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  • Articles  (27)
  • CHO cells  (27)
  • Springer  (27)
  • Oxford University Press
  • 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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  • 11
    Electronic Resource
    Electronic Resource
    Production of a membrane-bound proteins, the human gamma-glutamyl transferase, by CHO cells cultivated on microcarriers, in aggregates and in suspension (1994)
    Springer
    Cytotechnology 16 (1994), S. 121-129 
    Details
    ISSN: 1573-0778
    Keywords: CHO cells ; microcarriers ; aggregates ; suspension ; membrane-bound protein ; γ-glutamyl transferase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Recombinant Chinese Hamster Ovary (CHO) cells, engineered for the production of human gamma-glutamyl transferase (GGT), have been grown on Cytodex 1 microcarriers, as aggregates, or as single cells in suspension after adaptation. GGT is a membrane bound enzyme which was not secreted during the culture period. The maximal enzyme activity was found to be directly related to the achieved maximal cell density. Culture of CHO on microcarriers yielded the fastest growth, with a specific growth rate of 0.04 h−1, the highest cell density (near 1.3×106 cells ml−1), and the highest enzyme activity around 300 mU ml−1, which corresponded to a specific cellular level of 20 mU 10−5 cells. GGT could also be produced by growing CHO cells in suspension as single cells or as aggregates. Under these conditions, however, the specific CHO growth rate was significantly slower and the GGT level per cell was divided by a factor 6. Growing CHO cells without microcarriers also resulted in differences in cell metabolism, with a higher conversion yield of glutamine into ammonia, and a higher cell lysis. The catalytic kinetic constants of the enzyme were found identical for the three culture systems.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00754614
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  • 12
    Electronic Resource
    Electronic Resource
    Apoptosis in CHO cell batch cultures: examination by flow cytometry (1995)
    Springer
    Cytotechnology 17 (1995), S. 1-11 
    Details
    ISSN: 1573-0778
    Keywords: Apoptosis ; programmed cell death ; CHO cells ; batch culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Chinese hamster ovary cells grown under conditions which are optimal for the production of a genetically engineered protein in batch culture, lose significant viability shortly after entering the stationary phase. This cell death was investigated morphologically and was found to be almost exclusively via apoptosi. Furthermore, cells were analyzed by flow cytometry using a fluorescent DNA end-labeling assay to label apoptotic cells, in conjunction with cell cycle analysis using propidium iodide. Apoptotic cells could be detected by this method, and by the radioactive end-labeling of extracted DNA, on all days of culture from day 1 to day 7; however, the degree of apoptotic cell death increased dramatically when the cells entered the stationary phase, rising to 50–60% of the total cell number at the termination of the culture. Flow cytometric analysis showed that the majority of cells underwent apoptosis whilst in G1/G0 and formed an apoptotic population with high DNA FITC end-labeling and hypodiploid propidium iodide binding. Additionally, the ability or inability to secrete specific protein products did not appear to interfere with the development of the apoptotic population with time.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00749215
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  • 13
    Electronic Resource
    Electronic Resource
    Stable expression in Chinese hamster ovary cells of a homogeneous recombinant active fragment of human platelet glycoprotein Ibα (1995)
    Springer
    Cytotechnology 17 (1995), S. 133-141 
    Details
    ISSN: 1573-0778
    Keywords: CHO cells ; inhibition of glycosylation ; large-scale production ; recombinant GP Ib ; stable expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A fragment (residues His1-Val289) of the α chain of human platelet glycoprotein Ib containing the von Willebrand factor and thrombin binding sites has been expressed in Chinese hamster ovary cells. The secreted soluble recombinant protein had an apparent molecular mass of 42 kD and reacted with a conformation-dependent monoclonal antibody that only binds to native GP Ib, thus demonstrating its proper folding. The rather broad band obtained after immobilization of the recombinant fragment on nitrocellulose could be resolved into a very sharp band of molecular weight of about 35 kD by growing the cells in the presence of tunicamycin, and inhibitor of N-linked glycosylation. The recombinant GP Ibα fragments (with or without glycosylation) were purified by immunoaffinity chromatography. Both truncated forms bound vWF in the presence of botrocetin with comparable affinity as a proteolytic 42 kD fragment of purified human platelet GP Ib-IX. They were also retained on thrombin-Sepharose. We then selected a cell clone (B1) that produced over at least three months about 1.5 μg of recombinant protein per million cells per day. Using this clone a large-scale production finally yielded milligram amounts of the functionally active recombinant human GP Ibα fragment.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00749401
