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  • Articles  (120)
  • hybridoma  (120)
  • Process Engineering, Biotechnology, Nutrition Technology  (120)
  • 1
    Electronic Resource
    Electronic Resource
    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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  • 2
    Electronic Resource
    Electronic Resource
    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 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008143716374
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  • 3
    Electronic Resource
    Electronic Resource
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008167713696
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  • 4
    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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  • 5
    Electronic Resource
    Electronic Resource
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008169417980
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  • 6
    Electronic Resource
    Electronic Resource
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008155609072
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  • 7
    Electronic Resource
    Electronic Resource
    Hybridoma growth and productivity: effects of conditioned medium and of inoculum size (1999)
    Springer
    Cytotechnology 29 (1999), S. 1-10 
    Details
    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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  • 8
    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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  • 9
    Electronic Resource
    Electronic Resource
    Linoleic acid improves the robustness of cells in agitated cultures (1999)
    Springer
    Cytotechnology 30 (1999), S. 27-36 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008048126055
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  • 10
    Electronic Resource
    Electronic Resource
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008016020785
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  • 11
    Electronic Resource
    Electronic Resource
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008016806599
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  • 12
    Electronic Resource
    Electronic Resource
    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 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008080407581
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  • 13
    Electronic Resource
    Electronic Resource
    Effect of extracellular glutamine concentration on primary and secondary metabolism of a murine hybridoma: An in vivo 13C nuclear magnetic resonance study (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 172-186 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; futile cycling ; hollow fiber bioreactor ; glutamine ; NMR ; C-13 ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of changes in extracellular glutamine level on metabolism of a murine hybridoma was examined with in vivo nuclear magnetic resonance (NMR) spectroscopy. Cells were cultured in a hollow-fiber bioreactor at high cell density to allow intracellular metabolite levels to be determined on a metabolically relevant time scale. Steady infusions of [1-13C] glucose were used to label glycolytic and tricarboxylic acid cycle intermediates, which permitted continuous monitoring with NMR spectroscopy during changes in environmental glutamine level. Samples of the extracellular medium were also analyzed to determine the effect of glutamine on other metabolites associated with primary and secondary metabolism. The changes in glutamine concentration had several effects on primary and secondary metabolism, depending on the rate the changes were made. For a brief reduction in feed glutamine concentration from 4 to 0 mM (which produced a rapid change from 0.67 to ∼0 mM in residual glutamine), large changes were observed in the rate of consumption of metabolites normally associated with energy production. Antibody synthesis was strongly stimulated and nitrogen metabolism was significantly altered. For a more prolonged reduction from 2.4 to 1.2 mM (which produced a slower reduction from 0.30 to 0.08 mM in residual glutamine), much smaller changes were observed even though the concentration of glutamine at the reduced feed level was very low. Energy metabolism did not appear to be limited by glutamine at 0.08 mM, which suggests that significant futile cycling may occur in energy producing pathways when excess glucose and glutamine are available. However, this concentration of extracellular glutamine appeared to affect some anabolic pathways, which require amino groups from glutamine. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 172-186, 1998.
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  • 14
    Electronic Resource
    Electronic Resource
    Transition probability cell cycle model with product formation (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 387-399 
    Details
    ISSN: 0006-3592
    Keywords: population balance ; cell cycle ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A cell cycle population model based on the transition probability model of Smith and Martin (1973) has been extended to include product synthesis and export. The model handles two probable mechanisms. In the direct production model, the product is the protein. In the transcription model, the product is the specific mRNA. The protein is synthesized by translation of the specific mRNA and subsequently exported. In either case, the cell density is jointly distributed in the primary product and maturity age in the cell cycle. This extended model also is capable of describing a large range of conditions, including substrate dependent batch and continuous cultures. With the use of unity maturity-velocity (but the transition rate a function of limiting substrate), the model is shown to exhibit a negative growth association between the specific productivity of monoclonal antibodies from hybridomas and the dilution rates of a chemostat. Possibilities of maturity age dependent transcription and translation are considered, and the results show that these features can amplify the specific productivity negative association with specific growth rate. While this model may provide a partial elucidation of monoclonal antibody productivity in a chemostat, the present work provides a proper framework with which probable cell cycle dependent product formation can be analyzed rigorously with a comprehensive computational model. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:387-399, 1998.
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  • 15
    Electronic Resource
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    In hybridoma cultures, deprivation of any single amino acid leads to apoptotic death, which is suppressed by the expression of the bcl-2 gene (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 90-98 
    Details
    ISSN: 0006-3592
    Keywords: apoptosis ; necrosis ; bcl-2 ; amino acids ; cell culture ; cell death ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The transfection of murine hybridomas with the apoptosis suppressor gene bcl-2 has been reported to result in the extension of batch culture duration, leading to significant improvements in culture productivity. In the present study, the effect of deprivation, individually, of each amino acid found in culture medium was examined to characterize the chemical environment of the culture in terms of its propensity to induce apoptosis. When cells were deprived of each amino acid, individually for 48 h, the majority of cell deaths in each case occurred by apoptosis, with essential amino acids being clearly most effective. For nearly all the amino acids, the viability of the bcl-2 cell line cultures was greater than 70% after 48 h, representing a substantial improvement in viability over control cell line cultures. Time course studies revealed that the induction of death could be divided into two phases. Initially, following the deprivation of a single essential amino acid, there was a period of time during which all the control cell line cultures retained high viability. The duration of this phase varied from 15 h in the case of lysine deprivation, through to 40 h in the case methionine deprivation. In the second phase of deprivation, the cultures exhibited an abrupt and rapid collapse in viability. The time taken for the viability to fall to 50% was similar for each amino acid. In every case, the duration of both phases of the bcl-2 cultures was considerably extended. Specific utilization rates were increased during the control cultures relative to the bcl-2 cultures for both the growth phase (ranging between 2% and 57% higher than the bcl-2 cultures) and the death phase (ranging between 172% to 1900% higher than the bcl-2 culture). © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:90-98, 1998.
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  • 16
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    Metabolite-balancing techniques vs. 13C tracer experiments to determine metabolic fluxes in hybridoma cells (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 258-262 
    Details
    ISSN: 0006-3592
    Keywords: mass balance ; metabolic flux ; 13C tracer ; NMR spectroscopy ; mass spectroscopy ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The estimation of intracellular fluxes of mammalian cells using only mass balances of the relevant metabolites is not possible because the set of linear equations defined by these mass balances is underdetermined. In order to quantify fluxes in cyclic pathways the mass balance equations can be complemented with several constraints: (1) the mass balances of co-metabolites, such as ATP or NAD(P)H, (2) linear objective functions, (3) flux data obtained by isotopic-tracer experiments. Here, these three methods are compared for the analysis of fluxes in the primary metabolism of continuously cultured hybridoma cells. The significance of different theoretical constraints and different objective functions is discussed after comparing their resulting flux distributions to the fluxes determined using 13CO2 and 13C-lactate measurements of 1 - 13C-glucose-fed hybridoma cells. Metabolic fluxes estimated using the objective functions “maximize ATP” and “maximize NADH” are relatively similar to the experimentally determined fluxes. This is consistent with the observation that cancer cells, such as hybridomas, are metabolically hyperactive, and produce ATP and NADH regardless of the need for these cofactors. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:258-262, 1998.
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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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    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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    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 
    Details
    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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    URL: http://dx.doi.org/10.1023/A:1007914619219
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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 
    Details
    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 
    Details
    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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    Kinetic studies on hybridoma cells immobilized in fixed bed reactors (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 535-541 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; fixed bed ; metabolism ; kinetic model ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Cultures with immobilized hybridoma cells were performed in fixed bed systems. “Steady state” values for volume-specific substrate uptake and metabolite production rates were determined at various perfusion rates and superficial flow velocities of the medium within the carrier matrix. Data from fixed bed volumes between 50 and 600 ml did not show any difference. The volume-specific glutamine and glucose uptake rate turned out to be independent of the superficial flow velocity, but decreased with decreasing glutamine and glucose concentration. The volume-specific oxygen uptake rate increased with increasing superficial flow velocity and substrate concentration, respectively. A similar behavior was observed for the ratio between oxygen and glucose uptake rate. The production rate for monoclonal antibodies was neither affected by the substrate concentration nor by the superficial flow velocity. The metabolic parameters of the immobilized cells were put into kinetic equations and compared to those of suspended cells. It could be concluded that the metabolism of the immobilized cells is determined by the oxygen supply within the macroporous carriers. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 535-541, 1997.
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    Effect of serum-free and serum-containing medium on cellular levels of ER-based proteins in various mouse hybridoma cell lines (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 54 (1997), S. 165-180 
    Details
    ISSN: 0006-3592
    Keywords: monoclonal antibody ; hybridoma ; BiP ; PDI ; GRP94 ; serum-free medium ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: BiP, GRP94 and PDI, three endoplasmic reticulum (ER) based proteins are involved in the maturation of secretory proteins and might represent a bottleneck in the secretory pathway of monoclonal antibodies (MAB). With the three hybridoma cell lines tested, MAB production kinetics were significantly increased for the batch cultures done in serum-free medium (SFM) with respect to those done in serum-containing medium (SCM). It could be established that there was a correlation between the cellular levels of PDI and GRP94 and the specific MAB production rate. With respect to BiP, no correlation with the MAB production rate was observed. The non-producing myeloma cell line X63, used as a reference, showed increased cellular PDI levels when cultivated in SFM. However, in this cell, the cellular GRP94 levels were not significantly influenced by the medium composition.It was concluded that SFM induced an increase of cellular PDI levels and this elevation seemed to be responsible for the increase in the specific MAB production rates. On the other hand, only MAB producing cells showed an increase in the cellular GRP94 levels which might be a result of increased MAB sythesis. Indeed, I.13.17 cultivated in SFM supplemented with serum showed a significantly reduced (about 50%) specific MAB production rate in comparison to I.13.17 cultivated in non-serum supplemented SFM. The cellular PDI and BiP levels did not vary between these conditions of culture, whereas the cellular GRP94 level was about two-fold lower in I.13.17 cultivated in SFM when supplemented with serum than in I.13.17 cultivated in SFM without futher supplementation. These results are discussed with respect to the medium composition as well as in the context of apparent and potential bottlenecks within the secretory pathway of MAB. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 165-180, 1997.
