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  • Articles  (10)
  • Articles: DFG German National Licenses  (10)
  • cell culture  (5)
  • plasmid  (5)
  • 1995-1999  (10)
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  • Process Engineering, Biotechnology, Nutrition Technology  (10)
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  • Articles  (10)
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  • Articles: DFG German National Licenses  (10)
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  • 1
    Electronic Resource
    Electronic Resource
    Real-time monitoring of protein secretion in mammalian cell fermentation: Measurement of monoclonal antibodies using a computer-controlled HPLC system (BioCad/RPM) (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 48 (1995), S. 201-206 
    Details
    ISSN: 0006-3592
    Keywords: one-line monitoring ; fermentation ; cell culture ; monoclonal antibodies ; real-time immunoassays ; BioCad/RPM ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: On-line, “real-time” monitoring of product concentration is important for mammalian cell culture fermentation. The continuous measurement of monoclonal antibodies allows for instantaneous determination of cell productivity and effective manipulation of the fermentor operating conditions for optimal production. This article will present the evaluation and application of a BioCad/RPM system (Per Septive Biosystems) for rapid analysis of lgG concentration for hybridoma cell cultivation. Several commercial crossflow filtration devices are tested for low protein retention and fouling properties. A protein G column is used successfully for analyzing about 400 samples of lgG1, without significant loss in separation efficiency. The Immuno Detection system is integrated into a computer-controlled 15-L fermentor. This fermentor could be operated in batch and perfusion modes with cell densities up to 20 million cells/mL. A continuous cell-free sample stream obtained by a hollow fiber filter system is introduced to the BioCad/RPM for analysis. The speed of this system allows for real-time monitoring even at high densities with fast dynamics. A murine hybridoma cell (A10G10) is cultivated in batch and continuous reactors and antibody concentration is measured continuously with complete sterility. The results are compared to offline measurements with good agreement. © 1995 John Wiley & Sons, Inc.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260480306
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  • 2
    Electronic Resource
    Electronic Resource
    Sparging and agitation-induced injury of cultured animals cells: Do cell-to-bubble interactions in the bulk liquid injure cells? (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 399-409 
    Details
    ISSN: 0006-3592
    Keywords: cell damage ; cell culture ; bubble aeration ; agitation ; bubble coalescence and breakup ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: It has been established that the forces resulting from bubbles rupturing at the free air (gas)/liquid surface injure animal cells in agitated and/or sparged bioreactors. Although it has been suggested that bubble coalescence and breakup within agitated and sparged bioreactors (i.e., away from the free liquid surface) can be a source of cell injury as well, the evidence has been indirect. We have carried out experiments to examine this issue. The free air/liquid surface in a sparged and agitated bioractor was eliminated by completely filling the 2-L reactor and allowing sparged bubbles to escape through an outlet tube. Two identical bioreactors were run in parallel to make comparisons between cultures that were oxygenated via direct air sparging and the control culture in which silicone tubing was used for bubble-free oxygenation. Thus, cell damage from cell-to-bubble interactions due to processes (bubble coalescence and breakup) occurring in the bulk liquid could be isolated by eliminating damage due to bubbles rupturing at the free air/liquid surface of the bioreactor. We found that Chinese hamster ovary (CHO) cells grown in medium that does not contain shear-protecting additives can be agitated at rates up to 600 rpm without being damaged extensively by cell-to bubble interactions in the bulk of the bioreactor. We verified this using both batch and high-density perfusion cultures. We tested two impeller designs (pitched blade and Rushton) and found them not to affect cell damage under similar operational conditions. Sparger location (above vs. below the impeller) had no effect on cell damage at higher agitation rates but may affect the injury process at lower agitation intensities (here, below 250 rpm). In the absence of a headspace, we found less cell damage at higher agitation intensities (400 and 600 rpm), and we suggest that this nonintuitive finding derives from the important effect of bubble size and foam stability on the cell damage process. © 1996 John Wiley & Sons, Inc.
