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  • Articles  (70)
  • hybridoma  (67)
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  • Springer  (70)
  • Process Engineering, Biotechnology, Nutrition Technology  (70)
  • 1
    Electronic Resource
    Electronic Resource
    High density culture of mouse-human hybridoma cells using a perfusion culture apparatus with multi-settling zones to separate cells from the culture medium (1989)
    Springer
    Cytotechnology 2 (1989), S. 5-8 
    Details
    ISSN: 1573-0778
    Keywords: hybridoma ; monoclonal antibody ; perfusion culture ; mammalian cell culture ; serum-free culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Mouse-human hybridoma X87X cells were cultivated using a novel perfusion culture apparatus provided with three-settling zones to separate the cells from the culture medium by gravitational settling. The maximum viable cell density in a serum-free culture medium attained 3.0×107 cells/ml, when the specific perfusion rate was set to 2.3 vol day-1, and monoclonal antibody was continuously produced. These results were almost the same as those in the perfusion culture vessel with one settling zone and revealed that the process with a plurality of settling zones is a promising one for scale-up of a gravitation type of perfusion culture vessel.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00365409
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  • 2
    Electronic Resource
    Electronic Resource
    A serum-free medium for hybridoma cell culture (1993)
    Springer
    Cytotechnology 11 (1993), S. 169-174 
    Details
    ISSN: 1573-0778
    Keywords: cell culture ; hybridoma ; monoclonal antibody ; serum-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The effects of several different substances, including insulin, transferrin, ethanolamine, selenite and butyrate on the growth of murine hybridoma 2F7 cells, which secrete monoclonal antibody against small cell lung cancer, were investigated, and a serum-free medium SFMI was formulated. The effects of taurine, spermidine, progesterone and adenine on the cell growth were tested further on the basis of the medium SFMI, and a modified serum-free medium SFM II was established. On the basis of medium SFM II, the substitution tests of ferric citrate for transferrin were carried out, and it was found that transferrin could be replaced. The experiments suggested that the formulated serum-free medium was suitable for 2F7 cell growth and monoclonal antibody secretion, and thus facilitated subsequent purification of monoclonal antibody.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00749866
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  • 3
    Electronic Resource
    Electronic Resource
    Enhanced antibody production associated with altered amino acid metabolism in a hybridoma high-density perfusion culture established by gravity separation (1993)
    Springer
    Cytotechnology 11 (1993), S. 155-166 
    Details
    ISSN: 1573-0778
    Keywords: amino acids ; antibody production ; cell separator ; gravity ; hybridoma ; metabolism ; perfusion culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A high density hybridoma perfusion culture was established by separating and recycling cells from the product stream to the reactor using a simple external sedimentation-based separator — an inclined modified Erlenmeyer flask. After 3 weeks, when the optimal perfusion rate of 1.0 day−1 had been reached, viable cell density stabilized at around 10×106 cells ml−1, a level five times that obtained by simple batch culture. The efficiency of the separator was enhanced by cell flocculation. Specific antibody productivity, which was initially 0.4 μg 1×106 cells−1 h−1, decreased to half that value while cell density was increasing, but recovered to the initial level when the culture finally stabilized at a high cell density. During the final phase, when viable cell density and specific antibody production were high, there was a marked shift in metabolism. Consumption of the two most important substrates for energy generation, glucose and glutamine, caused their broth concentrations to decrease to 1.5 mM and 1 mM, respectively, from input medium concentrations of 25 mM and 10 mM, respectively. At the same time there was an increase in the specific production of glycine and aspartate, their broth concentrations reaching 1.5 mM and 0.02 mM, respectively. We suggest that this shift in metabolism results in enhanced production of ATP from glutamine. The specific glucose consumption and lactate production also indicate that there is a shift to more energy efficient metabolism. The mechanism whereby this leads to enhanced specific antibody production remains to be elucidated. Nevertheless, the combination of high cell density and enhanced productivity obtained with the present perfusion culture resulted in a high monoclonal antibody production −100 mg l−1 d−1.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00749005
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  • 4
    Electronic Resource
    Electronic Resource
    Determination of cell lysis and death kinetics in continuous hybridoma cultures from the measurement of lactate dehydrogenase release (1993)
    Springer
    Cytotechnology 11 (1993), S. 189-195 
    Details
    ISSN: 1573-0778
    Keywords: continuous culture ; death ; hybridoma ; lactate dehydrogenase ; lysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The death of the hybridoma VO 208 in a continuous culture at pH 7 and 6.8 was investigated by measuring both the appearance of visible dead cells which do not exclude the trypan blue dye and the release of lactate dehydrogenase (LDH) in the culture medium. The intracellular LDH was found to be completely released either when live cells lysed or when they were transformed into visible dead cells. No significant lysis of blue dead cells could be observed at the two different pH. Using a LDH balance over the culture system, cell lysis was found negligible at pH 7, but accounted for 20% of the total cell death at pH 6.8. A methodology is proposed to evaluate the rate constants of hybridoma lysis and total death. For the investigated cell line in continuous culture, the calculated total cell death rate constant was found to increase from 0.002 h−1 to 0.01 h−1 when decreasing the pH from 7 to 6.8.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00749869
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  • 5
    Electronic Resource
    Electronic Resource
    Cell suicide in starving hybridoma culture: survival-signal effect of some amino acids (1997)
    Springer
    Cytotechnology 23 (1997), S. 231-239 
    Details
    ISSN: 1573-0778
    Keywords: apoptosis ; hybridoma ; amino acids ; starvation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Two mouse hybridoma cell lines cultured in different basal media withthe iron-rich protein-free supplement were subjected to deliberatestarvation by inoculation into media diluted with saline to 50% or less.In the diluted media the growth was markedly suppressed and a largefraction of cells died by apoptosis. The cells could be rescued fromapoptotic death by individual additions of amino acids, such as glycine,L-alanine, L-serine, L-threonine, L-proline, L-asparagine, L-glutamine,L-histidine, D-serine, β-alanine or taurine. Amino acids withhydrophobic or charged side chains were without effect. The apoptosispreventing activity manifested itself even in extremely diluted media,down to 10% of the standard medium. The activity of L-alanine in theprotection of cells starving in 20% medium was shown also in semicontinuousculture. In the presence of 2 mM L-alanine the steady-state viable cell density more than doubled, with respect to control, andthe apoptotic index dropped from 37% in the control to 16%. It wasconcluded that the apoptosis-preventing amino acids acted as signalmolecules, rather than nutrients, and that the signal had a character ofa survival factor. The specificity of present results, obtained with twodifferent hybridomas, supports our view (Franěk and Chládková-Šrámková, 1995) that the membranetransport macromolecules themselves may play the role of therecognition elements in a signal transduction pathway controlling thesurvival of hybridoma cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/B:CYTO.0000010400.89582.b8
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  • 6
    Electronic Resource
    Electronic Resource
    Purification and characterization of immunoglobulin production stimulating factor derived from human B lymphoblastoid HO-323 cells (1990)
    Springer
    Cytotechnology 3 (1990), S. 189-197 
    Details
    ISSN: 1573-0778
    Keywords: immunoglobulin production stimulating factor ; human B lymphoblastoid cell line HO-323 ; hybridoma ; serum-free culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract An immunoglobulin (Ig) production stimulating factor (IPSF) for hybridomas was found in spent medium of the human B lymphoblastoid cell line, HO-323. The IPSF was purified by serial use of DEAE chromatography, ultrafiltration, gel filtration and HPLC-DEAE chromatography. Purified IPSF was estimated to be a 410 k macro molecule by gel filtration, and contained three types of isomers which were separated from each other by native polyacrylamide gel electrophoresis. All of the isomers were, however, assumed to have the same protein components by SDS polyacrylamide gel electrophoresis. The IPSF was effective for human-human and mouse-mouse hybridomas producing IgM, but not for IgG producers in the experimental condition used here. Human-human hybridoma HF10B4, cultured in IPSF-containing medium, produced 20 times more IgM than in IPSF-free medium under serum-free conditions. The IPSF showed very little proliferation stimulating activity on HF10B4 cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00143681
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  • 7
    Electronic Resource
    Electronic Resource
    Influence of bcl-2 on antibody productivity in high cell density perfusion cultures of hybridoma (1999)
    Springer
    Cytotechnology 30 (1999), S. 95-106 
    Details
    ISSN: 1573-0778
    Keywords: apoptosis ; Bcl-2 ; fixed-bed ; hollow fibre ; hybridoma ; perfusion ; protein-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Apoptosis is an active, genetically determined death mechanism which can be induced by a wide range of physiological factors and by mild stress. It is the predominant form of cell death during the production of antibodies from murine hybridoma cell lines. A number of studies have now demonstrated that the suppression of this death pathway, by means of over-expression of survival genes such as bcl-2, results in improved cellular robustness and antibody productivity during batch culture. In the present study, the influence of bcl-2 expression on hybridoma productivity in two high density perfusion bioreactor systems was investigated. In the first system, a fixed-bed reactor, the DNA content in the spent medium was 25% higher in the control (TB/C3-pEF) culture than that found in the bcl-2 transfected (TB/C3-bcl2) cultures at all perfusion rates. This is indicative of a higher level of cell death in the control cell line. The average antibody concentration for the TB/C3-pEF cell line was 14.9 mg L-1 at perfusion rates of 2.6 and 5.2 d-1. However, for the TB/C3-bcl2 cell line it was 33 mg L-1 at dilution rates of 2 and 4 d-1. A substantial increase in antibody concentration was also found in the Integra Tecnomouse hollow fibre reactor. The antibody titre in the TB/C3-bcl2 cassette was nearly 100% higher than that in the TB/C3-pEF cassette during the cultivation period which lasted 6 weeks. Clearly, these results demonstrate the positive impact of bcl-2 over-expression on production of antibody in hybridoma perfusion cultures.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008055702079
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  • 8
    Electronic Resource
    Electronic Resource
    Prolongation of murine hybridoma cell survival in stationary batch culture by Bcl-xL expression (2000)
    Springer
    Cytotechnology 34 (2000), S. 131-139 
    Details
    ISSN: 1573-0778
    Keywords: apoptosis ; bcl-xL ; cell growth ; cell viability ; hybridoma ; myeloma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract While the ectopic expression of the anti-apoptoticprotein Bcl-2 has been shown to significantly increaseboth cell viability and antibody production in batchculture, some cell lines are refractory to thesemanipulations. For example, the NS/O and theP3x63Ag8.653 murine myelomas, which express highendogenous levels of the Bcl-2 homologue Bcl-xL, areboth resistant to the anti-apoptotic effect of Bcl-2.This indicates that, in these cells, Bcl-2 and Bcl-xLmay be functionally redundant. In order to define therole which Bcl-xL plays in hybridoma cultures, we usedthe Sp2/0-Ag14 cell line. This murine hybridomaexpresses low levels of Bcl-xL and is highly sensitiveto apoptosis induction by cycloheximide (CHX) and byamino acid depletion. Bcl-xL-transfected Sp2/0-Ag14cells were more resistant than the wild type and theplasmid-containing cells to apoptosis induced by CHXand by glutamine depletion. Moreover, when compared tothe vector-transfected control, Bcl-xL-Sp2/0 cellsexhibited a substantial increase in viability instationary batch culture. Interestingly, Sp2/0-Ag14cells overexpressing Bcl-xL showed a growth behaviourthat was similar to the parent myeloma cell lineP3x63Ag8.653. Our results suggest that Bcl-xLexpression levels are sufficient to account for therelative robustness of some hybridoma cell lines instationary batch cultures.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008186302600
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  • 9
    Electronic Resource
    Electronic Resource
    Electron microscopy of hybridoma cells with special regard to monoclonal antibody production (1990)
    Springer
    Cytotechnology 4 (1990), S. 13-28 
    Details
    ISSN: 1573-0778
    Keywords: monoclonal antibody ; hybridoma ; electron microscopy ; endoplasmic reticulum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Electron microscopy of mouse hybridoma cell lines shows that the major difference between non, low and high producer cell lines is the amount of endoplasmic reticulum. Vesicular-tubular or cavernous structures of endoplasmic reticulum, which can survive long after cell death, are particularly abundant in producer cell lines. Immunogold labelling with anti-mouse IgG reveals that antibodies are predominantly located in these structures. The cell membrane undergoes structural changes during the late stages of batch culture with the disappearance of microvilli and the appearance of blebs and deep indentations. Necrosis disrupts the cytoplasmic structures and the nucleus is last to degrade.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00148807
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  • 10
    Electronic Resource
    Electronic Resource
    A comparison of different culture methods for hybridoma propagation and monoclonal antibody production (1990)
    Springer
    Cytotechnology 4 (1990), S. 69-76 
    Details
    ISSN: 1573-0778
