| 1. |
- Nilsang, Suthasinee, et al.
(författare)
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Three-dimensional culture for monoclonal antibody production by hybridoma cells immobilized in macroporous gel particles
- 2008
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Ingår i: Biotechnology progress (Print). - Malden, MA : Wiley-Blackwell Publishing Inc.. - 8756-7938 .- 1520-6033. ; 24:5, s. 1122-1131
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Tidskriftsartikel (refereegranskat)abstract
- Cell proliferation and long-term production of monoclonal antibody IgG(2b) by M2139 hybridoma cells immobilized in macroporous gel particles (MGPs) in packed-bed reactor were studied for a period of 60 days. The MGPs were made of supermacroporous gels produced in frozen conditions from crosslinked polyacrylamide and modified with gelatin which were housed in special plastic carriers (7 x 9 mm(2)). Cells were trapped in the interior part of MGPs by attaching to the void space of the gel matrix as three-dimensional (3D) cultivation using gelatin as a substrate layer. Optimizing productivity by hybridoma cell relies on understanding regulation of antibody production. In this study, the behavior of M2139 cells in two-dimensional cultures on multiwell plate surfaces was also investigated. The effect of three different medium such as basal medium Dulbecco's modified Eagle's medium (D-MEM) containing L-glutamine or L-glutamine + 2 mM alpha-ketoglutarate or L-alanyl-glutamine (GlutaMAXtrade mark) was studied prior to its use in 3D cultivation. The kinetics of cell growth in basal medium containing L-glutamine + alpha-ketoglutarate was similar to cells grown on GlutaMAX containing medium, whereas D-MEM containing L-glutamine showed lower productivity. With the maximal viable cell density (6.85 x 10(6) cells mL(-1)) and highest specific mAb production rate (3.9 mug mL(-1) 10(-4) viable cell day(-1)), D-MEM-GlutaMAX was further selected for 3D cultivation. Cells in MGPs were able to grow and secrete antibody for 30 days in packed-bed batch reactor, before a fresh medium reservoir was replaced. After being supplied with fresh medium, cells again showed continuous growth for another 30 days with mAb production efficiency of 50%. These results demonstrate that MGPs can be used efficiently as supporting carrier for long-term monoclonal antibody production. © 2008 American Institute of Chemical Engineers.
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| 2. |
- Basheer, Shabana, et al.
(författare)
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A membrane protein based biosensor: Use of a phosphate - H+ symporter membrane protein (Pho84) in the sensing of phosphate ions.
- 2011
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Ingår i: Biosensors & bioelectronics. - : Elsevier. - 0956-5663 .- 1873-4235. ; 27:1, s. 58-63
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Tidskriftsartikel (refereegranskat)abstract
- A label free biosensor for direct detection of inorganic phosphate based on potential-step capacitance measurements has been developed. The high-affinity Pho84 plasma membrane phosphate/proton symporter of Saccharomyces cerevisiae was used as a sensing element. Heterologously expressed and purified Pho84 protein was immobilized on a self-assembled monolayer (SAM) on a capacitance electrode. Changes in capacitance were recorded upon exposure to phosphate compared to the control substance, phosphate analogue methylphosphonate. Hence, even without the explicit use of lipid membranes, the Pho84 membrane protein could retain its capacity of selective substrate binding, with a phosphate detection limit in the range of the apparent in vivo K(m). A linear increase in capacitance was monitored in the phosphate concentration range of 5-25 mu M. The analytical response of the capacitive biosensor is in agreement with that the transporter undergoes significant conformational changes upon exposure to inorganic phosphate, while exposure to the analogue only causes minor responses.
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| 3. |
- Håkansson, Torbjörn, et al.