    Additional Material: 5 Ill.
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  • 3
    Electronic Resource
    Electronic Resource
    Plasmid-assisted morpholine degradation by Pseudomonas fluorescens CAS 102 (1997)
    Springer
    World journal of microbiology and biotechnology 14 (1997), S. 7-10 
    Details
    ISSN: 1573-0972
    Keywords: Biodegradation ; morpholine ; plasmid ; Pseudomonas fluorescens ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A fast-growing Pseudomonas fluorescens CAS102, isolated by enrichment technique from polluted soil, effectively utilized morpholine as the energy source. The strain was able to grow in high concentrations of morpholine but accumulation of ammonia inhibited its growth and complete mineralization. The molar conversion ratio of morpholine to ammonia was 1:0.82. The organism harboured a single, multiple antibiotic- and heavy metal-resistance 140kb plasmid which was resistant to curing. Transformation studies showed that the morpholine degradative phenotype was expressed only in Pseudomonas putida and not in Escherichia coli. Growth studies on different degradative intermediates of morpholine suggested that plasmid-encoded genes were involved in the heterocyclic ring cleavage and the remaining reactions were mediated by chromosomal genes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008855912907
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  • 4
    Electronic Resource
    Electronic Resource
    Rhizobium plasmids in bacteria-legume interactions (1996)
    Springer
    World journal of microbiology and biotechnology 12 (1996), S. 119-125 
    Details
    ISSN: 1573-0972
    Keywords: free-living metabolism ; genomic organization ; plasmid ; Rhizobium ; symbiosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The functional analysis of plasmids in Rhizobium strains has concentrated mainly on the symbiotic plasmid (pSym). However, genetic information relevant to both symbiotic and saprophytic Rhizobium life cycles, localized on other ‘cryptic’ replicons, has also been reported. Information is reviewed which concerns functional features encoded in plasmids other than the pSym: biosynthesis of cell surface polysaccharides, metabolic processes, the utilization of plant exudates, aromatic compounds and diverse sugars, and features involved symbiotic performance. In addition, factors which affect plasmid evolution through their influence on structural features of the plasmids, such as conjugative transfer and genomic rearrangements, is discussed. Based on the overall data, we propose that together the plasmids and the chromosome constitute a fully integrated genomic complex, entailing structural features as well as saprophytic and cellular functions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00364676
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  • 5
    Electronic Resource
    Electronic Resource
    Cationic liposomal delivery of plasmid to endothelial cells measured by quantitative flow cytometry (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 548-554 
    Details
    ISSN: 0006-3592
    Keywords: fluorescence ; transfection ; liposome ; flow cytometry ; plasmid ; cell cycle ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Cationic liposomes are potentially important gene transfer vehicles, capable of conjugating with anionic DNA by condensation. Flow cytometry was used to examine quantitatively the incorporation of DNA-liposome complex into murine capillary lung endothelial cells. The plasmid DNA, a pSV-β-galactosidase vector, was covalently labeled with ethidium monoazide by photoactivation. The cationic liposome consisted of egg phosphatidylcholine (90%), cholesterol (5%), and stearylamine (5%). The number of plasmid molecules contained within each cell as a function of exposure time was estimated from fluorescence intensity. Fluorescently labeled plasmid is detectable after 10 min and increases with continued exposure, but at a decreasing rate, up to 2160 min. After 2160 min each cell, on average, contains approximately 10,000 plasmid molecules. Following transfection, a single cell unimodal population was detected by flow cytometry, suggesting that all cells participate in transfection equally. Furthermore, cell cycle analysis indicates that the entry of DNA-liposome complex is independent of cell cycle. © 1996 John Wiley & Sons, Inc.
    Additional Material: 4 Ill.