    Keywords: hybridoma ; monoclonal ; productivity ; batch ; continuous ; perfusion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A major variable to consider in the production of biologicals from mammalian cell cultures is the mode of operation, be it a batch, continuous, perfusion, fed-batch or other production method. The final choice must consider a number of fundamental and economic issues. Here we present some antibody production data from different cell lines using different modes of production and discuss the important factors for consideration in choosing a production strategy. It was found that the productivity of batch cultures was lower than that obtained in continuous and perfused cultures, but that productivity could be improved by implementing suitable feeding strategies. The antibody productivity of one cell line, MCL1, during exponential phase was not affected by media type or glucose level. The maximum productivity of two cell lines in continuous culture was found to occur at dilution rates below the maximum, from 0.019 to 0.030 hr−1.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00148812
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  • 11
    Electronic Resource
    Electronic Resource
    Antibody production in packed bed reactors using serum-free and protein-free medium (1990)
    Springer
    Cytotechnology 4 (1990), S. 279-283 
    Details
    ISSN: 1573-0778
    Keywords: animal cell culture ; hybridoma ; monoclonal antibody ; packed bed reactor ; continuous culture ; perfusion ; protein-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The present work demonstrates the utility of packed bed reactors for the production of monoclonal antibody. We present data from a continuous process run for the production of over 100 grams of antibody, using serum-free medium. An additional pilot run also demonstrates the potential for continued antibody production under protein-free conditions, using a standard basal medium.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00563788
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  • 12
    Electronic Resource
    Electronic Resource
    Cell cycle kinetics of the accumulation of heavy and light chain immunoglobulin proteins in a mouse hybridoma cell line (1994)
    Springer
    Cytotechnology 14 (1994), S. 205-218 
    Details
    ISSN: 1573-0778
    Keywords: Cell cycle ; flow cytometry ; heavy chain ; hybridoma ; light chain ; monoclonal antibody ; population balance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Rates of accumulation of immunoglobulin proteins have been determined using flow cytometry and population balance equations for exponentially growing murine hybridoma cells in the individual G1, S and G2+M cell cycle phases. A producer cell line that secretes monoclonal antibodies, and a nonproducer clone that synthesizes only κ-light chains were analyzed. The pattern for the kinetics of total intracellular antibody accumulation during the cell cycle is very similar to the previously described pattern for total protein accumulation (Kromenaker & Srienc 1991). The relative mean rate of heavy chain accumulation during the S phase was approximately half the relative mean rate of light chain accumulation during this cell cycle phase. This indicates an unbalanced synthesis of heavy and light chains that becomes most pronounced during this cell cycle phase. The nonproducer cells have on average an intracellular light chain content that is 42% lower than that of the producer cells. The nonproducer cells in the G1 phase with low light chain content did not have a significantly higher rate of light chain accumulation relative to other G1 phase nonproducer cells. This is in sharp contrast to what was observed for the G1 phase producer cells. In addition, although the relative mean rate of accumulation of light chain was negative for G2+M phase nonproducer cells, the magnitude of this relative mean rate was less than half that observed for the producer cells in this cell cycle phase. This suggests that the mechanisms that regulate the transport of fully assembled antibody molecules through the secretion pathway differ from those which regulate the secretion of free light chains. The results reported here indicate that there is a distinct pattern for the cell cycle dynamics of antibody synthesis and secretion in hybridomas. These results are consistent with a model for the dynamics of secretion which suggests that the rate of accumulation of secreted proteins will be greatest for newborn cells due to an interruption of the secretion pathway during mitosis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00749617
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  • 13
    Electronic Resource
    Electronic Resource
    Optimization of serum-free fermentation processes for antibody production (1991)
    Springer
    Cytotechnology 5 (1991), S. 57-67 
    Details
    ISSN: 1573-0778
    Keywords: serum-free medium ; antibody production ; hybridoma ; amino acid analysis ; substrate utilization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Serum free fermentation procedures of cell cultures have got a wide application in production of biochemicals. But, cells cultured in serum free media in general are more sensitive to changes in culture condition, especially to nutrient limitation. There are no substances from serum which can support the cells when conditions are changing. In this study special attention is directed to amino acid utilization of mouse hybridoma in batch, chemostat and perfusion fermentations. Detailed data are presented which show the considerable difference of amino acid consumption rates in different fermentation modes. Already, in batch mode there are differences of the two investigated mouse hybridoma cell lines, although they are derived from the same myeloma line. In chemostat running at a dilution rate representing maximal growth rate most of the consumption rates are significant higher than in batch. On the other hand, in perfusion mode the rates are lower than in batch. This indicates clearly the different conditions of the fermentation modes. Therefore, it is necessary to develop serum free processes under the desired production conditions. An accurate analysis of the process is strongly recommended.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00365534
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  • 14
    Electronic Resource
    Electronic Resource
    Weaning of three hybridoma cell lines to serum free low protein medium (1991)
    Springer
    Cytotechnology 6 (1991), S. 65-78 
    Details
    ISSN: 1573-0778
    Keywords: hybridoma ; monoclonal antibody ; serum free culture ; low protein medium ; weaning protocol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A general weaning procedure is described which allowed a range of hybridomas to be weaned readily off serum without loss of antibody production. Initial work was carried out with one cell line only (SPO1 cells) and one serum substitute containing a final protein concentration of 40 mg l-1. The SPO1 cells were first adapted to a range of readily available basal media and then weaned off serum by a range of protocols. From this work an optimal weaning protocol and basal medium for weaning were determined. These were then used to wean the SPO1 cells and two other cell lines off serum with a second, protein free, serum substitute with varying concentrations of defined proteins added. All three cell lines investigated were readily weaned off serum by this protocol at protein concentrations as low as 1 mg l-1. No loss of antibody production was observed with any of the cell lines. The weaning procedure outlined is both simple and rapid and has been successfully adopted in our laboratory by relatively inexperienced cell culture technicians.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00353704
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  • 15
    Electronic Resource
    Electronic Resource
    Serum-free medium for murine and human lymphoid and hybridoma cell lines (1991)
    Springer
    Cytotechnology 6 (1991), S. 33-38 
    Details
    ISSN: 1573-0778
    Keywords: hybridoma ; monoclonal antibody ; myeloma ; serum-free and tissue plasminogen activator
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We established a serum-free medium of low protein content(125μg/ml) TYI 100, consisting of three hormones and five growth factors for the growth of lymphoid and hybridoma cell lines. In TYI 100 medium, mouse and human hybridomas grew equally well as in RPMI 1640 supplemented with 10% fetal bovine serum (10% FBS) without adaptation to the serum-free medium. TYI 100 medium allowed several passages of mouse hybridoma lines and the total cell number was more than in 10% FBS. TYI 100 medium also supported growth of myelomas and anchorage dependent cell lines, Bowes and CHO, well. TYI 100 medium is composed of inexpensive supplements and is therefor applicable to large scale culture.
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    URL: http://dx.doi.org/10.1007/BF00353700
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  • 16
    Electronic Resource
    Electronic Resource
    Adaptation of hybridoma cells to higher ammonia concentration (1991)
    Springer
    Cytotechnology 7 (1991), S. 103-112 
    Details
    ISSN: 1573-0778
    Keywords: adaptation ; ammonia ; hybridoma ; continuous culture ; serum-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Using two mouse-mouse hybridoma cell lines, the response to ammonia step and serial changes was investigated in batch and continuous cultures with serum-free medium. The inhibitory effect of ammonia on cell growth depended on the cultivation mode, and differed markedly between cell lines. The cell line, 4C10B6 producing IgG monoclonal antibody against Pseudomonas, showed a high adaptation ability to ammonia. The 4C10B6 cells could grow under ammonia concentration as high as 21 mmol/l NH4Cl with a viability of 80% in the continuous culture with serial increase in ammonia concentration. Whereas, in the batch culture with ammonia step change the cell growth completely ceased at 12 mmol/l NH4Cl. The other cell line, TO-405 producing IgG monoclonal antibody against hepatitis B surface antigen, could not adapt to ammonia, and the cell growth did not occur at 9 mmol/l NH4Cl even under the ammonia serial change.
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    URL: http://dx.doi.org/10.1007/BF00350916
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  • 17
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    Hybridoma growth and productivity: effects of conditioned medium and of inoculum size (1999)
    Springer
    Cytotechnology 29 (1999), S. 1-10 
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    ISSN: 1573-0778
    Keywords: batch culture ; conditioned medium ; growth ; hybridoma ; inoculum ; protein productivity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Apart from gas concentrations, temperature, and pH, generally only the initial conditions can be manipulated in batch culture. Inoculum size and initial conditioned medium concentration represent two important considerations for optimal batch production. Two hybridoma cell lines were used to assess the impact of these initial conditions on population growth and monoclonal antibody productivity in suspension batch culture. Varying initial cell concentration over the range of 1.0 × 105 cells mL-1 to 3.0 × 105 cells mL-1 did not affect maximum product titre or maximum volumetric cell-hours attained. Initial percent of conditioned medium up to 40 percent strongly impacted on population growth and productivity, with initial levels of 30 to 40% conditioned medium reducing or eliminating lag phase and increasing average viable cell density. However, specific productivity and product titre declined with increasing initial percent conditioned medium, even on a per volume of fresh medium basis. Glutamine and glucose depletion or ammonia toxicity could cause depressed product titres when conditioned medium is used. Glutamine and glucose levels can easily be replenished in conditioned medium at minimal cost, and ammonia can be removed. Specific productivity was higher during cyclic batch operating mode than during batch operating mode. This may be because cyclic batch operating mode results in an incidental volume of conditioned medium at the beginning of each cycle. A two stage, cyclic-batch/batch operating mode can be employed to fully utilize medium and maximize product titre.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008060802286
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  • 18
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    The effect of glucose and glutamine on the intracellular nucleotide pool and oxygen uptake rate of a murine hybridoma (2000)
    Springer
    Cytotechnology 34 (2000), S. 47-57 
    Details
    ISSN: 1573-0778
    Keywords: glucose ; glutamine ; hybridoma ; nucleotides oxygen
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The effects of media concentrations of glucose andglutamine on the intracellular nucleotide pools andoxygen uptake rates of a murine antibody-secretinghybridoma cell line were investigated. Cells takenfrom mid-exponential phase of growth were incubated inmedium containing varying concentrations of glucose(0–25 mM) and glutamine (0–9 mM). The intracellularconcentrations of ATP, GTP, UTP and CTP, and theadenylate energy charge increased concomitantly withthe medium glucose concentration. The total adenylatenucleotide concentration did not change over a glucose concentration range of 1–25 mM but therelative levels of AMP, ADP and ATP changed as theenergy charge increased from 0.36 to 0.96. Themaximum oxygen uptake rate (OUR) was obtained in thepresence of 0.1–1 mM glucose. However at glucoseconcentrations 〉1 mM the OUR decreased suggestinga lower level of aerobic metabolism as a result of theCrabtree effect.A low concentration of glutamine (0.5 mM) caused asignificant increase (45–128%) in the ATP, GTP,CTP, UTP, UDP-GNac, and NAD pools and a doubling ofthe OUR compared to glutamine-free cultures. Theminimal concentration of glutamine also caused anincrease in the total adenylate pool indicating thatthe amino acid may stimulate thede novosynthesis of nucleotides. However, all nucleotidepools and the OUR remained unchanged within the rangeof 0.5–9 mM glutamine.Glucose was shown to be the major substrate forenergy metabolism. It was estimated that in thepresence of high concentrations of glucose (10–25 mM),glutamine provided the energy for the maintenance ofup to 28% of the intracellular ATP pool, whereas theremainder was provided by glucose metabolism.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008154615643
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  • 19
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    High cell density perfusion culture of hybridoma cells recycling high molecular weight components (1988)
    Springer
    Cytotechnology 1 (1988), S. 171-178 
    Details
    ISSN: 1573-0778
    Keywords: high-density ; hybridoma ; monoclonal antibody ; perfusion culture ; serum-free ; suspension culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We have developed a high cell density and high product concentration culture system recycling high molecular weight components. The production of monoclonal antibodies in high concentration was performed by this culture system with mouse human hybridoma H2 and V6 cells in serum-free defined media. The concentration of IgG after 48 days culture of H2 cells in ITES-eRDF reached 2 mg/ml and the purity of IgG in culture fluid was 61%. In addition, high molecular weight components in serum-free media, such as transferrin or BSA, could be reduced to 5% of the original concentration.