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    Effects of glutamine supply on growth and metabolism of mammalian cells in chemostat culture (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 54 (1997), S. 272-286 
    Details
    ISSN: 0006-3592
    Keywords: glutamine limitation ; mammalian cells ; chemostat ; specific metabolic rates ; hybridoma ; medium optimization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Glutamine is a major source of energy, carbon, and nitrogen for mammalian cells. The amount of glutamine present in commercial mammalian cell media is, however, not necessarily balanced with cell requirements. Therefore, the effects of glutamine limitation on the physiology of two mammalian cell lines were studied in steady-state chemostat cultures fed with IMDM medium with 5% serum. The cell lines used were MN12, a mouse-mouse hybridoma, and SP2/0-Ag14, a mouse myeloma often used in hybridoma fusions. Cultures, grown at a fixed dilution rate of 0.03 h-1, were fed with media containing glutamine concentrations ranging from 0.5 to 4 mmol L-1. Biomass dry weight and cell number were linearly proportional to the glutamine concentrations fed, between 0.5 and 2 mmol L-1, and glutamine was completely consumed by both cell lines. From this it was concluded that glutamine was the growth-limiting substrate in this concentration range and that the standard formulation of IMDM medium contains a twofold excess of glutamine. In glutamine-limited cultures, the specific rates of ammonia and alanine production were low compared to glutamine-excess cultures containing 4 mmol L-1 glutamine in the feed medium. The specific consumption rates of nearly all amino acids decreased with increasing glutamine feed, indicating that, in their metabolic function, they may partially be replaced by glutamine. Both cell lines reacted similarly to differences in glutamine feeding in all aspects investigated, except for glucose metabolism, In SP2/0-Ag14 glutamine feed concentrations did not affect the specific glucose consumption, whereas in MN12 this parameter increased with increasing amounts of glutamine fed. This systematic study using controlled culture conditions together with a detailed analysis of culture data shows that, although cells may react similarly in many aspects, cell-line-specific characteristics may be encountered even with respect to fundamental physiological responses like the interaction of the glutamine and glucose metabolism. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 272-286, 1997.
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    Effect of growth factors and antigen on hybridoma cell culture dynamics (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 54 (1997), S. 357-364 
    Details
    ISSN: 0006-3592
    Keywords: cell culture ; hybridoma ; monoclonal antibody ; growth factor ; antigen ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The cell growth and monoclonal antibody production kinetics of hybridoma cell cultures continuously exposed to growth factors and the cognate antigen were investigated. The growth factors were the epidermal growth factor, fibroblast growth factor, and interleukin-2, whereas the antigen was the trinitrophenyl group conjugated to a carrier protein. The cultures were carried out in a protein-free medium in batch operation. During the entire cultivation period there was continuously available free, antibody-unbound antigen to interact with the cells. The produced antibody was measured with an ELISA after it was released from the antigen-protein conjugate by competitive elution with non-protein-conjugated antigen. Cultures with growth factors and without antigen increased the total antibody produced by up to 30%, whereas cell growth remained unaffacted. Soluble antigen-protein conjugates had no effect on the hybridoma cultures. In contrast, immobilized antigen-protein on sepharose beads in cultures with growth factors induced significant changes. Total antibody produced was higher by up to 40%. More importantly, the specific antibody production shifted from a growth-phase-independent to a growth-phase-dependent profile, with approximately twice as much specific antibody production during the late growth-early stationary phase relative to constant specific antibody production in the antigen-free, factor-free culture. The culture changes induced by the presence of immobilized antigen and growth factors were reversed when the antigen and the growth factors were removed from the cells' environment. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 357-364, 1997.
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    Effect of hypoosmotic stress on hybridoma cell growth and antibody production (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 565-570 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; hypoosmotic stress ; specific antibody productivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: To investigate the response of hybridoma cells to hypoosmotic stress, S3H5/γ2bA2 and DB9G8 hybridomas were cultivated in the hypoosmolar medium [Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% serum] resulting from sodium chloride subtraction. Both hybridomas showed similar responses to hypoosmotic stress in regard to cell growth and antibody production. The cell growth and antibody production at 276 mOsm/kg were comparable to those at 329 mOsm/kg (standard DMEM). Both cells grew well at 219 mOsm/kg, though their growth and antibody production were slightly decreased. When the osmolality was further decreased to 168 mOsm/kg, the cell growth did not occur. When subjected to hyperosmotic stress, both cells displayed significantly enhanced specific antibody productivity (qAb). However, the cells subjected to hypoosmotic stress did not display enhanced qAb. Taken together, both hyperosmotic and hypoosmotic stresses depressed the growth of S3H5/γ2bA2 and DB9G8 hybridomas. However, their response to hypoosmotic stress in regard to qAb was different from that to hyperosmotic stress. © 1997 John Wiley & Sons, Inc. Biotechnol Biong 55: 565-570, 1997.
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    The effect of dissolved oxygen on the metabolic profile of a murine hybridoma grown in serum-free medium in continuous culture (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 54 (1997), S. 153-164 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; oxygen ; serum-free medium ; continuous culture ; antioxidant ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The murine B-lymphocyte hybridoma, CC9C10 was grown at steady state under serum-free conditions in continuous culture at dissolved oxygen (DO) concentrations in the range of 10% to 150% of air saturation. Cells could be maintained with this range at high viability in a steady state at a dilution rate of 1 d-1, although with lower cell concentrations at higher DO. A higher specific antibody production measured at higher DO was matched by a decrease in the viable cell concentration at steady state, so that the volumetric antibody titre was not changed significantly. An attempt to grow cells at 250% of air saturation was unsuccessful but the cells recovered to normal growth once the DO was decreased.There was a requirement for cellular adaptation at each step-wise increase in dissolved oxygen. Adaptation to a DO of 100% was associated with an increase in the specific activities of glutathione peroxidase (×18), glutathione S-transferase (×11) and superoxide dismutase (×6) which are all known antioxidant enzymes. At DO above 100%, the activities of GPX and GST decreased possibly as a result of inactivation by reactive oxygen radicals.The increase in dissolved oxygen concentration caused changes in energy metabolism. The specific rate of glucose uptake increased at higher dissolved oxygen concentrations with a higher proportion of glucose metabolized anaerobically. Short-term radioactive assays showed that the relative flux of glucose through glycolysis and the pentose phosphate pathway increased whereas the flux through the tricarboxylic acid cycle decreased at high DO. Although the specific glutamine utilization rate increased at higher DO, there was no evidence for a change in the pattern of metabolism. This indicates a possible blockage of glycolytic metabolites into the TCA cycle, and is compatible with a previous suggestion that pyruvate dehydrogenase is inhibited by high oxygen concentrations.Analysis of the oxygen uptake rate of cell suspensions at steady state under all conditions showed a pronounced Crabtree effect which was manifest by a decrease (up to 40%) in oxygen consumption on addition of glucose. This indicates that the degree of aerobic metabolism in these cultures is highly sensitive to the glucose concentration. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 153-164, 1997.
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    Establishing apoptosis resistant cell lines for improving protein productivity of cell culture (1997)
    Springer
    Cytotechnology 23 (1997), S. 55-59 
    Details
    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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    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.
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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 
    Details
    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 
    Details
    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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    Hybridoma growth in a new generation hollow fibre bioreactor: antibody productivity and consistency (1997)
    Springer
    Cytotechnology 24 (1997), S. 109-119 
    Details
    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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    URL: http://dx.doi.org/10.1023/A:1007922004714
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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 
    Details
    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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    URL: http://dx.doi.org/10.1023/A:1007910831550
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    Dichloroacetate increases cell and antibody yields in batch cultures of a hybridoma cell line (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 377-382 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; batch culture ; dichloroacetate ; metabolism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have studied the effect of the pyruvate dehydrogenase (PDH) activator, dichloroacetate (DCA), on the growth, metabolism, and productivity of the PQXB ½ hybridoma cell line. In control batch cultures, cessation of growth and the onset of decline phase coincided with the time at which the media became exhausted of glutamine. Supplementation of the media with DCA (1 mM) extended the growth phase of this cell line by approximately 20 h without affecting its growth rate. This prolonged period of growth resulted in an increased maximum cell density (16%) and final antibody yield (55%). Repeat experiments showed these effects to be reproducible, with the increases in antibody yield being between 50 and 60%. DCA did not affect the specific rates of glucose utilization and lactate production. However, it decreased the specific glutamine consumption rate. This characteristic of DCA action appeared, at least in part, to provide an explanation for the extended growth phase exhibited by DCA-treated cultures, since it delayed the time at which the media became depleted of glutamine. The consumption and production kinetics for various nutrients and their metabolites in both control and DCA-treated cultures suggested that: (1) glutamine catabolism proceeded by a pathway involving conversion to glutamate by glutaminase followed by subsequent transamination by alanine aminotransferase, and (2) DCA decreased the specific glutamine consumption rate by directly or indirectly inhibiting the transamination. It is expected that the routine inclusion of DCA in media used for hybridoma cultivation will be valuable for enhancement of monoclonal antibody (Mab) yields on a laboratory scale. © 1996 John Wiley & Sons, Inc.
