2,3-dioxygenase (encoded by XyIE gene) production were, at first, studied in free E. coli W3101 continuous cultures in minimal media. It was found that decreasing specific growth rate increased the plasmid copy number and the catechol 2,3-dioxygenase activity but the stability decreased. In continuous culture with immobilized cells, an increase was shown in plasmid copy number and catechol 2,3-dioxygenase activity probably due to the distribution of growth in the gel beads. Besides mechanical properties of gel beads which may allow limited cell divisions, the increase in plasmid copy number is involved in enhanced plasmid stability in immobilized cells. In the same way, an experiment conducted in LB medium dealing with competition between pTG201-free and pTG201-containing E. coli B cells was described. It was shown that the competition was not more pronounced in gel bead compared to a free system. The effects of nutritional limitations on pTG201 plasmid stability and catechol 2,3-dioxygenase activity during chemostat cultivations in free and immobilized E. coli B cells were also investigated. It was found that immobilization of cells increased the stability of pTG201 even under glucose, nitrogen, or phosphate limited cultures. However in the case of magnesium depleted culture, pTG201 was shown to be relatively instable and a decrease in viable cell number during the immobilized continuous culture was observed. By contrast to the free system, the catechol 2,3-dioxygenase activity increased in immobilized cells under all culture conditions used. 相似文献
8.
Growth of Bacillus subtilis TN106[pAT5] and synthesis of plasmid-encoded protein (alpha-amylase) are investigated in batch, continuous, and fed-batch cultures using a defined medium containing glucose and/or starch as the carbohydrate source. The batch culture studies reveal that reduced availability of arginine hampers growth of recombinant cells (which lack an arginine synthesis gene) but promotes production of alpha-amylase and substitution of glucose by starch as the carbohydrate source leads to slower growth of recombinant cells and increased production of alpha-amylase per unit cell mass. Retention of recombinant cells over prolonged periods in continuous cultures is not possible without continuous application of antibiotic selection pressure owing to segregational plasmid instability. Fed-batch experiments with constant volumetric feed rate demonstrate that alpha-amylase production is enhanced at lower feed concentration of starch (sole carbohydrate source) and lower volumetric feed rate. Such slow addition of starch is however not conducive for growth of recombinant cells. The expression of the thermostable alpha-amylase gene carried on the recombinant plasmid pAT5 (derived from a plasmid isolated from a thermophilic bacterium) is promoted at higher temperatures, while growth of recombinant cells is depressed. In all batch and fed-batch experiments, production of alpha-amylase is observed to be inversely related to growth of recombinant cells. The efficacy of two-stage bioreactor operations, with growth of recombinant cells being promoted in the first stage and alpha-amylase production in the second stage, in attaining increased bulk alpha-amylase activity is demonstrated. (c) 1993 John Wiley & Sons, Inc. 相似文献
9.
A recombinant Saccharomyces cerevisiae C468/pGAC9 (ATCC 20690), which expresses Aspergillus awamori glucoamylase gene under the control of the yeast enolase I (ENO1) promoter and secretes glucoamylase into the extracellular medium, was used as a model system to investigate the effect of cell immobilization on bioreactor culture performance. Free suspension cultures in stirred-tank and airlift bioreactors confirmed inherent genetic instability of the recombinant yeast. An immobilized-cell-film airlift bioreactor was developed by employing cotton cloth sheets to immobilize the yeast cells by attachment. Enhanced enzyme productivity and production stability in the immobilized-cell system were observed. Experimental data indicated that the immobilized cells maintained a higher proportion of plasmid-bearing cells for longer periods under continuous operation. The higher plasmid maintenance with immobilized cells is possibly due to reduced specific growth rate and increased plasmid copy number. Double-selection pressure was used to select and maintain the recombinant yeast. The selected strain showed better production performance than the original strain. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 241-251, 1997. 相似文献
10.
Microencapsulation of recombinant Saccharomyces cerevisiae cells with invertase activity in liquid-core alginate capsules 总被引:1,自引:0,他引:1
As a means of integrating cell growth and immobilization, recombinant Saccharomyces cerevisiae cells with invertase activity were immobilized in liquid-core alginate capsules and cultured to a high density. S. cerevisiae cells of SEY 2102 (MAT alpha ura3-52 leu2-3, 112 his4-519) harboring plasmid pRB58 with the SUC2 gene coding for invertase were grown to 83 g/L of liquid-core volume inside the capsule on a dry weight basis. The cloned invertase was expressed well in the immobilized cells with slightly higher activity than the free cells in a batch culture. Invertase in the immobilized cells showed slightly more improved thermal stability than in the free cells. Storage in a Na-acetate buffer at 4 degrees C and 10 degrees C for 1 month resulted in 7% and 8% loss in activity, respectively. The sucrose hydrolysis reaction was stably maintained for 25 repeated batches for 7 days at 30 degrees C. Continuous hydrolysis of 0.3 M sucrose was carried out in a packed bed reactor with a conversion of more than 90% at a maximum productivity of 55.5 g glucose/L per hour for 7 days. In a continuous stirred tank reactor, the maximum productivity of 80.8 g glucose/L per hour was achieved at a conversion of 59.1% using 1.0 M sucrose solution, and 0.5 M sucrose solution was hydrolyzed for 1 week with a 95% conversion at a productivity of 48.8 g/L per hour. (c) 1996 John Wiley & Sons, Inc. 相似文献
11.