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    URL: http://dx.doi.org/10.1007/BF00146818
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    An immobilized hybridoma culture perfusion system for production of monoclonal antibodies (1988)
    Springer
    Cytotechnology 1 (1988), S. 331-337 
    Details
    ISSN: 1573-0778
    Keywords: hybridoma ; immobilization ; monoclonal antibodies ; perfusion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A fixed bed perfusion system for hybridoma cell immobilization is presented. The system consists of a culturing vessel (300 ml total volume) in which polyurethane (PU) sponges in the form of small cubes of about 5 mm sides are packed. Cells are immobilized by physical entrapment in the foam matrix. By entrapment of the cells in the pores of the matrix high cell concentration can be maintained in a mechanically protected environment. Medium is continuously circulated by an airlift pump mounted in the cell-free chamber (700 ml total volume). Medium flow rate, feeding rate, dissolved oxygen, pH, nutrient uptake and waste product formation can be easily monitored and controlled. Steady state conditions are established with medium dilution rates of 1.0–1.5 reactor volume per day. The steady state is characterized by a constant cell density, constant culture volume and constant glucose and lactate levels. Cell-free supernatant is collected continuously in a cold room adjacent to the 37°C culture room. Monoclonal antibodies (MAb) are produced at a concentration of 150–200 μg/ml for several weeks. An important feature of the system is the capacity to maintain a population of cells after the growth phase in a non-proliferating state for extended time periods expressing high titers of MAb.
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    URL: http://dx.doi.org/10.1007/BF00365078
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    Distinct volume distribution of viable and non-viable hybridoma cells: A flow cytometric study (1989)
    Springer
    Cytotechnology 2 (1989), S. 85-94 
    Details
    ISSN: 1573-0778
    Keywords: hybridoma ; cell volume ; cell culture ; flow cytometry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Light scattering properties of hybridoma cells were examined with flow cytometry. Viable and dead cells form two distinct populations. The distribution of the two populations changes during a batch culture. the concentration of dead cells measured by flow cytometry correlates well to that measured by hemacytometer. The distribution based on small-angle light scattering is similar to the distribution based on volume as measured by Elzone particle counter. It thus appears that viable cells form the population with a larger mean cell volume. The results also indicate that the volume of viable cells decreases during the cultivation while that of dead cells remains relatively constant.
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    URL: http://dx.doi.org/10.1007/BF00386140
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    Amino acid metabolism during batch culture of a murine hybridoma, AFP-27 (1996)
    Springer
    Cytotechnology 21 (1996), S. 111-120 
    Details
    ISSN: 1573-0778
    Keywords: hybridoma ; extracellular and intracellular amino acids ; glucose ; lactate ; batch culture ; enriched media
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract This paper presents batch culture data of the murine hybridoma, AFP-27, cultured in conventional basal media and in a nutrient-rich modified version. Expression of antibody was fivefold higher in the enriched formulation, with significant product secretion in the decline phase. Cultures were initiated at conventional inculation densities (1 ∼ 2 × 105 viable cells ml−1) and high inoculation densities (1.5 ∼ 1.7 × 106 viable cells ml−1). Amino acid levels have been reported for all cultures, with apparent differences described. Relative levels of intracellular amino acids are also reported, with significant accumulation of proline, glycine and alanine. The results have significance in the design of enriched media which are clearly beneficial for commercial production of antibodies from hybridomas.
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    URL: http://dx.doi.org/10.1007/BF02215661
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    The crystal violet nuclei staining technique leads to anomalous results in monitoring mammalian cell cultures (1996)
    Springer
    Cytotechnology 21 (1996), S. 73-80 
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    ISSN: 1573-0778
    Keywords: cell counting ; CHO ; crystal violet ; hybridoma ; trypan blue ; Vero-cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Nuclear counts determined by crystal violet staining from samples of stationary or microcarrier cultures of hybridomas, CHO or Vero cells were consistently and significantly higher than cell concentrations determined by the trypan blue or Coulter counter methods. This difference was attributed to the presence of a significant proportion of binucleated cells, which are assumed to be 35% of the cell population in the stationary phase of Vero cultures. The proportion of such cells during exponential growth was variable. However, continuous sub-culture of these cells induced a degree of synchrony during growth which resulted in a cyclic variation of the difference between the cell and nuclei counting techniques. This data indicates that care should be taken in interpreting cell culture profiles based solely on crystal violet nuclei staining counts.
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    URL: http://dx.doi.org/10.1007/BF00364838
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    Descriptive parameter evaluation in mammalian cell culture (1998)
    Springer
    Cytotechnology 26 (1998), S. 139-152 
    Details
    ISSN: 1573-0778
    Keywords: batch kinetics ; cell cycle ; cell-hours ; hybridoma ; population parameters ; productivity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Several methods exist for assessing population growth and protein productivity in mammalian cell culture. These methods were critically examined here, based on experiments with two hybridoma cell lines. It is shown that mammalian cell culture parameters must be evaluated on the same basis. In batch culture mode most data is obtained on a cumulative basis (protein product titre, substrate concentration, metabolic byproduct concentration). A simple numerical integration technique can be employed to convert cell concentration data to a cumulative basis (cell-hours). The hybridoma lines used in this study included a nutritionally non-fastidious line producing low levels of MAb and a nutritionally fastidious hybridoma with high productivity. In both cases the cell-hour approach was the most appropriate means of expressing the relationship between protein productivity and cell population dynamics. The cell-hour approach could be used as the basis for all metabolic population parameter evaluations. This method has the potential to be used successfully for both prediction and optimization purposes.
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    URL: http://dx.doi.org/10.1023/A:1007940119503
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    Modulation of cell cycle progression and of antibody production in mouse hybridomas by a nucleotide analogue (1998)
    Springer
    Cytotechnology 28 (1998), S. 65-72 
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    ISSN: 1573-0778
    Keywords: acyclic nucleoside phosphonate ; cell cycle ; hybridoma ; specific MAb production rate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The nucleotide analogue 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) has been identified as a powerful antiproliferative substance when acting on hybridoma cells. In the range of 10 nM to 100 nM concentrations this agent reduces cell growth rate, while its apoptosis-inducing activity is marginal. Marked induction of apoptosis can be observed at micromolar and higher order concentrations. In PMEG-supplemented media the cell cycle progression is perturbed, the flow-cytometric DNA profile shows a higher proportion of cells in the S and G2/M phases of the cell cycle. Concomitantly with the reduction of the growth rate, the specific monoclonal antibody production rate may rise by 20–27%. Addition of PMEG at the end of the exponential phase of a batch culture results in an enhancement of the final monoclonal antibody concentration.
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    URL: http://dx.doi.org/10.1023/A:1008017328061
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    Variable functions of bcl-2 in mediating bioreactor stress- induced apoptosis in hybridoma cells (1998)
    Springer
    Cytotechnology 28 (1998), S. 177-188 
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    ISSN: 1573-0778
    Keywords: apoptosis ; bcl-2 ; cell death ; hybridoma ; osmolarity ; pH ; shear ; stress
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract It has been demonstrated that the cell lines used for production of biopharmaceuticals are highly susceptible to apoptosis, and that over-expression of the bcl-2 oncogene can protect cells from death. Stress associated with the deprivation of nutrients has been shown to be the main cause of apoptosis in culture. We have extended these studies by investigating the mechanism of cell death under conditions of sub-optimal pH, shear stress and hyperosmolarity, and the protective action of bcl-2 over-expression. At pH 6, there was no clear evidence of protection from cell death. However, at pH 8, the viability of the bcl-2 transfected cells was about 20% higher relative to the control cells. Cultivation of control cells in a flat bottomed bioreactor with a magnetic stirrer bar without a pivot ring resulted in exposure of the cells to a high attrition effect. As a result, cell growth was retarded and a high level of cell death by apoptosis was observed. Under the same conditions, the bcl-2 transfected cell line exhibited a nearly five fold increase in viable cell number. This finding indicates that under apoptosis-suppressed conditions, shear stress can stimulate cell growth. Batch cultivation of both control and bcl-2 transfected cells in 350 and 400 mOsm media resulted in suppression of cell growth, athough the effect was most marked in the control cell line. Adaptation of control cells to 400 mOsm proved to be impossible to achieve. However, the bcl-2 transfected cells exhibited resistance to the osmotic stress resulting in long term adaptation to a high salt environment. Specific productivity of bcl-2 transfected cells grown in high osmolarity medium was 100% higher than that produced by non- adapted bcl-2 transfected cells grown in normal osmolarity medium. These results demonstrate that bcl-2 has a beneficial effect on hybridoma cultivation under a wide range of culture stresses.
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    URL: http://dx.doi.org/10.1023/A:1008002319400
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    High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge (1990)
    Springer
    Cytotechnology 3 (1990), S. 239-244 
    Details
    ISSN: 1573-0778
    Keywords: hybridoma ; perfusion culture ; monoclonal antibody ; serum-free culture ; centrifuge
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The influence of centrifugal force on the growth of cells was examined by exposing the cells of the mouse-human hybridoma X87 line to centrifugal force (100–500 G) for ten minutes twice a day and comparing the static culture with that of unexposed cells. In this experiment, both cell proliferation and specific antibody productivity were independent of the centrifugal effect, and gave the same results as in the case of no exposure to centrifugal force. High density cultivation of the mouse-human hybridoma X87 line was obtained by a perfusion system where the cells were separated from the culture medium by continuous centrifugation. In the serum-free culture, the maximum viable cell density exceeded 107 cells/ml, and monoclonal antibody was stably produced for 37 days. The results in this culture were equivalent to those obtained by intermittent centrifugal cell separation from the culture medium, and separation by gravitational settlement.
