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1.
The growth and antibody production of the SP2/0-derived hybridoma HB124 (ATCC) grown in media containing varying amounts of fetal bovine serum (FBS) were monitored using biochemical and flow cytometric methods. Hybridomas grown in 100 ml spinner flasks with RPMI-1640 containing varying amounts of serum demonstrated that cell growth, viability and IgG production show significant changes when serum content is decreased from 10.0 to 5.5 to 1.0 and 0.5%. A longer lag phase resulted when the lower serum content media were used. Cellular rates of glucose uptake showed a significant increase as serum levels were lowered. Similarly, exponential phase IgG production rates increased as the amount of serum was decreased, probably as a result of the decreased rate of exponential growth. Flow cytometric analysis showed a similar increase in cellular IgG content as medium serum levels declined. In contrast, the maximum IgG concentrations were found in flasks containing 1% FBS or above with the lowest concentration in the 0.5% FBS flask being due to the lower numbers of viable cells. Cells grown in microporous hollow fiber reactors were fed with medium containing serum which was decreased stepwise with time. Decreasing medium serum content stepwise from 10 to 2.5% resulted in increased antibody production. However, complete removal of serum from the medium resulted in a significant drop in antibody productivity. Cumulative antibody production was equivalent for cells grown entirely in medium containing 10% FBS and for those which experienced a drop to 2.5% FBS. To compare a defined serum-free medium preparation with medium containing 10% FBS, cells were again grown in batch suspension culture and analyzed. The growth rates were similar but there was a significant difference in IgG production rates. The serum-free culture exhibited both higher cellular production rates and higher IgG concentrations. These results indicate that decreasing medium serum content can adversely affect antibody yield because of lower cell viabilities, not because of lower production rates. Use of a defined serum-free medium, as done in this study, results in higher yields because of a higher IgG production rate as well as good cell growth and viability. 相似文献
2.
Rabbit IgG was purified, Fr-1-(G-200)2, and then separated by DEAE-cellulose column chromatography into three major fractions, Fr-I, -II, and -III. Binding affinities of the 125I-labeled IgG and its fractions to the rabbit fetal yolk sac membrane were studied. At a concentration of 2 mg/ml, rabbit IgG is bound to the extent of 9 mug/cm2 membrane, whereas the values for fractions Fr-I, -II, and -III are 13, 7, and 5 mug/cm2, respectively. The maximal amount of IgG bound appears to be approximately the same, i.e., 23 mug of IgG/cm2 membrane, for Fr-1-(G-200)2 and its three fractions. In contrast, bovine IgG does not bind to the membrane over the range of concentration tested. Binding constants for Fr-1-(G-200)2, fraction Fr-I, -II, and -III are estimated to be 5.4, 8.6, 4.0 and 2.0 times 10(4) M-1, respectively. The binding affinities of IgG fractions to the yolk sac membrane correlate with the chemical and physicochemical properties of the fractions. Also detailed in the text is the use of 22Na to facilitate quantitation of specific binding of the 125I-IgG to the membrane under equilibrium conditions. 相似文献
3.
Molinari R Torres JL Michaels AS Kilpatrick PK Carbonell RG 《Biotechnology and bioengineering》1990,36(6):572-580
A protein separation scheme combining affinity or ion exchange sorption with hollow fiber cross-flow filtration is described. Sorptive gel particles were loaded into the shell side of a hollow fiber membrane module. In the adsorption step, crude protein mixtures were passed through the lumen and permeating proteins passed through the membrane to bind on the gel particles in the shell. During elution, a buffer of adequate ionic strength to desorb the bound proteins was passed through the lumen and permeated through the shell. The eluant was then collected at the outlet to the shell of the hollow fiber module. The concept is illustrated by two examples: the purification of butyrylcholinesterase (EC 3.1.1.7) from raw horse serum using the affinity gel procainamide-Sepharose as the packing and the separation of carboxylesterase (EC 3.1.1.1) from beef liver homogenate using DEAE-Sephadex as the packing. The technique has the advantage of high volumetric throughputs typical of hollow fiber membrane modules as well as the high capacity characteristic of chromatographic packings. In addition, cross-flow filtration of particulates, agglomerates, and debris in passing protein from lumen to shell side can help eliminate the need for extensive pretreatment. 相似文献
4.