The up-regulation of cyclin-dependent kinase inhibitor p21 has been shown to enhance productivity of monoclonal antibodies and has been linked to various regulatory processes. To identify the potential role of p21 in adaptation to suspension and protein-free cultures, we studied the survival and growth of anchorage- and serum-dependent CHO cell lines that differed only in the period of p21-induced arrest. p21 overexpression led to rapid adaptation of cells to suspension and protein-free cultures. The period taken to achieve adaptation was correlated with the time the cells were arrested after transfer from the monolayer and serum-fed culture. Interestingly, cell aggregation associated with protein-free suspension culture was reduced in p21 culture in response to the loss of cellular adherence. The processes of adaptation to suspension and arrest did not decrease monoclonal antibody productivity. In contrast, following adaptation to protein-free growth media, an overall increase in specific productivity was observed. The ability of cells to survive in protein-free suspension cultures was due to the requirement of G1 cells to growth factors and to their relatively high resistance to the hydrodynamic forces. This improved process has the advantage of reducing the duration of critical path activity for developing CHO commercial cell lines from 72 to 36 days. 相似文献
12.
Rapid Purification of a New Humanized Single-chain Fv Antibody/Human Interleukin-2 Fusion Protein Reactive against HER2 Receptor 总被引:1,自引:0,他引:1
Overexpression of the proto-oncogene c-erbB-2, neuor HER2 has been shown in many human tumor cells,especially in breast cancer cells [1–5], which is thought tobe important in human carcinogenesis. c-erbB-2 geneencodes a 185 kD transmembrane glycoprotein … 相似文献
13.
The recombinant a and bsubunits for human coagulation factor XIII were transfected into Chinese hamster ovary (CHO) cells. CHO cells were amplified
and selected with methotrexate in adherent cultures containing serum, and CHO 1-62 cells were later selected in protein-free
medium. To develop a recombinant factor XIII production process in a suspension culture, we have investigated the growth characteristics
of CHO cells and the maintenance of factor XIII expression in the culture medium. Suspension adaptation of CHO cells was performed
in protein-free medium, GC-CHO-PI, by two methods, such as serum weaning and direct switching from serum containing media
to protein-free media. Although the growth of CHO cells in suspension culture was affected initially by serum depletion, cell
specific productivity of factor XIII showed only minor changes by the direct switching to protein-free medium during a suspension
culture. As for the long-term stability of factor XIII, CHO 1-62 cells showed a stable expression of factor XIII in protein-free
condition for 1000 h. These results indicate that the CHO 1-62cells can be adapted to express recombinant human factor XIII
in a stable maimer in suspension culture using a protein-free medium. Our results demonstrate that enhanced cell growth in
a continuous manner is achievable for factor XIII production in a protein-free medium when a perfusion bioreactor culture
system with a spin filter is employed.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
14.
The effects of dilution rates on the performance of a two-stage fermentation system for a recombinant Escherichia coli culture were studied. Dilution rate determines the apparent or averaged specific growth rate of a heterogeneous population of cells in the recombinant culture. The specific growht rate affects the genetic parameters involved in product formation in the second stage, such as plasmid stability, plasmid content, and specific gene expression rate. Kinetic models and correlations were developed for these parameters based on experimental data. Simulations of plasmid stability in the first stage showed that for longer fermentation periods, plasmid stability is better at higher dilution rates. However, the plasmid content is lower at these dilution rates. The optimal apparent specific growth rate for maximum productivity in the second stage was determined using two methods: (1) direct search for a constant specific growth rate, and (2) dynamic optimization using the maximum principle for a time-dependent specific growth rate profile. The results of the calculations showed that the optimum constant apparent specific growth rate for maximum over-all productivity is 0.40 h(-1). This coincides with the optimal specific growht rate for maximum plasmid content in the expressed stage. A 3.5% increase in overall productivity can be obtained by using a linear time dependent apparent specific growth rate control, mu(2)(t) = 0.0007t, in the course of the fermentation time. 相似文献
15.
Both the matrix structure of loofa sponge and the flocculating property of cells were necessary for efficient immobilization. The addition of chitosan to a reactor containing a bed of loofa sponge and a Candida brassicae cell suspension induced cell flocculation and the cells were efficiently immobilized. During ethanol production by the immobilized cells, the free cell concentration in the broth was controlled at the desired level by intermittent addition of chitosan to the reactor. The immobilized cell concentration increased but their specific ethanol productivity decreased with an increase in the chitosan concentration. The maximum ethanol productivity was obtained at a low chitosan concentration of 0·03 g/litre. With this optimal concentration, the cell concentration, ethanol yield and productivity were, respectively, 2, 1·3 and 3 times higher than those of the suspension culture. 相似文献
16.
