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  • Articles  (27)
  • CHO cells  (27)
  • Springer  (27)
  • Process Engineering, Biotechnology, Nutrition Technology  (27)
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  • Articles  (27)
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  • 1
    Electronic Resource
    Electronic Resource
    CHO cell growth and recombinant interferon-γ production: Effects of BSA, Pluronic and lipids (1995)
    Springer
    Cytotechnology 19 (1995), S. 27-36 
    Details
    ISSN: 1573-0778
    Keywords: BSA ; CHO cells ; interferon-γ ; linoleic acid ; lipids ; Pluronic F68
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The role of bovine serum albumin in mammalian cell cultures and the possibility of its substitution by other components in a serum-free medium has been investigated. In this study, BSA was shown to be important for growth and product formation in CHO cells expressing recombinant human interferon-γ. There were indications that its stimulating growth effect was dependent on the source of BSA used and probably was related to the purification procedure used for the production of the desired albumin fraction. Cell growth did not occur in the absence of BSA but at low concentration (1 mg ml−1) it was stimulated by the addition of a combination of a commercial lipid mixture plus Pluronic F68. However, under the latter conditions IFN-γ production was adversely effected. The importance of individual lipid components was investigated using a statistical approach based on a Plackett-Burman design. Linoleic acid was identified as a positive variable for cell growth while cholesterol was identified as a negative variable for both cell growth and IFN-γ production. When a combination of linoleic acid plus Pluronic F68 was included in the formulation of low BSA medium, cell growth was similar to that at high BSA concentration (5 mg ml−1) but the IFN-γ concentration was significantly reduced (ca. 45%).
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00749752
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  • 2
    Electronic Resource
    Electronic Resource
    Proteolytic cleavage of recombinant two-chain factor VIII during cell culture production is mediated by protease(s) from lysed cells. The use of pulse labelling directly in production medium (1997)
    Springer
    Cytotechnology 24 (1997), S. 227-234 
    Details
    ISSN: 1573-0778
    Keywords: CHO cells ; multicatalytic proteinase complex ; proteases ; protease inhibitors ; proteasome ; pulse labelling ; recombinant coagulation factor VIII ; SDS-PAGE
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract During the production by mammalian cells of recombinant factor VIII from which the B domain was deleted (rFVIII), proteolytic cleavages in the C-terminal part of the heavy chain were observed (Kjalke et al., 1995). By radioactive pulse labelling it was investigated whether the cleavages took place inside the cells during protein synthesis or after release in the medium. The rFVIII-producing CHO (Chinese hamster ovary) cells were cultured in the presence of 35S-methionine and then the cell lysate and the conditioned media were immunoprecipitated and analyzed by electrophoresis. By pulse labelling and chasing for various time periods, it was shown that the cleavages only took place after secretion of the protein from the cells. Adding cell lysate to uncleaved rFVIII caused cleavage of the heavy chain, as seen by loss of binding to a monoclonal antibody specific for intact rFVIII, indicating that the cleavage was performed by proteinase(s) released from the lysed cells. By incubating intact rFVIII with the multicatalytic proteinase (proteasome) present in cytoplasm and nucleus of eukaryotic cells, loss of binding to the monoclonal antibody was observed. This indicates that the multicatalytic proteinase, released from lysed rFVIII producing cells, could be responsible for the cleavage of rFVIII. Among several protease inhibitors tested, only bacitracin was found to diminish the extent of cleavage. Phosphatidylserine also protected rFVIII against cleavage, probably by binding to rFVIII.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007988713571
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  • 3
    Electronic Resource
    Electronic Resource
    Na-butyrate increases the production and α2,6-sialylation of recombinant interferon-γ expressed by α2,6- sialyltransferase engineered CHO cells (1999)
    Springer
    Cytotechnology 29 (1999), S. 55-64 
    Details
    ISSN: 1573-0778
