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  • 1
    Electronic Resource
    Electronic Resource
    Surface immobilization of anchorage-dependent mammalian cells (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 39 (1992), S. 697-706 
    Details
    ISSN: 0006-3592
    Keywords: anchorage-dependent mammalian cells ; immobilization ; fibers ; bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Anchorage-dependent HeLa cells were successfully cultured on two fibrous materials (A07 and R100) with porosities of 75-125 and 40 μm, void fractions of 92% and 81%, and fiber diameters of 7.6 and 10.2 μm, 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 ∼0.03-0.04 h-1, maximum cell density of 8 × 106-9 × 106 cells · mL-1, and yields of 0.4 × 108 cells · mM-1 on glucose and 2 × 108-3 × 108 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 × 106 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.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260390702
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  • 2
    Electronic Resource
    Electronic Resource
    Improvement of recombinant protein production with the human adenovirus/293S expression system using fed-batch strategies (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 613-623 
    Details
    ISSN: 0006-3592
    Keywords: adenovirus ; 293S cells ; protein production ; lactate ; osmolarity ; fed-batch ; glucose control ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The human adenovirus/293S cell expression system is used for the production of either recombinant protein or adenovirus vectors for use in gene therapy. In this work, the production of protein tyrosine phosphatase (PTP1C) was used as a model for the scale-up of both applications. Maximum specific production of 30 to 45 μg of active protein/106 cells was maintained upon infection with adenovirus vectors at cell densities between 2 × 106 to 3 × 106 cells/mL in a 3.5-L bioreactor. This was achieved by resuspending the culture in fresh medium at infection time. The pH was kept at 7.0 throughout the experiment and, at 24 h postinfection, glucose and essential amino acids were added. Attempts to replace the complete change of medium at the time of infection with nutrient supplementation of the used medium led to lower production levels, suggesting that protein expression was limited not by the absence of a key nutrient but by inhibitory factors. Two potentially inhibitory factors were investigated: lactic acid accumulation and increased osmolarity. Medium acidification such as that which would be brought about by lactic acid accumulation was shown to depress PTP1C production. The lactate molecule itself decreased the cell viability when added in concentrations of 20 mM or more. But the specific productivity was affected at higher lactate concentrations of 40 mM or more. Additions of glucose, amino acids, and NaHCO3 used to control pH, led to increases in osmolarity. Osmolarities above 400 mOsm lowered cell density. However, specific production was not significantly affected below 500 mOsm. But, at 500 mOsm, PTP1C production peak was shifted from 48 to 72 hpi. Because of the cell loss, this per cell yield increase did not translate into higher volumetric production. When glucose concentrations was kept at 5 mM by fed-batch addition, lactate production and increases in osmolarity were reduced. In shake flasks, this method permitted maximum production with cells resuspended either in fresh or spent medium at infection. This fed-batch process was implemented successfully at the 3.5-L scale. Fed-batch with glucose may provide a means to increase infected-cell density beyond 3 × 106 cells/mL.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 567-575 
    Details
    ISSN: 0006-3592
    Keywords: adenovirus ; adenoviral vectors ; 293 cells ; recombinant proteins ; serum-free medium ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This article describes the step-wise approach undertaken to select a serum-free medium (SFM) for the efficient production of a recombinant adenoviral vectors expressing β-galactosidase (Ad5 CMV-LacZ), in the complementing human embryonic kidney 293S cells. In the first step, a 293S-derived transfectoma, secreting a soluble epidermal growth factor receptor sEGFr (D2-22), was used to estimate the potential of selected serum-free formulations to support the production of a recombinant protein as compared to serum-containing medium. Assays showed that only one among six commercial serum-free formulations could support both sEGFr production and cell growth in static or suspension culture. In commercially available calcium-containing serum-free formulations, the cell aggregates reached up to 3 mm in diameter. In the second step, 293S cells were gradually adapted to a low-calcium version of the selected medium (LC-SFM). Cells were cloned, then screened according to their ability to grow at a rate and an extent comparable to parental cells in serum-containing medium (standard) as single cells or small aggregates. The 293SF-3F6 clone, first adapted to and then cloned in the selected serum-free medium, was selected for further experiments. Bioreactor run performed with the 293SF-3F6 clone showed similar growth curve as in the shake-flask controls. In the final step, the recombinant viral vector productivity of the 293S cells and the 293SF-3F6 clone was tested. The 293SF-3F6 cells infected by Ad5 CMV-LacZ in 3 L-scale bioreactor maintained the specific productivities of both β-galactosidase and adenoviral vector equivalent to the shake-flask controls in suspension culture. Results from this study clearly demonstrate that the 293SF-3F6 cell line thus selected may be used either for establishing stable transfected cell line or for the production of adenoviral vectors required for gene therapy studies. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 567-575, 1998.