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    URL: http://dx.doi.org/10.1007/BF00365487
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    Enhanced antibody production of human-human hybridomas by retinoic acid (2000)
    Springer
    Cytotechnology 33 (2000), S. 83-88 
    Details
    ISSN: 1573-0778
    Keywords: antibody production ; human monoclonal antibody ; hybridoma ; retinoic acid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The enhancement of human monoclonal antibody production by retinoic acid (RA) was evaluated usingthe human-human hybridoma cell line BD9 underserum-free culture condition. The amount of humanIgG secreted by BD9 hybriodmas was enhanced abouteight-fold by treatment with 10-7 M of RA for 4days. Northern blot analysis showed that both mRNAlevels of the IgG light and heavy chains were markedlyincreased by RA when compared with control without RAtreatment. On the other hand, it was found thatcontinuous treatment of cells with RA was not alwaysrequired to exhibit the enhancing effect, suggestingthat RA may act as a trigger for IgG gene expression. The comparison between extra- and intracellular IgGamounts by immunoblot analysis suggests that thesecretion rate of IgG may be accelerated by RAtreatment. These results suggest that RA may be aneffective culture additive for efficient production ofhuman monoclonal antibody using human-humanhybridomas.
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    URL: http://dx.doi.org/10.1023/A:1008155609072
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    Calcium alginate immobilized hybridomas grown using a fluidized-bed perfusion system with a protein-free medium (1994)
    Springer
    Cytotechnology 14 (1994), S. 109-117 
    Details
    ISSN: 1573-0778
    Keywords: calcium alginate ; hybridoma ; metabolism ; fluidized-bed ; perfusion ; protein-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Hybridoma SPO1 cells were immobilized in calcium alginate beads and were further grown in a fluidized-bed perfusion system with a protein-free medium. The presence of serum in the steps of entrapment was shown to be helpful for the preservation of cell viability. Each step during immobilization was investigated with respect to the extent of cell damage caused. The immobilization process using small beads caused a lower cell viability initially but allowed a higher rate of cell growth subsequently, compared to those in large beads. In a perfusion system for the continuous production of monoclonal antibodies (MAb), the viable cell density reached 2×107 cells per ml of beads with a viability of 40%. Compared with the cells in suspension culture, the immobilized SPO1 cells showed higher viable cell based specific rates of substrate uptake (glucose and glutamine) and of MAb production. A significant drop in the formation of lactate after the cell growth entered a steady state suggested a higher activity of the Tricarboxylic Acid Cycle in the cells when the cell density became high.
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    URL: http://dx.doi.org/10.1007/BF00758175
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    Induction of apoptosis in oxygen-deprived cultures of hybridoma cells (1994)
    Springer
    Cytotechnology 15 (1994), S. 117-128 
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    ISSN: 1573-0778
    Keywords: Animal cell culture ; anoxia ; apoptosis ; cell death ; hybridoma ; hypoxia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract It is now well documented that apoptosis represents the prevalent mode of cell death in hybridoma cultures. Apoptotic or programmed cell death occurs spontaneously in late exponential phase of batch cultures. Until lately, no specific triggering factors had been identified. Recently, we observed that glutamine, cystine or glucose deprivation induced apoptosis in both hybridoma and myeloma cell lines whereas accumulation of toxic metabolites induced necrotic cell death in these cells. Other triggering factors such as oxygen deprivation might also be responsible for induction of apoptosis. In the present study, induction of cell death by exposure to anoxia was examined in batch culture of the SP2/0-derived hybridoma D5 clone. The mode of cell death was studied by morphological examination of acridine orange-ethidium bromide stained cells in a 1.5 L bioreactor culture grown under anoxic conditions for 75 hours. Under such conditions, viable cell density levelled off rapidly and remained constant for 25 hours. After 45 hours of anoxia, cell viability had decreased to 30% and the dead cell population was found to be 90% apoptotic. In terms of cellular metabolism, anoxia resulted in an increase in the utilization rates of glucose and arginine, and in a decrease in the utilization rate of glutamine. The lactate production rate and the yield of lactate on glucose increased significantly while the MAb production rate decreased. These results demonstrate that glycolysis becomes the main source of energy under anoxic conditions. Cells incubated for 10 hours or less under anoxic conditions were able to recuperate almost immediately and displayed normal growth rates when reincubated in oxic conditions whereas cells incubated for 22 hours or more displayed reduced growth rates. Nonetheless, even after 22 h or 29 h of anoxia, cells reincubated in oxic conditions showed no further progression into apoptosis. Therefore, upon removal of the triggering signal, induction of apoptosis ceased.
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    URL: http://dx.doi.org/10.1007/BF00762386
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    Software sensors for the monitoring of perfusion cultures: Evaluation of the hybridoma density and the medium composition from glucose concentration measurements (1994)
    Springer
    Cytotechnology 15 (1994), S. 291-299 
    Details
    ISSN: 1573-0778
    Keywords: Estimator ; Extended Kalman Filter ; hybridoma ; kinetic model ; perfusion culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract New software sensors based on the Extended Kalman Filter technique have been developed for the monitoring of animal cell perfusion cultures. They use a kinetic model describing the growth, death and metabolism of hybridoma cells as a function of the medium composition. The model was initially validated on a batch culture and found to correctly predict the continuous perfusion culture kinetics, except for the production of ammonia and lactate. Using the measurement of a single component in the culture medium, in this case glucose, the Extended Kalman Filter provides an excellent evaluation of the time variation of the concentrations of living and dead cells, of glutamine and antibodies, during the whole perfusion culture for a retained cell density rising from 1 to 11×106 cells.ml−1 inside the reactor.
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    URL: http://dx.doi.org/10.1007/BF00762404
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    Design of a bubble-swawm bioreactor for animal cell culture (1994)
    Springer
    Cytotechnology 15 (1994), S. 301-309 
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    ISSN: 1573-0778
    Keywords: Aeration ; stirred bioreactor ; bubble-swarm ; hybridoma ; oxygen transfer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A stationary bubble-swarm has been used to aerate a mammalian cell culture bioreactor with an extremely low gas flow rate. Prolonging the residence time of the gas bubbles within the medium improved the efficiency of the gas transfer into the liquid phase and suppressed foam formation. An appropriate field of speed gradients prevented the bubbles from rising to the surface. This aeration method achieves an almost 90% transfer of oxygen supplied by the bubbles. Consequently, it is able to supply cells with oxygen even at high cell densities, while sparging with a gas flow of only 0.22·10−3–1.45·10−3 vvm (30–200 ml/h). The reactor design, the oxygen transfer rates and the high efficiency of the system are presented. Two repeated batch cultures of a rat-mouse hybridoma cell line are compared with a surface-aerated spinner culture. The used cell culture medium was serum-free, either with or without BSA and did not contain surfactants or other cell protecting agents. One batch is discussed in detail for oxygen supply, amino acid consumption and specific antibody production.
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    URL: http://dx.doi.org/10.1007/BF00762405
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    Selecting and designing cell lines for improved physiological characteristics (1994)
    Springer
    Cytotechnology 15 (1994), S. 11-16 
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    ISSN: 1573-0778
    Keywords: Chemostat ; cholesterol ; choline ; glutamine ; glutamine synthetase ; hybridoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We have developed several approaches to create cell lines with improved characteristics in cell culture. In some cases it has been possible to isolate natural variants with useful properties. Cholesterol independent variants of the mouse NSO myeloma cell line were isolated by cloning in a selective medium. A glutamine independent variant of a hyridoma was isolated by continuous (chemostat) culture under glutamine limited conditions in the presence of glutamate. Choline independent cells were isolated from a choline limited chemostat. In an alternative approach to modifying cell behaviour, we have used recombinant DNA techniques to introduce the glutamine synthetase (GS) gene to a hybridoma. This resulted in glutamine independence and increased productivity.
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    Enhanced specific antibody productivity of calcium alginate-entrapped hybridoma is cell line-specific (1994)
    Springer
    Cytotechnology 16 (1994), S. 1-15 
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    ISSN: 1573-0778
    Keywords: Flow cytometry ; hybridoma ; immobilization ; specific antibody productivity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract In order to determine whether the enhanced specific antibody productivity (q MAb ) of calcium alginate-entrapped hybridoma is cell line-specific, calcium alginate-entrapped hybridomas (4A2 and DB9G8) were cultivated under the condition where we had previously observed significantly enhancedq MAb of calcium alginate-entrapped S3H5/γ2bA2 hybridoma. Unlike S3H5/γ2bA2 hybridoma, neither 4A2 nor DB9G8 hybridomas showed persistently enhancedq MAb when they were entrapped in calcium alginate beads. The enhancedq MAb of entrapped 4A2 and DB9G8 hybridomas, which was 2–3 times higher than theq MAb of free-suspended cells in a control experiment, was observed only during the early stage of the culture. During the early stage of the culture, the viable cell concentration decreased probably due to cell damage during the entrapment process. As cell growth resumed, theq MAb decreased to the similar level ofq MAb of free-suspended cells within 5–7 days. Thus, we conclude that the enhancedq MAb of calcium alginate-entrapped hybridomas is cell line-specific.
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    URL: http://dx.doi.org/10.1007/BF00761774
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    Peptone, a low-cost growth-promoting nutrient for intensive animal cell culture (1994)
    Springer
    Cytotechnology 16 (1994), S. 17-26 
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    ISSN: 1573-0778
    Keywords: Cell culture ; peptone ; media ; intensive culture ; hybridoma ; spin-filter
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The effect of addition of peptone to serum-free and serum supplemented media for the growth of hybridoma cells in various systems was studied. Supplementation of defined medium with either proteose peptone or meat peptone resulted in significant increases in cell number and specific monoclonal antibody production in batch culture system. Other peptones were either inactive or less effective. In continuous culture, using medium supplemented with new born calf serum, the addition of peptone resulted in 125% and 150% increases in cell and antibody concentrations respectively. Similar increase in cell number (128%) was also obtained in spin-filter perfusion culture when medium was supplemented with peptone. By comparison, the substitution of a defined 1xMEM amino acids mixture resulted in only a 50% increase. At higher perfusion rates the cell number maintained in steady state using peptone supplement could be increased to 1.3×107 cells ml−1 while the serum concentration was reduced from 5% to 1% at a perfusion rate of 2.5 volumes per day.
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    Hybridoma cells in a protein-free medium within a composite gel perfusion bioreactor (1994)
    Springer
    Cytotechnology 16 (1994), S. 51-58 
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    ISSN: 1573-0778
    Keywords: Composite gel ; hybridoma ; protein-free medium ; perfusion ; metabolic activities ; growth pattern
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A composite gel system has been developed combining the chemical and physical properties of calcium alginate and agarose gels. The results of growing composite gel immobilized hybridoma SPO1 cells in a protein-free medium within a fluidized-bed perfusion bioreactor are presented in this paper. During the continuous operation of this system, the total cell density reached 3.9×107 cells per ml of beads (viability 79.6%). The specific productivity of monoclonal antibody of the immobilized hybridoma cells reached more than 1.5 μg per 106 viable cells per hour, compared with 0.5 for non-immobilized viable cells grown in a one liter agitated bioreactor with the same medium. Significant increases in cell metabolic activities, including substrate utilization and byproduct formation, were also observed. Leaching of materials from the beads was evident and the major fraction of released materials was alginate.
