An improved manufacturing process for Xyntha/ReFacto AF

Kelley, Brian; Jankowski, Marek; Booth, James A.

doi:10.1111/j.1365-2516.2009.02160.x

Cited by 50 publications

(39 citation statements)

References 14 publications

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“…These cleavages also release the a3 acidic domain from the N-terminus of the LC, which is required for Von Willebrand factor (VWF) binding to FVIII, as well as the remaining 14 amino acids of the B domain [13]. It has been reported that in a number of recombinant factor FVIII molecules [14]–[17], the cleavage at R1648 does not occur for a fraction of the secreted BDD rFVIII product, leading to the generation of non-processed single chain rFVIII isoform. Also, there are recent reports on different isoforms of SC rFVIII designed to improve in vivo activity and prolong half-life [18]–[20].…”

Section: Introductionmentioning

confidence: 99%

A Single Chain Variant of Factor VIII Fc Fusion Protein Retains Normal In Vivo Efficacy but Exhibits Altered In Vitro Activity

et al. 2014

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Recombinant factor VIII Fc (rFVIIIFc) is a fusion protein consisting of a single B-domain-deleted (BDD) FVIII linked recombinantly to the Fc domain of human IgG1 to extend half-life. To determine if rFVIIIFc could be further improved by maintaining the heavy and light chains within a contiguous single chain (SC), we evaluated the activity and function of SC rFVIIIFc, an isoform that is not processed at residue R1648. SC rFVIIIFc showed equivalent activity in a chromogenic assay compared to rFVIIIFc, but approximately 40% activity by the one-stage clotting assay in the presence of von Willebrand Factor (VWF), with full activity in the absence of VWF. Moreover, SC rFVIIIFc demonstrated markedly delayed thrombin-mediated release from VWF, but an activity similar to that of rFVIIIFc upon activation in FXa generation assays. Therefore, the apparent reduction in specific activity in the aPTT assay appears to be primarily due to delayed release of FVIII from VWF. To assess whether stability and activity of SC rFVIIIFc were affected in vivo, a tail vein transection model in Hemophilia A mice was utilized. The results demonstrated similar pharmacokinetic profiles and comparable efficacy for SC rFVIIIFc and rFVIIIFc. Thus, while the single chain configuration did not promote enhanced half-life, it reduced the rate of release of FVIII from VWF required for activation. This impaired release may underlie the observed reduction in the one-stage clotting assay, but does not appear to affect the physiological activity of SC rFVIIIFc.

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Section: Introductionmentioning

confidence: 99%

A Single Chain Variant of Factor VIII Fc Fusion Protein Retains Normal In Vivo Efficacy but Exhibits Altered In Vitro Activity

et al. 2014

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“…BDD-rFVIII was isolated from an rFVIII-expressing CHO cell line, which has secreted rFVIII that consistently contains HC fragments with varying portions of the B domain including one that entirely lacks the B domain. Its appearance in 1-D SDS-PAGE was similar to that of ReFacto/Xyntha [6] and N8 [19], as it contained two major bands representing the HC and LC (Fig. 1).…”

Section: Peg-rfviii 0854psr (Dop 5) Cy3 Peg-rfviii 09004psr (Dop 2) Cy5mentioning

confidence: 77%

“…Current substitution therapy uses plasma-derived and recombinant factor VIII (rFVIII) products, the latter comprising full-length and B-domain-deleted (BDD) variants [4][5][6]. BDD-rFVIII was developed because the B domain of FVIII is apparently not required for the catalytic activity of FVIIIa.…”

Section: Introductionmentioning

confidence: 99%

Structural analysis of the recombinant therapeutic product rFVIII and its PEGylated variants using 2‐D DIGE

et al. 2011

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2-D DIGE is a method that circumvents the gel-to-gel variability inherent in conventional 2-DE and is particularly useful for studying proteome changes in diverse applications such as developmental biology and tissue proteomics. We developed a 2-D DIGE protocol for recombinant factor VIII (rFVIII), a therapeutic protein used for the treatment of hemophilia A. The factor VIII heterodimer is composed of heterogeneous, heavily glycosylated heavy and light chains that are held together by a divalent cation. 2-DE of rFVIII led to a separation of the various fragments whose identity could be determined by Western blot. A comparison of two rFVIII batches by 2-D DIGE revealed their identical composition, whereas an rFVIII variant lacking its central B domain was congruent with the smallest heavy and light chain fragments of rFVIII only. A simpler pattern was obtained upon removal of the terminal sialic acids of rFVIII's glycans, due to a better focusing in the first dimension. 2-D DIGE was also well suited to structurally evaluate various PEGylated rFVIII conjugates. 2-D DIGE thus proved an excellent and straightforward method for structural analysis of rFVIII. Our data suggest that the method could serve as a tool for quality control of very complex pharmaceutically active ingredients.

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“…To our knowledge, this is the first reported use of a 15 nm nanofilter in the manufacturing process of a rFVIII product. The viral clearance resulting from the manufacturing process of rFVIIIFc can be compared with that of another rFVIII product produced in mammalian (CHO) cells [6]. The CHO-based rFVIII process achieved >11.4 logs for X-MLV, >14.0 logs for SuHV-1, >10.3 logs for Reo-3, and 5.2 logs for MMV.…”

Section: Discussionmentioning

confidence: 99%

Manufacturing process used to produce long-acting recombinant factor VIII Fc fusion protein

et al. 2015

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Recombinant factor VIII Fc fusion protein (rFVIIIFc) is a long-acting coagulation factor approved for the treatment of hemophilia A. Here, the rFVIIIFc manufacturing process and results of studies evaluating product quality and the capacity of the process to remove potential impurities and viruses are described. This manufacturing process utilized readily transferable and scalable unit operations and employed multi-step purification and viral clearance processing, including a novel affinity chromatography adsorbent and a 15 nm pore size virus removal nanofilter. A cell line derived from human embryonic kidney (HEK) 293H cells was used to produce rFVIIIFc. Validation studies evaluated identity, purity, activity, and safety. Process-related impurity clearance and viral clearance spiking studies demonstrate robust and reproducible removal of impurities and viruses, with total viral clearance >8-15 log10 for four model viruses (xenotropic murine leukemia virus, mice minute virus, reovirus type 3, and suid herpes virus 1). Terminal galactose-α-1,3-galactose and N-glycolylneuraminic acid, two non-human glycans, were undetectable in rFVIIIFc. Biochemical and in vitro biological analyses confirmed the purity, activity, and consistency of rFVIIIFc. In conclusion, this manufacturing process produces a highly pure product free of viruses, impurities, and non-human glycan structures, with scale capabilities to ensure a consistent and adequate supply of rFVIIIFc.

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An improved manufacturing process for Xyntha/ReFacto AF

Cited by 50 publications

References 14 publications

A Single Chain Variant of Factor VIII Fc Fusion Protein Retains Normal In Vivo Efficacy but Exhibits Altered In Vitro Activity

A Single Chain Variant of Factor VIII Fc Fusion Protein Retains Normal In Vivo Efficacy but Exhibits Altered In Vitro Activity

Structural analysis of the recombinant therapeutic product rFVIII and its PEGylated variants using 2‐D DIGE

Manufacturing process used to produce long-acting recombinant factor VIII Fc fusion protein

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