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  • 1
    Electronic Resource
    Electronic Resource
    Cell-dislodging methods under serum-free conditions (1997)
    Springer
    Applied microbiology and biotechnology 47 (1997), S. 482-488 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract In this work, a BHK21 clone producing a fusion protein consisting of a recombinant human IgG molecule with a cytokine tail, growing in a protein-free medium, was used to test several alternatives to avoid the use of serum for trypsin inactivation, currently used in cell dislodging. These included (1) trypsin inactivated with soybean trypsin inhibitor (STI); (2) cell dissociation solution instead of trypsin; (3) dispase instead of trypsin; (4) trypsin inactivated with fetal calf serum (positive control); (5) non-inactivated trypsin (negative control). Use of a centrifugation step was also tested for each alternative. Results indicate that the best method regarding cell growth, viability and adherent fraction is to use trypsin inactivated with STI followed by a centrifugation step. For all methods tested, the utilization of a centrifugation step always led to improved results. The optimal proportion for total trypsin inactivation is 1:1 trypsin (0.2% w/v) to STI (1 mg ml−1), equivalent to 2 mg trypsin to 1 mg STI. No toxic effect was observed for STI at the concentrations used. Long-term subculturing with this new, alternative dislodging method did not affect cell growth, viability and productivity.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002530050960
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  • 2
    Electronic Resource
    Electronic Resource
    Elucidation of the mechanism of lactic acid growth inhibition and production in batch cultures of Lactobacillus rhamnosus (1997)
    Springer
    Applied microbiology and biotechnology 48 (1997), S. 346-350 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Batch cultures of Lactobacillus rhamnosus were carried out at different pH values in order to study the limitation of growth and lactic acid production by the hydrogen ion, non-dissociated lactic acid and internal lactate concentrations. The effect of pH between 5 and 6.8 was studied at non-limiting concentrations of glucose; this is more significant for the lactic acid fermentation rate than for the maximum specific growth rate, as shown by the incomplete substrate consumption at lower values of medium pH and by the constant maximum cell mass obtained within the range of pH values studied. To check whether these results were a direct consequence of the different concentrations of the non-dissociated form of lactic acid at different external pH values, specific growth rates and lactic acid productions rates were calculated for each external pH value. The same specific growth rates were observed at the same non-dissociated lactic acid concentrations only at pH values of 5 and 5.5. For higher values of pH (pH 〉 6) the specific growth rate falls to zero as the non-dissociated lactic acid concentration decreases. This shows that generalisations made from studies performed within very narrow ranges of pH are not valid and that the non-dissociated form of lactic acid is not the only inhibiting species. The internal pH was measured experimentally for each external pH value in order to calculate the internal lactate ion concentration. This form is described to be the inhibitory one. The results obtained confirmed that the specific growth rate reached zero at approximately the same lactate concentration for all the pH values studied.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002530051060
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  • 3
    Electronic Resource
    Electronic Resource
    Metabolic responses to different glucose and glutamine levels in baby hamster kidney cell culture (1999)
    Springer
    Applied microbiology and biotechnology 51 (1999), S. 579-585 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract In this work, a BHK21 clone producing a recombinant antibody/cytokine fusion protein was used to study the dependence of cell metabolism on the glucose and glutamine levels in the culture medium. Results obtained indicate that both glucose and glutamine consumptions show a Michaelis-Menten dependence on glucose and glutamine concentrations respectively. A similar dependence is also observed for lactate and ammonia productions. The estimated value of the Michaelis constant for the dependence of lactate production on glucose (K Glc Lac) was 1.4 ± 0.1 mM and for the dependence of ammonia production on glutamine (K Gln Amm) was 0.25 ± 0.11 mM and 0.10 ± 0.03 mM, at glucose concentrations of 0.28 mM and 5.6 mM respectively. At very low glucose concentrations, the glucose to lactate yield decreased markedly, showing a metabolic shift towards lower lactate production. This␣metabolic shift was also confirmed by the significant increase in the specific oxygen consumption rate also observed at low glucose concentrations. Although it was␣highly dependent on glucose concentration, the oxygen consumption also increased with the increase in␣glutamine concentration. At very low glutamine concentrations, the glutamine to ammonia yield increased, showing a more efficient glutamine metabolism.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002530051435