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  • 6
    Electronic Resource
    Electronic Resource
    Noninvasive oxygen measurements and mass transfer considerations in tissue culture flasks (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 466-478 
    Details
    ISSN: 0006-3592
    Keywords: optical oxygen sensor ; tissue culture flask ; cell culture ; oxygen mass transfer ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Murine hybridomas were cultivated in tissue culture flasks. Dissolved oxygen tensions in the gas and liquid phases during cell growth were monitored. Oxygen levels were measured noninvasively by interrogating an oxygen-sensitive patch mounted on the interior surface of the tissue culture flask with an optrode from outside the tissue culture flask. Readings were made in tissue culture flasks with caps both cracked open and completely closed. Although the oxygen in the gas phase remained near atmospheric oxygen levels in both flasks, over time the liquid-phase oxygen tension at the bottom of the flasks reached zero during cell growth in both the open and closed tissue culture flasks. These results suggest that the widespread practice of cracking open tissue culture flask caps during cell growth with a view to supplying adequate oxygen to cells is ineffective and probably unnecessary.The mass transfer characteristics of the tissue culture flask were also studied. The dominant resistance to oxygen mass transfer to the sensor and the cells was through the liquid media. The mass transfer rates through the liquid layer under standard laboratory conditions were found to be greater than those predicted by diffusion alone. This suggests that mixing at a microscale occurs. Volumetric and specific oxygen consumption rates were also calculated from the sensor data. These consumption rates were comparable with values published elsewhere. © 1996 John Wiley & Sons, Inc.
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  • 7
    Electronic Resource
    Electronic Resource
    Activity and stability of a recombinant plasmid-borne TCE degradative pathway in suspended cultures (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 287-296 
    Details
    ISSN: 0006-3592
    Keywords: expression ; plasmid ; stability ; TCE ; continuous culture ; activity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The retention and expression of the plasmid-borne, TCE degradative toluene-ortho-monooxygenase (TOM) pathway in suspended continuous cultures of transconjugant Burkholderia cepacia 17616 (TOM31c) were studied. Acetate growth and TCE degradation kinetics for the transconjugant host are described and utilized in a plasmid loss model. Plasmid maintenance did not have a significant effect on the growth rate of the transconjugant. Both plasmid-bearing and plasmid-free strains followed Andrews inhibition growth kinetics when grown on acetate and had maximum growth rates of 0.22 h-1. The transconjugant was capable of degrading TCE at a maximum rate of 9.7 nmol TCE/min · mg protein, which is comparable to the rates found for the original plasmid host, Burkholderia cepacia PR131 (TOM31c). The specific activity of the TOM pathway was found to be a linear function of growth rate. Plasmid maintenance was studied at three different growth rates: 0.17/h, 0.1/h, and 0.065/h. Plasmid maintenance was found to be a function of growth rate, with the probability of loss ranging from 0.027 at a growth rate of 0.065/h to 0.034 at a growth rate 0.17/h. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 287-296, 1998.
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  • 8
    Electronic Resource
    Electronic Resource
    Growth factor and Bcl-2 mediated survival during abortive proliferation of hybridoma cell line (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 164-171 
    Details
    ISSN: 0006-3592
    Keywords: cell death ; apoptosis ; bcl -2 ; cell culture ; cell viability ; growth factors ; survival factors ; abortive proliferation ; hybridomas ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Cultures of the CRL-1606 hybridoma (ATCC) have been reported to undergo continuous proliferation with simultaneous death during nutrient limited fed-batch fermentations. The bcl-2 proto-oncogene has been shown to prevent cell death under a variety of otherwise death inducing conditions. We were interested in elucidating the nature of the massive death observed in cultures of CRL-1606, specifically with respect to the possible environmental causes, and the ability of overexpressed human bcl-2 (hbcl-2) to mitigate cell death. Abortive proliferation, or continuous proliferation in the presence of continuous death, could be induced in serum free cultures of CRL-1606 through the withdrawal of insulin provided the culture was competent for cell proliferation. Culture competency for proliferation was found to be solely determined by the presence of cell culture nutrients. Abortive proliferation was defective in cultures transfected with hbcl-2 and the enhanced viability observed resulted from an increased viable cell population and at the expense of the nonviable cell population normally found in untransfected cultures. Abortive proliferation was also observed in serum containing cultures upon serum shiftdowns. Like the insulin-supplemented serum free culture system, hbcl-2 transfected cultures exhibited defects in the abortive proliferation process. These results suggest that the massive death observed during nutrient-limited fed-batch fermentation originate, in part, from growth or survival factor limitations. Hence, approaches to design cell culture media that account for the cell's proliferation requirements without accounting for the cell's survival requirements may represent a cell death sentence. Given the transformed nature of the hybridomas, we conclude that the abortive proliferation of CRL-1606 is a consequence of inappropriate cell cycle entry in a survival factor limited environment. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 164-171, 1998.
