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Chun, Bok Hwan; Park, Song Yong; Chung, Namhyun; Bang, Won-Gi

doi:10.1023/a:1022301528118

Cited by 23 publications

(9 citation statements)

References 14 publications

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“…Levels of FVIII:Ag in the culture medium (Fig. 1c) ranged from 5 to 10 IU/ml which is comparable to typical published values for clonal cell lines producing FVIII-BDD [24]. Thus one-step transgene amplification under adherent conditions and direct selection of transiently transfected cells in the presence of 50 nM MTX resulted in oligoclonal cell lines, secreting FVIII-BDD in the range of 2–8 IU/ml.…”

Section: Resultssupporting

confidence: 83%

“…Expression level of FVIII is also boosted by sodium butyrate [28], probably due to inactivation of transcription silencer, present in the mRNA of FVIII [29]. A similar effect was described for sodium propionate addition [24]. At the same time, addition of sodium butyrate to the culture medium induces endoplasmic reticulum stress in the FVIII-producing cells, as was shown in [19] and damages the cells.…”

Section: Resultsmentioning

confidence: 79%

“…The use of the specialized plasmid vector p1.1 instead of the standard vector allowed us to increase the product titer 80-fold. Other studies of the FVIII-BDD producing cell lines also report lower levels of the active protein in the culture medium; for example Chun et al [24] reported a peak titer of 10 IU/ml of the FVIII BDD after treatment of the cell culture by the sodium propionate and around 2 IU/ml for the untreated culture. These data show that the plasmid used in the present study allows much higher secretion rates of the FVIII than standard CMV-based vectors.…”

Section: Discussionmentioning

confidence: 99%

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Stable high-level expression of factor VIII in Chinese hamster ovary cells in improved elongation factor-1 alpha-based system

et al. 2017

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BackgroundRecombinant factor VIII (FVIII), used for haemophilia A therapy, is one of the most challenging among the therapeutic proteins produced in heterologous expression systems. Deletion variant of FVIII, in which the entire domain B is replaced by a short linker peptide, was approved for medical use. Efficacy and safety of this FVIII deletion variant are similar to full-length FVIII preparations while the level of production in CHO cells is substantially higher.Typical levels of productivity for CHO cell lines producing deletion variant FVIII-BDD SQ, described elsewhere, are 0.5–2 IU/ml, corresponding to the concentration of FVIII of about 0.2 μg/ml. Using standard vectors based on the cytomegalovirus promoter (CMV) and the dihydrofolate reductase cDNA we have previously obtained the cell line secreting 0.5 IU/ml of FVIII-BDD, which roughly corresponds to the previously published data.ResultsAn expression system based on CHO genomic sequences including CHO-EEF1A promoter and Epstein-Barr virus terminal repeat fragment allowed us to achieve 80-fold increase in the production level as compared with the conventional expression system based on the CMV promoter.Immediately after the primary selection FVIII -producing cells secreted 5–10 IU/ml of FVIII-BDD, and after multi-stage methotrexate-driven amplification a stable clonal line 11A4H was selected, secreting 39 IU/ml of FVIII-BDD in the simple batch culturing conditions, which considerably exceeds known indicators for industrial producers of this protein. In contrast to other FVIII-BDD producing lines 11A4H accumulates low proportion of the secreted FVIII on the membrane. Its productivity may be further increased approximately two-fold by adding sodium butyrate and butylated hydroxyanisol to the culture medium.A five-stage purification process for the factor VIII was employed. It allowed isolation of the intact FVIII-BDD as was confirmed by mass spectrometry. Purified FVIII-BDD has a specific activity of 11,000 IU/mg, similar to known recombinant FVIII drugs.ConclusionsThe recombinant FVIII-BDD was produced in CHO cells without addition of any animal-derived materials, purified and characterized. Novel genetic constructions for the expression of heterologous proteins combined with optimized cultivation method allowed to obtain the secretion level of biologically active recombinant FVIII increased by almost ten times as compared with the previously published analogues.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-017-0353-6) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 83%

