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  • Articles  (58)
  • cell culture  (29)
  • continuous culture  (29)
  • 1995-1999  (58)
  • 1980-1984
  • 1925-1929
  • Process Engineering, Biotechnology, Nutrition Technology  (58)
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  • Articles  (58)
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  • 1
    Electronic Resource
    Electronic Resource
    Kinetics of lactose transport inKluyveromyces fragilis grown in a chemostat on diluted whey permeate (1995)
    Springer
    Journal of industrial microbiology and biotechnology 15 (1995), S. 45-48 
    Details
    ISSN: 1476-5535
    Keywords: Kluyveromyces fragilis ; lactose transport ; continuous culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Lactose transport was studied inKluyveromyces fragilis grown in lactose-limited chemostat cultures. Kinetic parameters were determined using a method based on genetic population evolution. Lactose transport was carried out via three carriers characterized respectively byK m of 0.1 mM, 3 mM and 15.5 mM. The synthesis of these lactose carriers and their capacity (V max) are dependent on the dilution rate (D). At D=0.12 h−1, the high affinity transporter is prominent. For intermediate dilution rate, only the high and the medium affinity systems are present. In cells growing at D=0.4 h−1, these carriers are absent but instead, the low affinity transporter is present. The effect on lactose transport of such metabolic inhibitors as CCCP, a proton ionophore, and Antimycin A, an energy inhibitor, were also investigated. The high affinity system is the most sensitive to the effect of these inhibitors. Lactose transport through this carrier is probably a mechanism dependent on the proton motive force.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01570012
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  • 2
    Electronic Resource
    Electronic Resource
    Effect of oxygen on denitrification in continuous chemostat culture withComamonas sp SGLY2 (1996)
    Springer
    Journal of industrial microbiology and biotechnology 16 (1996), S. 124-128 
    Details
    ISSN: 1476-5535
    Keywords: denitrification ; continuous culture ; oxygen ; Comamonas sp
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Continuous cultures ofComamonas sp SGLY2 were grown anaerobically prior to establishing steady states at different oxygen flow rates. At a low oxygen transfer rate, no dissolved oxygen accumulated in the medium and all nitrate was reduced to dinitrogen. Concurrently with the increase of dissolved oxygen concentration in the liquid phase, the rate of denitrification decreased. However, at a dissolved oxygen concentration near saturation (33 mg L−1), a part of the electron flow always diverted to nitrate with production of dinitrogen: the aerobic denitrification rate was equivalent to 35% of that calculated under anaerobic conditions. These experiments reflected the co-utilization of oxygen and N-oxides and the production of dinitrogen, up to saturated conditions, which implied synthesis and activity of the four denitrifying enzymes under various aeration conditions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01570072
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  • 3
    Electronic Resource
    Electronic Resource
    The prophylactic use of antibiotics in cell culture (1995)
    Springer
    Cytotechnology 19 (1995), S. 95-105 
    Details
    ISSN: 1573-0778
    Keywords: antibiotics ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract This article describes the historical development of the prophylactic use of antibiotics in cell culture as well as their effects on cells. The influence of antibiotics on cell morphology, cellular degeneration and cell death and cellular function is summarized. Cellular DNA as well as protein synthesis are affected which can lead to interference with, or even changes in, metabolic processes. Such effects must be considered in cell culture research. As antibiotics are used in multifold ways, the otherwise standardized conditions in cell culture are no longer comparable. The prophylactic use of antibiotics is rejected for scientific reasons.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00749764
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  • 4
    Electronic Resource
    Electronic Resource
    Kinetics of retroviral production from the amphotropic ΨCRIP murine producer cell line (1996)
    Springer
    Cytotechnology 22 (1996), S. 185-195 
    Details
    ISSN: 1573-0778
    Keywords: cell culture ; half-life ; packaging cells ; retrovirus ; titer ; ΨCRIP
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Rapidly expanding development and practice of gene therapy requires the availability of large quantities of high titer retroviral supernatants. One way to achieve high retroviral titers is through improved understanding of the kinetics of retroviral production and decay, and the subsequent development of improved cell culture methods. In the present study we investigated the effects of different operational modes on the retroviral production of the NIH 3T3 fibroblast derived amphotropic murine retroviral producing cell line pMFG/ΨCRIP. Semi-continuous culture (exchange of 50% of medium volume daily) was found to promote cell growth and enhance retroviral production. The rapid medium exchange resulted in significantly larger amounts of high titer supernatants and an extended production phase as compared to the batch control cultures. The specific viral productivity of the pMFG/ΨCRIP cells was in the range of 10 to 40 infectious viruses produced per thousand producer cells per day. The CV-1 African Green Monkey kidney cell line was used as the infection target. Lowering the serum level form 20% to 10% improved retroviral production slightly. However, at lower serum levels (1%, 5% and 10% (v/v)) growth of the producer cell line, and thus retroviral production, was directly proportional to the serum level. The half-life of the virus at 37°C was found to be 5.5 hours. Promoting the growth of producer cell lines can improve retroviral vectors titers and viral production. High cell density systems that allow for rapid cell growth and waste product removal are likely to be used to generate high-titer retroviral supernatants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00353938
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  • 5
    Electronic Resource
    Electronic Resource
    Use of cell cycle analysis to characterise growth and interferon-γ production in perfusion culture of CHO cells (1999)
    Springer
    Cytotechnology 30 (1999), S. 59-69 
    Details
    ISSN: 1573-0778
    Keywords: cell cycle ; CHO ; continuous culture ; flow cytometry ; perfusion culture ; spin-filter
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The importance of cell cycle analysis in cell culture development has been widely recognised. Whether such analysis is useful in indicating future performance of high cell density culture is uncertain. Using flow cytometric approach to address this question, we utilised the fraction of cells in the S phase to control specific growth rate and productivity in spin filter perfusion cultures and found a significant increase in the accumulated interferon-γ over that obtained from the nutrient-based controlled fed culture. While a general decrease with time exists in both percentage of S phase cells and specific growth rate, a clear oscillatory behaviour of both parameters is found in perfusion cultures.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008055904642
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  • 6
    Electronic Resource
    Electronic Resource
    Homogenisation and oxygen transfer rates in large agitated and sparged animal cell bioreactors: Some implications for growth and production (1996)
    Springer
    Cytotechnology 22 (1996), S. 87-94 
    Details
    ISSN: 1573-0778
    Keywords: cell culture ; mixing time ; oxygen demand ; oxygen transfer ; pH and dO2 sensitivity ; scale-up
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Because of concern for cell damage, very low agitation energy inputs have been used in industrial animal cell bioreactors, typical values being two orders of magnitude less than those found in bacterial fermentations. Aeration rates are also very small. As a result, such bioreactors might be both poorly mixed and also unable to provide the higher oxygen up-take rates demanded by more intensive operation. This paper reports experimental studies both of K L a and of mixing (via pH measurements) in bioreactors up to 8 m3 at Wellcome and of scaled down models of such reactors at Birmingham. Alongside these physical measurements, sensitivity of certain cell lines to continuously controlled dO2 has been studied and the oxygen up-take rates measured in representative growth conditions. An analysis of characteristic times and mixing theory, together with other recent work showing that more vigorous agitation and aeration can be used especially in the presence of Pluronic F-68, indicates ways of improving their performance. pH gradients offer a special challenge.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00353927
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  • 7
    Electronic Resource
    Electronic Resource
    Engineering challenges in high density cell culture systems (1996)
    Springer
    Cytotechnology 22 (1996), S. 3-16 
    Details
    ISSN: 1573-0778
    Keywords: cell culture ; process monitoring ; oxygenation ; CO2 transfer ; aggregation ; segregation ; diffusion, on-line monitoring