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  • 14
    Electronic Resource
    Electronic Resource
    Effects of temperature shift on cell cycle, apoptosis and nucleotide pools in CHO cell batch cultues (1997)
    Springer
    Cytotechnology 23 (1997), S. 47-54 
    Details
    ISSN: 1573-0778
    Keywords: apoptosis ; programmed cell death ; nucleotides ; energy charge ; CHO cells ; batch culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Temperature reduction in CHO cell batch culture may be beneficial in the production of recombinant protein and in maintenance of viability. The effects on cell cycle, apoptosis and nucleotide pools were studied in cultures initiated at 37°C and temperature shifted to 30 °C after 48 hours. In control cultures maintained at 37 °C, viable cells continued to proliferate until the termination of the culture, however, temperature reduction caused a rapid decrease in the percent of cells in S phase and accumulation of cells in G-1. This was accompanied by a concurrent reduction in U ratio (UTO/UDP-GNAc), previously shown to be a sensitive indicator of growth rate. Culture viability was extended following temperature shift, as a result of delayed onset of apoptosis, however, once initiated, the rate and manner of cell death was similar to that observed at 37 °C. All nucleotide pools were similarly degraded at the time of apoptotic cell death. Temperature reduction to 30 °C did not decrease the energy charge of the cells, however, the overall rate of metabolism was reduced. The latter may be sufficient to extend culture viability via a reduction in toxic metabolites and/or limitation of nutrient deprivation. However, the possibility remains that the benefits of temperature reduction in terms of both viability and productivity are more directly associated with cultures spending extended time in G-1.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007919921991
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  • 15
    Electronic Resource
    Electronic Resource
    Application of statistical design of experiments to the optimization of factor VIII expression by CHO cells (1991)
    Springer
    Cytotechnology 6 (1991), S. 233-240 
    Details
    ISSN: 1573-0778
    Keywords: CHO cells ; DOEHLERT matrix ; experimental design ; Factor VIII ; HADAMARD matrix ; mathematical model ; optimization ; serum-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Selection and optimization of the concentrations of chemically defined components effective on recombinant factor VIII:C production by CHO cells have been performed with statistically designed experiments. Screening of efficient compounds on factor VIII expression was performed by using HADAMARD method, while a precise optimization of the concentrations of two components was achieved with the help of DOEHLERT method. Increased specific activity of factor VIII and reduced cost medium have been obtained with a fewer number of experiments than compared to a less rational approach.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00624762
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  • 16
    Electronic Resource
    Electronic Resource
    A recombinant bait region mutant of human α2-macroglobulin exhibiting an altered proteinase-inhibiting spectrum (1999)
    Springer
    Cytotechnology 31 (1999), S. 53-60 
    Details
    ISSN: 1573-0778
    Keywords: alpha-2-macroglobulin ; α2M ; bait region ; CHO cells ; human ; lysyl endopeptidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Alpha 2-macroglobulin (α2M), a plasma glycoprotein produced in the liver, inhibits a variety of proteinases and thus considered to play important homeostatic roles in the body. This broad inhibitory spectrum has been explained by the trapping theory by which a proteinase recognizes a region of 25–30 amino acid peptide in α2M called bait region and cleaves it, leading to the conformational change of α2M, and to the subsequent entrapment and inhibition of the proteinase. We constructed α2M cDNAs with mutated DNA sequences in the bait region, and obtained recombinant CHO cell lines producing either wild type α2M, or mutant α2Ms, i.e., α2M/K692 and α2M/K696, each with substitution of Arg with Lys at codons 692 and 696, respectively. We tested if lysyl endopeptidase is not inhibited by wild type α2M, but could be inhibited by these engineered mutant α2Ms. Thus, recombinant α2M/K696 protein successfully inhibited lysyl endopeptidase activity, while recombinant α2M/K692 protein was not sensitive to lysyl endopeptidase, suggesting that not all bait region peptide bonds can equally be accessible and susceptible to proteinases. The present results not only provided the trapping theory with additional supportive evidence, but the first experimental evidence for the value of engineered α2M-derived proteinase inhibitor with an artificial proteinase inhibitory spectrum of potential industrial and/or therapeutic usefulness.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008011919876