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  • 9
    Electronic Resource
    Electronic Resource
    Activity of glutamate dehydrogenase is increased in ammonia-stressed hybridoma cells (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 447-453 
    Details
    ISSN: 0006-3592
    Keywords: ammonia ; cell culture ; metabolic flux ; glutamate dehydrogenase ; mass balance ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of added ammonia on the intracellular fluxes in hybridoma cells was investigated by metabolic-flux balancing techniques. It was found that, in ammonia-stressed hybridoma cells, the glutamate-dehydrogenase flux is in the reverse direction compared to control cells. This demonstrates that hybridoma cells are able to prevent the accumulation of ammonia by converting ammonia and α-ketoglutarate into glutamate. The additional glutamate that is produced by this flux, as compared to the control culture, is converted by the reactions catalyzed by alanine aminotransferase (45% of the extra glutamate) and aspartate aminotransferase (37%), and a small amount is used for the biosynthesis of proline (6%). The remaining 12% of the extra glutamate is secreted into the culture medium. The data suggest that glutamate dehydrogenase is a potential target for metabolic engineering to prevent ammonia accumulation in high-cell-density culture. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 447-453, 1998.
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  • 10
    Electronic Resource
    Electronic Resource
    Activity and stability of a recombinant plasmid-borne TCE degradative pathway in biofilm cultures (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 318-327 
    Details
    ISSN: 0006-3592
    Keywords: plasmid ; retention ; TCE ; biofilm ; segregational stability ; activity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The activity and stability of the TCE degradative plasmid TOM31c in the transconjugant host Burkholderia cepacia 17616 was studied in selective and non-selective biofilm cultures. The activity of plasmid TOM31c in biofilm cultures was measured by both TCE degradative studies and the expression of the Tom pathway. Plasmid loss was measured using continuous flow, rotating annular biofilm reactors, and various analytical and microbiological techniques. The probability of plasmid loss in the biofilm cultures was determined using a non-steady-state biofilm plasmid loss model that was derived from a simple mass balance, incorporating results from biofilm growth and plasmid loss studies. The plasmid loss model also utilized Andrew's inhibition growth kinetics and a biofilm detachment term.Results from these biofilm studies were compared to similar studies performed on suspended cultures of Burkholderia cepacia 17616-TOM31c to determine if biofilm growth has a significant effect on either plasmid retention or Tom pathway expression (i.e., TCE degradation rates). Results show that the activity and expression of the Tom pathway measured in biofilm cultures was significantly less than that found in suspended cultures at comparable growth rates. The data obtained from these studies fit the plasmid loss model well, providing plasmid loss probability factors for biofilm cultures that were equivalent to those previously found for suspended cultures. The probability of plasmid loss in the B. cepacia 17616-TOM31c biofilm cultures was equivalent to those found in the suspended cultures. The results indicate that biofilm growth neither helps nor hinders plasmid stability. In both the suspended and the biofilm cultures, plasmid retention and expression could be maintained using selective growth substrates and/or an appropriate plasmid-selective antibiotic. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:318-327, 1998.
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