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract High density cell culture systems offer the advantage of production of bio-pharmaceuticals in compact bioreactors with high volumetric production rates; however, these systems are difficult to design and operate. First of all, the cells have to be retained in the bioreactor by physical means during perfusion. The design of the cell retention is the key to performance of high density cell culture systems. Oxygenation and media design are also important for maximizing the cell number. In high density perfusion reactors, variable cell density, and hence the metabolic demand, require constant adjustment of perfusion rates. The use of cell specific perfusion rate (CSPR) control provides a constant environment to the cells resulting in consistent production. On-line measurement of cell density and metabolic activities can be used for the estimation of cell densities and the control of CSPR. Issues related to mass transfer and mixing become more important at high cell densities. Due to the difference in mass transfer coefficients for oxygen and CO2, a significant accumulation of dissolved CO2 is experienced with silicone tubing aeration. Also, mixing is observed to decrease at high densities. Base addition, if not properly done, could result in localized cell lysis and poor culture performance. Non-uniform mixing in reactors promotes the heterogeneity of the culture. Cell aggregation results in segregation of the cells within different mixing zones. This paper discusses these issues and makes recommendations for further development of high density cell culture bioreactors.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00353919
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  • 8
    Electronic Resource
    Electronic Resource
    Hybridoma cell behaviour in continuous culture under hyperosmotic stress (1999)
    Springer
    Cytotechnology 29 (1999), S. 71-84 
    Details
    ISSN: 1573-0778
    Keywords: bioreactor ; continuous culture ; hybridoma cells ; hyperosmolality ; monoclonal antibody production ; non-producing subpopulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract In this paper, we propose an alternative strategy to the ones proposed before (Oh et al., 1993; Øyaas et al., 1994a) to get real increases of global final antibody titer and production at hyperosmotic stress, by reducing the detrimental effect of such a stress on cell growth, and conserving the stimulating effect on antibody production. It consists of cultivating the cells in continuous culture and increasing the osmolality stepwise. In this way, the cells could progressively adapt to the higher osmolality at each step and antibody titers could be nearly doubled at 370 and 400 mOsm kg-1, compared to the standard osmolality of 335 mOsm kg-1. Surprisingly, the stimulation of antibody production was not confirmed for higher osmolalities, 425 and 450 mOsm kg- 1, despite the minor negative effect on cell growth. Intracellular IgG analysis by flow cytometry revealed at these osmolalities a significant population of non-producing cells. However, even when taking into account this non-producing population, a stimulating effect on antibody production could not be shown at these highest osmolalities. It seems to us that osmolality has a significant effect on the appearance of these non-producing cells, since they were not observed in continuous cultures at standard osmolality, of comparable duration and at an even higher dilution rate. The appearance of the non-producing cells coincides furthermore with modifications of the synthesised antibody, as shown by electrophoretic techniques. It is however not really clear if these two observations reflect actually the same phenomenon. Hyperosmolality affects the cell behaviour in continuous culture in multiple ways, independently of the growth rate, counting all at least partially for the observed stimulation of antibody production: acceleration of the amino acid, and in particular the glutamine metabolism, increase of the cell volume, increase of the intracellular pH and accumulation of cells in the G1 cell cycle phase.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008014909474
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  • 9
    Electronic Resource
    Electronic Resource
    The changing role of cell culture in the generation of transgenic livestock (1999)
    Springer
    Cytotechnology 31 (1999), S. 3-8 
    Details
    ISSN: 1573-0778
    Keywords: cell culture ; livestock ; milk ; nuclear transfer ; transgenic
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Transgenesis may allow the generation of farm animals with altered phenotype, animal models for research and animal bioreactors. Although such animals have been produced, the time and expense involved in generating transgenic livestock and then evaluating the transgene expression pattern is very restrictive. If questions about the ability and efficiency of expression could be asked solely in vitro rapid progress could be achieved. Unfortunately, experiments addressing transcriptional control in vitro have proved unreliable in their ability to indicate whether a transgene will be transcribed or not. However, initial studies suggest that cell culture may be able to predict in vivo post-transcriptional events. We review these issues and propose that strategies which engineer the transgene integration site could enhance the probability for efficient expression. This approach has now become feasible with the development of techniques allowing animals to be generated from somatic cells by nuclear transfer. The important step in this procedure is the use of cells grown in culture as the source of genetic information, allowing the selection of specific transgene integration events. This technology which has dramatically increased the potential use of transgenic livestock for both agricultural and biotechnological applications, is based on standard cell culture methodology. We are now at the start of a new era in large animal transgenics.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008044517150
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  • 10
    Electronic Resource
    Electronic Resource
    Analysis of the use of fortified medium in continuous culture of mammalian cells (1999)
    Springer
    Cytotechnology 31 (1999), S. 243-254 
    Details
    ISSN: 1573-0778
    Keywords: continuous culture ; growth inhibition ; osmolality ; perfusion culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Continuous culture is frequently used in the cultivation of mammalian cells for the manufacturing of recombinant protein pharmaceuticals. In such operations a large volume of medium is turned over each day, especially in the case where cell recycle, or perfusion cultivation, is practiced. In principle, the volumetric throughput of medium can be reduced by using a more concentrated feed while maintaining the same nutrient provision rate. Overall, the medium components are divided into two categories: ‘consumable nutrients' and ‘unconsumable inorganic bulk salts’. In such fortified medium, the concentrations of consumable nutrients, but not bulk salts, are increased. With a stoichiometrically-balanced medium, the large amount of nutrients fed into the culture is largely consumed by cells to give rise to residual concentrations of these nutrients in their optimal range. However, unless care is taken to initiate the continuous culture, overshoot of nutrients may occur during the transient period. The high nutrient concentration during overshoot may be inhibitory by itself, or the resulting high osmolality may retard the growth. Using a mathematical model that incorporates the growth inhibitory effect of high osmolality we demonstrate such a potentially catastrophic effect of nutrient and osmolality overshoot by simulation. To avoid overshoot a controlled nutrient feeding scheme should be devised at the initiation of continuous culture.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008026613975
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  • 11
    Electronic Resource
    Electronic Resource
    Glucoamylase production in continuous cultures of Aspergillus niger with special emphasis on growth parameters (1997)
    Springer
    World journal of microbiology and biotechnology 14 (1997), S. 113-118 
    Details
    ISSN: 1573-0972
    Keywords: Aspergillus ; continuous culture ; glucoamylase ; growth ; fungi ; nitrogen
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Maltose-limited continuous culture of Aspergillus niger was carried out with potassium nitrate to investigate growth and glucoamylase formation characteristics. Glucoamylase production was dependent on the specific growth rate. The maximal amount of glucoamylase (units/l and U/g dry weight) was obtained at μ=0.08h−1, and the maximum specific rate of production (units/g/dry weight per hour) was at μ=0.2h−1. The maintenance coefficients (ms and mATP) were higher than for some other fungi. Maximal growth yields on substrate, oxygen and ATP (Yxsm, YxO2m and Yxam) were very efficient (high) and the value of Yxam, which cannot exceed the theoretical maximal value, is obtained when a P/O ratio of 1:1 is assumed. This indicates that biomass formation is energetically inexpensive and most of the expended energy has to be invested in the process of glucoamylase excretion.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008840904246
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  • 12
    Electronic Resource
    Electronic Resource
    Kinetic characterization in batch and continuous culture of Escherichia coli mutants affected in phosphoenolpyruvate metabolism: differences in acetic acid production (1999)
    Springer
    World journal of microbiology and biotechnology 15 (1999), S. 587-592 
    Details
    ISSN: 1573-0972
    Keywords: Acetic acid production ; carbon metabolism ; continuous culture ; Escherichia coli ; metabolic engineering
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The growth kinetics of an Escherichia coli wild type strain and two derivative mutants were examined in batch cultures and in glucose-limited chemostats. One mutant (PB12) had an inactive phosphotranferase transport system and the other (PB25) had interrupted pykA and pykF genes that code for the two pyruvate kinase isoenzymes. In both batch and continuous culture, important differences in acetic acid accumulation and other metabolic activities were found. Compared to the wild type strain, we observed a reduction in acetic acid accumulation of 25 and 80% in PB25 and PB12 strains respectively, in batch culture. Continuous culture experiments revealed that compared to the other two strains, PB25 accumulated less acetic acid as a function of dilution rate. In continuous cultures, oxidoreductase metabolic activities were substantially affected in the two mutant strains. These changes in turn were reflected in different levels of biomass and CO2 production, and in oxygen consumption.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008934810150
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  • 13
    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.