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  • 17
    Electronic Resource
    Electronic Resource
    The gel microdrop secretion assay: Identification of a low productivity subpopulation arising during the production of human antibody in CHO cells (2000)
    Springer
    Cytotechnology 34 (2000), S. 27-37 
    Details
    ISSN: 1573-0778
    Keywords: CHO cells ; gel microdrops ; human antibody ; population parameters ; productivity ; stability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The long-term stability of high-level expression is the mostimportant factor to consider when choosing cell lines for the expression of recombinant proteins. Declining volumetricyields in large-scale fermentation can be caused by changes affecting the cell population as a whole such as loss in viability, depletion of nutrients or accumulation of metabolites affecting cell growth. Alternatively, geneticinstability may lead to the outgrowth of a less productive,metabolically favored sub-population. Currently a variety ofparameters are measured to monitor the condition of cells infermenters including glucose uptake, lactate accumulation andoxygen consumption; in addition, periodic viable cell countsallow the determination of the growth rate and viability of the population. All of these methods measure the condition ofthe cell population as a whole and changes must involve a significantly large proportion of the total culture in orderto be detectable. Here we report on a method that allows theevaluation of the productivity of individual cells. Using the gel microdrop secretion assay, we detected the appearance ofa sub-population of cells with lower productivity. Subsequentanalysis of the culture confirmed the existence of lower productivity cells with a lower vector copy number. Therefore,the single cell secretion assay proved to be a rapid method todetect and isolate a low productivity variant of the producer cell line.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008186113245
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  • 18
    Electronic Resource
    Electronic Resource
    Production of recombinant Von Willebrand factor by CHO cells cultured in macroporous microcarriers (1990)
    Springer
    Cytotechnology 4 (1990), S. 163-171 
    Details
    ISSN: 1573-0778
    Keywords: CHO cells ; macroporous microcarrier ; scale-up ; serum-free ; Von Willebrand factor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Recombinant Chinese hamster ovary cells producing Von Willebrand factor have been successfully grown in gelatin macroporous microcarriers (Cultispher-G). Serum-free cultures were maintained in 1, 4, and 10 liter fermentors for more than two months. Comparative studies with Cytodex-3 microcarriers have been performed in 1 liter fermentors. The lower specific Von Willebrand factor productivity of CHO cells cultivated on Cultispher-G were offset by higher cell densities (107−2×107 cells/ml). Volumetric Von Willebrand factor productivity was influenced by oxygen concentration, and remained stable during scale-up from 1 to 10 liter fermentors.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00365097
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  • 19
    Electronic Resource
    Electronic Resource
    Nutrient optimization for high density biological production applications (1991)
    Springer
    Cytotechnology 5 (1991), S. 15-30 
    Details
    ISSN: 1573-0778
    Keywords: high density ; cell culture ; serum-free medium ; hybridoma ; CHO cells ; virus production ; insect cells ; adoptive immunotherapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Conclusion At the 1989 annual meeting of the U.S. Tissue Culture Associations, Ricahrd am, a leading investigator in the serum-free nutrient requirements of cultured cells, commented on the process of medium development. He noted that a survey of major media manufacturers revealed that, among the top selling mammalian cell culture media formulations, most were nearly thirty years old. This commentary is noteworthy considering the tremendous changes in cell culture understanding and derived applications which have emerged over these three decades. Fastidious cell types relatively unknown to investigators of the 1950s and 1960s are now being cultivated in defined, serum-free environments. Culture environments range from limiting dilution clonal recoveries to maintenance cultures approaching tissue densities. While research applications continue to predominate, applications of cell culture have expanded to the engineered production of biopharmaceuticals, to replacement of animal models for toxicology testing, and to the preservation, activation and expansion of human cells, tissues and organs. It is likely that future nutrient medium development will be predicated upon the design of a minimal number of defined formulations of relatively generic utility to a broad class of cell types. Analytical techniques derived from those described herein will be exploited in the user laboratory and in collaboration with the supplier to optimize the nutrient composition for the desired biological response.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00365531