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    URL: http://dx.doi.org/10.1007/BF00761779
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    The culture of rat myeloma and rat hybridoma cells in a protein-free medium (1995)
    Springer
    Cytotechnology 17 (1995), S. 193-202 
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    ISSN: 1573-0778
    Keywords: Y0 ; hybridoma ; myeloma ; protein-free ; culture medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Y0 is a rat x rat hybridoma cell line, which does not secrete immunoglobulin, produced using a fusion partner derived from the Y3 (Y3,Ag.1.2.3) rat myoloma cell line. Y0 and Y3 have both been widely used as fusion partners in the production of rat x rat hybridomas. Y0 has also been used in recombinant gene technology. Y0 cells grown in shake flask culture, using RPMI 1640 medium with 4mM l-glutamine and 5% foetal bovine serum, reached a maximal cell density of 1.5×106 cells ml−1 with 86% viability. Y0 cells which has been adapted to grow in ABC protein-free medium reached a maximal density, in shake flask culture, of 8.75×105 cells ml−1 with 79% viability. An improved protein-free medium, designated W38 medium, was developed. In shake flask culture, W38 medium supported Y0 cell growth to a density of 2.02×106 cells ml−1 with 96% viability. Two Y3 hybridomas, YID 13.9.4 cells and SAM 618 cells were adapted to growth in W38 medium. For both hybridomas, cell growth and product yield in shake flask culture using W38 medium was superior to that obtained with serum-containing RPMI 1640 medium.
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    URL: http://dx.doi.org/10.1007/BF00749657
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    Cell suicide in starving hybridoma culture: survival-signal effect of some amino acids (1996)
    Springer
    Cytotechnology 21 (1996), S. 81-89 
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    ISSN: 1573-0778
    Keywords: apoptosis ; hybridoma ; amino acids ; starvation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Two mouse hybridoma cell lines cultured in different basal media with the iron-rich protein-free supplement were subjected to deliberate starvation by inoculation into media diluted with saline to 50% or less. In the diluted media the growth was markedly suppressed and a large fraction of cells died by apoptosis. The cells could be rescued from apoptotic death by individual additions of amino acids, such as glycine, L-alanine, L-serine, L-threonine, L-proline, L-asparagine, L-glutamine, L-histidine, D-serine, β-alanine or taurine. Amino acids with hydrophobic or charged side chains were without effect. The apoptosis preventing activity manifested itself even in extremely diluted media, down to 10% of the standard medium. The activity of L-alanine in the protection of cells starving in 20% medium was shown also in semicontinuous culture. In the presence of 2 mM L-alanine the steady-state viable cell density more than doubled, with respect to control, and the apoptotic index dropped from 37% in the control to 16%. It was concluded that the apoptosis-preventing amino acids acted as signal molecules, rather than nutrients, and that the signal had a character of a survival factor. The specificity of present results, obtained with two different hybridomas, supports our view (Franěk and Chládková-Šrámková, 1995) that the membrane transport macromolecules themselves may play the role of the recognition elements in a signal transduction pathway controlling the survival of hybridoma cells.
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    Establishing apoptosis resistant cell lines for improving protein productivity of cell culture (1997)
    Springer
    Cytotechnology 23 (1997), S. 55-59 
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    ISSN: 1573-0778
    Keywords: apoptosis resistant ; bag–1 ; bcl–2 ; COS–1 ; hybridoma ; protein production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The authors established apoptosis resistant COS–1, myeloma, hybridoma, and Friend leukemia cell lines by genetically engineering cells, aiming at more efficient protein production by cell culture. COS–1 cells, which are most widely used for eukariotic gene expression, were transfected with human bcl–2 gene. Both bcl–2 and mock transfected COS–1 cells were cultured at low (0.2%) serum concentration for 9 days. The final viable cell number of the bcl–2 transfected cells was ninefold of that of the mock transfectants. Both bcl–2 and mock transfectants were further transfected with the vector pcDNA-λ containing SV40 ori and immunoglobulin λ gene for transiently expressing λ protein. The bcl–2 expressing COS–1 cells produced more λ protein than the mock transfected COS–1 cells after 4 days posttransfection. Mouse myeloma p3-X63-Ag.8.653 cells, which are widely used as the partner for preparing hybridoma, and hybridoma 2E3 cells were transfected with human bcl–2 gene. Both bcl–2 transfected myeloma and hybridoma survived longer than the corresponding original cells in batch culture. The bcl–2 transfected 2E3 cells survived 2 to 4 four days longer in culture, producing 1.5- to 4-fold amount of antibody in comparison with the mock transfectants. Coexpression of bag–1 with bcl–2 improved survival of hybridoma 2E3 cells more than bcl–2 expression alone. The bag–1 and bcl–2 coexpressing cells produced more IgG than the the cells expressing bcl–2 alone. Apoptosis of Friend murine erythroleukemia(F-MEL) cells was suppressed with antisense c-jun expression. The antisense c-jun expressing cells survived 16 days at non-growth state.
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    Hybridoma growth in a new generation hollow fibre bioreactor: antibody productivity and consistency (1997)
    Springer
    Cytotechnology 24 (1997), S. 109-119 
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    ISSN: 1573-0778
    Keywords: antibody consistency ; hollow fibre bioreactor ; hybridoma ; monoclonal antibody
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract This paper analyses the performance of MAbMaxTM/TricentricTM, a new generation hollow fibre bioreactor, for hybridoma growth and antibody productivity, the down stream processing of monoclonal antibody harvests throughout the run and the further control of antibody quality consistency. Handling and process parameters were optimised using a mouse hybridoma, IgG1K secretor, and then confirmed with several other hybridomas. Cells were kept at optimal viability during an unusually long period of time and a continuously high production of antibodies was detected over several months. Foetal bovine serum concentration was reduced to 1\% and the effects of weaning of cells from serum were monitored in terms of cell metabolism and antibody productivity. Antibody harvests collected at regular intervals throughout the run (2 to 12 weeks) were purified using affinity chromatography on a recombinant protein A/G matrix and then analysed in terms of antigen binding properties, isoelectric forms and oligosaccharide structures, in order 1) to control antibody quality consistency as a function of time and serum concentration and 2) to compare antibody characteristics as a function of culture conditions, in vitro bioreactor cultivation versus in vivo mouse ascite cultivation.
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    Selective removal of ammonia from animal cell culture broth (1991)
    Springer
    Cytotechnology 6 (1991), S. 121-130 
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    ISSN: 1573-0778
    Keywords: ammonia removal ; hybridoma ; HBs monoclonal antibody ; zeolite
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Serum-free perfusion cultures of hybridoma TO-405 cells were carried out in spinner flasks coupled with zeolite A-3 packed beads. Ammonia was selectively removed from the culture broth by passing cell free permeate from ceramic cross flow filtration, through the zeolite packed bed. Ammonia concentration in the culture broth was effectively maintained between 1 to 4 mmol/l which was below the inhibitory concentration for cell growth. Maximum cell density levels of 107 cells/ml as well as improved percentage cell viability higher than in serum-supplemented cultures were feasible in this system. The possible effects of shear stress, generated by variation of the flow rates of the broth through the ceramic filter module, on the growth of the hybridoma cells were investigated. Backwashing, by reversing the direction of the permeate, was found necessary to prolong the life of the filter. Variation of the flow rates of the broth through the ceramic module between 0.29 m/s to 0.59 m/s did not cause immediate cell damage but growth was repressed at the higher flow rate. This study also showed that glutamine appears to be one of the factors limiting the growth of the hybridoma cells.
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    URL: http://dx.doi.org/10.1007/BF00373029
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    Co-expression of bcl-2 and bag-1, apoptosis suppressing genes, prolonged viable culture period of hybridoma and enhanced antibody production (1999)
    Springer
    Cytotechnology 31 (1999), S. 143-151 
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    ISSN: 1573-0778
    Keywords: antibody productivity ; apoptosis ; BAG-1 ; Bcl-2 ; cell survival ; hybridoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Human bcl-2 and bag-1 DNA were introduced into mouse hybridoma 2E3- O cells and expressed. The expression of bcl-2 in BCMGneo-bcl2 transfectants was confirmed by ELISA and that of bag-1 in pZeo-bag1 was confirmed by western blotting. In batch cultures, the over-expression of bcl-2 prolonged the culture period by 2 days and co-expression of bcl-2 and bag-1 prolonged the culture period by 3 days. The delayed increase in the dead cell number in culture of the bcl-2 and bag-1 cotransfectant indicated the additional antiapoptosis effect of bcl-2 and bag-1 cotransfection in comparison with the bcl-2 only transfection. The bcl-2 transfectants (2E3O-Bcl2) produced antibody twofold per batch culture in comparison with 2E3-O cells transfected with BCMGSneo (2E3O-Mock). Enhancement of this MoAb production was due to the improved survival of the cells and was not due to stimulation of antibody production rate per cell by Bcl-2 expression. And the bcl-2 and bag-1 co-transfectant (2E3O-Bcl2-BAG1) produced antibody approximately fourfold of 2E3O-Mock per batch culture. Enhancement of this MoAb production was due to the improved survival of the cells and was partly due to stimulation of MoAb production rate per cell in the non-growing phase by the cotransfection. The method to engineer hybridoma cells genetically with bcl-2 and bag-1 for increasing viability and productivity would be widely applied for improving antibody productivity of hybridoma cultures.
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    The inhibitory effect of glutamate on the growth of a murine hybridoma is caused by competitive inhibition of the x- C transport system required for cystine utilization (2000)
    Springer
    Cytotechnology 32 (2000), S. 31-43 
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    ISSN: 1573-0778
    Keywords: hybridoma ; glutamate ; cystine ; transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Glutamic acid was found to be growth inhibitory to a murinelymphocyte hybridoma in a concentration-dependent manner from 3to 12 mM glutamate. At 12 mM glutamate there was a 70% decreasein the specific growth rate of the cells. Attempts to alleviateinhibition or adapt cells to growth in glutamate-based mediawere unsuccessful. It is proposed that elevated glutamate levelsimpair adequate uptake of cystine, a critical amino acid for thesynthesis of glutathione. Glutathione is required by cells toprevent intracellular oxidative stress. The measured rate ofuptake of U-14C L-cystine into the cells was found to havethe following parameters: Km = 0.87 mM, Vmax = 0.9nmole/mg cell protein per min. The uptake was sodiumindependent and resembled the previously described x- ctransport system, with elevated glutamate levels causingextensive inhibition. Glutamate at a concentration of 1.4 mMcaused a 50% decrease in cystine uptake from the serum-freegrowth medium. Glutamate was taken up from the external medium(Km = 20 mM and Vmax = 12.5 nmole/mg cell protein permin) by the same transport system in a stereo specific, sodiumindependent manner. Of the amino acids examined, it was foundthat cystine and homocysteic acid were the most extensiveinhibitors of glutamate uptake and that inhibition was competitive. Metabolic profiles of the cells grown in culturescontaining enhanced glutamate levels revealed an overallincrease in net production of alanine, serine, asparagine andaspartate. A substantially increased specific consumption ofglutamate was accompanied by a decreased consumption of cystine,valine and phenylalanine.The combined kinetic and metabolic results indicate thatglutamate and cystine are taken up by the anionic transportsystem x- c. The increasing levels of glutamate in themedium result in a decreased transport of cystine by this systemdue to competitive inhibition by glutamate.