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  • 14
    Electronic Resource
    Electronic Resource
    Bcl-2 inhibits apoptosis and extends recombinant protein production in cells infected with Sindbis viral vectors (1996)
    Springer
    Cytotechnology 22 (1996), S. 169-178 
    Details
    ISSN: 1573-0778
    Keywords: apoptosis ; bcl-2 ; cell culture ; chloramphenicol acetyltransferase ; recombinant protein ; Sindbis virus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Viruses carrying foreign genes are often used for the production of recombinant proteins in mammalian cells and other eukaryotic expression systems. Though high levels of gene expression are possible using viral vectors, the host cell generally responds to the infection by inducing apoptotic cell death within several days, abruptly ending protein production. It has recently been demonstrated, however, that apoptosis can be suppressed in virally infected cells using anti-apoptotic genes, such as bcl-2. In this study, stably transfected rat carcinomal cell lines, AT3-bcl2 and AT3-neo, were infected with a Sindbis virus carrying the gene for chloramphenicol acetyltransferase (CAT) in an effort to determine the effect of bcl-2 on cell viability and recombinant protein production. Infected AT3-bcl2 cells consistently maintained viabilities close to 100% and a growth rate equivalent to that of uninfected cells (0.040 h-1). In contrast, the Sindbis viral vector induced apoptosis in the AT3-neo cells, which were all dead by three days post-infection. Though infected AT3-neo cells generated higher levels of heterologous protein, over 1000 mUnits per well, CAT activity fell to zero by two days post-infection. In contrast, chloramphenicol acetyltransferase was present in AT3-bcl2 cells for almost a week, reaching a maximum level of 580 mUnits per well. In addition, recombinant protein production in AT3-bcl2 cells was extended and amplified by the regular addition of virus to the culture medium, a process which resulted in expression for the duration of the cell culture process.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00353936
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  • 15
    Electronic Resource
    Electronic Resource
    The growth and phenotypic expression of human osteoblasts (1996)
    Springer
    Cytotechnology 22 (1996), S. 263-267 
    Details
    ISSN: 1573-0778
    Keywords: biodegradable ; bone regeneration ; cell culture ; human cell osteoblasts ; polymers
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The care of patients with a skeletal deficiency currently involves the use of bone graft or a non-biologic material such as a metal or polymer. There are alternate possibilities in development which involve the growth of bone cells (osteoblasts) on degradable polymer scaffolds. These tissue engineering strategies require production of the polymeric scaffold, cellular harvest followed by either ex vivo or in vivo growth of the cells on the scaffold, and exploration of the interaction between the cell and scaffold. Research into these strategies utilizes cells from a variety of species, but clinical applications will likely require human osteoblasts. This study explores the process whereby human osteoblasts are harvested under sterile conditions during joint replacement surgery from normally discarded cancellous bone, transported from the operating room to the lab, and grown in culture. This process is feasible, and the cells express their phenotype via the production of alkaline phosphatase and collagen in culture.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00353947
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  • 16
    Electronic Resource
    Electronic Resource
    Chinese hamster ovary cells produce sufficient recombinant insulin-like growth factor I to support growth in serum-free medium. Serum-free growth of IGF-I-producing CHO cells (1997)
    Springer
    Cytotechnology 24 (1997), S. 55-64 
    Details
    ISSN: 1573-0778
    Keywords: CHO ; IGF-I ; serum-free ; autocrine growth ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/106 cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007969502256
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  • 17
    Electronic Resource
    Electronic Resource
    Effects of CO2 and osmolality on hybridoma cells: growth, metabolism and monoclonal antibody production (1998)
    Springer
    Cytotechnology 28 (1998), S. 213-227 
    Details
    ISSN: 1573-0778
    Keywords: antibody production ; carbon dioxide ; cell metabolism ; continuous culture ; inhibition
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract CO2 partial pressure (pCO2) in industrial cell culture reactors may reach 150–200 mm Hg, which can significantly inhibit cell growth and recombinant protein production. Due to equilibrium with bicarbonate, increased pCO2 at constant pH results in a proportional increase in osmolality. Hybridoma AB2-143.2 cell growth rate decreased with increasing pCO2 in well-plate culture, with a 45% decrease at 195 mm Hg with partial osmolality compensation (to 361 mOsm kg- 1). Inhibition was more extensive without osmolality compensation, with a 63% decrease in growth rate at 195 mm Hg and 415 mOsm kg-1. Also, the hybridoma death rate increased with increasing pCO2, with 31- and 64-fold increases at 250 mm Hg pCO2 for 401 and 469 mOsm kg- 1, respectively. The specific glucose consumption and lactate production rates were 40–50% lower at 140 mm Hg pCO2. However, there was little further inhibition of glycolysis at higher pCO2. The specific antibody production rate was not significantly affected by pCO2 or osmolality within the range tested. Hybridomas were also exposed to elevated pCO2 in continuous culture. The viable cell density decreased by 25–40% at 140 mm Hg. In contrast to the well-plate cultures, the death rate was lower at the new steady state at 140 mm Hg. This was probably due to higher residual nutrient and lower byproduct levels at the lower cell density (at the same dilution rate), and was associated with increased cell-specific glucose and oxygen uptake. Thus, the apparent effects of pCO2 may vary with the culture system.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008010605287
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  • 18
    Electronic Resource
    Electronic Resource
    A simple and rapid reverse transcriptase assay for the detection of retroviruses in cell cultures (1999)
    Springer
    Cytotechnology 29 (1999), S. 221-227 
    Details
    ISSN: 1573-0778
    Keywords: cell culture ; polymerase chain reaction ; retrovirus ; reverse transcriptase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Reverse transcriptase (RT) is a good diagnostic tool for the detection of retroviruses. We have developed a simple and rapid assay for RT activity in culture supernatants. A 370-base RNA sequence from the tetracycline-resistance gene in pBR322 plasmid DNA was used as a template for RT-mediated cDNA synthesis. To detect the resultant cDNA, we used the nested polymerase chain reaction. A sensitivity test using purified recombinant RT of human immunodeficiency virus type 1 demonstrated that the detection limit of this method was 10-7–10-8 units of RT activity in 20 μl of a test sample (2 × 10-9–2 × 10-10 units ml-1). This method detected RT activity in unconcentrated supernatants of cell cultures infected with human T-cell leukemia virus, Moloney murine leukemia virus, Moloney murine sarcoma virus, or Rous sarcoma virus. This nonisotopic method provides results within 10 h and is useful for quality control to detect retroviruses in cell cultures.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008000210125
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    Production of recombinant h-AT III with mammalian cell cultures using capillary electrophoresis for product monitoring (1996)
    Springer
    Cytotechnology 21 (1996), S. 205-215 
    Details
    ISSN: 1573-0778
    Keywords: antithrombin III ; mammalian cell culture ; continuous culture ; capillary electrophoresis ; product monitoring ; recombinant protein ; temperature influence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The importance of mammalian cell cultures for biotechnological production processes is steadily increasing, despite the high demands of these organisms on their culture conditions. Efforts towards a more efficient bioprocess generally concentrate on maximizing the culture's life time, the cell number, and the product concentration. Here recombinant BHK 21 c13 cells are used to produce rh-AT III, an anticoagulant of high therapeutic value. The influence of the process mode (batch, repeated batch, continuous perfusion) and the process temperature (30°C vs. 37°C) on the above mentioned parameters is investigated. It is possible to increase the length of the culture from 140 h (batch) to more than 500 h (continuous perfusion culture), while concomitantly increasing the cell density from 0.72 106/ml (batch) to 2.27 106/ml (repeated batch) and 2.87 106/ml (continuous perfusion culture). The accumulation of toxic metabolites, such as lactate, can be curtailed by reducing the bioreactor temperature from 37°C to 30°C during the later part of the exponential growth phase. Fast and reliable product monitoring became essential during process optimization. Capillary zone electrophoresis (CZE) in uncoated fused silica capillaries was studied for that purpose and compared to the standard ELISA. Under optimized conditions an AT III quantification could be done within 2 min with CZE. The detection limit was 5 μg/ml. A relative standard deviation of less than 0.9% was calculated. The detection limit could be lowered by one order of magnitude by using a two dimensional system, where an liquid chromatographic (LC) system is coupled to the CZE. Concomitantly the resolution is improved. The two-dimensional analysis required 5 min. Membrane adsorbers (MA) were used as stationary phase in the LC-system, to allow the application of high flow rates (5–10 ml/min). The correlation between the LC-CZE analysis and the standard AT III-ELISA was excellent, with r2: 0.965. Using the assay for at line product monitoring, it is shown, that the process temperature is of no consequence for the productivity whereas the process mode strongly influences this parameter.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00365343
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    Processing of mutated human proinsulin to mature insulin in the non-endocrine cell line, CHO (1996)
    Springer
    Cytotechnology 21 (1996), S. 279-288 
    Details
    ISSN: 1573-0778
    Keywords: proinsulin processing ; CHO ; mutant human proinsulin ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Heterologous genes encoding proproteins, including proinsulin, generally produce mature protein when expressed in endocrine cells while unprocessed or partially processed protein is produced in non-endocrine cells. Proproteins, which are normally processed in the regulated pathway restricted to endocrine cells, do not always contain the recognition sequence for cleavage by furin, the endoprotease specific to the constitutive pathway, the principal protein processing pathway in non-endocrine cells. Human proinsulin consists of B-Chain — C-peptide — A-Chain and cleavage at the B/C and C/A junctions is required for processing. The B/C, but not the C/A junction, is recognised and cleaved in the constitutive pathway. We expressed a human proinsulin and a mutated proinsulin gene with an engineered furin recognition sequence at the C/A junction and compared the processing efficiency of the mutant and native proinsulin in Chinese Hamster Ovary cells. The processing efficiency of the mutant proinsulin was 56% relative to 0.7% for native proinsulin. However, despite similar levels of mRNA being expressed in both cell lines, the absolute levels of immunoreactive insulin, normalized against mRNA levels, were 18-fold lower in the mutant proinsulin-expressing cells. As a result, there was only a marginal increase in absolute levels of insulin produced by these cells. This unexpected finding may result from preferential degradation of insulin in non-endocrine cells which lack the protection offered by the secretory granules found in endocrine cells.
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    URL: http://dx.doi.org/10.1007/BF00365350
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    Foreign protein expression from S phase specific promoters in continuous cultures of recombinant CHO cells (1996)
    Springer
    Cytotechnology 22 (1996), S. 179-184 
    Details
    ISSN: 1573-0778
    Keywords: cell cycle ; CHO cells ; continuous culture ; SV40 promoter
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Foreign protein expression from the commonly used SV40 promoter has been found to be primarily during the S-phase of the cell cycle. Simple mathematical models with this cell cycle phase dependent expression of foreign protein suggest that the specific production rate will be proportional to the cell growth rate, which is particularly disadvantageous in high cell density fed-batch or perfusion bioreactors. In this study we investigate this predicted relationship between the production rate and growth rate by culturing recombinant CHO cells in a continuous suspension bioreactor. One CHO cell line, GS-26, has been stably transfected with the plasmid pSVgal, which contains the E. coli lac Z gene under the control of the SV40 promoter. This GS-26 cell line was grown in suspension cultures over a range of specific growth rates in batch and continuous modes. The intracellular β-galactosidase activity was assayed using a standard spectrophotometric method after breaking the cells open and releasing the enzyme. A strong growth associated relationship is found between the intracellular β-galactosidase content and the specific growth rate in batch and continuous cultures, as predicted.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00353937
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  • 22
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    Disposable bioreactor for cell culture using wave-induced agitation (1999)
    Springer
    Cytotechnology 30 (1999), S. 149-158 
    Details
    ISSN: 1573-0778
    Keywords: bioreactor ; cell culture ; disposable ; wave agitation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract This work describes a novel bioreactor system for the cultivation of animal, insect, and plant cells using wave agitation induced by a rocking motion. This agitation system provides good nutrient distribution, off-bottom suspension, and excellent oxygen transfer without damaging fluid shear or gas bubbles. Unlike other cell culture systems, such as spinners, hollow-fiber bioreactors, and roller bottles, scale-up is simple, and has been demonstrated up to 100 L of culture volume. The bioreactor is disposable, and therefore requires no cleaning or sterilization. Additions and sampling are possible without the need for a laminar flow cabinet. The unit can be placed in an incubator requiring minimal instrumentation. These features dramatically lower the purchase cost, and operating expenses of this laboratory/pilot scale cell cultivation system. Results are presented for various model systems: 1) recombinant NS0 cells in suspension; 2) adenovirus production using human 293 cells in suspension; 3) Sf9 insect cell/baculovirus system; and 4) human 293 cells on microcarrier. These examples show the general suitability of the system for cells in suspension, anchorage-dependent culture, and virus production in research and GMP applications.