(författare)
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Sulphate reducing bacteria to precipitate mercury after electrokinetic soil remediation
- 2008
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Ingår i: International journal of environmental science and technology. - 1735-1472 .- 1735-2630. ; 5:2, s. 267-274
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Tidskriftsartikel (refereegranskat)abstract
- Combined treatment with electroremediation and sulphate reducing bacteria (SRB) was tested in laboratory and pilot scale. The contaminated soil came from a chlor-alkali factory and contained about 100 mg/kg Hg. Iodide/iodine complexing agent was used to mobilize mercury. Mercury iodide complexes were moved to the anode solution using an electric field. The anode solution was then mixed with hydrogen sulphide (H2S) containing water, causing precipitation of mercury sulphide. The H2S was produced at site by a SRB reactor. Precipitation problems arising from the nature of the anode solution were expected, since this solution is highly acidic, very oxidised and may contain iodide/iodine that strongly complexes mercury and can hinder mercury sulphide precipitation. Mercury concentrations in the anode solution were up to 65.7 mg/L (field) and 15.4 mg/L (lab. scale). Reduction of mercury in the water was >93% at all times. Iodide did not hinder the process: Nonetheless, in the lab system, iodide concentration was high in the anode solution but mercury reduction was > 99.9%. The redox potential was sufficiently low for HgS precipitation during the experiments, except for a short period, when the mercury removal decreased to 94%. Sulphate reducing bacteria are shown as a viable tool for the treatment of mercury contaminated, acidic, oxidative, iodide containing water, such as that produced by electrokinetic remediation. A second SRB step or other water treatment is required to reduce the mercury concentration to environmentally acceptable levels. Redox. potential is the most sensitive factor in the system.
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| 4. |
- Kumar, Ashok, et al.
(författare)
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Affinity binding of cells to cryogel adsorbents with immobilized specific ligands : effect of ligand coupling and matrix architecture
- 2005
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Ingår i: Journal of Molecular Recognition. - : Wiley. - 0952-3499 .- 1099-1352. ; 18:1, s. 84-93
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Tidskriftsartikel (refereegranskat)abstract
- The capture of human acute myeloid leukemia KG-1 cells expressing the CD34 surface antigen and the fractionation of human blood lymphocytes were evaluated on polyvinyl alcohol (PVA)-cryogel beads and dimethyl acrylamide (DMAAm) monolithic cryogel with immobilized protein A. The affinity ligand (protein A) was chemically coupled to the reactive PVA-cryogel beads and epoxy-derivatized monolithic cryogels through different immobilization techniques and the binding efficiency of the cell surface receptors specific antibody-labeled cells to the gels/beads was determined. The binding of cells to monolithic cryogel was higher (90-95%) compared with cryogel beads (76%). B-lymphocytes, which bound to the protein A-cryogel beads, were separated from T-lymphocytes with yields for the two cell types 74 and 85%, respectively. About 91% of the bound B-cells could be recovered without significantly impairing their viability. Our results show differences in the percentage of cell-binding to the immunosorbents caused by ligand density, flow shear forces and bond strength between the cells and the affinity surface once distinct chemical coupling of protein A, size of beads, sequence of antibody binding to protein A adsorbents, morphology and geometry of surface matrices were compared.
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| 5. |
- Lacayo, Martha, et al.
(författare)
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A toxaphene-degrading bacterium related to Enterobacter cloacae, strain D1 isolated from aged contaminated soil in Nicaragua
- 2005
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Ingår i: Systematic and Applied Microbiology. - : Elsevier BV. - 0723-2020 .- 1618-0984. ; 28:7, s. 632-639
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Tidskriftsartikel (refereegranskat)abstract
- Enterobacter sp. strain D1 is a facultative anaerobic, Gram-negative heterotrophic bacterium isolated from toxaphene-contaminated soil. This organism was identified and characterized through phylogenetic and taxonomic studies. Based on 16S rDNA analysis, the strain D1 was clustered closely with the species Enterobacter cloacae subsp. dissolvens (LMG 2683) and E. cloacae (ATCC 13047T). Strain D1 resembled these E. cloacae strains with respect to various biochemical and nutritional characteristics, but also exhibited differences. Moreover, strain D1 is able to grow and survive with toxaphene supplied in the medium in the range 3-96 mg/L. Amongst the chemical components of toxaphene, octachlorocamphenes, nonachlorobornanes and decachlorobornanes were seen to be rapidly metabolized, although levels of hexachlorocamphenes and heptachlorobornanes were found to be slowly degraded, and subsequently accumulated during the last stage of the cultivation.