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    Material balance studies on animal cell metabolism using a stoichiometrically based reaction network (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 579-590 
    Details
    ISSN: 0006-3592
    Keywords: fed batch ; hybridoma ; material balance ; reaction network ; stoichiometric analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A detailed reaction network of mammalian cell metabolism contains hundreds of enzymatic reactions. By grouping serial reactions into single overall reactions and separating overlapped pathways into independent reactions, the total number of reactions of the network is significantly reduced. This strategy of manipulating the reaction network avoids the manipulations of a large number of reactions otherwise needed to determine the reaction extents. A stoichiometric material balance model is developed based on the stoichiometry of the simplified reaction network. Closures of material balances on glucose and each of the 20 amino acids are achieved using experimental data from three controlled fed-batch and one-batch hybridoma cultures. Results show that the critical role of essential amino acids, except glutamine, is to provide precursors for protein synthesis. The catabolism of some of the essential amino acids, particularly isoleucine and leucine, is observed when an excess amount of these amino acids is available in the culture medium. It was found that the reduction of glutamine utilization (for reducing ammonia production) is accompanied by an increase in the uptake of nonessential amino acids (NAAs) from the culture medium. This suggests that NAAs are necessary even though they are not essential for cell growth. A glutamine balance shows that less than 20% of the glutamine nitrogen is utilized for essential roles, such as protein and nucleotide syntheses. A relatively constant percentage (about 45%) of the glutamine nitrogen is utilized for NAA biosynthesis, despite the fact that the absolute amount varies among the four experiments. As to the carbon skeleton of glutamine, a significant portion enters the tricarboxylic acid (TCA) cycle. A material balance on glucose shows that most of the glucose (81%) is converted into lactate when glucose is in excess. On the other hand, when glucose is limited, lactate production is considerably reduced, while a major portion of glucose (48%) enters the TCA cycle. The fraction of glucose used for the synthesis of cellular components ranges from 9 to 28%. © 1996 John Wiley & Sons, Inc.
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    Energy metabolism and ATP balance in animal cell cultivation using a stoichiometrically based reaction network (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 591-601 
    Details
    ISSN: 0006-3592
    Keywords: animal cell metabolism ; ATP balance ; energy metabolism ; reaction network ; stoichiometric analysis ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A metabolic reaction network is developed for the estimation of the stoichiometric production of adenosine triphosphate (ATP) in animal cell culture. By using the material balance data from fed-batch and batch cultures of hybridoma cells, the stoichiometric ATP productions are determined with estimated effective P/O ratios of 2 for NADH and 1.2 for FADH2. A significant percentage of the ATP requirement (16-41%) in hybridoma cells is generated directly from free energy release without the participation of oxygen. The oxidative phosphorylation of NADH accounts for about 60% of the total ATP production in the fed-batch cultures and about 47% in the batch culture. The oxidative phosphorylation of FADH2 accounts for less then 20% of the total ATP production in all cases.A fractional model is devised to analyze the contribution of each nutrient to the ATP production. Results show that a majority of the ATP is produced from glucose metabolism (60-76%). Less than 30% of the ATP is derived from glutamine, and less than 11% is derived from other essential amino acids. The analysis also shows that the glycolytic pathway generates more ATP in the batch (41%) than in the fed-batch (〈27%) cultures. The TCA cycle provides 51-68% of the total ATP production. The calculated stoichiometric oxygen consumption differs among the batch and fed-batch cultures, depending on the glucose concentration. This result suggests that the relationship between the oxygen uptake rate (OUR) and cell growth may change with the culture conditions. However, the calculated respiratory quotient (RQ) is relatively constant in all cases.A linear relationship is obtained between the specific ATP production rate and the specific cell growth rate. The maximum ATP yield and the maintenance ATP requirement are determined based on this linear relationship. The biosynthetic ATP demand estimated from the dry cell weight and cell composition is significantly lower than that calculated from the maximum ATP yield, indicating that the non-growth-associated ATP demand may contain other factors than what is considered in the estimation of the biosynthetic ATP demand. © 1996 John Wiley & Sons, Inc.
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    Use of the weighted jackknife method to calculate the variance in cellular-specific protein secretion rate: Application to monoclonal antibody secretion rate kinetics in response to osmotic stress (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 184-196 
    Details
    ISSN: 0006-3592
    Keywords: specific secretion rate ; animal cell culture ; hybridoma ; osmotic stress ; variance of specific secretion rate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The specific secretion rate (q, μg protein secreted/viable cell-h) and its variance are very useful to compare the capability of cell lines for protein secretion. An assessment of specific secretion rate variability is also beneficial and important when the specific secretion rate is to be used as an on-line process parameter to monitor culture production behavior or for in-process decisionmaking. Experimental errors in mammalian cell culture (e.g., protein concentration measurement and cell counting) and estimation error in the method of calculating q contribute to the total variance of the specific secretion rate. Although the variance of q is essential for comparing the differences between cell lines and the response of the same cell line to different nutrient or environmental conditions, few methods for calculating the variance of the specific secretion rate have been reported. As a model system, we have used the weighted jackknife method and the delta method to calculate the variance in the specific secretion rate of a murine monoclonal antibody (qmAb) determined by a differential method. These methods were applied to calculate qmAb and its standard deviation to determine the change in qmAb kinetics during batch culture of the 9.2.27 hybridoma in response to growth in hyperosmotic media or osmotic stress. Without osmotic stress, during exponential growth in DMEM + 5% FBS spinner culture, the estimate of qmAb decreases at least threefold. Results indicate that the 9.2.27 hybridoma responds to hyperosmotic media (400 mOsm, 470 mOsm) by significantly reducing the degree of qmAb decrease in the exponential phase, thus maintaining a higher qmAb through the stationary phase. The trend of qmAb during the batch cultures studied is further confirmed by t-test. Osmotic stress is statistically shown to be able to alter significantly the hybridoma-specific mAb secretion kinetics during batch culture. Determination of the variance of specific secretion rate using the weighted jackknife method offers a powerful approach for establishing the confidence limits of specific protein secretion rate between cell cultures in different nutritional or osmotic environments. © 1996 John Wiley & Sons, Inc.
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    On-line estimation of viable cells in a hybridoma culture at various DO levels using ATP balancing and redox potential measurement (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 277-283 
    Details
    ISSN: 0006-3592
    Keywords: cell culture ; on-line viable cell concentration ; ATP balance ; redox potential ; hybridoma ; dissolved oxygen ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Two on-line methods for the estimation of viable cell number in hybridoma cultivation were investigated. One used an empirical correlation between redox potential and animal cell density. The other was based on an ATP balance with ATP steady-state assumption. Oxygen uptake rate measurement provided the amount of ATP which was produced by oxidation of NADH. Oxygen uptake rate was measured either by stationary liquid phase balance with surface aeration or by gas balance during bubble aeration with headspace flushing with an inert gas. The amount of ATP produced through the glycolysis was estimated based on the amount of lactate produced. In cultures, in which pH was controlled via manipulation of the gas phase composition, the flow of CO2 was linearly correlated with the lactate concentration. At constant dissolved oxygen levels, the viable cell density was proportional to the estimated ATP production rate, during exponential growth and during later phases. The estimated specific ATP production rate, however, varied from 2.2 pmol cell-1 h-1 at 10% air saturation to 4.5 pmol cell-1 h-1 at 100% air saturation. Specific rates of glutamine, glucose, and lactate followed the shape of the specific ATP production rate, whereas the specific oxygen uptake rate was minimal at around 50% air saturation. © 1996 John Wiley & Sons, Inc.
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    On-line measurement of oxygen uptake in cell culture using the dynamic method (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 443-448 
    Details
    ISSN: 0006-3592
    Keywords: on-line ; oxygen uptake rate ; OUR ; cell culture ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Measurement of oxygen uptake rate is useful in assessing growth, viability, and metabolic activity. In cell culture, however, the oxygen demand is extremely small (typically 0.1-0.3 mM O2L-h) and is very difficult to measure accurately using conventional offgas analysis. In many industrial submerged cell culture systems, dissolved oxygen levels are controlled between preset limits by intermittent sparging of air or oxygen. This article describes a computational method for the automatic online determination of oxygen uptake from the dynamic dissolved oxygen probe response. Experimental measurements show that for a typical hybridoma culture, specific oxygen demand is 0.15 mM O2/109 cells/h. © 1996 John Wiley & Sons, Inc.
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    The crystal violet nuclei staining technique leads to anomalous results in monitoring mammalian cell cultures (1996)
    Springer
    Cytotechnology 21 (1996), S. 73-80 
    Details
    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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    Cell suicide in starving hybridoma culture: survival-signal effect of some amino acids (1996)
    Springer
    Cytotechnology 21 (1996), S. 81-89 
    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 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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    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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    Evaluation of a simple protein free medium that supports high levels of monoclonal antibody production (1996)
    Springer
    Cytotechnology 21 (1996), S. 95-109 
    Details
    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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    On-line gas analysis in animal cell cultivation: II. Methods for oxygen uptake rate estimation and its application to controlled feeding of glutamine (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 45 (1995), S. 54-62 
    Details
    ISSN: 0006-3592
    Keywords: oxygen uptake rate ; animal cell cultivation ; hybridoma ; monoclonal antibody ; glutamine ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Different methods for oxygen uptake rate (OUR) determinations in animal cell cultivation were investigated using a high quality mass spectrometer. Dynamic measurements have considerable disadvantages because of disturbances of the growing cells by the necessary variations of dissolved oxygen concentration. Only infrequent discrete measurements are possible using this method. Stationary liquid phase balance yielded better results with much higher frequency. Gas phase balancing has the advantage of not requiring dissolved oxygen measurement and knowledge of KLa, both of them are easily biased. It was found that simple gas phase balancing is either very inaccurate (error larger than expected signal) or very slow, with gas phase residence times of several hours. Therefore, a new method of aeration was designed. Oxygen and CO2 transfer are mainly achieved via sparging. The gas released to the headspace is diluted with a roughly 100-fold stream of an inert gas (helium). Through this dilution, gas ratios are not changed for O2, CO2, Ar, and N2. The measurement of lower concentrations (parts per million and below) is easy using mass spectrometry with a secondary electron multiplier. With this new method an excellent accuracy and sufficient speed of analysis were obtained. All these on-line methods for OUR measurement were tested during the cultivation of animal cells. The new method allowed better study of the kinetics of animal cell cultures as was shown with a hybridoma cell line (HFN 7.1, ATCC CRL 1606) producing monoclonal antibodies against human fibronectin. With the aid of these methods it was possible to find a correlation between a rapid decrease in oxygen uptake rate (OUR) and glutamine concentration. The sudden decrease in OUR can be attributed to glutamine depletion. This provided a basis for the controlled addition of glutamine to reduce the formation of ammonia produced by hydrolysis. This control method based on OUR measurement resulted in increased cell concentration and threefold higher product concentration. © 1995 John Wiley & Sons, Inc.