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    URL: http://dx.doi.org/10.1023/A:1008025016272
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    Enhanced CEA production associated with aspirin in a culture of CW-2 cells on some polymeric films (1999)
    Springer
    Cytotechnology 31 (1999), S. 233-242 
    Details
    ISSN: 1573-0778
    Keywords: cell culture ; carcinoembryonic antigen ; aspirin ; enhanced production ; Langmuir-Blodgett film
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Human colorectal adenocarcinoma tumor (CW2) cells were cultivated in RPMI 1640 media containing 0–7.5 mM aspirin and 10% fetal bovine serum for the production of carcinoembryonic antigen (CEA). By adding aspirin to the media, the production of CEA per cell increased by up to one hundred fold compared to cultivation in normal media containing no aspirin, even though the total cell concentration decreased with the increase in aspirin in the media. The production of CEA was also investigated for CW2 cells cultured on silk fibroin, poly(γ-benzyl-L-glutamate) and poly(γ-benzyl-L-glutamate)/poly(ethylene oxide) diblock copolymer films prepared by the Langmuir-Blodgett and casting methods. The highest production of CEA per cell was observed for the CW2 cells on poly(γ-benzyl-L-glutamate) and its diblock copolymer films prepared by the Langmuir-Blodgett method in the medium containing 5 mM aspirin after 168 hr of inoculation. This originates from the fact that the cell density on the films in the medium containing 5 mM aspirin was the lowest under these conditions. It is suggested that CW2 cells produce CEA more effectively when the cell growth is suppressed by addition of toxic chemicals such as aspirin or by culture on unfavorable films for cell growth.
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    URL: http://dx.doi.org/10.1023/A:1008030730814
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    Engineering Chinese hamster ovary (CHO) cells to achieve an inverse growth – associated production of a foreign protein, β-galactosidase (1998)
    Springer
    Cytotechnology 28 (1998), S. 73-80 
    Details
    ISSN: 1573-0778
    Keywords: adenovirus major late promoter ; β-galactosidase ; Chinese hamster ovary cells ; continuous culture ; G1 phase expression ; inverse-growth associated production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Protein synthesis in mammalian cells can be observed in two strikingly different patterns: 1) production of monoclonal antibodies in hybridoma cultures is typically inverse growth associated and 2) production of most therapeutic glycoproteins in recombinant mammalian cell cultures is found to be growth associated. Production of monoclonal antibodies has been easily maximized by culturing hybridoma cells at very low growth rates in high cell density fed- batch or perfusion bioreactors. Applying the same bioreactor techniques to recombinant mammalian cell cultures results in drastically reduced production rates due to their growth associated production kinetics. Optimization of such growth associated production requires high cell growth conditions, such as in repeated batch cultures or chemostat cultures with attendant excess biomass synthesis. Our recent research has demonstrated that this growth associated production in recombinant Chinese hamster ovary (CHO) cells is related to the S (DNA synthesis)-phase specific production due to the SV40 early promoter commonly used for driving the foreign gene expression. Using the stably transfected CHO cell lines synthesizing an intracellular reporter protein under the control of SV40 early promoter, we have recently demonstrated in batch and continuous cultures that the product synthesis is growth associated. We have now replaced this S-phase specific promoter in new expression vectors with the adenovirus major late promoter which was found to be active primarily in the G1-phase and is expected to yield the desirable inverse growth associated production behavior. Our results in repeated batch cultures show that the protein synthesis kinetics in this resulting CHO cell line is indeed inverse growth associated. Results from continuous and high cell density perfusion culture experiments also indicate a strong inverse growth associated protein synthesis. The bioreactor optimization with this desirable inverse growth associated production behavior would be much simpler than bioreactor operation for cells with growth associated production.
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    URL: http://dx.doi.org/10.1023/A:1008069312131
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    Preparation of conditioned medium to stimulate anthocyanin production using suspension cultures of Fragaria ananassa cells (1999)
    Springer
    World journal of microbiology and biotechnology 15 (1999), S. 635-637 
    Details
    ISSN: 1573-0972
    Keywords: Anthocyanin ; cell culture ; conditioned medium ; strawberry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A conditioned medium (CM) prepared from strawberry suspension cultures greatly stimulated anthocyanin accumulation. CM separated by dialysis membrane showed a significant increase (p 0.05) in anthocyanin synthesis at a fraction smaller than 10,000 Da. The stimulation by CM was eliminated when the CM was treated with alkali.
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    URL: http://dx.doi.org/10.1023/A:1008946506287
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    Structural and functional characterization of a stable, 4-chlorosalicylic-acid-degrading, bacterial community in a chemostat (1995)
    Springer
    World journal of microbiology and biotechnology 11 (1995), S. 643-645 
    Details
    ISSN: 1573-0972
    Keywords: Chemostat ; 4-chlorosalicylic acid ; continuous culture ; degradation ; microbial consortium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A mixed, stable microbial community, obtained by continuous enrichment of a sediment core using 4-chlorosalicylic acid as sole source of carbon and energy, contained 10 different bacterial species, including Klebsiella pneumonia, Pseudomonas fluorescens, P. mendocina and P. cichorii. The members of the community were grown separately on various chlorinated compounds which were readily degraded.
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    URL: http://dx.doi.org/10.1007/BF00361007
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    Continuous cultivation of the yeast Candida utilis at different dilution rates on pineapple cannery effluent (1999)
    Springer
    World journal of microbiology and biotechnology 15 (1999), S. 115-117 
    Details
    ISSN: 1573-0972
    Keywords: Candida utilis ; pineapple cannery effluent ; continuous culture ; steady-state
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Candida utilis was grown on a pineapple cannery effluent in a chemostat at dilution rates ranging between 0.05 and 0.65 h−1 to establish optimal conditions for biomass production and chemical oxygen demand (COD) reduction. Sucrose, fructose and glucose were the main sugars in the effluent. Maximum value for cell yield coefficient and productivity were (0.686, gx/gs) and (2.96, gx/l/h) at a dilution rate of 0.425 and 0.475 h−1, respectively, while maximum COD reduction (98%) was attained at a dilution rate of 0.1 h−1. The maintenance coefficient attained a value of (0.093, gs/gx/h). An increase in dilution rate produced a higher protein content of the biomass.
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    URL: http://dx.doi.org/10.1023/A:1008870228213
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    Reassessment of the bioenergetic yield of Arthrospira platensis using continuous culture (1999)
    Springer
    World journal of microbiology and biotechnology 15 (1999), S. 235-238 
    Details
    ISSN: 1573-0972
    Keywords: Arthrospira platensis ; bioenergetic yield ; continuous culture ; irradiance ; specific growth rate ; mixotrophy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Reassessement of bioenergetic growth yield of Arthrospira platensis was performed by using continuous culture under both autotrophic and mixotrophic conditions. Continuous culture was carried out at dilution rates of 0.017, 0.023 and 0.030 h−1. Under these dilution rates bioenergetic yields ranged between 4.45–6.03 × 10−3 g biomass kJ−1 and between 5.42–7.46 × 10−3 g biomass kJ−1, under autotrophic and mixotrophic conditions respectively. A maximum bioenergetic yield of 8.1 × 10−3 g biomass kJ−1 using an autotrophic culture can be calculated. Pigment accumulation (chlorophyll a and carotenoids) may be related to light irradiance, reaching a maximum pigment concentration under light saturation irradiance. Phycocyanin concentration increased during light limitation.