To improve protein separation, a novel integrated device combining membrane filtration and chromatography has been developed. The device basically consists of a hollow fiber filtration module whose shell side is filled with chromatographic resin beads. However, there is an essentially impermeable coated zone near the hollow fiber module outlet. The integrated device enjoys the advantages of both membrane filtration and chromatography; it also allows one to load the chromatographic media directly from the fermentation broth or lysate and separate the adsorbed proteins through the subsequent elution step in a cyclic process. Interfacial polymerization was carried out to coat the bottom section of the hollow fiber membrane; the rest of the hollow fiber membrane remained unaffected. Myoglobin (Mb) and alpha-lactalbumin (alpha-LA) were primarily used as model proteins in a binary mixture; binary mixtures of Mb and bovine serum albumin (BSA) were also investigated. Separation behaviors of binary protein mixtures were studied in devices having either an ultrafiltration (UF) or a microfiltration (MF) membrane. Experimental results show that the breakthrough time and the protein loading capacities were dramatically improved after introducing the impermeable coating in both UF and MF modules. For a synthetic yeast fermentation broth feed, four loading-washing-elution-reequilibration-based cyclic runs for separation of Mb and alpha-LA were performed in the device using a MF membrane with a coated zone without cleaning in between. The Mb and alpha-LA elution profiles for the four consecutive runs were almost superimposable. Due to lower transmembrane flux in this device plus the periodical washing-elution during the chromatographic separation, fouling was not a problem, unlike in conventional microfiltration. 相似文献
5.
Coffinier Y Vijayalakshmi MA 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2004,808(1):51-56
In this study, we attempted a limited combinatorial approach for designing affinity ligands based on mercaptoheterocyclic components. The template, divinyl sulfone structure (DVS), which was grafted on poly(ethylene vinyl alcohol) (PEVA) hollow fiber membrane, has served for the tethering of different heterocyclic compounds as pyridine, imidazole, purine and pyrimidine rings. Their ability to adsorb specifically IgG in a salt independent manner out of pure IgG solution, mixture of IgG/albumin and human plasma was demonstrated. Mercapto methyl imidazole (MMI) has shown the best adsorption of IgG in terms of binding capacity. No subclass discrimination was observed on all tested ligands except for mercapto methyl pyrimidine where the major IgG subclass adsorbed was IgG3. MMI gave an IgG binding capacity of 100 microg/cm2 of hollow fiber membrane surface area. 相似文献
6.
Invertase was ionically immobilized on the poly(ethylene-co-vinyl alcohol) hollow fiber inside surface, which was aminoacetalized with 2-dimethylaminoacetaldehyde dimethyl acetal. Immobilization and enzyme reaction were carried out by letting the respective solutions pass or circulate through the inside of the hollow fiber, and the activity of invertase was determined by the amount of glucose produced enzymatically from sucrose. Immobilization conditions were examined with respect to the enzyme concentration and to the time, and consequently the preferable conditions at room temperature were found to be 5 mug/mL of enzyme concentration and 4 h of immobilization time. Under those conditions the immobilization yield and the ratio of the activity of the immobilized invertase to that of the native one were 89 and 80%, respectively. For both repeating and continuous usages, the activity fell to ca. 60% of the initial activity in the early stage and after that almost kept that value. The apparent Michaelis constant K(m) (') for the immobilized invertase decreased with increasing the flow rate of the substrate solution, to be close to the value for the native one. Furthermore, the possibility of the separation of the enzymatically formed glucose from the reaction mixture through the hollow fiber membrane was preliminarily examined. 相似文献
7.
Takazawa Y Tokashiki M Murakami H Yamada K Omura H 《Biotechnology and bioengineering》1988,31(2):168-172
Mouse-human hybridoma 4H11 cells producing anti-Pseudomonas sp. monoclonal antibody (IgA) grew in a serum-free medium supplemented with insulin, transferrin, ethanolamine, and selenite (ITES). The hybridoma could be applied to high-density culture in a serum-free medium supplemented with ITES, 0.5% BSA, egg yolk VLDL, and artificial blood FC-43 in a culture vessel equipped with hollow-fiber modules for medium exchange. Total cell density reached 1.1 x 10(7) cells/mL (viable cell density was 7.6 x 10(6) cells/mL), and the IgA productivity was around 20 mug/10(6) cells/day in the serum-free medium, which corresponded to the levels in serum-supplemented medium. 相似文献
8.