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    Correlation between steady-state cell concentration and cell death of hybridoma cultures in chemostat (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 45 (1995), S. 18-26 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; cell death ; chemostat ; autoinhibitor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In the present study, the steady-state cell density (X) of chemostat cultures of murine hybridoma was varied by the concentration of glucose and glutamine in culture medium and the dissolved oxygen partial pressure. Except at low glutamine and low oxygen levels, the specific death rate (kd) of the cultures was found to decrease with increasing dilution rate (D). However, the plot of kd vs. X/D yielded linear relation, which suggests that cell death was due to a non-growth-linked inhibitory product of the cells. The kd value measured at low glutamine and low oxygen levels remained practically unchanged over a wide range of D between 0.020 and 0.029 h-1. The kd for low oxygen cultures was always lower than the values obtained in low glucose and low glutamine cultures. A low-molecular-weight component of possibly less than 3000 MW was detected to be cell-death-inducing in the supernatant of exponentially growing cultures. It was neither lactate nor ammonium. The autoinhibitor was not cell-line specific. © 1995 John Wiley & Sons, Inc.
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    Starvation-induced programmed death of hybridoma cells: Prevention by amino acid mixtures (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 45 (1995), S. 86-90 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; nutrition ; cell death ; apoptosis ; monoclonal antibody ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Association of the availability of nutrients with the phenomenon of programmed cell death - apoptosis - was investigated using hybridoma cells cultured in protein-free medium under conditions of starvation, i.e., in RPMl-1640 medium diluted to 50% with saline. Amino acid mixtures, such as MEM essential amino acids or MEM nonessential amino acids were found to prevent starvation death significantly when added to the diluted medium in 1 to 2 mM concentrations, the MEM vitamin mixture was ineffective, and glutamine displayed a moderate growth-supporting effect. The specific monoclonal antibody production rate in cultures supplemented with amino acid mixtures was strikingly low, whereas supplementation with glutamine alone or simultaneously with other amino acids resulted in a specific antibody production rate comparable with the rate observed in undiluted medium. © 1995 John Wiley & Sons, Inc.
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    Relationship between loss of heavy chains and the appearance of nonproducing hybridomas (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 47 (1995), S. 270-275 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; antibody ; heavy chain ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: One drawback to the in vitro production of monoclonal antibodies is the loss of productivity exhibited by hybridomas over time, which has been shown to correspond to the appearance of a nonproducing subpopulation. In this study, we monitored the presence of antibody components, both intra- and extracellular, between producing and nonproducing hybridomas. A nonproducing cell population appeared which lacked heavy chain, while all cultures continued to produce light chain, indicating that the loss in antibody production resulted from the absence of heavy chain and occurred before protein assembly or secretion. © 1995 John Wiley & Sons, Inc.
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    In-situ removal of ammonium and lactate through electrical means for hybridoma cultures (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 47 (1995), S. 308-318 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; cell growth ; antibody production ; toxic waste removal ; electrical technique ; electrokinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Ammonium and lactate are two known toxic products detrimental to mammalian cell growth and productivity. An electrokinetic technique, utilizing an electrophoretic mechanism, was developed to remove these cellular wastes in-situ from suspension hybridoma (ATCC CRL-1606) cultures to enhance cell growth and productivity. This technique applies continuously a dc electric field to selectively remove the electrically charged wastes. The experiments were shown to be successful in the removal of externally added 10 rnM ammonium and 45 mM lactate while maintaining the chemostatic condition of culture medium in a cell-free condition under an electric current density of 50 A/m2. Toxic levels of ammonium were added, ranging from 7.5 to 12.5 mM, at the start of the hybridoma culture, and the applied dc electric fields were able to completely remove these added materials. This in turn released the inhibition and restored the cell growth. Finally, this electrokinetic technique was applied to the batch and glutamine fed-batch hybridoma cultures. At an applied electric current density of 50 A/m2, this was able to completely remove cell-produced ammonium and increased the cell growth and antibody titer by 30% to 50%, respectively, compared to the control experiment in the absence of the electric field. Lastly, the applied electric current density of 50 A/m2 did not affect cellular functionalities such as glucose and glutamine consumption and antibody productivity.© 1995 John Wiley & Sons, Inc
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    Overexpression of bcl-2, apoptosis suppressing gene: Prolonged viable culture period of hybridoma and enhanced antibody production (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 48 (1995), S. 118-122 
    Details
    ISSN: 0006-3592
    Keywords: apoptosis ; bcl-2 ; hybridoma ; cell survival ; antibody productivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Human bcl-2 DNA was introduced into mouse hybridoma 2E3 cells and expressed at a high level by using BCMGSneo vector, which reportedly amplifies as multiple copies in the cells independently of their chromosomes. The high expression of bcl-2 in BCMGSneo-bcl-2 transfectants was confirmed by western blotting. In batch cultures, the overexpression of bcl-2 raised the maximum viable cell density by 45%, delayed the initiation of apoptosis by 2 days, and prolonged the viable culture period by 4 days. The delayed initiation of apoptosis was detected by emergence of the ladder pattern on DNA electrophoresis and increase of the dead cell number. The bcl-2 transfectants produced lgG1 fourfold per batch culture in comparison with 2E3 cells transfected with BCMGSneo but not with bcl-2: a little less than twofold due to the improved survival of the cells and more than twofold due to the enhanced lgG1 production rate per cell of the bcl-2 transfectants. The method to engineer hybridoma cells genetically with bcl-2 using BCMGSneo vector for increasing viability and productivity would be widely applied for improving antibody productivity of hybridoma cultures. © 1995 John Wiley & Sons, Inc.
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    A combined cell-cycle and metabolic model for the growth of hybridoma cells in steady-state continuous culture (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 48 (1995), S. 49-65 
    Details
    ISSN: 0006-3592
    Keywords: cell cycle ; apoptosis ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The Model presented in this work demonstrates the combination of cell-cycle model with a model describing the growth and conversion kinetics of hybridoma cells in a steady-state continuous culture. The cell-cycle model is based upon a population balance model as described by Cazzador et al. and assumes the existence of a cycling-and apoptotic-cell population, which together form the viable-cell population. In this part the fraction of apoptotic cells, the age distribution of the cycling and apoptotic-cell population, the mean volume and biomass content per cell of the cycling, apoptotic, and viable cells, and the specific growth and death rates of the cells are calculated. The metabolic part consists of a Monod-type growth equation, four elemental balances, an equation assuming a constant yield of ammonia on glutamine, an equation for product formation, and the relation of Glacken for energy production. Furthermore, a maintenance-energy model for the consumption of glucose and glutamine is introduced, which combines the approaches of Herbert and Pirt into one model in a way similar to Beeftink et al. For energy consumption a Pirt model is assumed. The model is capable of predicting trends in steady-state vaues of a large number of variables of interest like specific growth rate, specific death rate, viability, cell numbers, mean viable-cell volume, and concentrations and conversion rates of product, glucose, glutamine, lactate, and ammonia. Also the concentrations and conversion rates of oxygen and carbon dioxide are qualitatively predicted. The values of the model predictions are generally close to experimental data obtained from literature. © 1995 John Wiley & Sons, Inc.
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    URL: http://dx.doi.org/10.1002/bit.260480109
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    Protein-free human-human hybridoma cultures in an intercalated-spiral alternate-dead-ended hollow fiber bioreactor (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 47 (1995), S. 384-400 
    Details
    ISSN: 0006-3592
    Keywords: hollow fiber ; bioreactor ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Batch cell cultures of a human-human hybridoma line in a convective flow dominant intercalated-spiral altetnate-dead-ended hollow fiber are compared with those using conventional axial-flow hollow fiber bioreactors and a stirred-tank bioreactor. Relatively short-term fed-batch and perfusion cell cultures were also employed for the intercalated-spiral bioreactor. When operating conditions of a batch intercalated-spiral bioreactor were properly chosen, the cell growth and substrate consumption paralleled that of a batch stirred-tank culture. The results verified the premise of the intercalated-spiral hollow fiber bioreactor that nutrient transport limitations can be eliminated when the convective flux through the extracapillary space is sufficiently high.© John Wiley & Sons, Inc.
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    Enhancement of monoclonal antibody production by immobilized hybridoma cell culture with hyperosmolar medium (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 48 (1995), S. 699-705 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; hyperosmotic stress ; immobilization ; antibody productivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: To determine the effect of hyperosmotic stress on the monoclonal antibody (MAb) production by calcium-alginate-immobilized S3H5/γ2bA2 hybridoma cells, the osmolalities of medium in the MAb production stage were varied through the addition of NaCI. The specific MAb productivity (qMAb) of immobilized cells exposed to abrupt hyperosmotic stress (398 mOsm/kg) was increased by 55% when compared with that of immobilized cells in the control culture (286 mOsm/kg). Furthermore, this enhancement of qMAb was not transient. Abrupt increase in osmolality, however, inhibited cell growth, resulting in no increase in volumetric MAb productivity (rMAb). On the other hand, gradual increase in osmolality allowed further cell growth while maintaining the enhanced qMAb immobilized cells. The qMAb immobilized cells at 395 mOsm/kg was 0.661 ± 0.019 μg/106 cells/h, which is almost identical to that of immobilized cells exposed to abrupt osmotic stress. Accordingly, the rMAb was increased by ca. 40% when compared with that in the control immobilized cell culture. This enhancement in iMAb of immobilized S3H5/γ2bA2 hybridoma cells by applying gradual osmotic stress suggests the potential of using hyperosmolar medium in other perfusion culture systems for improved MAb production. © 1995 John Wiley & Sons, Inc.