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    URL: http://dx.doi.org/10.1023/A:1008841605798
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    Determination of the respiration quotient in mammalian cell culture in bicarbonate buffered media (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 45 (1995), S. 524-535 
    Details
    ISSN: 0006-3592
    Keywords: respiration quotient ; carbon dioxide evolution rate ; continuous culture ; cell metabolism ; bicarbonate buffer ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The determination of the respiration quotient (RQ = CER/OUR) has not been used so far as a tool for understanding animal cell metabolism. This is due to problems in measuring the carbon dioxide evolution rate (CER) rather than the oxygen uptake rate (OUR). The determination of the CER is complicated by the use of bicarbonate in the medium. Using liquid and gas balances we have derived an equation for continuous culture to quantify the amount of CO2 that comes from the bicarbonate in the feed. Under cell-free conditions, values predicted by this equation agree within 4% with the experimental results. In continuous culture using hybridoma cells, the CO2 from the feed, as determined by an IR-gas analyzer, was found to represent a significant amount of the total measured CO2 in the off-gas (50% in a suboptimal, and 30% in high-growth medium). Furthermore, the problem of CO2 loss from the medium during medium preparation and storage was solved using both a theoretical and an experimental approach. RQ values in continuous culture were evaluated for two different growth media. Small but significant differences in RQ were measured, which were matched by differences in specific antibody rates and other metabolic quotients. In a medium with Primatone RL, an enzymatic hydrolysate of animal cell tissue that causes a more than twofold increase in cell density, the RQ was found to be 1.05, whereas in medium without Primatone RL (but containing amino acids equivalent in composition and concentration to Primatone RL) the RQ was found to be 0.97. We suggest the RQ to be a useful parameter for estimating the physiological state of cells. Its determination could be a suitable tool for both the on-line control of animal cell cultivations and the understanding of cell metabolism. © 1995 John Wiley & Sons, Inc.
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    URL: http://dx.doi.org/10.1002/bit.260450610
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    Preferential release of one daughter cell during division of immobilized chinese hamster ovary cells (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 45 (1995), S. 374-378 
    Details
    ISSN: 0006-3592
    Keywords: cell culture ; patterened surfaces ; cell adhesion ; hydrogel ; polyHEMA ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Chinese hamster ovary (CHO) cells were attached to tiny adhesive sites in poly-2-hydroxyethyl methacrylate(polyHEMA-) coated glass, and their divison properties were examined. The adhesive sites were produced by placing a metal mask, containing 8-μm-diameter holes arranged in a regular pattern, on top of the coated glass and exposing the sandwich to glow discharge treatment. This treatment produced an ordered array of circular cavities in the polyHEMA down to the glass. These adhesive sites were smaller in diameter than a newborn CHO cell, so that, upon division, there would theoretically be room for only one of the two new daughter cells to remain attached. It was found that individual CHO cells attached to, and grew upon, the sites, and that division normally resulted in the releas of one of the two new daughters. It is concluded that this culture technique has applications in research on the mammalian cell cycle, cell partitioning, and cellular senescence. © 1995 John Wiley & Sons, Inc.
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    URL: http://dx.doi.org/10.1002/bit.260450412
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    Sustained and constitutive high levels of protein production in continuous cultures of bacillus subtilis (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 47 (1995), S. 520-524 
    Details
    ISSN: 0006-3592
    Keywords: bacillus subtilis ; plasmid ; continuous culture ; CAT ; recombinant cultures ; acid formation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The feasibility of continuous production of proteins in chemostat cultures of Bacillus subtilis was investigated. An expression system consisting of the bacterium B. subtilis BR151 carrying plasmid p602/19 was used. The plasmid contains the cat (chioramphenicol acetyltrans-ferase) gene downstream of a strong vegetative T5 promoter. It was found that, at a dilution rate of 0.2 h-1 production of relatively high levels of CAT protein (about 4% ofcellular protein) can be sustained. But, experiments at a higher dilution rate of 0.4 h-1 were unproductive because of high acidformation and washout. Combination of low cell yield, which results from excessive acid formation, and low dilution rate led to a low volumetric CAT productivity. Our recent work with the nonrecombinant cells, has demonstrated that uptake of small amounts of citrate significantly reduces or entirelyeliminates the acid formation. This superior performance in the presence ofcitrate was hypothesized, based on strong experimental evidence, to be the result of a reduction in glycolysis flux through a sequence of events leading to a reduction in pyruvate kinase and phosphof- ructokinase activities, the regulatory enzymes of glycol-ysis. In this study, it is demonstrated that cofeeding of glucose and citrate substantially reduces theorganic acid formation and significantly increases the recombinant culture productivity. The combination of high specific CAT activity and cell density resulted in a total of six- to tenfold higher culture productivitywhen citrate and glucose were cometabolized than when glucose was the only carbon source. © 1995 John Wiley & Sons Inc.
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    Heterogeneity within populations of recombinant Chinese hamster ovary cells expressing human interferon-γ (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 46 (1995), S. 147-158 
    Details
    ISSN: 0006-3592
    Keywords: CHO cell ; cell aggregation ; recombinant human interferon-γ ; mammalian cell culture ; cell culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The Chinese hamster ovary (CHO) cell line has great commercial importance in the production of recombinant human proteins, especially those for therapeutic use. Much attention has been paid to CHO cell population physiology in order to define factors affecting product fidelity and yield. Such studies have revealed that recombinant proteins, including human interferon-γ (IFN-γ), can be heterogeneous both in glycosylation and in proteolytic processing. The type of heterogeneity observed depends on the growth physiology of the cell population, although the relationship between them is complex. In this article we report results of a cytological study of the CHO320 line which expresses recombinant human IFN-γ. When grown in suspension culture, this cell line exhibited three types of heterogeneity: (1) heterogeneity of the production of IFN-γ within the cell population, (2) heterogeneity of the number of nuclei and mitotic spindles in dividing cells, and (3) heterogeneity of cellular environment. The last of these arises from cell aggregates which form in suspension culture: Some cells are exposed to the culture medium; others are fully enclosed within the mass with little or no direct access to the medium. Thus, live cells producing IFN-γ are heterogeneous in their environment, with variable access to O2 and nutrients. Within the aggregates, it appears that live cells proliferate on a dead cell mass. The layer of live cells can be several cells deep. Specific cell-cell attachments are observed between the living cells in these aggregates. Two proteins, known to be required for the formation of certain types of intercellular junctions, spectrin and vinculin, have been localized to the regions of cell-cell contact. The aggregation of the cells appears to be an active process requiring protein synthesis. © 1995 John Wiley & Sons, Inc.
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    URL: http://dx.doi.org/10.1002/bit.260460208
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    Importance of spore mutants for fed-batch and continuous fermentation of Bacillus subtilis (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 47 (1995), S. 696-702 
    Details
    ISSN: 0006-3592
    Keywords: Bacillis subtilis ; spore mutant ; fed-batch ; continuous culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: To alleviate plasmid instability and to prolong the production phase of subtilisin, integrable plasmid and spore mutants are used. Compared with batch-type shake flask cultures, spore mutants' ability to produce subtilisin can be well pronounced in fed-batch and continuous cultures. Hence, the two culture methods make it possible to identify the peculiar characteristics of the spore mutants unobtainable in batch culture. Spore mutants can enhance subtilisin productivity and prolong subtilisin production time in fed-batch culture as well as enable us to use very low dilution rates (〈0.1 h-1) without losing productivity in continuous culture, thereby improving the conversion yield of the nitrogen source. At 0.05 h-1 the spollG mutant of Bacillus subtilis DB104 (Δnpr Δapr) (Emr) spollG (Bimr):: pMK101 (Cmr) showed a subtilisin yield about ten times higher than that from wild-type DB104 (Δnpr Δapr)::pMK101 (Cmr). © 1995 John Wiley & Sons, Inc.
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    URL: http://dx.doi.org/10.1002/bit.260470610
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    Cell cycle and cell size dependence of susceptibility to hydrodynamic forces (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 46 (1995), S. 88-92 
    Details
    ISSN: 0006-3592
    Keywords: cell cycle ; hydrodynamic forces ; apoptosis ; cell culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Exposure of animal cells to intense hydrodynamic forces exerted in turbulent capillary flow, and by controiled agitation and aeration, resulted in preferential destruction of S and G2 cells and the extent of destruction of these cells was dependent upon the intensity of the action. The loss of these cells was possibly due to their larger size. However, the appearance of large numbers of membrane-bound vesicular structures similar to apoptotic bodies as well as cells with low DNA stainability (in a sub-G1 peak) suggested that the action of adverse hydrodynamic forces on these large cells may at least in part be to induce an apoptotic response. © 1995 John Wiley & Sons, Inc.
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    URL: http://dx.doi.org/10.1002/bit.260460112
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    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.
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    URL: http://dx.doi.org/10.1002/bit.260480306
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    Ammonium toxicity in different cell lines (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 530-537 
    Details
    ISSN: 0006-3592
    Keywords: ammonium ; cell culture ; cell cycle ; cell death ; cell growth ; Jurkat cells, GH4 cells ; LLC-PK1 cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The toxic effect of ammonium upon a variety of cell lines of lymphoid (Jurkat), pituitary (GH4), and renal (LLC-PK1) origin was studied. Millimolar concentrations of the ion mildly affected the growth of GH4 cells and prevented the growth of LLC-PK1 cells. The ion did not lead to the death of LLC-PK1 cells but it produced morphologic changes in these cells. The effects of ammonium upon Jurkat cells were different because cells died after accumulating at S phase. Cell death was due to apoptosis and might be related to ammonium-induced calcium mobilization from intracellular stores. These results indicate that the toxic effects caused by ammonium accumulation are different depending upon the cell type. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 530-537, 1997.