Anchorage-dependent HeLa cells were successfully cultured on two fibrous materials (A07 and R100) with porosities of 75-125 and 40 mum, void fractions of 92% and 81%, and fiber diameters of 7.6 and 10.2 mum, respectively, in 100-mL spinner flasks and 2-L stirred tank bioreactors. The matrix was formed into a fixed vertical spiral configuration. All cultures displayed rapid (=2-3 h) attachment of inoculated cells (>/=95%) to the matrix, uniform coverage of the immobilizing area with viable cells, and no significant amount of cell debris in the medium. Spinner flask cultures indicated that the denser material R100 showed better results in terms of final cell density. The growth of HeLa cells on material R100 in both culture systems was similar to that observed in tissue culture dishes (specific growth rate approximately 0.03-0.04 h(-1), maximum cell density of 8 x 10(6)-9 x 10(6) cells . mL(-1), and yields of 0.4 x 10(8) cells . mM(-1) on glucose and 2 x 10(8)-3 x 10(8) cells . mM(-1) on glutamine). Scale-up of this culture technique in a 2-L bioreactor under perfusion with pH and dissolved oxygen (DO) control yielded cell densities of up to 1.6 x 10(6) cells . mL(-1). Two other anchorage-dependent mammalian cells (ADC) known to be cultured with difficulty in roller bottles or with micro carriers were easily grown on material R100 in spinner flasks. The performance of this culture technique was compared to other ADC culture systems. 相似文献
9.
Streptomyces aureofaciens (ATCC 12416c) was grown in the interstitial region formed by a parallel arrangement of three hollow silicone tubules contained within a microporous polypropylene hollow fiber. Liquid-soluble nutrients were supplied by diffusion across the polypropylene fiber to the interstitial cell-containing region whereas air or oxygen was provided by diffusion from the silicone tubule lumina to the cell mass. In this bioreactor, S. aureofaciens grew to high cell densities (greater than 10(11) cells/cm(3)) and the culture so-obtained continously synthesized the secondary metabolite tetracycline. The volumetric productivity of tetracycline based on the interstitial volume was 90 mug/ml/h and based on the total reactor volume was 5.5 mug/mL/h. The high surface area-to-volume ratio afforded by the cylindrical configuration together with spatially distinct conduits to continuously transport liquids and gases, each of which may be nutrients or products of biosynthesis, to or from a tissuelike cell mass provides an alternative to the conventional air- or oxygen-sparged fermentation vessel. High volumetric reactor productivities may be achieved by virute of the concentrated stationary cell mass and by the appropriate selection of fiber sizes and materials so as to ensure adequate supplies of liquid and gaseous substrates to, as well as removal of metabolites from, most cells in the culture. This reactor topology is quite general and may be adapted to most microbial as well as mammalian and plant cell systems. 相似文献
10.
We established a serum-free medium of low protein content(125g/ml) TYI 100, consisting of three hormones and five growth factors for the growth of lymphoid and hybridoma cell lines. In TYI 100 medium, mouse and human hybridomas grew equally well as in RPMI 1640 supplemented with 10% fetal bovine serum (10% FBS) without adaptation to the serum-free medium. TYI 100 medium allowed several passages of mouse hybridoma lines and the total cell number was more than in 10% FBS. TYI 100 medium also supported growth of myelomas and anchorage dependent cell lines, Bowes and CHO, well. TYI 100 medium is composed of inexpensive supplements and is therefor applicable to large scale culture.Abbreviations FBS
Fetal Bovine Serum
- IMDM
Iscove's Modification of Dulbecco's Medium
- PBS
Phosphate-Buffered Saline
- TPA
Tissue Plasminogen Activator 相似文献
11.
To enhance oxygen transfer in surface-aeration bioreactors, stabilized foams were generated to increase the gas-liquid interfacial area by slowly introducing coarse bubbles into media containing fetal bovine serum. The bubble sparging rates were so low (i.e., 20 and 50 mL/h) that the contribution to oxygen transfer from these bubbles was due to foaming instead of bubbling. Furthermore, no physical cell damage caused by bubble sparging was observed. Oxygen transfer coefficients, kLa, in the bioreactors were measured in cell-free media. Without the foam-stabilizing agent (i.e., serum), no appreciable change in kLa was observed due to the bubble sparging. On the other hand, with serum, kLa increased with increasing serum content and bubble sparging rate and corresponded well with the degree of foaming. With 10% fetal bovine serum and a bubble sparging rate of 50 mL/h, kLa increased approximately 90% compared with no foaming. The enhancing effect of foam on oxygen transfer in surface aeration bioreactors has been further demonstrated with hybridoma cultures simultaneously grown in three identical bioreactors with and without stabilized foams. 相似文献
12.