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    Surface IgG content of murine hybridomas: Direct evidence for variation of antibody secretion rates during the cell cycle (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 47 (1995), S. 535-540 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; flow cytometry ; cell cycle ; surface lgG ; antibody secretion rate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Previous experiments have shown that population average surface lgG content is correlated with the specific antibody production rates of batch hybridoma cultures. Therefore, surface associated lgG content of single hybridoma cells might indicate antibody secretion rates of individual cells. Moreover, the surface lgG content should reflect the pattern of secretion rates during the cell cycle. To probe for lgG secretion rates during the cellcycle, a double staining procedure has been developed allowing simultaneousflow cytometric analysis of surface lgG content and DNA content of murine hybridoma cells. Crosslinking of the surface associated immunofluorescence with the cell by paraformaldehyde fixation permits subsequent DNA staining without loss of immunofluorescence. The optimized protocol has been used to determine the pattern of the surface lgG fluorescence as a function of the cell cycle position. It is highest during the G2+M cell cycle phase and the experimental data are in excellent agreement with the previously predicted secretion pattern during the cell cycle. © 1995 John Wiley & Sons Inc.
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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 
    Details
    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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    Modified monoclonal antibody production kinetics kappa/gamma mRNA levels, and metabolic activities in a murine hybridoma selected by continuous Culture (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 44 (1994), S. 753-764 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; subclone ; continuous culture ; batch culture ; igG-mRNA ; biosynthetic activities ; antibody production ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: During long-term continuous culture of the hybridoma cell line 11317, a better-producing subclone (I1317-SF11), giving improved productivity, has been selected. The comparison of the original cell line (I1317-DC) with this subclone revealed that although the growth patterns of both clones were similar, both in continuous and in batch cultures, considerable differences could be seen between the clones with respect to monoclonal antibody (MAB) accumulation, MAB production rate, the levels of mRNA coding for heavy and light chains of IgG, and some metabolic activities. In continuous culture as well as in batch culture, I1317-SF11 showed increased levels of mRNA coding for kappa and gamma chains compared with I1317-DC and/or a modified ratio of the mRNA species when compared to that in I1317-DC. Using pulse experiments, it could be established that the biosynthesis of both chains was augmented in I1317-SF11. Although the kappa and gamma mRNA levels were modified or inversed for I1317-SF11, the cells always synthesized more kappa than gamma chains. The overall increase in the synthetic activity of I1317-SF11 is suggested as one reason for the considerable increase of IgG productivity and product accumulation in continuous culture as well as in repeated batch cultures. Tests concerning metabolic activity revealed that I1317-SF11 had a predominantly glycolytic metabolism independent of growth requirements, whereas for I1317-DC the metabolism became increasingly glycolytic with increased growth. The antibody yield coefficient of I1317-SF11 on glutamine was significantly higher than that of I1317-DC for the continuous culture, whereas the antibody coefficients on glucose were almost similar for both clones under the different culture conditions used. Both antibody coefficients were considerablly influenced by the specific growth rate.All these facts together lead to the conclusion that subclone I1317-SF11 uses more of the energy available, or it was the energy and/or precursors available for the synthesis and production of MAB more efficiently than the thesis and production of MAB more efficiently than the original cell line. Although the levels of mRNA coding for heavy and light chains of IgG were modified, it could be confirmed that the overall regulation of MAB-synthesis and -production occurs post-translationally and that at higher growth rates, more biosynthetic activity is diverted to biomass production. © 1994 John Wiley & Sons, Inc.
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    Bubble bed reactor: A reactor design to minimize the damage of bubble aeration on animal cells (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 44 (1994), S. 1246-1254 
    Details
    ISSN: 0006-3592
    Keywords: bubble aeration ; oxygen transfer ; hybridoma ; cell damage ; foam reduction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A new bubble aeration system was designed to minimize cell killing and cellular damage due to sparging. The residence time of the bubbles in the developed bubble bed reactor was prolonged dramatically by floating them in a countercurrent produced by an impeller. The performance of the new reactor bubble aeration system, implemented in a laboratory reactor, was tested in dynamic aeration experiments with an without cells. An efficiency up to 95% in oxygen transfer could be achieved, which enables a much lower gas flow rate compared with conventional bubble aeration reactors. The low gas flow rate is important to keep cell damage by bubbles as low as possible. A laser light sheet technique used to find the optimal flow pattern in the reactor. The specific power dissipation of the impeller is a good measure to predict cell damage in a turbulent flow. Typical values for the power dissipation measured in the bubble bed reactor were in the range of 0.002 to 0.013 W/kg, which is far below the critical limit for animal cells. The growth of a hybridoma cell line was studied in cell cultivation experiments. A protein-free medium without supplements such as serum or Pluronic F68 was used to exclude any effect of cell-protecting factors, No difference in the specific growth rate and the yield of the antibodies was observed in cell grown in the bubble free surface aeration in the spinner flask. In contrast to the spinner flask, however, the bubble bed reactor design could be scaled up. © 1994 John Wiley & Sons, Inc.
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    Effect of temperature on nucleotide pools and monoclonal antibody production in a mouse hybridoma (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 44 (1994), S. 1235-1245 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; monoclonal antibody ; temperature ; nucleotides ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The specific monoclonal antibody productivity (qMab) of a murine hybridoma (CC9C10) increased with incubation temperature in the range 33°C to 39°C. qMab was constant at each temperature and was independent of the phase of culture. The qMab increased 97% at 39°C and decreased by 21% at 33°C compared with controls at 37°C. Specific rates of substrate (glucose and glutamine) utilization and byproduct (lactate and ammonia) formation also increased with temperature but the yield coefficient, YLac/Llc' was constant for 33°C to 39°C and YAmm/Gin was constant for 37°C to 39°C. YAmm/Gin at 33°C was lower than the control. Changes in specific nucleotide concentrations and ratios were monitored by analysis of intracellular nucleotide pools. The NTP ratio, (ATP + GTP)/(UTP + CTP), increased and the U-ratio (UTP/UDP-GNac) decreased during the course of each culture, whereas the adenylate energy charge, (ATP + 0.5ADP)/(ATP + ADP + AMP), remained relatively constant at a value 0.8. The relative content of UDP-/N acetyl galactosamine, UDP-N acetyl glucosamine, and NAD increased with incubation temperature, whereas the relative ATP content, SA(ATP + ADP + AMP)/SU (UTP + UDP-sugars) ratio, purine/pyrimidine, ATP/GTP, and U-ratio decreased at higher incubation temperatures. It is possible that these nucleotide parameters may have a regulatory role in the changes of qMab observed at the higher temperatures. © 1994 John Wiley & Sons, Inc.
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    Induction of apoptosis in nutrient-deprived cultures of hybridoma and myeloma cells (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 44 (1994), S. 1140-1154 
    Details
    ISSN: 0006-3592
    Keywords: ammonia ; apoptosis ; hybridoma ; lactate ; myeloma ; nutrient deprivation ; programmed cell death ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In the present study, cell death was investigated in cultures of NS/0 myelomas and SP2/0-derived D5 hybridomas through morphological examination of cells stained with acridine orange and ethidium bromide. The relative contribution of elevated levels of lactic acid and ammonia, as well as deprivation of glutamine, cystine, and glucose on the induction of necrosis or apoptosis, was investigated. In batch culture of D5 hybridoma cells, induction of apoptotic cell death correlated with the exhaustion of glutamine, while in the case of NS/0 myelomas, it coincided with exhaustion of cystine. To determine whether limiting nutrients were the actual triggering factors for apoptosis in batch culture, exponentially growing cells were resuspended in glutamine or cystine-free media. Within 30 to 40 h, viability decreased to 50% and the nonviable cell population displayed typical apoptotic morphology, with crescents of condensed chromatin around the periphery of the nucleus, or with the entire nucleus present as one or a group of featureless, brightly staining spherical beads. Similarly, D5 hybridomas and NS/0 myelomas cultivated in glucose-free medium died mainly from apoptosis. Cells were also cultivated in fresh medium supplemented with elevated concentrations of ammonia (3.0 mM) and/or lactate (35 mM, 50 mM). This resulted in decreased viabilities and necrotic death in both cell lines. From these results, we conclude that D5 hybridomas and NS/0 myelomas deprived of essential nutrients die by apoptosis, whereas incubation in the presence of elevated levels of metabolic byproducts such as ammonia and lactate will induce necrotic cell death in these cells. © 1994 John Wiley & Sons, Inc.
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    Ammonia inhibition of hybridomas propagated in batch, fed-batch, and continuous culture (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 43 (1994), S. 434-438 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; continuous culture ; ammonia ; growth inhibition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The nature and temporal development of ammonia inhbition were investigated in batch, fed-batch, and continuous cultures. Significant inhibition was observed when cells were inoculated in serum-containing or chemically defined medium containing more than 2 mM of ammonia. In contrast, no inhibition was observed at greater than 10 mM when the ammonia concentration was gradually increased over the span of a batch culture by feeding ammonium chloride. Strong growth inhibition was observed after each of five step changes (2.8 → 3.7 → 4.0 → 4.9 → 7.7 → 13.5 mM) in continuous culture. Following a period of adaptation at each higher value, the viable cell density stabilized at a new lower value. The lowering in viable cell density was caused by an increase in specific death rate and a decreased cell yield on glucose, glutamine, and oxygen. Increased ammonia concentration had little or no effect on the steady-state specific growth kinetics or specific antibody productivity. © 1994 John Wiley & Sons, Inc.
