The B-domain of Factor VIII reduces cell membrane attachment to host cells under serum free conditions

Kolind, Mille; Nørby, Peder Lisby; Flintegaard, Thomas Veje; Berchtold, Martin W.; Johnsen, Laust Bruun

doi:10.1016/j.jbiotec.2010.04.010

Cited by 12 publications

(17 citation statements)

References 28 publications

(23 reference statements)

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“…In the case of full-length FVIII, membrane-bound fraction was estimated as ¾ of all extracellular FVIII [16] and in the case of FVIII-BDD it may increase up to 90% [31]. Membrane-bound FVIII-BDD may be liberated during the cultivation by the addition of vWF, annexin V or water-soluble phosphatidylserine analogue — o-phospo-L-serine [32].…”

Section: Resultsmentioning

confidence: 99%

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: Resultsmentioning

confidence: 99%

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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“…Whereas the expression of rFVIII in serum containing medium, which includes vWF (von Willebrand Factor) that acts as a natural FVIII stabilizer, usually results in high yields, the expression of this complex protein in serum free medium imposes further challenges due to the absence of vWF [3]. There is evidence showing that under serum free conditions, rFVIII binds to the phospholipids on the cell membrane resulting in degradation [20].…”

Section: Discussionmentioning

confidence: 99%

“…There is evidence showing that under serum free conditions, rFVIII binds to the phospholipids on the cell membrane resulting in degradation [20]. Therefore, to protect, stabilize and release rFVIII into the culture medium, the coexpression with vWF has been performed [3]. In another approach to solve this problem, Winge [21] shows that large amounts of active rFVIII could be released from serum-free recombinant HEK293 cells through exposure to a solution of high ionic strength.…”

Section: Discussionmentioning

confidence: 99%

“…However, efficient production of human rFVIII has been shown to be difficult [2-4]. The rFVIII expression levels are significantly lower than those of other recombinant proteins comparable in structure, size, and complexity [3]. Also, the expression is two or three orders of magnitude lower than other recombinant proteins produced with similar strategies [2].…”

Section: Introductionmentioning

confidence: 99%

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Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

et al. 2011

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BackgroundHemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37°C.ResultsWe observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34°C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection.ConclusionSerum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability.

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“…Recently, we demonstrated that the use of serum free medium in rFVIII production brought a new aspect of media optimisation into play, because of product yield limitations due to product attachment to the cell surface of the production cells (Kolind et al, 2010). The fact that rFVIII binds to phospholipids in vivo and more specifically to PS (Andree et al, 1992) has been recognised for several years.…”

Section: Discussionmentioning

confidence: 99%

Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction

Kolind

Nørby

Berchtold

et al. 2011

Journal of Biotechnology

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The B-domain of Factor VIII reduces cell membrane attachment to host cells under serum free conditions

Cited by 12 publications

References 28 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

Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction

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