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  • 1
    Electronic Resource
    Electronic Resource
    Effect of pyridine on the fatty acid composition of Pimelobacter sp. (1996)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 141 (1996), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract The fatty acid composition of Pimelobacter sp. is of the complex type. When pyridine was used as a sole inhibitory substrate, the fatty acid composition of Pimelobacter sp. was quite different from that within other readily available substrates. When compared with the fatty acid composition in a complex medium, the proportion of isopalmitic acid (iso-C16:0) drastically decreased from 68.4% to 7.7%, while the proportion of anteisoheptadecanoic acid (anteiso-C17:0) remained almost constant at ca. 7%. Concomitantly, this decrease of the branched-chain fatty acid was accompanied by the increase of the straight-chain, especially long-chain, fatty acid such as heptadecanoic (C17:0), octadecenoic (Cig:i), 10-methylheptadecanoic (10-me-18) and 10-methyloctadecanoic (10-me-19) acids. Consequently, in response to membrane active organic solvents, Pimelobacter sp. was found to regulate its membrane fatty acid composition in a fashion different from Gram-negative bacteria.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6968.1996.tb08375.x
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  • 2
    Electronic Resource
    Electronic Resource
    Expansion of Human Bone Marrow Progenitor Cells in a High Cell Density Continuous Perfusion System (1993)
    [s.l.] : Nature Publishing Company
    Nature biotechnology 11 (1993), S. 368-372 
    Details
    ISSN: 1546-1696
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: [Auszug] We describe here a continuous perfusion bioreactor system that enables a population of unselected human mononuclear bone marrow cells obtained from adult donors to expand up to 20 to 25-fold over a two-week period. Colony-forming units of granulocyte-macrophage (CFU-GM) progenitor cells expand 10 ...
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1038/nbt0393-368
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  • 3
    Electronic Resource
    Electronic Resource
    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.
    Additional Material: 11 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260380804
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  • 4
    Electronic Resource
    Electronic Resource
    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.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260410307
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  • 5
    Electronic Resource
    Electronic Resource
    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.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260420917
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  • 6
    Electronic Resource
    Electronic Resource
    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.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260480618
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  • 7
    Electronic Resource
    Electronic Resource
    Characterization of chimeric antibody producing CHO cells in the course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 73-84 
    Details
    ISSN: 0006-3592
    Keywords: chimeric antibody ; CHO cells ; dihydrofolate reductase ; flow cytometry ; gene copy number ; stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Recombinant Chinese hamster ovary (CHO) cells expressing a high-level of chimeric antibody against S surface antigen of hepatitis B virus were obtained by co-transfection of heavy and light chain cDNA expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level such as 0.02, 0.08, 0.32, 1.0, and 4.0 μM. The highest producer (HP) subclone was isolated from each MTX level and was characterized with respect to cell growth and antibody production in the corresponding level of MTX. The specific growth rate of the HP subclone was inversely proportional to the MTX level. On the other hand, its specific antibody productivity (qAb) rapidly increased with increasing MTX level up to 0.08 μM, and thereafter, it gradually increased to 20 μg/106 cells/day at 4 μM MTX. Southern blot analysis showed that the enhanced qAb at higher MTX level resulted from immunoglobulin (Ig) gene amplification. The stability of the HP subclones isolated at 0.02, 0.08, 0.32, and 1.0 μM MTX in regard to antibody production was investigated during long-term culture in the absence of MTX. The qAb of all subclones significantly decreased during the culture. However, the relative extent of decrease in qAb was variable among the subclones. The HP subclone isolated at 1 μM MTX was most stable and could retain 59% of the initial qAb after 80 days of cultivation. Southern blot analysis showed that this decrease in qAb of the subclones resulted mainly from the loss of Ig gene copies during long-term culture. Despite the decreased qAb, the HP subclone isolated at 1 μM MTX could maintain high volumetric antibody productivity over three months because of improved cell growth rate during long-term culture. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:73-84, 1998.