    Additional Material: 5 Ill.
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  • 4
    Electronic Resource
    Electronic Resource
    Detection of β- 1,3-glucanase activity after native polyacrylamide gel electrophoresis: Application to tobacco pathogenesis-related proteins (1989)
    Weinheim : Wiley-Blackwell
    Electrophoresis 10 (1989), S. 527-529 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: β-1,3-Glucanase (laminarinase) activity was detected after polyacrylamide gel electrophoresis under native conditions by using laminarin as substrate. Following incubation of gels, laminarin was stained with Aniline Blue. Under UV illumination, lysis zones appeared as dark bands against a fluorescent background. As low as 0.001 unit of commercial Penicillium laminarinase could be observed after incubating the polyacrylamide gel for 45 min at pH 5.0. Extracts of commercial Penicillium laminarinase exhibited four bands with lytic activity towards laminarin. Analysis of intercellular fluid extracts of tobacco mosaic virus-infected tobacco leaves revealed four β-1,3-glucanases corresponding to three acidic pathogenesis-related proteins, b4 (2), b5 (N) and b6b (0), and one basic protein. The presence of laminarin in gels retarded the migration of some proteins with β1, 3-glucanase activity. This change in electrophoretic mobility could be used as a complementary affinity test for identifying proteins with β-1,3-glucanase activity.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150100714
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  • 5
    Electronic Resource
    Electronic Resource
    Production of ethanol by Saccharomyces cerevisiae under high-pressure conditions (1987)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 30 (1987), S. 74-80 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A research project was initiated to examine the possibility of using supercritical carbon dioxide for in situ recovery of ethanol during its production by yeast Saccharomyces cerevisiae. As a preliminary step, it was necessary to study the behavior of ethanol production under high-pressure conditions, up to 7 MPa (1000 psi). The results show that pressure has a significant inhibiting effect on the production of ethanol. There is a significant decrease in the initial rate of production as well as in the final ethanol concentration as pressure is increased. This decrease is more significant when carbon dioxide is used to pressurize the fermentor. The pressure affects the ability of the cells to produce ethanol in a reversible way. When the fermentor is returned to atmospheric conditions, the reaction resumes its normal fermentation rate.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260300111
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  • 6
    Electronic Resource
    Electronic Resource
    Detection of β-glucanase activity on various β-1,3 and β- 1,4-glucans after native and denaturing polyacrylamide gel electrophoresis (1991)
    Weinheim : Wiley-Blackwell
    Electrophoresis 12 (1991), S. 69-74 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: β-Glucanases were detected after polyacrylamide gel electrophoresis under native and denaturing conditions using various β-1, 3- and β-1, 4-glucans, including mixed glucans (laminarin, pachyman, carboxymethyl cellulose, lichenan and barley β-glucan). After electrophoresis and incubation of gels, substrates incorporated into polyacrylamide gels were stained with specific fluorochromes, Sirofluor for β-1, 3 linkages and Calcofluor White M2R for β-1, 4 linkages. Under UV illumination, lysis zones appeared as dark bands against a fluorescent background. Enzymes of bacterial, fungal and plant sources could be revealed sequentially in gles containing mixed β-(1.3) (1,4)-glucans by staining first with sirofluor followed by staining with Calcofluor White M2R. Active profiles were more diverse when substrates were stained with sirofluor. The use of purified sirofluor at pH 11.5 compared with Aniline Blue at pH 8.6 allowed better detection of β-1, 3-glucanase activities. In gels containing laminarin stained with sirofluor, bands exhibiting a more intense fluorescence than the background fluorescence were observed in addition to dark nonfluorescent bands. It is postulated that these two types of β-1, 3-glucanase activities differ by their enzymatic action (partial versus extensive hydrolysis). Analysis of fungal extracts using denaturing gels embedded with various β-glucans displayed lysis bands migrating between 32 and 35 kDa.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150120113
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