Ribeiro MB Vijayalakshmi M Todorova-Balvay D Bueno SM 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2008,861(1):64-73
The purification of IgG from human plasma was studied by comparing two affinity membranes complexed with Ni(II), prepared by coupling iminodiacetic acid (IDA) and Tris(2-aminoethyl)amine (TREN) to poly(ethylenevinyl alcohol), PEVA, hollow fiber membranes. The Ni(II)-TREN-PEVA hollow fiber membrane had lower capacity for human IgG than the complex Ni(II)-IDA-PEVA, but with similar selectivity. The IgG in peak fractions eluted from the Ni(II)-IDA-PEVA with a stepwise concentration gradient of Tris-HCl pH 7.0 (100-700 mM) reached a purity of 98% (based on IgG, IgM, IgA, albumin, and transferrin nephelometric analysis). Adsorption IgG data at different temperatures (4-37 degrees C) were analyzed using Langmuir model resulting in a calculated maximum capacity at 25 degrees C of 204.6 mg of IgG/g of dry membrane. Decrease in Kd with increasing temperature (1.7x10(-5) to 5.3x10(-6) M) indicated an increase in affinity with increased temperature. The positive value of enthalpy change (26.2 kJ/mol) indicated that the adsorption of IgG in affinity membrane is endothermic. Therefore, lower temperature induces adsorption as verified experimentally. 相似文献
13.
Most bio-industrial mammalian cells are cultured in serum-free media to achieve advantages, such as batch consistency, suspended growth, and simplified purification. The successful development of a serum-free medium could contribute to a reduction in the experimental variation, enhance cell productivity, and facilitate biopharmaceuticals production using the cell culture process. Commercial serum-free media are also becoming more and more popular. However, the cell line secrets its own recombinant product and has special nutritional requirements. How can the composition of the proprietary medium be adjusted to support the specific cell’s metabolism and recombinant protein? This article uses statistical strategies to modify the commercial medium. A design of experiments is adopted to optimize the medium composition for the hybridoma cell in a serum-free condition. The supplements of peptone, ferric citrate, and trace elements were chosen to study their impact on hybridoma growth and antibody production using the response surface methodology. The stimulatory effect of the developed formulation on hybridoma growth was confirmed by the steepest ascent path. The optimal medium stimulated the hybridoma growth and antibody production in three diverse systems: a static plate, an agitated spinner flask, and a hollow fiber reactor. The cells in the developed serum-free medium had a better antibody production as compared to that in the commercial medium in the hollow fiber reactor. Our results demonstrated that the facile optimization for medium and antibody production was successfully accomplished in the hybridoma cells. 相似文献
14.
A dual-layer coaxial hollow fiber (DLHF) bioreactor for cell immobilization developed to overcome nutrients transport limitation is presented. Cells were contained in the annular space between two coaxial hollow fibers, and nutrients were supplied by a forced convective transport from the shell side through the annular space to the lumen side. With judicious selection of the membrane materials, a low operating transmembrane pressure of 50 kPa, and using E. coli as the model organism, a high cell density of 10(11) cells/mL annular space volume and a high cell viability of (up to 80%) were obtained. 相似文献
15.
16.
Encapsulation of viable cells within polyacrylate membranes 总被引:1,自引:0,他引:1
A new, semipermeable hydrogel membrane for encapsulating viable cells has been developed. The encapsulation was performed by consecutively introducing droplets of a suspension of hybridoma cells in a solution of a polyanionic acrylic copolymer into aqueous solutions of three polycationic polymers. As a result of interpolymeric ionic interactions and some chemical reactions, a polyelectrolyte complex membrane was formed at the interface between each droplet and the polycationic polymeric solutions. The hybridoma cells, used as a model system, were derived by fusing spleen cells from immunized BALB/c mice with the NS-1 murine plasmacytoma cell line. The cells divided and gradually filled the microcapsules over a period of 8 days. Prior to encapsulation of the hybridoma cells, the polyanions were tested for toxicity and inhibition of cell growth. A direct relationship was observed between hybridoma cell viability in the acrylic polyanion/RPMI-1640/10% (w/v) fetal calf serum (FCS) solutions and the kinematic viscosities of the solutions of the polyanions. Antihuman IgM was produced by the encapsulated hybridoma cells and immunoassay showed that the antibody concentration was 3 mug/ml of the total culture medium. 相似文献
17.