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  • 20
    Electronic Resource
    Electronic Resource
    Foreign protein expression from S phase specific promoters in continuous cultures of recombinant CHO cells (1996)
    Springer
    Cytotechnology 22 (1996), S. 179-184 
    Details
    ISSN: 1573-0778
    Keywords: cell cycle ; CHO cells ; continuous culture ; SV40 promoter
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Foreign protein expression from the commonly used SV40 promoter has been found to be primarily during the S-phase of the cell cycle. Simple mathematical models with this cell cycle phase dependent expression of foreign protein suggest that the specific production rate will be proportional to the cell growth rate, which is particularly disadvantageous in high cell density fed-batch or perfusion bioreactors. In this study we investigate this predicted relationship between the production rate and growth rate by culturing recombinant CHO cells in a continuous suspension bioreactor. One CHO cell line, GS-26, has been stably transfected with the plasmid pSVgal, which contains the E. coli lac Z gene under the control of the SV40 promoter. This GS-26 cell line was grown in suspension cultures over a range of specific growth rates in batch and continuous modes. The intracellular β-galactosidase activity was assayed using a standard spectrophotometric method after breaking the cells open and releasing the enzyme. A strong growth associated relationship is found between the intracellular β-galactosidase content and the specific growth rate in batch and continuous cultures, as predicted.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00353937
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  • 21
    Electronic Resource
    Electronic Resource
    Genetic engineering of α2,6-sialyltransferase in recombinant CHO cells and its effects on the sialylation of recombinant interferon-γ (1996)
    Springer
    Cytotechnology 22 (1996), S. 197-203 
    Details
    ISSN: 1573-0778
    Keywords: sialyltransferase ; glycosylation ; CHO cells ; transfection ; interferon
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The CHO cell line has achieved considerable commercial importance as a vehicle for the production of human therapeutic proteins, but is known to lack a functional copy of the gene coding for α2,6-sialyltransferase (EC 2.4.99.1). The cDNA for rat α2,6-ST was expressed in a recombinant CHO cell line making interferon-γ, using a novel in vitro amplification vector. The enzyme was expressed efficiently, and resulted in up to 60% of the total sialic acids on interferon-γ being linked in the α2,6-conformation. This sialic acid linkage distribution was more akin to that seen in natural human glycoproteins. In the most successful cell clones, expression of α2,6-sialyltransferase improved the overall level of sialylation by up to 56%, and had no adverse effects on cell growth, IFN-γ productivity or other aspects of IFN-γ glycosylation. These experiments demonstrate how the glycosylation machinery of rodent cells can be genetically manipulated to replicate human tissues.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00353939
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  • 22
    Electronic Resource
    Electronic Resource
    Stable, recombinant expression of human insulin-like growth factor binding protein-1 (hIGFBP-1) in Chinese hamster ovary (CHO) cells (1997)
    Springer
    Cytotechnology 24 (1997), S. 193-200 
    Details
    ISSN: 1573-0778
    Keywords: CHO cells ; DHFR ; IGFBP-1 ; stability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Stable expression of human insulin-like growth factor of binding protein-1 (hIGFBP-1)at high levels has been achieved in Chinese hamster ovary (CHO) cells by co-transfection and subsequent co-amplification of expression vectors containing the hIGFBP-1 cDNA and a dihydrofolate reductase (DHFR) cDNA gene into DHFR-deficient cells. Stepwise selection of the DHFR+ transformants in increasing concentrations of methotrexate (MTX) generated cells which had high copy numbers of the hIGFBP-1 gene (around 100 copies in cells amplified in medium containing 100 nM MTX). Expression of hIGFBP-1 in mixed clones was found to increase with increasing copy number and an apparent correlation between intra- and extracellular levels of hIGFBP-1 produced by these cells was observed. It was further observed that continuous cultivation over eight months in medium supplemented with 100 nM MTX increased the production of hIGFBP-1 25 times. The productivity did not increase further after five more months cultivation in MTX containing medium. A subcloning of this cell line gave clones with an even higher productivity. Further amplification in 500 nM or 1 uM MTX did not increase the hIGFBP-1 production.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007999512662