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| 6. |
- Lacayo, Martha, et al.
(författare)
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Degradation of toxaphene by Bjerkandera sp. strain BOL13 using waste biomass as a cosubstrate
- 2006
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 71:4, s. 549-554
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Tidskriftsartikel (refereegranskat)abstract
- The white-rot fungus Bjerkandera sp. strain BOL13 was capable of degrading toxaphene when supplied with wood chips, wheat husk or cane molasses as cosubstrates in batch culture experiments. Approximately 85% of toxaphene was removed when wheat husk was the main substrate. The production of lignin peroxidase was only stimulated when wheat husk was present in the liquid medium. Although xylanase was always detected, wheat husk supported the highest xylanase production. A negligible amount of beta-glucosidase and cellulase were found in the batch culture medium. To the best of our knowledge, this is the first reported case of toxaphene degradation by white-rot fungi.
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| 7. |
- Lacayo-Romero, Martha, et al.
(författare)
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Degradation of toxaphene in aged and freshly contaminated soil
- 2006
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Ingår i: Chemosphere. - : Elsevier BV. - 0045-6535 .- 1879-1298. ; 63:4, s. 609-615
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Tidskriftsartikel (refereegranskat)abstract
- Degradation of toxaphene in soil from both newly contaminated (from Sweden) and aged spills (from Nicaragua) were studied. The newly contaminated soil contained approximately 11 mg kg(-1) toxaphene while the aged Nicaraguan soil contained approximately 100 mg kg(-1). Degradation was studied in anaerobic bioreactors, some of which were supplied with lactic acid and others with Triton X-114. In this study we found that the lower isomers Parlar 11, 12 were degraded while the concentration of isomer Parlar 15 increased. This supported an earlier evaluation which indicated that less chlorinated isomers are formed from more heavily isomers. Lactic acid when added to the soil, interfere with the degradation of toxaphene. Lactic acid was added; several isomers appeared to degrade rather slowly in newly contaminated Swedish soil. The Swedish soil, without any external carbon source, showed the slowest degradation rate of all the compounds studied. When Triton X-114 at 0.4 mM was added, the degradation rate of the compounds increased. This study illustrates that biodegradation of toxaphene is a complex process and several parameters have to be taken into consideration. Degradation of persistent pollutants in the environment using biotechnology is dependent on bioavailability, carbon sources and formation of metabolites.
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| 8. |
- Welander, Ulrika, et al.
(författare)
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Decolorization of synthetic and real textile wastewater by the use of white-rot fungi
- 2006
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Ingår i: Enzyme and Microbial Technology. - : Elsevier Inc. - 0141-0229. ; 38:1, s. 94-100
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Tidskriftsartikel (refereegranskat)abstract
- Batch and continuous reactors inoculated with white-rot fungi were operated in order to study decolorization of textile dyes. Synthetic wastewater containing either Reactive Blue 4 (a blue anthraquinone dye) or Reactive Red 2 (a red azo dye) was used during the first part of the study while real wastewater from a textile industry in Tanzania was used in the later part. Trametes versicolor was shown to decolorize both Reactive Blue 4 and Reactive Red 2 if glucose was added as a carbon source. Reactive Blue 4 was also decolorized when the fungus was allowed to grow on birch wood discs in a continuous biological rotating contactor reactor. The absorbance at 595 nm, the wavelength at which the dye absorbs at a maximum, decreased by 70% during treatment. The initial dye concentration in the medium was 200 mg/l and the hydraulic retention time in the reactor 3 days. No glucose was added in this experiment. Changes of the absorbance in the UV range indicated that the aromatic structures of the dyes were altered. Real textile wastewater was decolorized by Pleurotus flabellatus growing on luffa sponge packed in a continuous reactor. The reactor was operated at a hydraulic retention time of 25 h. The absorbance at 584 nm, the wavelength at which the wastewater absorbed the most, decreased from 0.3 in the inlet to approximately 0.1 in the effluent from the reactor.