    Keywords: α2,6-sialyltransferase ; CHO cells ; interferon-γ ; sialylation ; sodium butyrate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A non-human like glycosylation pattern in human recombinant glycoproteins expressed by animal cells may compromise their use as therapeutic drugs. In order to correct the CHO glycosylation machinery, a CHO cell line producing recombinant human interferon- γ (IFN) was transformed to replace the endogenous pseudogene with a functional copy of the enzyme α2,6-sialyltransferase (α2,6-ST). Both the parental and the modified CHO cell line were propagated in serum-free batch culture with or without 1 mM sodium butyrate. Although Na-butyrate inhibited cell growth, IFN concentration was increased twofold. The IFN sialylation status was determined using linkage specific sialidases and HPLC. Under non- induced conditions, IFN expressed by α2,6-engineered cells contained 68% of the total sialic acids in the α2,6- conformation and the overall molar ratio of sialic acids to IFN was 2.3. Sodium butyrate addition increased twofold the molar ratio of total sialic acids to IFN and 82% of total sialic acids on IFN were in the α2,6-conformation. In contrast, no effect of the sodium butyrate was noticed on the sialylation of the IFN secreted by the α2,6-ST deficient parental cell line. This study deals for the first time with the effect of Na-butyrate on CHO cells engineered to produce human like sialylation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008080432681
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  • 4
    Electronic Resource
    Electronic Resource
    Quantitative analysis of transcription and translation in gene amplified Chinese hamster ovary cells on the basis of a kinetic model (1999)
    Springer
    Cytotechnology 29 (1999), S. 93-102 
    Details
    ISSN: 1573-0778
    Keywords: CHO cells ; gene expression ; kinetic model ; protein secretion ; transcription ; translation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The elevation of expression levels for secreted glycoproteins by gene amplification in mammalian cells shows a saturation behavior at high levels of gene amplification. At high expression levels a drop in the secretion efficiency for the recombinant protein occurs (Schröder and Friedl, 1997), coinciding with the appearance of misfolded protein in the cell. In this communication we investigated whether additional limitations exist at the levels of transcription and translation. Four Chinese hamster ovary (CHO) cell lines expressing different amounts of human antithrombin III (ATIII) were used as a model system. A tenfold increase in the ATIII cDNA copy number from the lowest to the highest producing cell line coincided with a 38-fold increase in ATIII mRNA levels, and an 80-fold increase in the amount of intracellular ATIII levels. The data was analyzed using a simple kinetic model. The following conclusions were derived: I. The transcriptional activity for the recombinant protein is not saturated. II. Translation itself is not saturated either, but may be downregulated as secretion efficiency drops. III. Two explanations for the previously reported drop in secretion efficiency for the recombinant protein with increasing expression level are possible: A. Protein degradation is an alternative fate for translated ATIII and the fraction of ATIII degraded after translation increases as expression level is increased. B. Translation is downregulated as the secretory apparatus becomes exhausted to maintain cell viability.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008077603328
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  • 5
    Electronic Resource
    Electronic Resource
    Cell growth and protein formation on various microcarriers (1999)
    Springer
    Cytotechnology 29 (1999), S. 151-158 
    Details
    ISSN: 1573-0778
    Keywords: CHO cells ; comparison ; microcarriers
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A large number of microcarriers are commercially available. The capability of cells to successfully proliferate on microcarriers varies with cell lines and media. Choosing the right microcarrier for a particular cell line is more than a choice of a microcarrier. It is part of an integrated process design. A detailed picture of cell growth and product formation will not only be essential in identifying the kind of microcarrier, but also in determining other parts of the process, such as operation mode and media. Our initial screening on thirteen microcarriers showed that cultures on some microcarriers reached a low cell density but high cell-specific productivity, and high density microcarrier cultures have a low specific productivity. The result is a similar product output per unit volume and time for these two types of cultures. An ideal culture system shall have increased volumetric productivity at elevated cell density. This requires the process goal to be incorporated as early as cell line construction and screening. A high output process can then be realized through high density culture.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008053421462