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    Enhancement of survivability of mammalian cells by overexpression of the apoptosis-suppressor gene bcl-2 (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 166-175 
    Details
    ISSN: 0006-3592
    Keywords: bcl-2 ; apoptosis ; cell culture ; metabolic engineering ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Cell lines derived from the hemopoetic lineages are widely used as hosts for the production of biologicals. These cell lines have been demonstrated to undergo high levels of the active death program commonly referred to as apoptosis. The effects of overexpression of the apoptosis suppressor gene bcl-2 on the properties of a Burkitt lymphoma were compared with the control cell line (transfected with a negative control plasmid) under a variety of conditions relevant to cell culture production technology. In stationary batch cultures, there was a clear reduction in both the rate of total cell death and the level of apoptosis during the decline phase of the bcl-2 transfected cell cultures as compared with that of the control cell cultures. Nutrient analysis revealed that the onset of death during the control cell cultures occurred following complete exhaustion of glutamine. However, the bcl-2 transfected cell cultures continued to grow even though glutamine had been exhausted, and a significant decline in viability only occurred when glucose had also been completely exhausted.When cells were cultured in suspension without prior adaptation, the bcl-2 transfected cells grew significantly better, suggesting that the bcl-2 gene protected the cells from apoptosis triggered by either the lack of substrate or the hydrodynamic environment. Fluorescence microscopy revealed that death of the control cells was almost entirely by apoptosis, whereas death was almost exclusively by necrosis in the delayed decline phase of the transfected cell cultures. In both instances, death occurred before total exhaustion of glucose and glutamine.The induction of apoptosis following growth arrest is a major impediment to the development of culture strategies that optimize specific productivity by reducing the growth rate. Results presented here suggest that suppression of apoptosis by bcl-2 under the condition of excess thymidine allows the maintenance of cells in a growth-arrested state for much longer than would otherwise be possible.When cells were transferred to a range of commercial serum-free media, cell growth was, in all cases, much better for the bcl-2 transfected cell line. Moreover, when cells were cultivated in glutamine-free medium, the control cells exhibited a decrease in viable cell number within the first 24 h whereas, for the bcl-2 transfected cell cultures, viable cell number did not exhibit any clear decrease until after 75 h. Clearly, these results indicate that the metabolic engineering approach can be used to alter advantageously the survival and proliferative capacity of cells in cell culture environments. © 1996 John Wiley & Sons, Inc.
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    Biooxidation capacity of the extremely thermoacidophilic archaeon Metallosphaera sedula under bioenergetic challenge (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 617-624 
    Details
    ISSN: 0006-3592
    Keywords: thermoacidophile ; chemolithotroph ; heat shock ; chemical stress ; continuous culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The biooxidation capacity of an extremely thermoacidophilic archaeon Metallosphaera sedula (DSMZ 5348) was examined under bioenergetic challenges imparted by thermal or chemical stress in regard to its potential use in microbial bioleaching processes. Within the normal growth temperature range of M. sedula (70-79°C) at pH 2.0, upward temperature shifts resulted in bioleaching rates that followed an Arrhenius-like dependence. When the cells were subjected to supraoptimal temperatures through gradual thermal acclimation at 81°C (Han et al., 1997), cell densities were reduced but 3 to 5 times faster specific leaching rates (Fe3+ released from iron pyrite/cell/h) could be achieved by the stressed cells compared to cells at 79°C and 73°C, respectively. The respiration capacity of M. sedula growing at 74°C was challenged by poisoning the cells with uncouplers to generate chemical stress. When the protonophore 2,4-dinitrophenol (5-10 μM) was added to a growing culture of M. sedula on iron pyrite, there was little effect on specific leaching rates compared to a culture with no protonophore at 74°C; 25 μM levels proved to be toxic to M. sedula. However, a significant stimulation in specific rate was observed when the cells were subjected to 1 μM nigericin (+135%) and 2 μM (+63%); 5 μM levels of the ionophore completely arrested cell growth. The ionophore effect was further investigated in continuous culture growing on ferrous sulfate at 74°C. When 1 μM nigericin was added as a pulse to a continuous culture, a 30% increase in specific iron oxidation rate was observed for short intervals, indicating a potential positive impact on leaching when periodic chemical stress is applied. This study suggests that biooxidation rates can be increased by strategic exposure of extreme thermoacidophiles to chemical or thermal stress, and this approach should be considered for improving process performance. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 617-624, 1998.
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    Regulation of growth and adhesion of cultured cells by insulin conjugated with thermoresponsive polymers (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 339-344 
    Details
    ISSN: 0006-3592
    Keywords: cell culture ; tissue engineering ; thermoresponsive polymer ; cell adhesion ; insulin conjugate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We developed a new biomaterial for use in cell culture. The biomaterial enabled protein-free cell culture and the recovery of viable cells by lowering the temperature without the aid of supplements. Insulin was immobilized and a thermoresponsive polymer was grafted onto a substrate. We investigated the effect of insulin coupling on the lower critical solution temperature (LCST) of the thermoresponsive polymer, poly(N-isopropylacrylamide-co-acrylic acid), using polymers that were ungrafted, or coupled with insulin. The insulin conjugates were precipitated from an aqueous solution at high temperatures, but they were soluble at low temperatures. The LCST was not significantly affected by the insulin coupling. The thermoresponsive polymer was grafted to glow-discharged polystyrene film and covalently conjugated with insulin. The surface wettability of the conjugate film was high at low temperatures and low at high temperatures. The amounts of immobilized insulin required to stimulate cell growth were 1-10% of the amount of free insulin required to produce the same effect. The maximal mitogenic effect of immobilized insulin was greater than that of free insulin. About half of the viable cells was detached from the film only by lowering the temperature. The recovered cells proliferated normally on new culture dishes. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 339-344, 1997.
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    Study of stability of recombinant plasmids during the continuous culture of Bacillus stearothermophilus NUB3621 in nonselective medium (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 507-514 
    Details
    ISSN: 0006-3592
    Keywords: Bacillus stearothermophilus ; continuous culture ; plasmid stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The optimal culture conditions for Bacillus stearothermophilus NUB3621 (BGSC 9A5) in chemostat were studied. The results obtained showed that the optimal culture conditions in terms of biomass concentration and maximum growth rate were 65°C, pH 6.8 to 7.2. Dissolved oxygen became growth limiting at pO2 levels below 10%. Furthermore, this strain was transformed with three new hybrid vectors (pPAM2, pPCH2, or pPLY2) constructed by cloning in pRP9, a plasmid based on the thermophilic replicon, pBC1, and three heterologous genes: the α-amylase gene from Bacillus licheniformis, the cholesterol oxidase gene from Streptomyces sp., and the lipase gene from Pseudomonas fluorescens. The influence of several fermentative conditions on segregational and structural stability of the recombinant B. stearothermophilus NUB3621 transformants was studied.The parameters of plasmid loss, that is, rate of plasmid loss (R) and specific growth rate difference (δμ), were calculated. B. stearothermophilus NUB3621 carrying pRP9 showed great segregational stability in all the assayed conditions, exceeding more than 300 generations without significant plasmid loss, whereas NUB3621 carrying pPAM2, pPCH2, or pPLY2 exhibited relatively low plasmid stability. The segregational instability of the recombinant constructs increased by increasing the fermentation temperature, decreased by increasing the dilution rate, and was not affected by the level of dissolved oxygen. On the other hand, plasmid maintenance decreased in minimal medium if compared with the results obtained in complex medium. Restriction analyses carried out on cultures of NUB3621 carrying pRP9, pPAM2, pPCH2, or pPLY2, grown for 200 generations on nonselective media, revealed that all the clones tested contained the parental plasmids. These results indicate that the heterologous inserts did not affect the structural stability of the recombinant plasmids. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 507-514, 1997.
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    Continuous culture study of the expression of hepatitis B surface antigen and its self-assembly into virus-like particles in Saccharomyces cerevisiae (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 578-586 
    Details
    ISSN: 0006-3592
    Keywords: Saccharomyces cerevisiae ; hepatitis B surface antigen (HBsAg) ; continuous culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have studied the growth rate dependence of hepatitis B surface antigen (HBsAg) p24s monomer and lipoprotein particle synthesis produced in Saccharomyces cerevisiae using galactose-limited continuous culture. The hepatitis B virus S gene, which encodes the p24s monomer, is transcribed under the control of the GAL 10p on a chimeric 2-μm plasmid harbored in a haploid yeast strain. Monomers autonomously form lipoprotein aggregates (particles) in vivo using only host-cell-derived components. Steady states were evaluated in a range from 0.015 h-1 to washout (0.143 h-1). Both p24s monomer and HBsAg particle levels, at steady state, varied in an inverse linear manner with growth rate. A consistent excess of total p24s monomer to HBsAg particle, estimated at five- to tenfold by mass, was found at all dilution rates. The average copy number of the 2-μm plasmid (carrying LEU2 selection) remained constant at 200 copies per cell from washout to 0.035 h-1. Surprisingly, the average copy number was undetectable at the lowest dilution rate tested (0.015 h-1), even though HBsAg expression was maximal. Total p24s monomer and HBsAg particle values ranged twofold over this dilution rate range. No differences in the trends for HBsAg expression and average copy number could be detected past the critical dilution rate where aerobic fermentation of galactose and ethanol overflow were observed. HBsAg expression in continuous culture was stable for at least 40 generations at 0.100 h-1. © 1996 John Wiley & Sons, Inc.