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| 9. |
- Ahlqvist, Josefin, et al.
(författare)
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Monitoring the production of inclusion bodies during fermentation and enzyme-linked immunosorbent assay analysis of intact inclusion bodies using cryogel minicolumn plates
- 2006
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 354:2, s. 229-237
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Tidskriftsartikel (refereegranskat)abstract
- A novel minicolumn chromatgraphic method to monitor the production of inclusion bodies during fermentation and anenzyme-linked immunosorbent assay (ELISA) system allowing direct analysis of the particles with surface-displayed antigens are described. A 33-kDa protein containing 306 amino acids with three sulfur bridges produced its inclusion bodies wits labeled with polyclonal antibodies against 15 amino acid (anti-A15) and 17 amino acid (anti-B17) residues at the N- and C-terminal ends of the protein, respectively. Labeled particles were bound to macroporous Monolithic protein A-cryogel adsorbents inserted into the open-ended wells of a 96-well plate (referred to as protein A-cryogel minicolumn plate). The concept behind this application is that the binding degree of inclusion bodies from lysed fermentation broth to the cryogel minicolumns increases with an increase in their concentration during fermentation. The technique allowed LIS to monitor the increase in the production levels of the inclusion bodies as the fermentation process progressed. The system also has a built-in quality parameter to ensure that the target protein has been fully expressed. Alternatively, inclusion bodies immobilized on phenyl-cryogel minicolumn plate were used in indirect ELISA based on anti-A15 and anti-B17 antibodies against terminal amino acid residues displayed oil the surface of inclusion bodies. Drainage-protected properties of the cryogel minicolumns allow performance of successive reactions with tested immunoglobulin G (IgG) samples and enzyme-conjugated secondary I-G and of enzymatic reaction within the adsorbent. (c) 2006 Elsevier Inc. All rights reserved.
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| 10. |
- Kumar, Ashok, et al.
(författare)
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Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices
- 2006
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Ingår i: Biotechnology and Bioengineering. - Hoboken, NJ : Wiley. - 1097-0290 .- 0006-3592. ; 93:4, s. 636-646
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Tidskriftsartikel (refereegranskat)abstract
- An integrated cell cultivation and protein product separation process was developed using a new type of supermacroporous polyacrylamide gel, called cryogel (pAAm-cryogel) support matrix. Human fibrosarcoma HT1080 and human colon cancer HCT116 cell lines were used to secrete urokinase (an enzyme of immense therapeutic utility) into the culture medium. The secreted protein was isolated from the circulating medium using a chromatographic capture column. A pAAm cryogel support with covalently coupled gelatin (gelatin-pAAm cryogel) was used for the cultivation of anchorage dependent cells in the continuous cell culture mode in 5% carbon dioxide atmosphere. The cells were attached to the matrix within 4-6 h of inoculation and grew as a tissue sheet inside the cryogel matrix. Continuous urokinase secretion into the circulating medium was monitored as a parameter of growth and viability of cells inside the bioreactor. No morphological changes were observed in the cells eluted from the gelatin-cryogel support and re-cultured in T-flask. The gelatin-pAAm cryogel bioreactor was further connected to a pAAm cryogel column carrying Cu(II)-iminodiacetic acid (Cu(II)-IDA)-ligands (Cu(II)-IDA-pAAm cryogel), which had been optimized for the capture of urokinase from the conditioned medium of the cell lines. Thus an automated system was built, which integrated the features of a hollow fiber reactor with a chromatographic protein separation system. The urokinase was continuously captured by the Cu(II)-IDA-pAAm cryogel column and periodically recovered through elution cycles. The urokinase activity increased from 250 PU/mg in the culture fluid to 2,310 PU/mg after recovery from the capture column which gave about ninefold purification of the enzyme. Increased productivity was achieved by operating integrated bioreactor system continuously for 32 days under product inhibition free conditions during which no back-pressure or culture contamination was observed. A total 152,600 Plough units of urokinase activity was recovered from 500 mL culture medium using 38 capture columns over a period of 32 days. (c) 2006 Wiley Periodicals, Inc.
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