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  • 23
    Electronic Resource
    Electronic Resource
    Increased production of recombinant hIGFBP-1 in PEG induced autofusion of Chinese hamster ovary (CHO) cells (1997)
    Springer
    Cytotechnology 24 (1997), S. 183-191 
    Details
    ISSN: 1573-0778
    Keywords: CHO cells ; fusion ; IGFBP-1 ; polyethylene glycol ; serum-free
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A recombinant Chinese hamster ovary (CHO) cell clone, S1, stably expressing human insulin-like growth factor binding protein-1 (hIGFBP-1), was treated with polyethylene glycol (PEG), resulting in cell fusion, in order to further enhance the protein expression by increasing the gene copy number and/or the amount of organelles important to the protein expression/-secretion. Both the fused cell line, Peg1, and its mother cell line, S1, were adapted to serum-free growth in suspension and were characterised with respect to growth and productivity. Peg1 was easier to adapt to the serum-free suspension conditions and had a higher viability during the adaptation period than S1. Furthermore, Peg1 showed a stable productivity of hIGFBP-1 that was twice as high as that for S1 under both adherent and suspension conditions. A considerable difference in the specific productivity (up to 3–4 times) was noticed during the growth phase. PEG fusion experiments have earlier been studied in our laboratory with CHO cells producing recombinant factor VIII and our results correlates very well with the results obtained with the factor VIII producing cells. Surprisingly, it was possible to obtain high producing recombinant cell lines, which were stable for more than 4 months.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007931623160
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  • 24
    Electronic Resource
    Electronic Resource
    Efficient and inducible production of human interleukin 6 in Chinese hamster ovary cells using a novel expression system (1997)
    Springer
    Cytotechnology 25 (1997), S. 53-60 
    Details
    ISSN: 1573-0778
    Keywords: CHO cells ; coactivator ; recombinant protein production system ; transactivator
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract High level and inducible production of human interleukin 6 (hIL-6) was achieved using a novel expression system in Chinese hamster ovary (CHO) cells. In this system, the transcription of hIL-6 gene under the control of PhCMV*-1 promoter composed of tetracycline operator sequences and a minimal promoter is activated by a chimeric transactivator (tTA) composed of tetracycline repressor and transactivating domain of VP16 protein of herpes simplex virus. The transcription of tTA gene, which is also under the control of PhCMV*-1 promoter, is activated by itself via a positive feedback cycle. The expression of both genes is further enhanced by potentiating the VP16 transactivating domain of tTA transactivator with pX protein of hepatitis B virus. In the presence of tetracycline, the tTA transactivators can not bind to PhCMV*-1 promoter, therefore, the expression of hIL-6 and tTA gene is suppressed, and the pX will not activate basal transcription. In the absence of tetracycline, tTA transactivators bind to PhCMV*-1 promoter and activate efficient transcription of hIL-6 and tTA gene, and the transcription is further enhanced by pX via VP16 transactivating domain. Using this strategy, we isolated a clone (UX1) producing hIL-6 at a rate about 1425 ng/106 cells/day. Furthermore, the hIL-6 production is stringently regulated by tetracycline. This results suggested a novel strategy to establish highly efficient, inducible and cell type independent recombinant protein production system by using an artificial promoter to recruit transactivators and coactivators which can synergistically activate transcription.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007972002180
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  • 25
    Electronic Resource
    Electronic Resource
    The effect of osmolarity on metabolism and morphology in adhesion and suspension chinese hamster ovary cells producing tissue plasminogen activator (2000)
    Springer
    Cytotechnology 32 (2000), S. 171-179 
    Details
    ISSN: 1573-0778
    Keywords: adherent CHO cells ; CHO cells ; morphology of CHO cells ; osmolarity ; suspended CHO cells ; tissue plasminogen activator