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    On-line estimation of viable cells in a hybridoma culture at various DO levels using ATP balancing and redox potential measurement (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 277-283 
    Details
    ISSN: 0006-3592
    Keywords: cell culture ; on-line viable cell concentration ; ATP balance ; redox potential ; hybridoma ; dissolved oxygen ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Two on-line methods for the estimation of viable cell number in hybridoma cultivation were investigated. One used an empirical correlation between redox potential and animal cell density. The other was based on an ATP balance with ATP steady-state assumption. Oxygen uptake rate measurement provided the amount of ATP which was produced by oxidation of NADH. Oxygen uptake rate was measured either by stationary liquid phase balance with surface aeration or by gas balance during bubble aeration with headspace flushing with an inert gas. The amount of ATP produced through the glycolysis was estimated based on the amount of lactate produced. In cultures, in which pH was controlled via manipulation of the gas phase composition, the flow of CO2 was linearly correlated with the lactate concentration. At constant dissolved oxygen levels, the viable cell density was proportional to the estimated ATP production rate, during exponential growth and during later phases. The estimated specific ATP production rate, however, varied from 2.2 pmol cell-1 h-1 at 10% air saturation to 4.5 pmol cell-1 h-1 at 100% air saturation. Specific rates of glutamine, glucose, and lactate followed the shape of the specific ATP production rate, whereas the specific oxygen uptake rate was minimal at around 50% air saturation. © 1996 John Wiley & Sons, Inc.
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    Growth kinetics of a bacteriophage in continuous culture (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 217-221 
    Details
    ISSN: 0006-3592
    Keywords: Qβ phage ; molecular evolution ; phage display ; continuous culture ; cellstat ; wall growth ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Lytic coliphage Qβ was grown in continuously cultured host bacteria using a cascade of stirred flow reactors. The apparatus was constructed so that the steady stream of exponentially growing bacterial cells passing through the stirred flow reactors served to prevent coevolution brought about by host-parasite interactions. Wall growth was the primary cause for deviation from ideal continuous culture conditions and is largely dependent on the surface structure of the host bacteria. Using an Escherichia coli strain deficient in adhesive type I pili expression, the desynchronization of single burst events could easily be followed over the course of four infection latency periods. Computer simulations based on a two-stage model for the Qβ infection cycle were in perfect agreement with the experimental data. Applications of the optimized system to strategies of molecular evolution are discussed. © 1996 John Wiley & Sons, Inc.
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    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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    Continuous cultures limited by a gaseous substrate: Development of a simple, unstructured mathematical model and experimental verification with Methanobacterium thermoautotrophicum (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 645-658 
    Details
    ISSN: 0006-3592
    Keywords: Methanobacterium thermoautotrophicum ; gaseous substrate limitation ; continuous culture ; mathematical modeling ; amperometric measurement of dissolved H2 concentration ; reaction calorimetry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This article presents a simple, unstructured mathematical model describing microbial growth in continuous culture limited by a gaseous substrate. The model predicts constant gas conversion rates and a decreasing biomass concentration with increasing dilution rate. It has been found that the parameters influencing growth are primarily the gas transfer rate and the dilution rate. Furthermore, it is shown that, for correct simulation of growth, the influence of gaseous substrate consumption on the effective gas flow through the system has to be taken into account.Continuous cultures of Methanobacterium thermoautotrophicum were performed at three different gassing rates. In addition to the measurement of the rates of biomass production, product formation, and substrate consumption, microbial heat dissipation was assessed using a reaction calorimeter. For the on-line measurement of the concentration of the growth-limiting substrate, H2, a specially developed probe has been used. Experimental data from continuous cultures were in good agreement with the model simulations. An increase in gassing rate enhanced gaseous substrate consumption and methane production rates. However, the biomass yield as well as the specific conversion rates remained constant, irrespective of the gassing rate. It was found that growth performance in continuous culture limited by a gaseous substrate is substantially different from “classic” continuous culture in which the limiting substrate is provided by the liquid feed. In this report, the differences between both continuous culture systems are discussed.
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    Plasmid stability of recombinant Pseudomonas sp. B13 FR1 pFRC20P in continuous culture (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 62-70 
    Details
    ISSN: 0006-3592
    Keywords: plasmid stability ; recombinant microorganism ; continuous culture ; Pseudomonas sp. B13 FR1 pFRC20P ; degradation of aromatic compounds ; chlorobenzoate ; methylbenzoate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Plasmid stability of recombinant Pseudomonas sp. B13 FR1 pFRC20P, a strain capable of mineralizing 3- and 4-chlorobenzoate and 4-methylbenzoate, was investigated in continuous culture. The hybrid cosmid pFRC20P enables the strain to mineralize 4-methylbenzoate. Rapid plasmid loss was observed under nonselective conditions using 3-chlorobenzoate as the substrate. Plasmid stability decreased with increasing dilution rate. Despite the growth advantage of the generated plasmid free cells a total depletion of plasmid bearing cells was not observed. After approximately 50 generations the fraction of plasmid bearing cells reached a constant level of 10%, which was stably maintained during the next 25 generations. Cells from this stage were used to inoculate a new culture that resulted in a stable level of 50% plasmid bearing cells. By a temporary substrate change to selective conditions (4-methylbenzoate), this level could be further increased to 70%. Literature models on plasmid stability could not be applied to describe the experimental data. Therefore, a new but unstructured model was developed to describe the experimental results. The model is based on the existence of three subpopulations: a plasmid free one, an original plasmid bearing one with a growth disadvantage compared to plasmid free cells, and a second plasmid bearing subpopulation with increased stability that is generated from the original one and has a growth rate comparable to the plasmid free cells. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 62-70, 1998.
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    Capture of human monoclonal antibodies from cell culture supernatant by ion exchange media exhibiting high charge density (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 60 (1998), S. 689-698 
    Details
    ISSN: 0006-3592
    Keywords: purification ; cation exchange chromatography ; cell culture ; cell culture medium ; serum free ; therapeutic antibodies ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A shortcut purification sequence for therapeutic proteins should consist of three steps: capture, purification, and polishing. Special emphasis has been put on direct capture of human monoclonal antibodies from culture supernatants with ion-exchangers avoiding pretreatment steps such as desalting, dilution, and other means to reduce the ionic strength. CM-HyperD, a cation-exchanger composed of an inorganic macroporous support filled with a viscoelastic gel with a high charge density was used. Capture of monoclonal antibodies from clarified hybridoma cell culture grown in media supplemented with fetal calf serum was investigated. Screening of different pH conditions and buffers for the load step showed that monoclonal antibodies were efficiently bound by CM-HyperD at pH 4.0 and 5.0 at an ionic strength equivalent to culture supernatant. Combination of negative purification with Q-Sepharose FF and capturing with CM-HyperD gave sufficient yield and resolution. Implementation of wash steps with higher conductivity did not improve the purity, but decreased the yield. Interestingly, high flow rates improved the purity. When antibodies were captured from serumfree culture supernatant the antibody could be eluted in a single peak with substantial reduction of contaminants. Capturing of antibodies by ion-exchange sorbents from culture supernatant is possible despite the high salt content. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 689-698, 1998.
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    The effect of dissolved oxygen on the metabolic profile of a murine hybridoma grown in serum-free medium in continuous culture (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 54 (1997), S. 153-164 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; oxygen ; serum-free medium ; continuous culture ; antioxidant ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The murine B-lymphocyte hybridoma, CC9C10 was grown at steady state under serum-free conditions in continuous culture at dissolved oxygen (DO) concentrations in the range of 10% to 150% of air saturation. Cells could be maintained with this range at high viability in a steady state at a dilution rate of 1 d-1, although with lower cell concentrations at higher DO. A higher specific antibody production measured at higher DO was matched by a decrease in the viable cell concentration at steady state, so that the volumetric antibody titre was not changed significantly. An attempt to grow cells at 250% of air saturation was unsuccessful but the cells recovered to normal growth once the DO was decreased.There was a requirement for cellular adaptation at each step-wise increase in dissolved oxygen. Adaptation to a DO of 100% was associated with an increase in the specific activities of glutathione peroxidase (×18), glutathione S-transferase (×11) and superoxide dismutase (×6) which are all known antioxidant enzymes. At DO above 100%, the activities of GPX and GST decreased possibly as a result of inactivation by reactive oxygen radicals.The increase in dissolved oxygen concentration caused changes in energy metabolism. The specific rate of glucose uptake increased at higher dissolved oxygen concentrations with a higher proportion of glucose metabolized anaerobically. Short-term radioactive assays showed that the relative flux of glucose through glycolysis and the pentose phosphate pathway increased whereas the flux through the tricarboxylic acid cycle decreased at high DO. Although the specific glutamine utilization rate increased at higher DO, there was no evidence for a change in the pattern of metabolism. This indicates a possible blockage of glycolytic metabolites into the TCA cycle, and is compatible with a previous suggestion that pyruvate dehydrogenase is inhibited by high oxygen concentrations.Analysis of the oxygen uptake rate of cell suspensions at steady state under all conditions showed a pronounced Crabtree effect which was manifest by a decrease (up to 40%) in oxygen consumption on addition of glucose. This indicates that the degree of aerobic metabolism in these cultures is highly sensitive to the glucose concentration. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 153-164, 1997.