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  • 6
    Electronic Resource
    Electronic Resource
    Productivity enhancement of recombinant protein in CHO cells via specific promoter activation by oncogenes (1999)
    Springer
    Cytotechnology 31 (1999), S. 103-109 
    Details
    ISSN: 1573-0778
    Keywords: CHO cells ; human interleukin 6 ; oncogene ; promoter activation ; recombinant protein production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract To construct a recombinant protein highly producing cell lines, we have previously developed the Oncogene Activated Production (OAP) system by using BHK-21 cells. Here we verified the availability of the OAP system in CHO cells. We firstly generated ‘primed’ ras amplified CHO cells, ras clone I, by introducing human c-Ha-ras oncogene into CHO cells. This ras clone I enables quick and easy establishment of recombinant protein hyper producing cell lines by introduction reporter gene of interest. Then we generated I13 by introducing human interleukin 6 (hIL-6) gene as a reporter gene, which showed enhanced productivity rate as compared to A7 established by conventional method. Furthermore, we found that hIL-6 production level of I13 was slightly improved by raising the CO2 concentration from 5 to 8% possibly because of the enhanced growth rate. We further introduced the E1A oncogene, which has been shown to have a synergistic effect on the recombinant protein production of the ras-amplified BHK-21 cells, then evaluated the productivity. When culture in 5% CO2 condition, only the slight effect can be seen. However when cultured in 8% CO2 condition, not only cell number, but also productivity increased significantly, resulted in great augmentation of hIL-6 production, maximum production being 88.6 μg/ml/3 days. This study demonstrates that recombinant protein production level reached commercially desirable level by utilizing our OAP system in CHO cells and optimizing the culture condition.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008048928053
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  • 7
    Electronic Resource
    Electronic Resource
    Enhanced erythropoietin heterogeneity in a CHO culture is caused by proteolytic degradation and can be eliminated by a high glutamine level (2000)
    Springer
    Cytotechnology 34 (2000), S. 83-99 
    Details
    ISSN: 1573-0778
    Keywords: CHO cells ; erythropoietin ; glycosylation ; glutamine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The molecular heterogeneity of recombinant humanerythropoietin (EPO) increased during the course of abatch culture of transfected Chinese hamster ovary(CHO) cells grown in serum-free medium. This wasshown by both an increased molecular weight and pIrange of the isolated EPO at the end of the culture. However, analysis of the N-glycan structures of themolecule by fluorophore-assisted carbohydrateelectrophoresis (FACE) and HPLC anion exchangechromatography indicated a consistent pattern ofglycosylation. Seven glycoforms were identified, thepredominant structure being a fully sialylatedtetra-antennary glycan. The degree of sialylationwas maintained throughout the culture. Analysis ofthe secreted EPO indicated a time-dependent increasein the molecular weight band width of the peptideconsistent with proteolytic degradation. A highglutamine concentration (16–20 mM) in the culturedecreased the apparent degradation of the EPO.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008137712611
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  • 8
    Electronic Resource
    Electronic Resource
    Influence of alterations in culture condition and changes in perfusion parameters on the retention performance of a 20 μm spinfilter during a perfusion cultivation of a recombinant CHO cell line in pilot scale (2000)
    Springer
    Cytotechnology 34 (2000), S. 141-150 
    Details
    ISSN: 1573-0778
    Keywords: CHO cells ; culture condition ; modelparticles ; operation cut off ; perfusion cultivation ; retention ; spinfilter ; tip speed