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    Fed-batch cultivation of animal cells using different medium design concepts and feeding strategies (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 43 (1994), S. 1175-1189 
    Details
    ISSN: 0006-3592
    Keywords: fed-batch ; medium design ; animal cell culture ; ammonia ; lactate ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In animal cell cultivation, cell density and product concentration are often low due to the accumulation of toxic end-products such as ammonia and lactate and/or the depletion of essential nutrients. A hybridoma cell line (CRL-1606) was cultivated in T-flasks using a newly devised medium feeding strategy. The goals were to decrease ammonia and lactate formation by the design of an initial medium which would provide a starting environment to achieve optimal cell growth. This was followed by using a stoichiometric equation governing animal cell growth and then designing a supplemental medium for feeding strategy used to control the nutritional environment. The relationship between the stoichiometric demands for glutamine and nonessential amino acids was also studied. Through stoichiometric feeding, nutrient concentrations were controlled reasonably well. Consequently, the specific production rate of lactate was decreased by fourfold compared with conventional fed-batch culture and by 26-fold compared with conventional batch culture. The specific production rate of ammonia was decreased by tenfold compared with conventional fed-batch culture and by 50-fold compared with conventional batch culture. Most importantly, total cell density and monoclonal antibody concentration were increased by five- and tenfold respectively, compared with conventional batch culture. © 1994 John Wiley & Sons, Inc.
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    Cell cycle kinetics and monoclonal antibody productivity of hybridoma cells during perfusion culture (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 44 (1994), S. 361-367 
    Details
    ISSN: 0006-3592
    Keywords: cell cycle ; flow cytometry ; perfusion culture ; hybridoma ; monoclonal antibody productivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The flow-cytometric (FCM) analysis of bivariate DNA/lgG distributions has been conducted to study the cell cycle kinetics and monoclonal antibody (MAb) production during perfusion culture of hybridoma cells. Three different perfusion rates were employed to demonstrate the dependency of MAb synthesis and secretion on cell cycle and growth rate. The results showed that, during the rapid growth period of perfusion culture, the level of intracellular igG contents of hybridoma cells changed significantly at each perfusion rate, while the DNA histograms showing cell cycle phases were almost constant. Meanwhile, during the reduced growth period of perfusion culture, the fraction of cells in the S phase decreased, and the fraction cells in the G1/G0 phase increased with decreasing growth rate. The fraction of cells in the G2/M phase was relatively constant during the whole period of perfusion culture. Positive correlation was found between mean intracellular IgG contents and the specific MAb production rate, suggesting that the deletion of intracellular IgG contents by a flow cytometer could be used as a good indicator for the prediction of changes in specific MAb productivity following manipulation of the culture condition. © 1994 John Wiley & Sons, Inc.
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    Growth characteristics in fed-batch culture of hybridoma cells with control of glucose and glutamine concentrations (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 44 (1994), S. 95-103 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; antibody production ; glutamine ; glucose ; fed-batch culture ; adaptive control ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An online system using HPLC was developed for the measurement of glucose, glutamine, and lactate in a culture broth. Using the system, the glucose and glutamine concentrations were controlled simultaneously by an adaptive-control algorithm within the ranges of 0.2 to 2.0 and 0.1 to 0.6 g/L, respectively. When the glucose concentration was controlled at the low level of 0.2 g/L, the intracellular lactate dehydrogenase activity decreased by one-half and the lactate concentration by one-third, whereas the uptake rates of serine and glycine were about twice as high, compared with the amounts when the glucose concentration was controlled at 1.0 g/L. On the other hand, ammonia production increased when the glucose concentration was kept low. To reduce the production of inhibitory metabolites such as ammonia and lactate and improve the antibody production rate in a hybridoma cell culture, the concentrations of glucose and glutamine were controlled at 0.2 and 0.1 g/L, respectively. With these low concentrations of glucose and glutamine, the cell concentration (4.1 × 106 cells/mL) and antibody production (172 mg/L) both increased about twofold compared with the amounts when the glucose was controlled at higher levels. From these results, simultaneous control of the glucose and glutamine concentrations was shown to be useful in the production of antibody by hybridoma cell cultivation. © 1994 John Wiley & Sons, Inc.
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    Enhancement of monoclonal antibody yield by hybridoma fed-batch culture, resulting in extended maintenance of viable cell population (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 44 (1994), S. 1099-1106 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; fed batch ; materials balancing ; fermentation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Hybridoma batch cultures were extended using feed formulations based on nutrient consumption measured during different batch culture phases when (a) growth but negligible antibody production was taking place; (b) maximum antibody production rate and declining viable cell growth rate were observed. Strategy (a) was the more successful (2.8-fold compared with 1.8-fold antibody titer increase) and maintained cell viability for longer. Analysis of the effects of omitting individual amino acids yielded results which were consistent with those from the feeding experiment © 1994 John Wiley & Sons, Inc.
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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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    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.
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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 
    Details
    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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    Induction of apoptosis in oxygen-deprived cultures of hybridoma cells (1994)
    Springer
    Cytotechnology 15 (1994), S. 117-128 
    Details
    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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    Design of a bubble-swawm bioreactor for animal cell culture (1994)
    Springer
    Cytotechnology 15 (1994), S. 301-309 
    Details
    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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    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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    Selecting and designing cell lines for improved physiological characteristics (1994)
    Springer
    Cytotechnology 15 (1994), S. 11-16 
    Details
    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 
    Details
    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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    Peptone, a low-cost growth-promoting nutrient for intensive animal cell culture (1994)
    Springer
    Cytotechnology 16 (1994), S. 17-26 
    Details
    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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    Cell Culture conditions determine the enhancement of specific monoclonal antibody productivity of calcium alginate-entrapped S3H5/γ2bA2 hybridoma cells (1993)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 330-340 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; Immobilization ; monoclonal antibody productivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Immobilization offers several intrinsic advantages over free suspension cultures for the production of monoclonal antibodies. An important advantage of immobilization is the improved specific monoclonal antibody (MAb) productivity (qMAb) that can be obtained. However, there are conflicting reports in the literature on the enhancement of the qMAb with immobilization. The discrepancies between these reports can be attributed to the different to either the cultivation methods used for immobilized cell or to difference between the cell lines used in the various studies. We show that these differences may be attributed to the different cultivation methods used for one model hybridoma cell line. S3H5/ϒ2bA2 hybridoma cells entrapped in different sizes of calcium alginate beads were cultivated in both T- and spinner flasks in order to determine whether cultivation methods (T- and spinner flasks) and bead size influence the qMAb Free-suspended cell cultures inoculated with cells recovered from alginate beads were also carried out in order to determine whether changes in the qMab of the entrapped cells are reversible.The cultivation methods was found to influence significantly the qMAb of the entrapped cells. When the entrapped cells in 1-mn diameter beads were cultivated in T-flasks, the qMAb was not increased by 200% as previously observed in an entrapped cell culture using 1-mm-diameter alginate beads in spinner flasks. The qMAb of the entrapped cell was approximately 58% higher than that of the free-suspended cells in a control experiment. Unlike the cultivation method, the bead size in the range of 1- to 3-mm diameter did not significantly influence the qMAb, regardless of cultivations methods. The changes in qMAb of an entrapped cells were reversible. When the free-suspended cells recovered from the T- and spinner flasks were sub-cultured in T- and spinner flasks enhanced qMAb of the entrapped cells in both cases decreased to the level of the free-suspended cell in a control experiments. Taken together, these results shows that the method of cultivation of hybridoma cells immobilized in alginate beads determines the extent of enhancement of the qMAb. © 1993 John Wiley & Sons, Inc.
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    Stability of antibody productivity is improved when hybridoma cells are entrapped in calcium alginate beads (1993)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 42 (1993), S. 1131-1135 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; instability ; immobilization ; monoclonal antibody productivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Loss of monoclonal antibody (MAb) productivity in long-term, free-suspended cell culture is often attributed to the appearance of a nonproducing population of hybridoma cell (NP) in the culture which has a growth advantage over the producing population (P). However, when an NP appears in long-term culture of entrapped cells, it may not be able to take over the whole culture in a short period of time due to the limited growth of the entrapped cells. In order to examine the hypothesis that entrapped cells can have improved stability of MAb productivity due to limited cell growth, free-suspended cell culture and calcium alginate-entrapped cell culture with inocula consisting of a P and an NP were compared with regard to stability of MAb productivity in a repeated fed-batch culture. In free-suspended cell culture, the NP appeared to take over the whole culture within three batches, and thereby MAb production completely disappeared. In entrapped cell culture, an NP appeared to outgrow the P rapidly only during an exponential growth phase, resulting in a significant decrease in specific MAb productivity, qMAb, from 11.58 μg/106 cell/day to 2.76 μg/106 cell/day. However, when the cell growth was limited in entrapped cell culture, the NP no longer outgrew the P rapidly, as indicated by the stable value of qMAb. In addition, when the cells recovered from the alginate beads by citrate buffer treatment were subcultured in free-suspended cell culture, MAb production rapidly deteriorated and completely disappeared within two batches. Thus, the P present at a small fraction of viable cell concentration in the beginning of the free-suspended cell culture, which were previously entrapped in alginate beads, seemed to be outgrown rapidly by the NP. Taken together, the results obtained from these experiments support the hypothesis that the limited cell growth in entrapped cell culture, which keeps an NP from taking over the whole culture, is responsible, in part, for the improved stability of MAb productivity. © 1993 John Wiley & Sons, Inc.