    Additional Material: 15 Ill.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Microencapsulated human bone marrow cultures: A potential culture system for the clonal outgrowth of hematopoietic progenitor cells (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 43 (1994), S. 734-739 
    Details
    ISSN: 0006-3592
    Keywords: bone marrrow cultures ; hematopoietic progenitor cells ; microencapsulation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Currently the most successful methods for culturing human hematopoietic cells employ some form of perfused bioreactor system. However, these systems do not permit the clonal outgrowth of single progenitor cells. Therefore, we have investigated the use of alginate-poly-L-lysine microencapsulation of human bone marrow, combined with rapid medium exchange, as a system that may overcome this limitation for the purpose of studying the kinetics of progenitor cell growth. We report that a 12 to 24-fold multilineage expansion of adult human bone marow cells was achieved in about 16 to 19 days with this system and that visually identifiable colonies within the capsules were responsible for the increase in cell number. The colonies that represented the majority of cell growth originated from cells that appeared to be present in a frequency of about 1 in 4000 in the encapsulated cell population. These colonies were predominantly granulocytic and contained greater than 40,000 cells each. Large erythroid colonies were also present in the capsules, and they often contained over 10,000 cells each. Time profiles of the erythroid progenitor cell density over time were obtained. Burst-forming units erythroid (BFU-E) peaked around day 5, and the number of morphologically identifiable erythroid cells (erythroblasts through reticulocytes) peaked on day 12. We also report the existence of a critical inoculum density and how growth was improved with the use of conditioned medium derived from a microcapsule culture initiated above the critical inoculum density. Taken together, these results suggest that microencapsulation of human hematopoietic cells allows for outgrowth of progenitor, and possible preprogenitor, cells and could serve as a novel culture system for monitoring the growth and differentiation kinetics of these cells.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260430807
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  • 9
    Electronic Resource
    Electronic Resource
    Stability of transfectomas producing chimeric antibody against the pre-S2 surface antigen of hepatitis B virus during a long-term culture (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 47 (1995), S. 243-251 
    Details
    ISSN: 0006-3592
    Keywords: transfectoma ; chimeric antibody ; stability ; flow cytometry ; RT-PCR ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: To design the scheme of large-scale production of chimeric antibody for the postexposure prophylaxis of hepatitis B virus (HBV) infection, the stability of transfectomas (H69K-1 and 6-31) in regard to antibody production was examined during a long-term, repeated fed-batch culture without selection pressure using antibiotics. Although the H69K-1 transfectoma was more stable than the 6-31 transfectoma, both displayed gradual decreases in specific antibody productivity (qAb) for the first several weeks of cultivation. During this period, qAb was de-creased by 40% to 50%. This loss of qAb was due mainly to the appearance of a nonproducing population (NP) of transfectoma, which was monitored throughout the culture by flow cytometry and the limiting dilution method. However, an NP did not overtake the culture and was balanced with a producing population (P) of transfectoma, resulting in stable antibody production. The subclones of NP obtained at the end of long-term culture were further characterized by reverse transcription-polymerase chain reaction assay of the heavy and light chain mRNA. All the subclones of NP derived from H69K-1 transfectoma had only light chain mRNA. On the other hand, an NP in the 6-31 transfectoma culture was heterogeneous. Some subclones of NP derived from 6-31 transfectoma had only heavy chain mRNA and other subclones had only light chain mRNA. Taken together, the results obtained here suggest that selection pressure is necessary for a long-term, continuous culture, because stable antibody production in a long-term culture was achieved only after a significant loss of antibody productivity. Accordingly, a batch culture appears to be more appropriate for large-scale chimeric antibody production without selection pressure. © 1995 John Wiley & Sons, Inc.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260470216
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  • 10
    Electronic Resource
    Electronic Resource
    Clonal variability within dihydrofolate reductase-mediated gene amplified Chinese hamster ovary cells: Stability in the absence of selective pressure (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 60 (1998), S. 679-688 
    Details
    ISSN: 0006-3592
    Keywords: chimeric antibody ; CHO cells ; clonal analysis ; dihydrofolate reductase ; gene amplification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Recombinant Chinese hamster ovary (rCHO) cells expressing a high level of chimeric antibody were obtained by cotransfection of heavy- and light-chain cDNA expression vectors into dihydrofolate reductase-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level up to 1.0 μM. To determine the clonal variability within the amplified cell population in regard to antibody production stability, 20 subclones were randomly isolated from the amplified cell population at 1.0 μM MTX (CS13-1.0 cells). Clonal analysis showed that CS13-1.0 cells were heterogeneous with regard to specific growth rate (μ) and specific antibody productivity (qAb), although they were derived from a single clone. The μ and qAb of 20 subclones were in the range of 0.51 to 0.72 day-1 and 10.9 to 19.1 μg/106 cells/day, respectively. During 8 weeks of cultivation in the absence of selective pressure, the μ of most subclones did not change significantly. On the other hand, their qAb decreased significantly. Furthermore, the relative decrease in qAb varied among subclones, ranging from 30% to 80%. Southern and Northern blot analyses showed that this decreased qAb resulted mainly from the loss of amplified immunoglobulin (Ig) gene copies and their respective cytoplasmic mRNAs. For the sake of screening convenience, an attempted was made to correlate the initial properties of subclones (such as μ, qAb, and Ig gene copies) with their antibody production stability during long-term culture. Among these initial properties examined, only qAb of subclones could help to predict their stability to some extent. The subclones with high qAb were relatively stable with regard to antibody production during long-term culture in the absence of selective pressure (P 〈 0.005, ANOVA). Taken together, the clonal heterogeneity in an amplified CHO cell population necessitates clonal analysis for screening stable clones with high qAb. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 679-688, 1998.
    Additional Material: 9 Ill.
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