An acid proteolytic activity has been found in cell culture supernatants from long-term cultivations of hybridoma cells in hollow fibre bioreactors using serum free medium. The proteolytic activity has now been further characterized and the main results were: (1) the proteolytic activity showed a maximum around pH 3 and declined essentially to zero at pH 8; (2) the activity was specifically inhibited by pepstatin A; (3) the acid proteases consisted of two sets of closely spaced bands with apparent molecular weights of 40-45K and 90-105K, respectively; (4) the protease bands (40-45K and 90-105K) were reactive with anti-human cathepsin D; (5) the IEP values of the acid proteases ranged from pH 4.55-6.5. Furthermore, IgG incubation with the acid proteases isolated from hybridoma cells yielded fragments similar to those found in serum-free hollow fibre cell culture supernatants. These results indicated that the IgG fragments are the result of degradation by cathepsin D like proteases released after cell death or cell lysis. 相似文献
18.
The heterogeneous nature of hollow fiber reactors for cell cultivation requires special considerations for proper design and operation. Downstream concentration of high-molecular-weight proteins has been measured in the shell side of ultrafiltration hollow fiber bioreactors. This distribution resulted from shell-side convective fluxes which caused a concentration polarization of proteins retained by the ultrafiltration membranes (nominal 3 x 10(4) D cutoff). Measurements of the axial hybridoma cell distribution also revealed a downstream concentration of viable cells during the first month of perfusion operation. This is believed to result from the shell-side convective flow and its influence on the inoculum and high-molecular-weight growth factor distributions. The heterogeneous distribution of cells leads to reduced cell numbers and reactor productivities. The mechanisms responsible for these phenomena have been investigated and their implications in process design and operation are considered. The heterogeneous protein and cell distributions on the shell side of hollow fiber bioreactors have been reduced significantly by periodic alternation of the direction of recycle flow and the reactor antibody productivities have been doubled. 相似文献
19.
Serpa G Augusto EF Tamashiro WM Ribeiro MB Miranda EA Bueno SM 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,816(1-2):259-268
The large scale production of monoclonal antibodies (McAbs) has gaining increased relevance with the development of the hybridoma cell culture in bioreactors creating a need for specific efficient bioseparation techniques. Conventional fixed bead affinity adsorption commonly applied for McAbs purification has the drawback of low flow rates and colmatage. We developed and evaluated a immobilized metal affinity chromatographies (IMAC) affinity membrane for the purification of anti-TNP IgG(1) mouse McAbs. We immobilized metal ions on a poly(ethylene vinyl alcohol) hollow fiber membrane (Me(2+)-IDA-PEVA) and applied it for the purification of this McAbs from cell culture supernatant after precipitation with 50% saturation of ammonium sulphate. The purity of IgG(1) in the eluate fractions was high when eluted from Zn(2+) complex. The anti-TNP antibody could be eluted under conditions causing no loss of antigen binding capacity. The purification procedure can be considered as an alternative to the biospecific adsorbent commonly applied for mouse IgG(1) purification, the protein G-Sepharose. 相似文献
20.
Murine hybridoma cells that produce monoclonal antibody directed against human fibronectin have been cultured in VITAFIBER II and VITAFIBER V hollow fiber bioreactors using defined, serum-free WRC 935 medium. During a two-week growth period, following inoculation of the bioreactors, the cells proliferated to an extent where the bioreactor was filled with cultured cells. Using a 5 sq. ft. VITAFIBER V bioreactor, over 15 grams of antibody were produced during the 40 days of the experiment. This antibody was greater than 95% IgG. During the production period, this packed mass of cells produced 579 +/- 15 mg IgG per day. Because the medium is formulated for air equilibration and high cell densities, WRC 935 medium is especially useful for production of gram quantities of monoclonal antibodies using continuous feed hollow fiber bioreactor cell culture systems. 相似文献