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    Improvement in the antibody productivity of human-human hybridomas by transfection with Tac gene (1990)
    Springer
    Cytotechnology 4 (1990), S. 29-37 
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    ISSN: 1573-0778
    Keywords: hybridoma ; monoclonal antibody ; rIL-2 ; Tac gene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The presence of a highly purified recombinant interleukin-2 (rIL-2) increased the production of immunoglobulin (IgM or IgG) by human-human hybridomas to 1.5–2.0 times the production by untreated cells. However, these cells did not react with anti-Tac (IL-2 receptor α) antibody. To enhance the response of the hybridoma cells to rIL-2, Tac gene was introduced by co-transfection with Tac gene expression plasmid pTB459 and G418 resistant gene expression plasmid pRSVneo. Tac cDNA transfected hybridoma (HBW-4.16.459-6-126) was induced to produce 6 times as much IgG by rIL-2 as was the control. This antibody production promoting phenomenon mediated by rIL-2 was depressed by anti-Tac antibody.
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    Interleukin-6 is antiproliferative to a mouse hybridoma cell line and promotive for its antibody productivity (1992)
    Springer
    Cytotechnology 10 (1992), S. 15-23 
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    ISSN: 1573-0778
    Keywords: antibody productivity ; growth suppression ; hybridoma ; interleukin-6 ; specific productivity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Monoclonal antibody production by hybridoma cells at moderately slowed growth states would be favorable for commercial scale production since cells can devote their resources to performing the differentiated function, immunoglobulin production. We found that a purified recombinant human interleukin-6, which had been reported to support or stimulate proliferation of B cell hybridoma/plasmacytoma cells, suppressed growth of a hybridoma cell line in serum-free medium. In the presence of the interleukin, the growth-suppressed cells were viable for remarkably long periods in batch culture, and after removal of the interleukin from the culture medium, they started to proliferate at their normal growth rate. As the concentration of the interleukin increased in the culture, the growth rate decreased and the specific antibody productivity (antibody production rate per cell) increased to 5-fold of control at 10 U ml−1 (2 ng ml−1) of the interleukin.
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    Deletion of the κ genes from mouse hybridomas established with NS-1 myelomas (1997)
    Springer
    Cytotechnology 24 (1997), S. 213-218 
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    ISSN: 1573-0778
    Keywords: gene deletion ; hybrid antibody ; hybridoma ; immunoglobulin light chain ; monoclonal antibody
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Monoclonal antibodies (mAbs) of the IgG class produced by mouse hybridomas raised with NS-1 myelomas have been shown to contain two types of immunoglobulin light (κ) chains derived from the myelomas and antigen-stimulated spleen lymphocytes, and the hybridomas produce three mAb species with light chain heterogeneity (Abe and Inouye, 1993). In the present study, 9 hybridoma lines secreting homogeneous mAbs have been isolated from 63 lines cloned from an established hybridoma line producing three mAbs. They secrete homogeneous mAbs containing light chains derived from either myeloma or spleen cells. They contain either κ gene derived from the respective cells, and the other gene was deleted during the cultivation. The deletion frequency of the κ gene of myelomas is 3 times higher than that of spleen cells, although 80–85% of hybridomas reach the stable state containing both κ genes.
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    A novel acid proteinase relased by hybridoma cells (1990)
    Springer
    Cytotechnology 3 (1990), S. 157-169 
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    ISSN: 1573-0778
    Keywords: acid proteinase ; cell culture ; hybridoma ; immunoglobulin cleavage ; lysosomal proteinases ; recycling reactor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract An acid proteinase has been detected in culture supernate of the 9.2.27 murine hybridoma. This enzyme extensively degrades albumin and transferrin during short incubations at pH 3 and below. Limited proteolysis of the 9.2.27 IgG2a appears to occur in the culture supernate. Proteolysis is enhanced at low pH in the presence of urea or 1 M acetic acid. The proteinase activity accumulates in continuous perfusion, total cell recycle cultures, beginning during exponential growth of the hybridoma. It is destroyed by boiling and blocked by pepstatin, but not by inhibitors of cysteine or serine proteinases or by EDTA. The low pH optimum may distinguish this enzyme from the known rat and mouse aspartic acid proteinases including cathepsin D and cathepsin E.
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    Hybridoma growth limitations: The roles of energy metabolism and ammonia production (1990)
    Springer
    Cytotechnology 3 (1990), S. 215-229 
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    ISSN: 1573-0778
    Keywords: hybridoma ; ammonia ; inhibition ; energetics ; glutaminolysis ; ammonia removal
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Energy metabolism and the production of ammonia in hybridoma cell culture and its inhibitory effects on cell growth are reviewed. The interactive roles of glucose and glutamine metabolism affect the rate of production of ammonia, and these interactions are described. It is shown that growth inhibition usually occurs between 2–4 mM ammonia although some cell lines have been shown to adapt to much higher concentrations, particularly in continuous culture. In batch cultures cell growth appears to be particularly susceptible to increased ammonia concentrations during the early stages of growth; ammonia increased the rate of cell death in the late stage of batch growth. The specific productivity of monoclonal antibodies is much less sensitive to the released ammonia than is growth; lower volumetric productivities relate to the lower viable cell concentrations which are achieved at the high ammonia levels. Techniques to prevent ammonia accumulation or remove ammonia selectively have been relatively unsuccessful to date.
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    Essential role of optimal protein synthesis in preventing the apoptotic death of cultured B cell hybridomas (1993)
    Springer
    Cytotechnology 13 (1993), S. 99-105 
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    ISSN: 1573-0778
    Keywords: amino acids ; apoptosis ; cell viability ; hybridoma ; protein synthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The monoclonal antibody productivity of cell culture systems is strongly dependent on the maintenance of hybridoma cell viability. We report that partial (〈50%) and transient (3 h) inhibition of protein synthesis by cycloheximide or deprivation of an essential amino acid induces apoptosis (programmed cell death) in B cell hybridomas. This unusual mechanism of apoptosis induction is likely to play a significant role in limiting cell viability in batch and perfusion cultures of hybridomas and emphasizes the importance of constantly maintaining a near optimal rate of macromolecular synthesis by optimization of all culture parameters. Inhibition of apoptosis in hybridomas by cell engineering and other technologies should permit, in the near future, a significant increase in the antibody productivity of existing cell culture systems.
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    Nutrient optimization for high density biological production applications (1991)
    Springer
    Cytotechnology 5 (1991), S. 15-30 
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    ISSN: 1573-0778
    Keywords: high density ; cell culture ; serum-free medium ; hybridoma ; CHO cells ; virus production ; insect cells ; adoptive immunotherapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Conclusion At the 1989 annual meeting of the U.S. Tissue Culture Associations, Ricahrd am, a leading investigator in the serum-free nutrient requirements of cultured cells, commented on the process of medium development. He noted that a survey of major media manufacturers revealed that, among the top selling mammalian cell culture media formulations, most were nearly thirty years old. This commentary is noteworthy considering the tremendous changes in cell culture understanding and derived applications which have emerged over these three decades. Fastidious cell types relatively unknown to investigators of the 1950s and 1960s are now being cultivated in defined, serum-free environments. Culture environments range from limiting dilution clonal recoveries to maintenance cultures approaching tissue densities. While research applications continue to predominate, applications of cell culture have expanded to the engineered production of biopharmaceuticals, to replacement of animal models for toxicology testing, and to the preservation, activation and expansion of human cells, tissues and organs. It is likely that future nutrient medium development will be predicated upon the design of a minimal number of defined formulations of relatively generic utility to a broad class of cell types. Analytical techniques derived from those described herein will be exploited in the user laboratory and in collaboration with the supplier to optimize the nutrient composition for the desired biological response.
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    Comparison of specific rates of hybridoma growth and metabolism in batch and continuous cultures (1992)
    Springer
    Cytotechnology 10 (1992), S. 147-155 
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    ISSN: 1573-0778
    Keywords: batch culture ; continuous culture ; hybridoma ; kinetics ; specific rates
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract For the mouse hybridoma cell line VO 208, kinetics of growth, consumption of glucose and glutamine, and production of lactate, ammonia and antibodies were compared in batch and continuous cultures. At a given specific growth rate, different metabolic activities were observed: a 40% lower glucose and glutamine consumption rate, but a 70% higher antibody production rate in continuous than in batch culture. Much higher metabolic rates were also measured during the initial lag phase of the batch culture. When representing the variation of the specific antibody production rate as a function of the specific growth rate, there was a positive association between growth and antibody production in the batch culture, but a negative association during the transient phase of the continuous culture. The kinetic differences between cellular metabolism in batch and continuous cultures may be result of modifications in the physiology and metabolism of cells which, in continuous cultures, were extensively exposed to glucose limitations.
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    Evaluation of a simple protein free medium that supports high levels of monoclonal antibody production (1996)
    Springer
    Cytotechnology 21 (1996), S. 95-109 
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    ISSN: 1573-0778
    Keywords: adaptation ; hybridoma ; monoclonal antibody ; protein free medium ; suspension culture ; weaning
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A simple protein free medium was formulated and tested in suspension culture using three hybridoma cell lines. The medium, referred to as CDSS (Chemically Defined Serum Substitutes), consisted of the basal medium DMEM:Ham F12, 1:1, with HEPES (D12H), plus pluronic F68, trace elements, ferric citrate, ascorbic acid, and ethanolamine. No protein or lipid components were added. All three cell lines were weaned off serum using CDSS and a commercially available protein free medium PFHM-II. Data shown here indicated that normally cells took 1–7 weeks to wean off serum and an additional 2–7 weeks to adapt to suspension culture. After adaptation the cells were able to grow well in suspension culture using both protein free media and in the main performed better than serum containing controls. The stability of the three hybridoma cells for antibody production following freeze/thaw procedures and long term subculturing was also tested. All three lines were frozen using our protein free CDSS medium (containing 0.75% bovine serum albumin and 10% dimethyl sulfoxide) in liquid nitrogen for up to one year. Cells thawed from these stocks recovered well and were able to maintain good growth and antibody production characteristics. One line was shown to grow using our protein free CDSS medium in suspension culture for 12 weeks without loss of antibody productivity.
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    Optimization of monoclonal antibody production: combined effects of potassium acetate and perfusion in a stirred tank bioreactor (1997)
    Springer
    Cytotechnology 24 (1997), S. 47-54 
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    ISSN: 1573-0778
    Keywords: hybridoma ; monoclonal antibody ; stirred tank perfusion culture ; potassium acetate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract To increase the yield of monoclonal antibody in a hybridoma culture, it is important to optimize the combination of several factors including cell density, antibody productivity per cell, and the duration of the culture. Potassium acetate enhances the production of antibodies by cells but sometimes depresses cell density. The production of anti-(human B-type red blood cell surface antigen) antibody by Cp9B hybridoma was studied. In batch cultures, potassium acetate inhibited Cp9B cells growth and decreased the maximal cell density but the productivity of antibody per cell was increased. The balance of the two effects resulted in a slight decline of antibody production. In a stirred tank bioreactor, the inhibitory effect of potassium acetate on cell density was overcome by applying the perfusion technique with the attachment of a cell-recycling apparatus to the bioreactor. In such a reactor, potassium acetate at 1 g l-1 did not cause a decrease in the cell density, and the antibody concentration in the culture supernatant was increased from 28 μg ml-1 to 38 μg ml-1. Potassium acetate also suppressed the consumption of glucose and the accumulation of lactate in batch cultures, but the glucose and lactate levels were kept stable by applying the perfusion technique in the stirred tank bioreactor.