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Since 1969 much attention has been devoted to the useof spinfilter systems for retention of mammalian cellsin continuous perfusion cultivations. Previousinvestigations dealt with hydrodynamic conditions,fouling processes and upscaling. But hydrodynamicconditions and fouling processes seem to have asecondary importance in spinfilter performance duringauthentic perfusion cultivations. Obviously,alterations in culture condition are more relevantespecially during long-term processes. Therefore, ourpratical approach focussed on the performance qualityof a commercially available 20 μm spinfilterduring a perfusion cultivation of a recombinant CHOcell line in pilot scale regarding the followingissues: 1) retention of viable cells in thebioreactor; 2) removal of dead cells and cell debrisfrom the bioreactor; 3) alterations in culturecondition; and 4) changes in perfusion mode.Furthermore, we tested the performance of 20 μmspinfilters in 2 and 100 l pilot scale using solidmodel particles instead of cells. Our investigationsshowed that retention of viable cells in pilot scalewas independent of spinfilter rotation velocity andperfusion rate; the retention increased from 75 to 95%corresponding to operation time, enlarging celldiameter and enhanced formation of aggregates in theculture during the perfusion cultivation. By means ofthe Cell Counter and Analyzer System (CASY) anoperation cut off of 13 μm was determined forthis spinfilter. Using solid model particles in 2 lscale, optimal retention was achieved at a tip speedof 0.43 m s-1 (141 rpm) – furtherenhancement of spinfilter rotation velocity up to0.56 m s-1 (185 rpm) decreased the retentionrapidly. In pilot scale best retention performance wasobtained with tip speeds of 0.37 m s-1(35 rpm) and 1.26 m s-1 (120 rpm). Hence,significant retention in pilot scale could already beachieved with low agitation. Therefore, the additionof shear force protectives could be avoided so thatthe purification of the target protein from thesupernatant would be facilitated.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008183508439
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  • 9
    Electronic Resource
    Electronic Resource
    Effect of lipid supplements on the production and glycosylation of recombinant interferon-γ expressed in CHO cells (1994)
    Springer
    Cytotechnology 15 (1994), S. 209-215 
    Details
    ISSN: 1573-0778
    Keywords: Glycosylation ; recombinant ; interferon ; CHO cells ; lipids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The effects of lipids on the glycosylation of recombinant human interferon-γ expressed in a Chinese Hamster Ovary cell line were investigated in batch culture. Lipids form an essential part of the N-glycosylation pathway, and have been shown to improve cell viability. In control (serum-free) medium the proportion of fully-glycosylated interferon-γ deteriorated reproducibly with time in batch culture, but the lipoprotein supplement ExCyte was shown to minimise this trend. Partially substituting the bovine serum albumin content of the medium with a fatty-acid free preparation also improved interferon-γ glycosylation, possibly indicating that oxidised lipids carried on Cohn fraction V albumin may damage the glycosylation process.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00762395
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  • 10
    Electronic Resource
    Electronic Resource
    Observations on the influence of glutamine, asparagine and peptone on growth and t-PA production of Chinese hamster ovary (CHO) cells (1994)
    Springer
    Cytotechnology 16 (1994), S. 37-42 
    Details
    ISSN: 1573-0778
    Keywords: Ammonium ; asparagine ; CHO cells ; glutamine ; peptone ; t-PA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract When a transfected CHO cell, that produces tissue-type Plasminogen Activator, t-PA, was transferred from a medium based on 5% Fetal Calf Serum, FCS, to a medium based on 0.8% casein peptone with variable glutamine and asparagine content, it was observed, that the growth of the cells changed from anchorage dependant to suspension culture giving more reproducible cultivations. In the FCS culture t-PA was unstable, observed as a decline in t-PA concentration after 250 h. This decline in t-PA concentration was not observed in the serum free culture, although there was a decline in productivity after 200 h. This change in production profile may be attributed to either no proteolytic attack from serum or by scavenging of proteolytic activities produced by the cells from the peptone peptides. Increasing amounts of glutamine/asparagine gave higher production of t-PA in synchrony with an increasing production of ammonia/ammonium ions. Ammonia inhibition does not seem to be a key factor for this cell line as seen with many others.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00761777
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