Abstract
IntroductionSince the early 1990s, recombinant human clotting factor VIII (rhFVIII) produced in hamster cells has been available for haemophilia A treatment. However, the post-translational modifications of these proteins are not identical to those of native human FVIII, which may lead to immunogenic reactions and the development of inhibitors against rhFVIII. For the first time, rhFVIII produced in a human host cell line is available.AimWe describe here the establishment of the first human production cell line for rhFVIII and the manufacturing process of this novel product.Methods and resultsA human cell line expressing rhFVIII was derived from human embryonic kidney (HEK) 293 F cells transfected with an FVIII expression plasmid. No virus or virus-like particles could be detected following extensive testing. The stringently controlled production process is completely free from added materials of animal or human origin. Multistep purification employing a combination of filtration and chromatography steps ensures the efficient removal of impurities. Solvent/detergent treatment and a 20 nm pore size nanofiltration step, used for the first time in rhFVIII manufacturing, efficiently eliminate any hypothetically present viruses. In contrast to hamster cell-derived products, this rhFVIII product does not contain hamster-like epitopes, which might be expected to be immunogenic.ConclusionsHEK 293 F cells, whose parental cell line HEK 293 has been used by researchers for decades, are a suitable production cell line for rhFVIII and will help avoid immunogenic epitopes. A modern manufacturing process has been developed to ensure the highest level of purity and pathogen safety.
Highlights
Since the early 1990s, recombinant human clotting factor VIII produced in hamster cells has been available for haemophilia A treatment
The main reason for using a human cell line is the presence of the translational machinery and all the subcellular compartments necessary to generate correctly folded and fully active factor VIII (FVIII) [19], with an authentic human pattern of post-translational modifications (PTMs). This is of particular importance as PTMs have been demonstrated to impact on recombinant human clotting factor VIII (rhFVIII) activity (Sandberg H, Kannicht C, Stenlund P, Dadaian M, Oswaldsson U, Cordula C, Walter O, manuscript submitted for publication)
Random amplified polymorphic DNA (RAPD) analysis yielded almost identical fragment patterns to those obtained with human embryonic kidney (HEK) 293 DNA and DNA extracted from the master cell bank (MCB) and research cell bank (RCB), whereas no similarity in banding patterns was observed with mouse, ovine, monkey or hamster DNA (Fig. 2)
Summary
Since the early 1990s, recombinant human clotting factor VIII (rhFVIII) produced in hamster cells has been available for haemophilia A treatment. The post-translational modifications of these proteins are not identical to those of native human FVIII, which may lead to immunogenic reactions and the development of inhibitors against rhFVIII. RhFVIII produced in a human host cell line is available. Aim: We describe here the establishment of the first human production cell line for rhFVIII and the manufacturing process of this novel product. Solvent/detergent treatment and a 20 nm pore size nanofiltration step, used for the first time in rhFVIII manufacturing, efficiently eliminate any hypothetically present viruses. In contrast to hamster cell-derived products, this rhFVIII product does not contain hamster-like epitopes, which might be expected to be immunogenic. A modern manufacturing process has been developed to ensure the highest level of purity and pathogen safety
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