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    Characterization and fed-batch culture of hybridoma overexpressing apoptosis suppressing gene bcl-2 (1997)
    Springer
    Cytotechnology 24 (1997), S. 135-141 
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    ISSN: 1573-0778
    Keywords: antibody productivity ; apoptosis ; bcl-2 ; fed batchculture ; hybridoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Mouse hybridoma 2E3 transfected with human bcl-2 gene survived longer with increasing expression level of bcl-2 when cultured in DME medium supplemented with 9% serum. One of the transfectants, 2E3BCMGbcl-2, overexpressed bcl-2 and could maintain viable cell density higher than the initial density for more than four days at a low 0.5% serum concentration. In comparison a mock transfectant 2E3BCMG remained viable for only one day. However, both hybridomas died out within a day in serum-free medium. These results suggested that bcl-2 needed a small amount of some serum components to suppress apoptosis of the hybridoma. Overexpression of bcl-2 also suppressed apoptosis of the hybridoma induced by glutamine deprivation. When hybridoma 2E3BCMGbcl-2 was inoculated in DME medium supplemented with 9% serum and cultured for 10 d with additional 2% serum feed at day 4 of the culture, viable cell density increased 2-fold and antibody produced 3-fold, in comparison with mock transfected 2E3 cultured in the same manner. The mock transfectant with additional feed of serum at day 4 of the culture showed no difference in viable cell density and antibody production. These results suggested that the mock transfectant committed to apoptosis before day 4 of the culture and the additional serum at day 4 could not reverse the commitment.
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    Effect of Bcl-2 Expression on Hybridoma Cell Growth in Serum-Supplemented, Protein-Free and Diluted Media (1998)
    Springer
    Cytotechnology 26 (1998), S. 219-225 
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    ISSN: 1573-0778
    Keywords: apoptosis ; Bcl-2 ; diluted medium ; hybridoma ; protein-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Two transfected hybridoma cell lines TB/C3-bcl2 (overexpressing the Bcl-2 protein) and TB/C3-pEF (control cell line), were compared in batch suspension cultures using a medium supplemented either with horse serum or with a protein-free, iron-rich supplement. The membrane intact index (percentage of cells with intact membranes determined by trypan blue staining) of the TB/C3-bcl2 cell line decreased much slower than that of the control cell line during the dying phase of the cultures. No significant difference in antibody, lactate and ammonia production as well as glucose and glutamine consumption was noted in the exponential phase of the experiments. Both cell lines were also compared in batch experiments using media diluted with saline to further investigate the effect of Bcl-2 under sub-optimal conditions. The Bcl-2 overexpressing cell line again exhibited a higher membrane intact index at increasing dilution steps.
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    Repeated hybridoma batch culture with cell recycle (1993)
    Springer
    Cytotechnology 13 (1993), S. 51-53 
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    ISSN: 1573-0778
    Keywords: cell recycle ; filtration ; hybridoma ; monoclonal antibody
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract At the end of a hybridoma batch culture, the cells are usually discarded after separation from the culture broth. If, however, they are aseptically recycled into the reactor, the production process can be resumed simply by the addition of fresh medium. This cycle can then be repeated several times consecutively. In a test case, with a mouse hybridoma, we found antibody yields for each cycle in the same range as for a standard batch. In a 15 1 stirred tank reactor we could, within 6 days, produce 2.8 g of monoclonal antibody (MAb). This type of reactor operation allowed a doubling in the reactor volumetric productivity (mg/l/day).
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    Linoleic acid improves the robustness of cells in agitated cultures (1999)
    Springer
    Cytotechnology 30 (1999), S. 27-36 
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    ISSN: 1573-0778
    Keywords: agitation ; fatty acids ; hybridoma ; linoleic acid ; lipid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The murine hybridoma (CC9C10) was subjected to high shear rates in a spinner flask to determine the effect of various culture additives on cell survival. At 500 rpm, the half-life of the viable cell concentration in a low protein serum-free medium was 50 min. Both bovine serum albumin and Pluronic F-68 had a significant effect in protecting cells under these conditions. The effects of the two supplements were additive, so that in the presence of both supplements there was minimal cell damage at 500 rpm. The survival rate of cells grown in media supplemented with linoleic acid improved significantly under high stirring rates. Cells grown for one passage in 50 μM linoleic acid and stirred at 500 rpm had a significantly higher survival rate than control cells. For cells grown over 5 passages in 25 μM linoleic acid, the survival rate at 470 rpm was ×3 greater than that determined for control cells. This difference gradually decreased at higher stirring rates up to 610 rpm when the half-life of the viable cell population was reduced to ∼10 min. Supplementation of cultures with linoleic acid has previously been shown to result in incorporation into all three cellular lipid fractions - polar, non-polar and free fatty acid (Butler et al., 1997). Our explanation for the increased survivability of the cells at high agitation rates in the presence of linoleic acid is that the structural lipid components of the cell including the outer membrane attained a higher unsaturated/saturated ratio which was more robust than that of control cells.
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    Effectiveness of vitamin A acetate for enhancing the production of lung cancer specific monoclonal antibodies (1999)
    Springer
    Cytotechnology 31 (1999), S. 77-83 
    Details
    ISSN: 1573-0778
    Keywords: antibody production ; human monoclonalantibody ; hybridoma ; lung cancer ; vitamin A acetate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The antibody productivity of the human–human hybridoma cell line AE6, which produces the lung cancer specific human monoclonal antibody AE6F4, was enhanced fourfold upon stimulation with 1 μg/ml of vitamin A acetate for one day. The enhancement lasted for about two weeks, and could be repeated by another stimulation with vitamin A acetate. The enhancing effect of vitamin A acetate was influenced by the cell density. Enhancement was clearly observed when the cell density was under 106 cells/ml. However, when the cell density was over 107 cells/ml, enhancement was observed weakly or not at all. Although the enhancing effect of vitamin A acetate is not unique to AE6 cells, not all human–human hybridoma cell lines show increased productivity upon VA acetate stimulation. This study suggests that the response to vitamin A acetate may be related to the properties of a particular fusion partner which the hybridoma cell inherits. The efficacy of vitamin A acetate for production of human monoclonal antibodies using human–human hybridomas is discussed.
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    URL: http://dx.doi.org/10.1023/A:1008016020785
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    Effect of feed rate on growth rate and antibody production in the fed-batch culture of murine hybridoma cells (2000)
    Springer
    Cytotechnology 32 (2000), S. 229-242 
    Details
    ISSN: 1573-0778
    Keywords: fed batch ; hybridoma ; macromolecular composition ; monoclonal antibody ; substrate limitation ; target specific growth rate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Batch and fed-batch cultures of a murine hybridomacell line (AFP-27) were performed in a stirred tankreactor to estimate the effect of feed rate on growthrate, macromolecular metabolism and antibodyproduction. Macromolecular composition was foundto change dynamically during batch culture ofhybridoma cells possibly due to active production ofDNA, RNA and protein during the exponential phase.Antibody synthesis is expected to compete with theproduction of cellular proteins from the amino acidpool. Therefore, it is necessary to examine therelationship between cell growth in terms of cellularmacromolecules and antibody production. In this study,we searched for an optimum feeding strategy bychanging the target specific growth rate in fed-batchculture to give higher antibody productivity whileexamining the macromolecular composition. Concentratedglucose (60 mM) and glutamine (20 mM) in DR medium(1:1 mixture of DMEM and RPMI) with additional aminoacids were fed continuously to the culture and thefeed rate was updated after every sampling to ensureexponential feeding (or approximately constantspecific growth rate). Specific antibody productionrate was found to be significantly increased in thefed-batch cultures at the near-zero specific growthrate in which the productions of cellular DNA, RNA,protein and polysaccharide were strictly limited byslow feeding of glucose, glutamine and other nutrients. Possible implications of these results are discussed.
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    URL: http://dx.doi.org/10.1023/A:1008169417980
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    Comparison of cell growth in T-flasks, in micro hollow fiber bioreactors, and in an industrial scale hollow fiber bioreactor system (2000)
    Springer
    Cytotechnology 34 (2000), S. 111-119 
    Details
    ISSN: 1573-0778
    Keywords: cell culture ; hollow fiber bioreactor ; hybridoma ; micro bioreactor ; optimization ; T-flask
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract In this article, cell growth in a novel micro hollow fiberbioreactor was compared to that in a T-flask and theAcuSyst-Maximizer®, a large scale industrial hollowfiber bioreactor system. In T-flasks, there was relativelylittle difference in the growth rates of one murine hybridomacultured in three different media and for three other murinehybridomas cultured in one medium. However, substantialdifferences were seen in the growth rates of cells in themicro bioreactor under these same conditions. These differencecorrelated well with the corresponding rates of initial cellexpansion in the Maximizer. Quantitative prediction of thesteady-state antibody production rate in the Maximizer was moreproblematic. However, conditions which lead to faster initialcell growth and higher viable cell densities in the microbioreactor correlated with better performance of a cell line inthe Maximizer. These results demonstrate that the microbioreactor is more useful than a T-flask for determining optimalconditions for cell growth in a large scale hollow fiberbioreactor system.
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    URL: http://dx.doi.org/10.1023/A:1008167713696
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    Influence of inoculum age on hybridoma culture kinetics (1991)
    Springer
    Cytotechnology 5 (1991), S. 165-171 
    Details
    ISSN: 1573-0778
    Keywords: growth ; hybridoma ; inoculum age ; kinetics ; production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract To determine the influence of the inoculum age on the kinetics of hybridoma growth and metabolism, spinner flasks have been inoculated with cells previously propagated in T flasks for 43, 52, 62 and 71 hr respectively. Increasing the age of the inoculum is found to result in a longer lag phase, in a lower maximum specific growth rate and in a reduced maximal cell density. During the growth phase specific rates of glucose and glutamine uptake and of ammonia and lactate production are similar. However, with the older inoculum, much higher metabolic activities are observed during the lag phase. The production of antibodies is delayed with increasing inoculum age, but the final antibody concentrations are similar, which indicates a higher specific antibody production rate when inoculating with older cells.
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    URL: http://dx.doi.org/10.1007/BF00365433
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    Cell cycle dependency of monoclonal antibody production in asynchronous serum-free hybridoma cultures (1991)
    Springer
    Cytotechnology 5 (1991), S. 243-254 
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    ISSN: 1573-0778
    Keywords: hybridoma ; serum-free medium ; flow cytometry ; cell cycle kinetics ; bromodeoxyuridine labelling
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The cell cycle kinetics of F3(B6) mouse hybridoma was examined by immunocytochemical staining of bromodeoxyuridine incorporated into the DNA of exponentially growing cells in three different cultures: one supplemented with 10% fetal bovine serum and two adapted to serum-free media, TABIES and BITES. The serum-free cultures, particularly the BITES, had longer cycling times and higher specific antibody production rate. Both observations were correlated to the prolongation of the G1 phase traverse time and substantiated with a starvation blocking experiment.