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    A three-phase pattern in growth, monoclonal antibody production, and metabolite exchange in a hybridoma bioreactor culture (1993)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 42 (1993), S. 133-139 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; kinetics ; curve fitting ; fermentation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The use of partial cubic spline data interpolation for the calculation of volumetric metabolite exchange rates suggested the existence of three distinct metabolic phases during bioreactor culture of a hybridoma cell line. During phase 1, a rapid amino acid uptake rate and ammonia release rate were observed. The growth rate was low and glutamine synthetase activity fell. In phase 2, maximum growth rate and minimum glutamine assimilation and ammonium production rates were observed. Attempts to corroborate the apparent ammonia assimilation in this phase using 15NH4Cl resulted in low incorporation rates into alanine and glutamine. Maximum glutamine synthetase activity took place during this period. Maximum antibody production rate was observed during phase 3 during which peaks in glutamine assimilation, ammonia release, and glutamine synthetase activity were observed. The apparent existence of the three phases prompted us to carry out Northern blot analysis of glutamine synthetase RNA at appropriate times during the process. This revealed a pattern of appearance and dis-appearance of mRNA consistent with the three phases indicated by the fermentation parameters. © 1993 John Wiley & Sons, Inc.
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    Substantial overproduction of antibodies by applying osmotic pressure and sodium butyrate (1993)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 42 (1993), S. 601-610 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; antibody productivity ; osmolarity ; butyrate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Much of the current cell technology has enabled increased antibody production levels due to judicious nutrient feeding to raise cell densities and design better bioreactors. This study demonstrates that hybridomas can be hyperstimulated to produce higher immunoglobulin (lg) levels by suppressing cell growth and increasing culture longevity through adaptation to higher osmolarity media and addition of sodium butyrate. Prior to adaptation, cells placed in higher osmotic pressures (350 and 400 mOsm) were severely suppressed in growth down to 25% of the control (300 mOsm), although total lg titers achieved were similar to the control, approximately 140 mg/L. After a week of adaptation to 350 and 400 mOsm media, cell growth was not as dramatically suppressed, but considerably higher lg levels were attained at these elevated osmolarities. The highest yield of 265 mg/L was obtained at 350 mOsm compared to 140 mg/L at 300 mOsm, while maximum viable cell numbers dropped from 35 × 105 cells/mL to 31 × 105 cells/mL and culture longevity was extended by 20 h more than the control. Sodium butyrate, known to enhance protein production in other cell types, was then supplemented at a range of concentrations between 0.01 and 0.4 mM to the 350 mOsm culture to further enhance the lg levels. Butyrate at a concentration of 0.1 mM, in combination with osmotic pressure at 350 mOsm, further elevated the lg levels to 350 mg/L. Concomitantly, maximum viable cell numbers were reduced to 22 × 105 cells/mL, but culture longevity was extended by 40 h in the 0.1 mM butyrate supplemented culture compared to the control condition. Specific antibody productivity, qMab, continued to stay high during the stationary phase and was further elevated during the decline phase: thus, overall lg levels can be increased by 2.3 times by combining osmotic pressure and butyrate treatment. © 1993 John Wiley & Sons, Inc.
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    Loss of antibody productivity is highly reproducible in multiple hybridoma subclones (1993)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 42 (1993), S. 247-250 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; antibody productivity ; kinetics ; instability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An immunoglobulin G (IgG2b) producing hybridoma cell line (S3H5/γ2bA2) was cloned and subcloned. Twenty subclones were grown in parallel while being adapted in a stepwise fashion to serum-free medium. Following adaptation to serum-free medium, it was found that 16 of the 20 subclones remained at a relatively constant proportion of nonproducing cells. Three of the remaining subclones transiently deviated from this balance but eventually returned toward this population composition. One subclone continued to lose productivity. A population balance was reached at approximately 8% of the population being nonproducers. The loss of antibody productivity was thus highly reproducible. © 1993 John Wiley & Sons, Inc.
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    Repeated fed-batch culture of hybridoma cells in nutrient-fortified high-density medium (1993)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 42 (1993), S. 1229-1237 
    Details
    ISSN: 0006-3592
    Keywords: repeated fed-batch culture ; nutrient fortifications ; high-density culture ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Long-term high-density cultivation of the hybridoma 2c3.1 was successfully carried out in a repeated fed-batch mode using high-density media that were constructed to meet in vitro cell growth limitations. The high-density culture was possible in a range of 0.5 ∼ 1.0 × 107 cells/mL in MBRI 40-02 medium for over 2500 h by the repeated supplementation of the most fortified medium, MBRI 40-03, and consequently, distinct enhancement of MAb production was achieved. MAb concentrations were maintained around 1 g/L for about 1000 h of the process and the maximum MAb concentration was around 1.56 g/L. The result supported strongly the fact that the nutritional fortification was the most critical factor for high-density cell culture in vitro. The mean chromosome number of the hybridoma 2c3.1 was maintained stably for about 1500 h, whereas gradual loss of the MAb activity was apparent during the long-term cultivation. © 1993 John Wiley & Sons, Inc.
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    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.
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    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.
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    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.
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    Repeated hybridoma batch culture with cell recycle (1993)
    Springer
    Cytotechnology 13 (1993), S. 51-53 
    Details
    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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    Essential role of optimal protein synthesis in preventing the apoptotic death of cultured B cell hybridomas (1993)
    Springer
    Cytotechnology 13 (1993), S. 99-105 
    Details
    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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    URL: http://dx.doi.org/10.1007/BF00749936
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    Effects of lactate concentration on hybridoma culture in lactate-controlled fed-batch operation (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 39 (1992), S. 556-564 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; effects of lactate concentration ; inhibition by osmotic pressure ; fed-batch culture ; antibody production rate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: To investigate the effects of lactate on cell growth and antibody production, a new method of maintaining the lactate concentration constant in a fed-batch culture is described. When the pH was initially adjusted by sodium hydroxide, the specific growth rate decreased and specific death rate increased with an increase of lactate concentration. To investigate whether the inhibition was due to the lactate concentration itself or to the osmotic pressure, the effect of the osmotic pressure adjusted by sodium chloride was compared with that of sodium lactate. When the osmotic pressure was adjusted to same condition as that of sodium lactate using sodium chloride, the specific growth data showed the same degree of growth inhibition. It was thus evident that the inhibition to cell growth was mainly due to osmotic pressure while lactate production from glucose was found to be inhibited by the lactate itself compared with sodium chloride. The specific antibody production rate had a maximum value within a certain range of lactate concentration. Moreover, specific antibody production rate had a unified relationship with the kinetic parameter μ, in spite of the different causes of inhibition by lithium lactate and sodium lactate. A certain “trade-off” relationship between growth and antibody production existed at higher growth rates.
    Additional Material: 9 Ill.
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    URL: http://dx.doi.org/10.1002/bit.260390511
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    Mechanical agitation of hybridoma suspension cultures: Metabolic effects of serum, pluronic F68, and albumin supplements (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 40 (1992), S. 1045-1055 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; mechanical agitation ; metabolism ; FBS ; pluronic ; BSA ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Metabolic effects of the medium supplements, fetal bovine serum (FBS), Pluronic F68, and bovine serum albumin (BSA) were compared for agitated bioreactor cultures of hybridoma cells. Agitation speeds up to 600 rpm, without entrainment of gas bubbles by sparging or vortex formation, allowed examination of cell interactions with turbulent fluid forces. For cultures in FBS-supplemented RPMI media, there was no significant effect of intense turbulent fluid shear on cell growth, metabolism, or antibody, production. Serum-free cultures (Pluronic F68 or BSA supplements) at 600 rpm demonstrated greatly increased glycolysis rates during exponential growth relative to controls. Nutrient limitations caused increased rates of decline of the viable cell concentrations and a reduction in final antibody titers by around 70%. The Pluronic F68 and BSA supplements did not lead to cell protection by modifying metabolism under conditions of intense turbulent fluid shear. Supplementing the protein-free medium with FBS reduced glycolysis rates in exponential growth phase, but this did not prevent a high rate of viable cell decline and low antibody titers. We concluded that FBS does not have a metabolic effect on cells subjected to intense turbulent fluid shear. Although the agitation conditions employed in this study were more intense than generally required for agitated bioreactor culture of hybridomas, we have demonstrated the importance of considering metabolic effects of turbulent fluid forces on cultures using nutrient-rich basal media, in addition to the considerations of gas bubble effects described by other workers. © 1992 John Wiley & Sons, Inc.
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    Cell cycle model for growth rate and death rate in continuous suspension hybridoma cultures (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 40 (1992), S. 359-368 
    Details
    ISSN: 0006-3592
    Keywords: cell cycle ; hybridoma ; death ; cell arrest ; growth ; monoclonal antibody ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: As a result of recent advances in flow cytometry, renewed interest is shown in modeling the kinetic behavior of cells in culture on the basis of cell cycle parameters. An important but often overlooked kinetic variable in hybridoma cultures is the cell death rate. Not only the overall cell growth but also the kinetics of nutrient metabolism and monoclonal antibody production have been shown to depend on the cell death rate in continuous suspension hybridoma cultures. The present study shows that the death rate in hybridoma cultures is proportional to the fraction of cells arrested in the G1 phase of the cell cycle. The steady-state cell age distributions in the various phases of the division cycle have been calculated analytically. A simple mathematical model has been used to produce the profiles of the cycling and arrested cell fractions with respect to the dilution rate. The calculated steady-state growth rate, death rate, and viability profiles are shown to be in agreement with recently published experimental data from continuous suspension hybridoma cultures. © 1992 John Wiley & Sons, Inc.
    Additional Material: 11 Ill.