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  • 4
    Electronic Resource
    Electronic Resource
    An integrated strategy for the process development of a recombinant antibody-cytokine fusion protein expressed in BHK cells (1999)
    Springer
    Applied microbiology and biotechnology 52 (1999), S. 345-353 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Recombinant fusion proteins offer important new therapeutic approaches for the future. This report describes the use of three different genetic strategies (i.e. “mono-”, “bi-” and “tri-cistronic” vectors) to achieve stable secretion from BHK cells of a glycosylated antibody-cytokine fusion protein designed for use in anti-tumour therapy. It describes selection of a robust and effective production cell line based on stability of secretion of the product, quality of mRNA and protein products and performance in in vitro bioassays for potency. The data obtained at this stage were utilised in the selection of a suitable candidate production cell line. The relative productivity and general performance of the cells in stirred tank and fixed bed culture systems indicated that a variety of cell culture technologies provided robust tools for production of a highly selected cell clone. Consistency of the product glycosylation was determined by analysis of released oligosaccharides using matrix-assisted laser desorption ionisation – time of flight mass spectrometry and high-performance anion exchange chromatography. These investigations showed consistent expression of three glycoforms of the fusion protein which varied in their relative proportions in different culture systems and at different time points in a fixed bed reactor with continuous perfusion. In conclusion, this study dealt with a range of important scientific and technical issues which are essential for regulatory approval and commercial success of a recombinant protein and elucidates some useful markers for process development for similar recombinant biologicals.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002530051530
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  • 5
    Electronic Resource
    Electronic Resource
    Operational patterns affecting lactic acid production in ultrafiltration cell recycle bioreactor (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 45 (1995), S. 320-327 
    Details
    ISSN: 0006-3592
    Keywords: cell recycle reactor ; ultrafiltration tubular membranes ; high lactic acid productivities ; best operational conditions ; different dilution rates ; start-up strategy ; membrane permeability ; long-term fermentations ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Lactic acid production with cell recycling on an ultrafiltration tubular membrane reactor was studied; higher lactic acid concentrations as well as productivities were obtained under long-term fermentations compared with other high cell density systems. Different operational conditions, namely dilution rates and start-up modes, were assessed. Performances were very different at the three different dilution rates tested (D = 0.20 h-1, D = 0.40 h-1, or D = 0.58 h-1). The different behaviours are discussed and factors responsible for them are presented. The best way to operate for lactic acid production is chosen, the dilution rate of D = 0.40 h-1 being the one providing the best overall performance. On the other hand, results show that of the two start-up modes tested, continuous start (membrane open) permits higher permeabilities throughout the operational runs than batch start (membrane closed). Operational stability was found to be directly associated with membranes that work at “steady state,” the membrane permeability being kept around 15 L/m2 h. Optimized cell bleed can improve time of operation if such membrane permeability can be maintained for a longer time. A comparison of results with those obtained in other lactic acid production systems is presented; such comparison shows that this tubular ultrafiltration membrane cell recycle reactor presents three important advantages: (1) concomitant lactic acid concentrations and productivities; (2) long periods of operation at reasonable permeabilities; and (3) good mechanical stability permitting the use of steam sterilization. © 1995 John Wiley & Sons, Inc.