Section: Resultsmentioning

confidence: 79%

Section: Discussionmentioning

confidence: 99%

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Stable high-level expression of factor VIII in Chinese hamster ovary cells in improved elongation factor-1 alpha-based system

et al. 2017

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“…ELISA: ELISA was performed as described in [8]. Antibody capture ELISA was used for the testing of anti FVIII mAbs, and a concentrate of plasma-derived FVIII (a generous gift from Dr. A.L.…”

Section: Experimental Partmentioning

confidence: 99%

Stable Expression of Recombinant Factor VIII in CHO Cells Using Methotrexate-Driven Transgene Amplification

et al. 2012

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Prophylaxis and treatment of inherited clotting disorder hemophilia A requires regular administration of factor VIII. Recombinant factor VIII, which is produced in CHO or BHK cells, is equivalent to the plasma-derived one and is prevalent in current clinical practice in developed countries. Development of a biosimilar recombinant FVIII requires the creation of a highly productive clonal cell line and generation of monoclonal antibodies suitable for affinity purification of the product. Methotrexate-driven transgene amplification of genetic cassettes that code full-length and truncated variants of FVIII under the control of the CMV promoter was studied. It was shown that the expression level of the truncated variant of FVIII is 6.5 times higher than that of the full-length molecule. The transgene amplification procedure was sufficient for a twofold increase of the expression level in the transfected cells pool and subsequent selection of the clonal line, stably producing truncated FVIII at the level of 0.52 IU/ml during cultivation in a chemically defined protein-free culture medium. Four generated mouse monoclonal antibodies toward the heavy chain of FVIII were found suitable for binding the truncated variant of FVIII directly from the conditioned medium and elution of the FVIII with a more than 85% yield and normal pro-coagulant activity. The producer cell line and monoclonal antibodies obtained are sufficient for the development of upstream and downstream processes of biosimilar FVIII production. Generation of more productive cell lines by the use of stronger, nonviral promoters and shorter cDNA of FVIII will be the subject of further studies.

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“…Factor VIII is an important cofactor in the blood coagulation cascade. Deficiency or dysfunction of factor VIII causes hemophilia A [17,18] . There are many barriers to the production of recombinant human coagulation factor VIII (rhFVIII).…”

Section: Introductionmentioning

confidence: 99%

Development of PCL-PEG nanofibrous mats as alternative carriers for recombinant Chinese hamster ovary cells

Shakibaie¹,

Rahbar

Tabandeh

et al. 2012

Journal of Polymer Engineering

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In this study, six polyblended nanofiber mats, composed of poly ε-caprolactone (PCL)-polyethylene glycol (PEG) mixed at different proportions in an electrospinning solution were fabricated, and then utilized alongside commercial polyester microcarriers for the mass cultivation of recombinant Chinese hamster ovary (rCHO) cells. The SEM micrographs showed that the rCHO cells were attached to the nanofiber mats with significant differences in their affinities, which can be related to the chemical structure and hydrophilicity of the nanofiber mats. The cell densities of the rCHO cells grown on the polyblended PCL-PEG nanofiber mat (80:20)14 % were much higher when compared with the commercial polyester microcarrier. Moreover, recombinant protein production increased by approximately 20 % when cells were grown on the PCL-PEG nanofiber mat (80:20)14 % . The results of this research indicated a potential for the replacement of the commercial polyester microcarrier with the PCL-PEG polyblended nanofiber mats, in order to achieve mass cultivation of recombinant animal cells in industrial bioreactors.

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Abstract: Sodium propionate, as well as sodium butyrate, enhanced the production of recombinant B-domain-deleted, factor VIII (rFVIIIdB) by Chinese hamster ovary cells growing in a spinner-flask with a protein-free medium by more than six-fold. The two acids, however, had different cytotoxicities.

Cited by 23 publications

References 14 publications

Stable high-level expression of factor VIII in Chinese hamster ovary cells in improved elongation factor-1 alpha-based system

Stable high-level expression of factor VIII in Chinese hamster ovary cells in improved elongation factor-1 alpha-based system

Stable Expression of Recombinant Factor VIII in CHO Cells Using Methotrexate-Driven Transgene Amplification

Development of PCL-PEG nanofibrous mats as alternative carriers for recombinant Chinese hamster ovary cells

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