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    Effect of low serum concentrations (0%-2.5%) on growth, production, and shear sensitivity of hybridoma cells (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 40 (1992), S. 179-182 
    Details
    ISSN: 0006-3592
    Keywords: animal-cell culture ; hybridoma ; reduced serum content ; shear sensistivity ; bubble-column design ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In a stirred culture of hybridoma cells, the effects of serum reduction from 2.5% to 0% on growth and monoclonal antibody porduction have been investigated. The shear sensitivity of cells from the same culture has been tested in a bubble column. Serum reduction does not greatly affect viable-cell concentrations, but cell specific monoclonal-antibody production rate shows a decreasing trend. A gradual increase in sensitivity for sparging, which is nor the result of a long-term biological effect, has beeen measured in a bubble column at decreasing fetal calf serum concentrations. Finally, the hypothetical killing-volume model describing the death rate of insect cells in bubble columns has now been completely validated for the pertinent hybridoma-cell line.
    Additional Material: 5 Ill.
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    URL: http://dx.doi.org/10.1002/bit.260400125
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  • 88
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    Population balance between producing and nonproducing hybridoma clones is very sensitive to serum level, state of inoculum, and medium composition (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 39 (1992), S. 354-360 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; nonproducer ; instability ; antibody secretion rate ; flow cytometer ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Secreting and nonsecreting hybridoma populations derived from the murine hybridoma cell line 167.4G5.3 were each grown in batch culture in low serum and serum-free media. Under serum-free conditions, a secreting population gained on a predominantly nonsecreting population and competed with the existing antibody-deficient cells effectively. It was found that this competition was sensitive to state of inoculum and medium composition. We conclude that the competition between a secreting and nonsecreting, or more generally, a producing and nonproducing, population is important; the appearance of the latter may not be a significant setback in terms of expected product titer.
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    URL: http://dx.doi.org/10.1002/bit.260390315
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    Analysis of mammalian viable cell biomass based on cellular ATP (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 39 (1992), S. 859-864 
    Details
    ISSN: 0006-3592
    Keywords: cell volume ; intracellular ATP ; hybridoma ; biomass ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Analysis of cellular ATP as a means of measuring viable biomass loading was investigated in hybridoma cell culture. ATP analysis by the luciferin-luciferase assay was compared with trypan blue-stained hemocytometer counts. The cell-specific ATP content varied between 2 and 6 fmol per viable cell over a batch culture. ATP levels were highest during exponential growth, and decreased during the stationary and decline phases. Electronic counting and volume measurements were performed to assay the viable cell biomass. Cell sorting, using fluorescein diacetate, was used to separate viable and nonviable cells in cultures with between 35% and 90% viable cells. Viable cells contained over 2 orders of magnitude greater cell-specific ATP than nonviable cells. Cell-specific ATP correlated directly with the viable cell volume rather than viable cell numbers. Over the range of batch culture conditions, ATP analysis should provide a more accurate measurement of hybridoma viable biomass than hemocytometer counts.
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    URL: http://dx.doi.org/10.1002/bit.260390807
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    Effect of temperature on hybridoma cell cycle and MAb production (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 40 (1992), S. 427-431 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; cell cycle ; monoclonal antibody ; temperature ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The kinetics of growth and antibody formation of an anti-interleukin-2 producing hybridoma line were studied in suspension culture at temperatures ranging from 34°C to 39°C. Flow cytometry was used to determine the effect of temperature on the cell cycle. Maximum cell density and monoclonal antibody yield were observed at 37°C. The specific monoclonal antibody production rate was approximately constant throughout each batch experiment. Lower temperatures caused cells to stay longer in the G1-phase of the cell cycle, but temperature had only a marginal effect on the specific antibody production rate. Arresting of cells in the G1-phase by means of temperature was, therefore, not suited for enhanced monoclonal antibody production. Rather, antibody production for this hybridoma was directly linked to viable cell concentration. © 1992 John Wiley & Sons, Inc.
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    URL: http://dx.doi.org/10.1002/bit.260400312
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    Monoclonal antibody production in dialyzed continuous suspension culture (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 39 (1992), S. 504-510 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; continuous culture ; dialysis ; monoclonal antibody ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Hybridoma cell growth and monoclonal antibody production in dialyzed continuous suspension culture were investigated using a 1.5-L Celligen bioreactor. Medium supplemented with 1.5% fetal bovine serum was fed directly into the reactor at a dilution rate of 0.45 d-1. Dailysis tubing with a molecular weight cut-off (MWCO) of 1000 was coiled inside the bioreactor. Fresh medium containing no serum or serum substitues passed through the dialysis tubing at flow rates of 2 to 5 L/d. The objective was to remove low molecular weight inhibitors, such as lactic acid and ammonia, by diffusion through the tubing, while continuoulsy replenishing essential nutrients by the same mechanism. Due to the low MWCO of the dialysis tubing high molecular weight components such as growth factors and antibody were not removed by the dialyzing stream. In the batch start-up phase, the monoclonal antibody (MAb) titer was almost 3 times that achieved in typical batch cultures (i.e., 170 to 180 mg/L). During dialyzed continuous operation, a substantial increase (up to 40%) in cell density, monoclonal antibody (MAb) titer, and reactor MAb productivity was observed, as compared with a conventional continuous suspension culture. The cell viability and the specific MAb productivity remained practically constant at different dialysis rates. This finding suggests that the steady state growth and death rate in continuous suspension hybridoma cultures are not direct functions of the nutrient or inhibitor concentrations.
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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 
    Details
    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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    Comparison of specific rates of hybridoma growth and metabolism in batch and continuous cultures (1992)
    Springer
    Cytotechnology 10 (1992), S. 147-155 
    Details
    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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    Production of monoclonal antibody using free-suspended and immobilized hybridoma cells: Effect of serum (1991)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 38 (1991), S. 821-830 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; immobilization ; serum ; flow cytometry ; antibody productivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of serum on cell growth and monoclonal antibody (MAb) productivity was studied in a repeated fedbatch mode using both free-suspended and immobilized S3H5/γ2bA2 hybridoma cells. In the suspension culture, serum influenced the cell growth rate but not the specific MAb productivity. The average specific growth rate of the suspension culture in medium containing 10% serum was approximately 0.99 ± 0.12 day-1 (±standard deviation), while that in medium containing 1% serum was approximately 0.73 ± 0.12 day-1. The specific MAb productivity was almost constant at 3.69 ± 0.57 μg/106 cells/day irrespective of serum concentration reached a maximum at ca. 1.8 × 106 cells/mL of medium in 10% serum medium, and the cell concentration was gradually reduced to 1%. The specific MAb productivity of the immobilized cells was more than three times higher than that of the free-suspended cells. The amount of serum in the medium did not influence the specific MAb production rate of the immobilized cells. The maintenance of high cell concentration and the enhanced specific MAb productivity of the immobilized cell culture resulted in a higher volumetric MAb productivity. In addition, MAb yield in the immobilized cell culture with medium containing 1% serum was 2.2 mg/mL of serum, which was approximately three times higher than that in the suspension culture.
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    Cell-cycle-dependent protein accumulation by producer and nonproducer murine hybridoma cell lines: A population analysis (1991)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 38 (1991), S. 665-677 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; flow cytometry ; cell cycle ; population balance ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Single-cell rates of accumulation of cellular protein have been determined as a function of total protein content using flow cytometry and population balance equations for exponentially growing murine hybridoma cells in the individual G1, S1 and G2 + M cell cycle phases. A novel flow cytometric technique for the identification of hybridoma cells in mitosis was developed and implemented. The data were obtained from a producer cell line which synthesizes and secretes high levels of monoclonal antibodies, and from a nonproducer clone which does not synthesize and secrete substantial amounts of antibody. The results indicate that the kinetics of single-cell protein accumulation in these two cell lines are considerably different. In particular, low protein content G1 phase producer cells were characterized by a rate of protein accumulation which was approximately five times higher than the mean rate observed for higher protein content producer cells cycle phase. In contrast, the rate of accumulation of protein increased continuously with totalprotein content for the G1 phase nonproducer cells. S phase hybridoma cells were characterized by a considerably lower rate of protein accumulation which did not vary much with protein content for either cell line. Finally, G2 + M phase producer cells demonstrated a negative rate of protein accumulation which indicates that the rates of protein synthesis. It was hypothesized that these differences in total protein accumulation are caused by differences in monoclonal antibody accumulation. The distribution of rates suggests the need for a segregated approach to the modeling of the kinetics of antibody production in hybridomas.
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    Kinetic study of hybridoma cell growth in continuous culture: II. Behavior of producers and comparison to nonproducers (1991)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 38 (1991), S. 1020-1028 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; cell culture ; continuous culture ; kinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A hybridoma cell line, AFP-27-P, was cultivated in continuous culture under glucose-limited conditions. The viable cell concentration, dead-cell concentration, and cell volume all varied with the dilution rate. A model previously developed for a nonproducing clone of the same cell line, AFP-27-NP, was extended to describe the behavior of the cells. The relationship between the specific growth rate and glucose concentration is described by a function similar to the Monod model. A threshold glucose concentration and a minimum specific growth rate are incorporated; the model is meaningful only at glucose concentration and a minimum specific growth rate are incorporated; the model is meaningful only at glucose concentrations and specific growth rates above these levels. The relationship between the death rate and the glucose concentration is described by an inverted Monod-type function. Furthermore, the yield coefficient based on glucose is constant in the lower range of specific growth rates and changes to a new constant value in the upper range of specific growth rates. No maintenance term for glucose consumption is used; in the plot of specific glucose consumption rate vs. specific growth rate, the line intercepts the specific growth rate at a value close to the minimum growth rate. The productivity of antibody as a function of the specific growth rate is described by a mixed type model with a noon-growth-associated term and a negative-growth-associated term. The values for the model parameters were determined from regression analysis of the steady state data.
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    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.
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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 
    Details
    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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    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.
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    URL: http://dx.doi.org/10.1007/BF00353704
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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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