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The effects of constant osmolarity, between 300 and500 mOsm/kg, on the metabolism of Chinese HamsterOvary (CHO) cells producing tissue plasminogenactivator (tPA) were compared between adhesion andsuspension cultures. In both suspension and adhesionculture, the specific rates of glucose consumption(νG), lactate production (qL), and tPAproduction (qtPA) increased as osmolarityincreased, while these rates decreased when osmolaritywas higher than the respective critical levels. However, specific growth rate (μ) decreased withincrease in osmolarity and this slope grew steeper inthe osmolarity range higher than the critical level. The decrease in μ in the adhesion culture was morerapid than that in the suspension culture. Thecritical osmolarity for adhesion culture (400 mOsm/kg)was lower than that for suspension culture (450 mOsm/kg). These results indicated that the adhesionculture was more sensitive to increase of osmolaritythan the suspension culture, while the specific ratesobtained from the adhesion cultures were in general1.5- to 3-fold higher than those obtained from thesuspension cultures. Cell volume increased asosmolarity increased in both the suspension andadhesion cultures, as reported previously forsuspension culture of hybridoma cells, but there wasno morphological change in the suspension culture. Incontrast, cell height decreased and cell adhesion areamarkedly increased as osmolarity increased in theadhesion culture. This morphological change inadhesion cultures may be one reason for the highersensitivity of adherent cells to the increase ofosmolarity than suspended cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008171921282
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  • 26
    Electronic Resource
    Electronic Resource
    Bead-to-bead transfer of chinese hamster ovary cells using macroporous microcarriers (1994)
    Springer
    Cytotechnology 14 (1994), S. 67-80 
    Details
    ISSN: 1573-0778
    Keywords: bead-to-bead transfer ; CHO cells ; macroporous microcarrier
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Chinese hamster ovary cells (CHO-K1) were cultivated in macroporous gelatin microcarriers (CultiSpher G and CultiSpher S) in spinner flasks and a 5 1 bioreactor. Near-to-confluent cultures were harvested by bead-to-bead transfer where intact microcarriers with cells were transferred from a spinner flask to another spinner flask or to the bioreactor with naked microcarrier beads. Successful bead-to-bead transfer was achieved in various split ratios. The duration of attachment seemed to be important where the direct contact of beads to each other can be achieved by intermittent stirring. Repeated transfers were performed and at least four transfers in spinner flasks were achieved. Two variations of bead-to-bead transfer were performed in the 5 1 bioreactor either by seeding the bioreactor with near-to-confluent beads cultivated in spinner flasks orin situ transfer by adding fresh beads to the bioreactor. As in the spinner case, attachment was achieved by intermittent stirring where donor beads were in close proximity to the acceptor beads. Again successful transfers were obtained as evidenced by the good growth on acceptor beads where cell yields were in the range of 3100–4500 cells/bead. The results suggest that bead-to-bead transfer of CHO-K1 cells can be easily performed and do provide an alternative route to applications where dissolution techniques may not offer an efficient solution.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00772197
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  • 27
    Electronic Resource
    Electronic Resource
    Effect of endogenous methylglyoxal on Chinese hamster ovary cells grown in culture (1996)
    Springer
    Cytotechnology 22 (1996), S. 33-42 
    Details
    ISSN: 1573-0778
    Keywords: metabolic engineering ; CHO cells ; methylglyoxal ; glyoxalase I
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Methylglyoxal is a ketoaldehyde that reacts readily under physiological conditions with biologically relevant ligands, such as amine and sulfhydryl groups. It is produced in mammalian cells primarily as a by-product of glycolysis. The level of glucose, L-glutamine and fetal bovine serum in culture media was found to significantly affect levels of intracellular methylglyoxal in Chinese hamster ovary cells. Medium with 25 mM glucose and 5 mM L-glutamine caused an increase in free methylglyoxal levels of 90 to 100% relative to medium containing 5 mM glucose and 2 mM L-glutamine. Both of these media compositions are representative of those found in commercially available media. Pseudomonas putida glyoxalase I was expressed in Chinese hamster ovary cells to enhance methylglyoxal detoxification. The Chinese hamster ovary cell clones showed an 80 to 90% decrease in free methylglyoxal levels. The colony-forming ability of these cells was compared to wild-type Chinese hamster ovary cells under conditions found to cause elevated methylglyoxal levels. The wild-type cells showed a 10% decrease in colony-forming ability relative to the clones. This decrease was found to be statistically significant (P〉0.99) by analysis of variance. The variation in colony-forming ability amongst the clones was statistically insignificant. More importantly, the clones shoed increased colony-forming ability relative to the wild-type cells under conditions of higher methylglyoxal production with fair to good statistical significance (P〉0.75 to P〉0.95). This result is the first quantifiable evidence that endogenously produced methylglyoxal can negatively affect cell function under conditions found in animal cell culture.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00353922
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