    Additional Material: 10 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260450406
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  • 6
    Electronic Resource
    Electronic Resource
    Nitrite inhibition of denitrification by Pseudomonas fluorescens (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 46 (1995), S. 194-201 
    Details
    ISSN: 0006-3592
    Keywords: denitrification kinetics ; nitrite inhibition model ; Pseudomonas fluorescens ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Using a pure culture of Pseudomonas fluorescens as a model system nitrite inhibition of denitrification was studies. A mineral media with acetate and nitrate as sole electron donor and acceptor, respectively, was used. Results obtained in continuous stirred-tank reactors (CSTR) operated at pH values between 6.6 and 7.8 showed that growth inhibition depended only on the nitrite undissociated fraction concentration (nitrous acid). A mathematical model to describe this dependence is put forward. The maximum nitrous acid concentration compatible with cell growth and denitrification activity was found to be 66 μg N/L. Denitrification activity was partially associated with growth, as described by the Luedeking-Piret equation. However, when the freshly inoculated reactor was operated discontinuosly, nitrite accumulation caused growth uncoupling from denitrification activity. The authors suggest that these results can be interpreted considering that (a) nitrous acid acts as a proton uncoupler; and (b) cultures continuoulsy exposed to nitrous acid prevent the uncoupling effect but not the growth inhibition. Examination of the growth dependence on nitrite concentration at pH 7.0 showed that adapted cultures (grown on CSTR) are less sensitive to nitrous acid inhibition than the ones cultivated in batch. © 1995 John Wiley & Sons, Inc.
    Additional Material: 12 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260460303
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  • 7
    Electronic Resource
    Electronic Resource
    Competition between nitrate and nitrite reduction in denitrification by Pseudomonas fluorescens (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 46 (1995), S. 476-484 
    Details
    ISSN: 0006-3592
    Keywords: denitrification ; substrate limitation ; competition ; kinetic model ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A pure culture of Pseudomonas fluorescens was used as a model system to study the kinetics of denitrification. An exponentially growing culture was harvested and resuspended in an anoxic acetate solution buffered with K/Na phosphate at pH values of 6.6, 7.0, 7.4, and 7.8. The temperature was kept at 28°C in all assays. Nitrate pulses of approximately 0.2 mg N/L caused nitrite to accumulate due to a faster rate of nitrate reduction over nitrite reduction. The rate of nitrate reduction was observed to depend on its concentration as predicted by the Michaelis-Menten equation. At nonlimiting nitrate concentrations, nitrite reduction was described by the same equation. Otherwise, nitrite reduction also depended on nitrate concentration. Consequently, nitrate and nitrite reductions compete with each other for the oxidation of common electron donors. A kinetic model for nitrate competitive inhibition of nitrite reduction is proposed. The model was used to interpret the nitrate and nitrite profiles observed at the four pH values: the optimum pH value was 7.0 in both cases; the affinity for nitrite was also not affected by the medium pH in the range of values 6.6 to 7.4 (KmNO3 = 0.04 mg N/L); the affinity for nitrite was also not affected by the medium pH in the range of values 6.6 to 7.4 (KmNO2 = 0.06 mg N/L), but it decreased sharply for the pH value of 7.8. Although the ratio between the two maximum reduction rates (Vmax NO2/Vmax NO3) is constant, nitrite accumulation depends on the medium pH value. Therefore, the regulation mechanism that shifts the electron flow between the two terminal reductases is readily reversible and does not change their relative maximum reduction rates. © 1995 John Wiley & Sons, Inc.
    Additional Material: 12 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260460512
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  • 8
    Electronic Resource
    Electronic Resource
    Two-dimensional versus three-dimensional culture systems: Effects on growth and productivity of BHK cells (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 429-432 
    Details
    ISSN: 0006-3592
    Keywords: BHK ; aggregates ; porous microcarriers ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The influence of surface growth (two-dimensional microcarriers) and three-dimensional growth (aggregates and macroporous supports) in agitated, suspended batch culture systems upon growth and productivity of BHK was compared. Cultures using three porous microcarriers (CultiSpher G, Cellsnow EX, and Cytocell), one nonporous microcarrier (Cytodex 3) and natural aggregates were performed in stirred tanks using two different agitation rates (60 and 100 RPM). With the exception of Cytocell, cell growth, viability, and productivity were similar when three-dimensional structures (porous microcarriers and aggregates) were used. Nonporous microcarriers only compared well at 60 RPM as growth ceased under overagitation. These results suggest that cultures less susceptible to fluid shear are advantageous for scale-up. © 1996 John Wiley & Sons, Inc.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
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