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  • 1
    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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  • 2
    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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  • 3
    Electronic Resource
    Electronic Resource
    Studies of Baby Hamster Kidney Natural Cell Aggregation in Suspended Batch Cultures (1994)
    Oxford, UK : Blackwell Publishing Ltd
    Annals of the New York Academy of Sciences 745 (1994), S. 0 
    Details
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1749-6632.1994.tb44368.x
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  • 4
    Electronic Resource
    Electronic Resource
    Expression of recombinant antibody and secreted alkaline phosphatase in mammalian cells. Influence of cell line and culture system upon production kinetics (1994)
    Springer
    Applied microbiology and biotechnology 40 (1994), S. 851-856 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The growth and productivity of an Sp2/0 cell line, F3b10, expressing a recombinant antibody (rAb) and BHK21 cells expressing either the same rAb from the same plasmids (BHK.IgG) or secreted alkaline phosphatase (SEAP) (BHK.SEAP) were investigated. The F3b10 line was grown as a single cell suspension. The BHK lines were grown either as suspended natural aggregates or on Cytodex 3 microcarriers. The data for F3b10 showed that the cell-specific rAb production rate (Qs rAb) increased in parallel with increases in the specific growth rate (μ). A similar result was obtained for suspended aggregate cultures of both recombinant BHK cell lines. In contrast, for microcarrier cultures of both BHK cell lines, Qs product increased as μ decreased. This report shows that the relationship between cell growth and Qs product for the cell lines and products studied is dependent upon the culture process. In systems where recombinant cells are growing as a single cell suspension or within a natural suspension aggregate, Qs product increased with increases in μ. In such systems, the cells have a rounded morphology. When cells were grown on microcarriers, Qs product decreased as μ increased. Cells growing attached to a surface are flat and elongated. The observed differences in the relationship of Qs product to μ are correlated with changes in cell morphology. The relationship between Qs product and μ is also affected by the choice of cell line.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00173987
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  • 5
    Electronic Resource
    Electronic Resource
    Expression of recombinant antibody and secreted alkaline phosphatase in mammalian cells. Influence of cell line and culture system upon production kinetics (1994)
    Springer
    Applied microbiology and biotechnology 40 (1994), S. 851-856 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract. The growth and productivity of an Sp2/0 cell line, F3b10, expressing a recombinant antibody (rAb) and BHK21 cells expressing either the same rAb from the same plasmids (BHK.IgG) or secreted alkaline phosphatase (SEAP) (BHK.SEAP) were investigated. The F3b10 line was grown as a single cell suspension. The BHK lines were grown either as suspended natural aggregates or on Cytodex 3 microcarriers. The data for F3b10 showed that the cell-specific rAb production rate (QsrAb) increased in parallel with increases in the specific growth rate (μ). A similar result was obtained for suspended aggregate cultures of both recombinant BHK cell lines. In contrast, for microcarrier cultures of both BHK cell lines, Qsproduct increased as μ decreased. This report shows that the relationship between cell growth and Qsproduct for the cell lines and products studied is dependent upon the culture process. In systems where recombinant cells are growing as a single cell suspension or within a natural suspension aggregrate, Qsproduct increased with increases in μ. In such systems, the cells have a rounded morphology. When cells were grown on microcarriers, Qsproduct decreased as μ increased. Cells growing attached to a surface are flat and elongated. The observed differences in the relationship of Qsproduct to μ are correlated with changes in cell morphology. The relationship between Qsproduct and μ is also affected by the choice of cell line.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002530050079
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  • 6
    Electronic Resource
    Electronic Resource
    Changes in animal cell natural aggregates in suspended batch cultures (1994)
    Springer
    Applied microbiology and biotechnology 41 (1994), S. 203-209 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Some anchorage-dependent animal cells can form natural aggregates in stirred tanks. Baby hamster kidney (BHK) natural aggregates are described and characterized. Total cell concentration and viability could be obtained after aggregate mechanical aissociation, with negligible cell lysis and no change in cell membrane permeability. During a normal batch run, aggregates were formed immediately after inoculation, a few spherical aggregates increasing size during the initial growth phase. At the end of the growth phase, an increase in aggregate concentration was observed, without a considerable increase in aggregate diameter. At the end of the batch run, 160 h after inoculation, aggregates disintegrated into smaller, non-spherical units, following a sharp viability decrease. Cell concentrations of 1. 2 · 106 cells/ml were obtained, with 60% of the total cells being in aggregates; the cell concentration in aggregates achieved 5 · 108 cells/ml, with a porosity of 55%. Viability was consistently in the range 85–90%, both for aggregate and suspended cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00186960
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  • 7
    Electronic Resource
    Electronic Resource
    Changes in animal cell natural aggregates in suspended batch cultures (1994)
    Springer
    Applied microbiology and biotechnology 41 (1994), S. 203-209 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Some anchorage-dependent animal cells can form natural aggregates in stirred tanks. Baby hamster kidney (BHK) natural aggregates are described and characterized. Total cell concentration and viability could be obtained after aggregate mechanical dissociation, with negligible cell lysis and no change in cell membrane permeability. During a normal batch run, aggregates were formed immediately after inoculation, a few spherical aggregates increasing in size during the initial growth phase. At the end of the growth phase, an increase in aggregate concentration was observed, without a considerable increase in aggregate diameter. At the end of the batch run, 160 h after inoculation, aggregates disintegrated into smaller, non-spherical units, following a sharp viability decrease. Cell concentrations of 1.2⋅106 cells/ml were obtained, with 60% of the total cells being in aggregates; the cell concentration in aggregates achieved 5⋅108 cells/ml, with a porosity of 55%. Viability was consistently in the range 85–90%, both for aggregate and suspended cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002530050131
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  • 8
    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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  • 9
    Electronic Resource
    Electronic Resource
    High cell density reactor for the production ofLactobacillus plantarum (1991)
    Springer
    Journal of industrial microbiology and biotechnology 7 (1991), S. 63-69 
    Details
    ISSN: 1476-5535
    Keywords: Flocculation ; Fluidized bed reactor ; Lactobacillus plantarum ; Lactic starter culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The production of a flocculent strain ofLactobacillus plantarum was performed in a high cell density reactor: a fluidized bed reactor (FBR) with a settler and an external cell recirculation. Two variables were assessed, the recirculation rate (R) and the dilution rate (D). The effect of the latter is much more important than the effect of the former in ensuring a quick start up in the flocculation process. The cell volumetric productivities obtained with this system increase directly with dilution rate and recirculation rate. The values of cell volumetric productivities obtained are considerably higher than those obtained in continuous stirred tank reactors (CSTR) and much higher than in batch reactors.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01575605
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  • 10
    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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