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    Effect of growth factors and antigen on hybridoma cell culture dynamics (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 54 (1997), S. 357-364 
    Details
    ISSN: 0006-3592
    Keywords: cell culture ; hybridoma ; monoclonal antibody ; growth factor ; antigen ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The cell growth and monoclonal antibody production kinetics of hybridoma cell cultures continuously exposed to growth factors and the cognate antigen were investigated. The growth factors were the epidermal growth factor, fibroblast growth factor, and interleukin-2, whereas the antigen was the trinitrophenyl group conjugated to a carrier protein. The cultures were carried out in a protein-free medium in batch operation. During the entire cultivation period there was continuously available free, antibody-unbound antigen to interact with the cells. The produced antibody was measured with an ELISA after it was released from the antigen-protein conjugate by competitive elution with non-protein-conjugated antigen. Cultures with growth factors and without antigen increased the total antibody produced by up to 30%, whereas cell growth remained unaffacted. Soluble antigen-protein conjugates had no effect on the hybridoma cultures. In contrast, immobilized antigen-protein on sepharose beads in cultures with growth factors induced significant changes. Total antibody produced was higher by up to 40%. More importantly, the specific antibody production shifted from a growth-phase-independent to a growth-phase-dependent profile, with approximately twice as much specific antibody production during the late growth-early stationary phase relative to constant specific antibody production in the antigen-free, factor-free culture. The culture changes induced by the presence of immobilized antigen and growth factors were reversed when the antigen and the growth factors were removed from the cells' environment. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 357-364, 1997.
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    Influence of the degree and mode of light limitation on growth characteristics of the Rhodobacter capsulatus continuous cultures (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 605-612 
    Details
    ISSN: 0006-3592
    Keywords: phototrophic bacteria ; Rhodobacter capsulatus ; continuous culture ; light limitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The influence of the degree and mode of light limitation on growth characteristics of turbidostat cultures of Rhodobacter capsulatus was investigated using mass and energy balance regularities. Light limitation was achieved by increasing the steady-state biomass concentration at constant incident light intensity (∼100 W/m2) or by decreasing the incident light intensity at constant steady-state biomass concentration (∼500 mg of dry biomass/L). It was shown that under conditions of light limitation of Rh. capsulatus, the content of P and N in the biomass as well as the biomass degree of reduction were determined by the growth rate of the cultures. The energetic yield of biomass of Rh. capsulatus and total bacteriochlorophyll a content increased when light limitation increased. These parameters were higher in the cultures, in which light limitation was achieved by lowering the incident light intensity at low biomass concentration. This seems to be due to different distribution of light within the photobioreactor when dissimilar modes of light limitation were used.
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    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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    Kinetic models for the growth of Escherichia coli with mixtures of sugars under carbon-limited conditions (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 99-107 
    Details
    ISSN: 0006-3592
    Keywords: Monod kinetics ; mixed substrate growth ; continuous culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In natural environments, heterotrophic microorganisms encounter complex mixtures of carbon sources, each of which is present only at very low concentrations. Under such conditions no significant growth could be expected if cells utilized only one of the available carbon compounds as suggested by the principle of diauxic growth. Indeed, there is much evidence that microbial cells utilize many carbon sources simultaneously. In order to predict bacterial growth under such conditions we developed a model describing the specific growth rate as a function of the individual concentrations of several simultaneously utilized carbon substrates. Together with multisubstrate models previously published, this model was evaluated for its ability to describe growth of Escherichia coli during the simultaneous utilization of mixtures of sugars in carbon-limited continuous culture. Using the μmax and Ks constants determined for single substrate growth with six different sugars, the model was able for most experiments to adequately describe the specific growth rate of the culture, i.e., the experimentally set dilution rate, from the measured concentrations of the individual sugars. The model provides an explanation why bacteria can still grow relatively fast under environmental conditions where the concentrations of carbon substrates are usually extremely low. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:99-107, 1998.
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    On-line measurement of oxygen uptake in cell culture using the dynamic method (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 443-448 
    Details
    ISSN: 0006-3592
    Keywords: on-line ; oxygen uptake rate ; OUR ; cell culture ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Measurement of oxygen uptake rate is useful in assessing growth, viability, and metabolic activity. In cell culture, however, the oxygen demand is extremely small (typically 0.1-0.3 mM O2L-h) and is very difficult to measure accurately using conventional offgas analysis. In many industrial submerged cell culture systems, dissolved oxygen levels are controlled between preset limits by intermittent sparging of air or oxygen. This article describes a computational method for the automatic online determination of oxygen uptake from the dynamic dissolved oxygen probe response. Experimental measurements show that for a typical hybridoma culture, specific oxygen demand is 0.15 mM O2/109 cells/h. © 1996 John Wiley & Sons, Inc.
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    In hybridoma cultures, deprivation of any single amino acid leads to apoptotic death, which is suppressed by the expression of the bcl-2 gene (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 90-98 
    Details
    ISSN: 0006-3592
    Keywords: apoptosis ; necrosis ; bcl-2 ; amino acids ; cell culture ; cell death ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The transfection of murine hybridomas with the apoptosis suppressor gene bcl-2 has been reported to result in the extension of batch culture duration, leading to significant improvements in culture productivity. In the present study, the effect of deprivation, individually, of each amino acid found in culture medium was examined to characterize the chemical environment of the culture in terms of its propensity to induce apoptosis. When cells were deprived of each amino acid, individually for 48 h, the majority of cell deaths in each case occurred by apoptosis, with essential amino acids being clearly most effective. For nearly all the amino acids, the viability of the bcl-2 cell line cultures was greater than 70% after 48 h, representing a substantial improvement in viability over control cell line cultures. Time course studies revealed that the induction of death could be divided into two phases. Initially, following the deprivation of a single essential amino acid, there was a period of time during which all the control cell line cultures retained high viability. The duration of this phase varied from 15 h in the case of lysine deprivation, through to 40 h in the case methionine deprivation. In the second phase of deprivation, the cultures exhibited an abrupt and rapid collapse in viability. The time taken for the viability to fall to 50% was similar for each amino acid. In every case, the duration of both phases of the bcl-2 cultures was considerably extended. Specific utilization rates were increased during the control cultures relative to the bcl-2 cultures for both the growth phase (ranging between 2% and 57% higher than the bcl-2 cultures) and the death phase (ranging between 172% to 1900% higher than the bcl-2 culture). © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:90-98, 1998.
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    Improvement of antibiotic titers from Streptomyces bacteria by interactive continuous selection (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 185-196 
    Details
    ISSN: 0006-3592
    Keywords: streptomycin ; Streptomyces ; strain improvement ; continuous culture ; feedback control ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have applied a technique of interactive continuous selection (ICS) to the isolation of streptomycin-resistant mutants of the streptomycin-producing organism, Streptomyces griseus. A series of mutants, each with a different colonial morphology and expressing successively greater resistance to streptomycin, was isolated during the course of selection. Takeover of the mutants has been correlated with changes in on-line estimates of streptomycin concentration such that these estimates may be used as a real-time measure of the genetic state of the cell population. When grown in the medium employed for ICS, mutants expressed increased antibiotic production titers; the best mutant produced 10 to 20 times more streptomycin than the parent strain. Absolute improvements in the maximum specific growth rate and intrinsic resistance to streptomycin did not account for the observed growth advantage of all mutants. Rather, each mutant exhibited relative increases in specific growth rate at increasing concentrations of streptomycin. © 1996 John Wiley & Sons, Inc.
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    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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    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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    Multiplicity and stability analysis of microorganisms in continuous culture: Effects of metabolic overflow and growth inhibition (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 251-261 
    Details
    ISSN: 0006-3592
    Keywords: continuous culture ; metabolic overflow ; multiplicity ; stability analysis ; dynamics ; growth inhibition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Metabolic overflow (enhanced uptake of substrate and secretion of intermediates) is a phenomenon often observed for cells grown under substrate excess. Growth inhibition by substrate and/or product is also normally found for this kind of culture. An effort is made in this work to analyze the dynamic behavior of a continuous culture subject to metabolic overflow and growth inhibition by substrate and/or product. Analysis of a model system shows that in a certain range of operating conditions three nonwashout steady state solutions are possible. Local stability analysis indicates that only two of them are stable thus leading to multiplicity and hysteresis. Further analysis of the intrinsic effects of different terms describing the metabolic overflow and growth inhibitions reveals that for the model system and the parameters considered, the combined effects of product inhibition and an enhanced formation rate of product under substrate excess cause the multiplicity and hysteresis. Growth inhibition by substrate and/or an enhanced substrate uptake appear not to be necessary conditions. The combined effects of enhanced product formation and product inhibition can also lead to unusual dynamic behavior such as a prolonged time period to reach a steady state, oscillatory transition from one steady state to another, and sustained oscillations. Using the occurrence of multiplicity and oscillation as criteria, the operating regime of a continuous culture can be divided into four domains: one with multiplicity and oscillation, one with unique steady state but possible oscillatory behavior, the other two with unique and stable steady state. The model predictions are in accordance with recent experimental results. The results presented in this work may be used as guidelines for choosing proper operating conditions of similar culture systems to avoid undesired instability and multiplicity. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 251-261, 1998.
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