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    URL: http://dx.doi.org/10.1007/BF00556294
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    Development of a serum-free and heat-sterilizable medium and continuous high-density cell culture (1991)
    Springer
    Cytotechnology 5 (1991), S. 35-51 
    Details
    ISSN: 1573-0778
    Keywords: glutamine peptide ; high-density cell culture ; human diploid fibroblast ; hybridoma ; lymphoblastoid cell line ; serum-free
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We tried to establish a new serum-free and heat-sterilizable medium, based on our serum-free medium in which many lymphoblastoid cells and hybridoma could grow as well as in a conventional serum-containing medium. As is well-known, L-glutamine (L-Gln) is one of the most heat-labile but essential components for cell growth. As a substitute for L-Gln, dipeptide such as Gly-L-Gln or L-Ala-L-Gln, which was quite stable even after autoclaving, was found to be utilizable for mammalian cell growth. The L-Gln dipeptide-containing serum-free medium was quite stable in a solution even after storing at 37°C for 4 months. In the serum-free medium containing L-Ala-L-Gln, mouse hybridola could grow and produce more antibody than in RPMI 1640+10% FBS. It has been proved that BSA and transferrin, which are also heat-labile but essential for the growth of various cell lines, can be substituted by heat-stable alpha-cyclodextrin and cholesterol, and Fe-gluconate, respectively. Insulin has also proved to be heat stable in a solution of Fe-gluconate. We thus established a new serum-free medium, all the components of which could be heat-sterilizable. Moreover, by adding EGF and BSA but without the adhesion factor included in FBS, the serum-free medium was found to support a long-term serial culture of a human diploid fibroblast. Finally, with this auotoclavable serum-free medium in a perfusion culture apparatus, we were able to continuously cultivate a human lymphoblastoid cell line. The production rate of IgM was found to be markedly increased by feeding the serum-free medium enriched by glucose, bicarbonate, L-Cys, and approtinin. The cell density reached as high as 2×108/ml in the serum-free medium. Although the working volume in the reactor was only 1 1, the rate of IgM production reached 480 mg/day. The new heat-sterilizable serum-free medium has several advantages, because L-Gln peptide is a heat-stable and available precursor of L-Gln.
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    URL: http://dx.doi.org/10.1007/BF00573879
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    Altered reactivity of immunoglobulin produced by human-human hybridoma cells transfected by pSV2-neo gene (1991)
    Springer
    Cytotechnology 6 (1991), S. 219-226 
    Details
    ISSN: 1573-0778
    Keywords: immunoglobulin ; hybridoma ; antigen specificities ; glycosylation ; pSV2-neo
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The HB4C5 and HF10B4 cell lines are human-human hybridomas producing human IgM monoclonal antibodies (MAbs) reactive to porcine carboxypeptidase A (CPase), but not to double stranded DNA (ds DNA). We obtained G418-resistant HB4C5 and HF10B4 cells by an introduction of pSV2-neo DNA. Almost all of the G418-resistant clones produced MAbs reactive to not only the CPase but the ds DNA. The results of the inhibition ELISA suggested that the cross-reactivity of the antibodies from G418-resistant clones to CPase and ds DNA was responsible for the alteration on their antigen specificity. HB4C5 and HF10B4 cells and their G418-resistant clones produced antibodies having glycosylated λ chain. The antibodies produced by tunicamycin-treated G418-resistant subclones of HB4C5 and HF10B4 lost the ability to bind to ds DNA, but retained the ability to bind to CPase. These results suggest that an introduction of pSV2-neo DNA into these hybridomas alters the specificities of their MAbs, and that the alteration to antigen binding specificities of their MAbs may be associated with glycosylation of the MAbs by these hybridomas.
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    URL: http://dx.doi.org/10.1007/BF00624760
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    The medium cycle bioreactor (MCB): Monoclonal antibody production in a new economic production system (1991)
    Springer
    Cytotechnology 7 (1991), S. 63-74 
    Details
    ISSN: 1573-0778
    Keywords: economy ; medium ; recycling ; re-use ; supplementation ; hybridoma ; antibody production ; productivity ; protein retention ; substrate utilization ; amino acids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The perfusion mode of a continuous cell culture bioreactor was modified to establish a closed loop system. Eighty percent of the spent medium was re-used twice. The medium cycle bioreactor unit was operated sterile and uncomplicated without a technical retention system for the high molecular weight substances. Therefore, only 20% of the actual medium was necessary to run the recycling process. During seven days culture time in a two liter scale 5 grams of IgG1 type monoclonal antibody was produced. During that period the cell specific productivity was constant. Renewal of proteins was omitted because the protein content in the system persisted at a high level. Therefore, self-conditioning substances of the cells were retained in the system as well as the expensive medium components (proteins with catalytic or stimulating function). Seventy to 80% of medium costs and medium quantity were saved for each medium recycling step. Only cheap metabolites that are consumed by the cells had to be supplemented. Uptake rates of glucose and amino acids were calculated to establish a suitable supplementation mixture for the recirculated medium.
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    URL: http://dx.doi.org/10.1007/BF00350912
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    Effects of dissolved oxygen levels and the role of extra- and intracellular amino acid concentrations upon the metabolism of mammalian cell lines during batch and continuous cultures (1998)
    Springer
    Cytotechnology 26 (1998), S. 185-197 
    Details
    ISSN: 1573-0778
    Keywords: CHO ; dissolved oxygen (DO) ; essential amino acids ; hybridoma ; intracellular amino acids ; Monod constants (KS
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The effects of dissolved oxygen and the concentration of essential amino acids upon the metabolism of two mammalian cell lines (rCHO producing human active (t-PA) and a mouse-mouse hybridoma) were investigated in batch, chemostat, and perfusion cultures. Intracellular amino acid concentrations were measured for both cell lines during repeated batch cultures and the KS-values for the essential amino acids were calculated using Monod equations via computer simulation. The KS-values were in the range of 10 mmol L−1 and the pool of most intracellular amino acids remained constant at about 10–100 fold higher in concentration than in the medium. No significant differences were observed between the hybridoma and CHO cell. The specific nutrient uptake rates corresponded with the cell specific growth rate and the effects of reduced dissolved oxygen concentrations only became evident when the DO dropped below 5% of air saturation (critical concentration below 1%). Nevertheless, a correlation between nutrient concentration and specific oxygen uptake was detected.
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    URL: http://dx.doi.org/10.1023/A:1007917409455
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    Enhanced monoclonal antibody production by gradual increase of osmotic pressure (1999)
    Springer
    Cytotechnology 29 (1999), S. 27-33 
    Details
    ISSN: 1573-0778
    Keywords: adaptation ; antibody production rate ; hybridoma ; intracellular amino acids ; osmotic pressure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The time length required for the adaptation of AFP-27 hybridoma cells to high osmotic pressure and the effect of a gradual increase of osmotic pressure on monoclonal antibody production were investigated. When the cells were subjected to an increase of osmotic pressure from 300 mOsmol kg-1 to 366 mOsmol kg- 1, the intracellular content of osmoprotective free amino acids reached a maximum level 6 h after the osmotic pressure was increased to 366 mOsmol kg-1. The same time period of 6 h incubation at 366 mOsmol kg-1 was required to obtain a high growth rate of AFP-27 cells at 440 mOsmol kg-1 when the cells were subjected to a two-step increase of osmotic pressure from 300 mOsmol kg-1 to 366 mOsmol kg-1 and then to 440 mOsmol kg-1. The time length for the physiological adaptation of the cells to 366 mOsmol kg-1 was consequently estimated to be 6 h. Osmotic pressure during batch cultivation was gradually increased from 300 mOsmol kg-1 to 400 mOsmol kg-1 with an adaptation time of at least 6 h. The specific growth rates following a gradual increase of osmotic pressure were higher than those at a constant osmotic pressure of 400 mOsmol kg-1, while the specific monoclonal antibody production rate increased with the increase in the mean osmotic pressure. As a result, the cells grown under a gradual increase of osmotic pressure produced higher amounts of monoclonal antibodies than did those grown under constant osmotic pressure.
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    URL: http://dx.doi.org/10.1023/A:1008016806599
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    Characterization of proteases produced by newly isolated and identified proteolytic microorganisms from fermented fish (Budu) (1992)
    Springer
    World journal of microbiology and biotechnology 8 (1992), S. 284-286 
    Details
    ISSN: 1573-0972
    Keywords: Bacillus ; budu ; fermentation ; fish ; proteases
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Eight different strains ofBacillus were isolated from fermented fish (Budu) and their proteolytic enzyme activities were determined after 18 h cultivation at room temperature (35° C). Four isolates possessed high protease activities. Optimum pH for these enzymes was between 7.0 and 8.0 and the optimal temperature was 55° C. The proteases retained 40% of their original activity after 20 min at 55° C but lost all activity at 65° C. Three of the four isolates were identified asBacillus subtilis, the fourth asBacillus licheniformis.
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    URL: http://dx.doi.org/10.1007/BF01201880
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    Iridoviruses associated with epizootic haematopoietic necrosis (EHN) in aquaculture (1997)
    Springer
    World journal of microbiology and biotechnology 13 (1997), S. 367-373 
    Details
    ISSN: 1573-0972
    Keywords: Amphibian ; aquaculture ; epizootic haematopoietic necrosis virus ; fish ; frog virus 3 ; Iridoviridae ; ranavirus ; reptile
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Systemic infections of teleost fishes caused by iridoviruses have recently been recognized in Australia, Asia, Europe and the USA. These iridoviruses are different from those of the established genera Lymphocystivirus and Goldfish Virus 1-like Viruses of the family Iridoviridae. The agents exhibit similar physicochemical properties, are antigenically related and prove to be of high virulence to different teleost fishes in aquaculture. The first iridovirus, epizootic haematopoietic necrosis virus, responsible for an epizootic outbreak of haematopoietic necrosis in redfin perch, was reported in Australia. Some years later, similar iridovirus epizootics occurred in sheatfish and catfish in Europe. The Australian and the European isolates proved to be antigenically related and showed properties in common with frog virus 3, the type species of the genus Ranavirus of the Iridoviridae. Further iridovirus isolates from fish, amphibians and reptiles exhibited a close relationship with each other and with frog virus 3. It is important to note that the Australian amphibian iridovirus, Bohle iridovirus, was experimentally transmitted to teleost fish inducing high mortalities. The occurrence of similar viruses in different host species in the aquatic environment and their inter-species transmission emphasize the importance of health control in aquaculture.
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    URL: http://dx.doi.org/10.1023/A:1018563930712
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    Effect of introduced Pseudomonas fluorescens strains on the uptake of nitrogen by wheat from 15N-enriched organic residues (1999)
    Springer
    World journal of microbiology and biotechnology 15 (1999), S. 417-423 
    Details
    ISSN: 1573-0972
    Keywords: 15N ; nitrogen mineralization ; Pseudomonas fluorescens ; rhizosphere ; Triticum aestivum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The effects of an antibiotic-producing Pseudomonas fluorescens strain (F113) carrying the marker gene cassette lacZY and a marked, non-producing strain (F113G22) on the uptake of nitrogen from 15N-enriched organic residues incorporated into a sandy soil were investigated in microcosm studies. Strain F113 produces the antibiotic 2,4-diacetylphloroglucinol (DAPG), whilst its modified derivative strain F113G22 has DAPG production deleted by Tn5 mutagenesis. Uptake of nitrogen by wheat (Triticum aestivum) from 15N-enriched organic residues was estimated using stable isotope-ratio mass spectrometry of shoot and root material of 17-day-old plants. In addition, plant growth and active microbial biomass in soil were monitored. In contrast to results obtained in our previous study on pea (Pisum sativum), it was found that in wheat, inoculation with either strain F113 or F113G22 decreased the proportion of nitrogen derived from 15N-labelled organic residues incorporated into soil as compared to non-inoculated controls. It is therefore suggested that these strains decreased mineralization of organic residues in the rhizosphere of wheat, making less inorganic N (15N) available for plant uptake. The results of this study indicate that the effects of introduced Pseudomonas fluorescens strains on nitrogen mineralization in the rhizosphere are plant-species dependent, and highlight the importance of testing microbial inocula on a range of plant species.
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    URL: http://dx.doi.org/10.1023/A:1008999112121
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