A selection of mouse hybridoma cell lines showed a variation of approximately two orders of magnitude in intracellular monoclonal
antibody contents. The different levels directly influenced apparent specific monoclonal antibody productivity during the
death phase but not during the growth phase of a batch culture. The pattern of changes in specific productivity during culture
remained basically similar even though at different levels for all cell lines tested. Arresting the cells in the G1 phase using thymidine increased the specific productivity, cell volume and intracellular antibody content but at the same
time led to decreased viability. In continuous culture DNA synthesis decreased with decreasing dilution rate though without
an accompanying change in cell cycle and cell size distributions. The data shows both the decrease in viability and intracellular
antibody content to be important factors which influence the negative association between specific antibody productivity and
growth rate. In high cell density perfusion culture, when the cell cycle was prolonged by slow growth, viability was low and
dead, but not lysed, cells were retained in the system, the specific antibody productivity was nearly two fold higher than
that obtained in either batch or continuous cultures. The results imply that the prolongation of G1 phase and the increase in death rate of cells storing a large amount of antibody together cause an apparent increase in specific
antibody productivity. 相似文献
17.
M Nasri S Sayadi J N Barbotin P Dhulster D Thomas 《Applied and environmental microbiology》1987,53(4):740-744
The stability of pTG201 plasmid was examined by continuous culture in three genetically different Escherichia coli hosts. Two types of experiment were carried out, one with free cells and one with immobilized cells. When cells were cultivated in free continuous culture in the absence of antibiotic selection, the plasmid was maintained with various degrees of stability in the three host organisms. By contrast, in continuous culture with immobilized cells, plasmid pTG201 was stably maintained in the three strains. We showed that the increase in pTG201 stability in immobilized cells is due neither to plasmid transfer between immobilized cells nor to an increase of the plasmid copy number of immobilized cells. We also showed that plasmid-free cells, when coimmobilized and grown in competition with plasmid-containing cells, cannot overrun the culture. 相似文献
18.
Influence of immobilization on the stability of pTG201 recombinant plasmid in some strains of Escherichia coli.
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The stability of pTG201 plasmid was examined by continuous culture in three genetically different Escherichia coli hosts. Two types of experiment were carried out, one with free cells and one with immobilized cells. When cells were cultivated in free continuous culture in the absence of antibiotic selection, the plasmid was maintained with various degrees of stability in the three host organisms. By contrast, in continuous culture with immobilized cells, plasmid pTG201 was stably maintained in the three strains. We showed that the increase in pTG201 stability in immobilized cells is due neither to plasmid transfer between immobilized cells nor to an increase of the plasmid copy number of immobilized cells. We also showed that plasmid-free cells, when coimmobilized and grown in competition with plasmid-containing cells, cannot overrun the culture. 相似文献
19.
P de Taxis du Po?t Y Arcand R Bernier J N Barbotin D Thomas 《Applied and environmental microbiology》1987,53(7):1548-1555
Stability of the plasmid pKK223-200 in Escherichia coli JM105 was studied for both free and immobilized cells during continuous culture. The relationship between plasmid copy number, xylanase activity, which was coded for by the plasmid, and growth rate and culture conditions involved complex interactions which determined the plasmid stability. Generally, the plasmid stability was enhanced in cultured immobilized cells compared with free-cell cultures. This stability was associated with modified plasmid copy number, depending on the media used. Hypotheses are presented concerning the different plasmid instability kinetics observed in free-cell cultures which involve the antagonistic effects of plasmid copy number and plasmid presence on the plasmid-bearing/plasmid-free cell growth rate ratio. Both diffusional limitation in carrageenan gel beads, which is described in Theoretical Analysis of Immobilized-Cell Growth, and compartmentalized growth of immobilized cells are proposed to explain plasmid stability in immobilized cells. 相似文献
20.
The isolation of strains of Bacillus subtilis showing improved plasmid stability characteristics by means of selective chemostat culture 总被引:1,自引:0,他引:1
A pUB110-derived plasmid encoding chloramphenicol resistance, kanamycin resistance and high-temperature alpha-amylase showed a high degree of segregational instability when inserted into Bacillus subtilis. In an attempt to obtain stable derivatives, the organism was grown in chemostat culture in the presence of chlorampheniol. It was periodically found necessary to increase the concentration of chloramphenicol in the medium feed in order to avoid plasmid loss. Strains were isolated after 19 and 160 generations, which showed high levels of plasmid stability. This characteristic appeared to be genotypic. No detectable difference in plasmid copy number was found between the original and the improved strains. The stability characteristics resided in the host, rather than in the plasmid. Stable isolates possessed elevated MICs for both chloramphenicol and kanamycin. Their maximum specific growth rates were higher than that of the original strain, and similar to that of the plasmid-free parent strain. 相似文献