Limits...
Turoctocog alfa (NovoEight®)--from design to clinical proof of concept.

Ezban M, Vad K, Kjalke M - Eur. J. Haematol. (2014)

Bottom Line: Viral inactivation is ensured by a detergent inactivation step as well as a 20-nm nano-filtration step.Tyr1680 was also fully sulphated in turoctocog alfa resulting in strong affinity (low nm Kd ) for binding to von Willebrand factor (VWF).The non-clinical data thus confirm the haemostatic effect of turoctocog alfa and, together with the comprehensive clinical evaluation, support the use as FVIII replacement therapy in patients with haemophilia A.

View Article: PubMed Central - PubMed

Affiliation: Novo Nordisk A/S, Maaloev, Denmark.

Show MeSH

Related in: MedlinePlus

Molecular structure of full-length FVIII and turoctocog alfa. The upper part of the figure illustrates full-length FVIII coding for 2332 amino acid residues. The middle part of the figure shows the structure of turoctocog alfa. The molecule consists of the heavy chain containing the A1-A2 domains, a truncated B-domain coding for 21 amino acids of the naturally occurring B-domain (amino acid 741–750 fused with 1638–1648) and the light chain (A3-C1-C2 domains). The lower part of the figure shows the sequence coding for the 21 amino acid residue truncated B-domain (light blue), which represents 10 amino residues from the N-terminal of the B-domain linked to 11 amino acid residues from the C-terminal of the B-domain. Cleavage sites, tyrosine sulphation sites and the epitope detected by F25 during purification are indicated in this region 13. Reproduced from Thim et al. 13 with permission from John Wiley and Sons.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4232928&req=5

fig01: Molecular structure of full-length FVIII and turoctocog alfa. The upper part of the figure illustrates full-length FVIII coding for 2332 amino acid residues. The middle part of the figure shows the structure of turoctocog alfa. The molecule consists of the heavy chain containing the A1-A2 domains, a truncated B-domain coding for 21 amino acids of the naturally occurring B-domain (amino acid 741–750 fused with 1638–1648) and the light chain (A3-C1-C2 domains). The lower part of the figure shows the sequence coding for the 21 amino acid residue truncated B-domain (light blue), which represents 10 amino residues from the N-terminal of the B-domain linked to 11 amino acid residues from the C-terminal of the B-domain. Cleavage sites, tyrosine sulphation sites and the epitope detected by F25 during purification are indicated in this region 13. Reproduced from Thim et al. 13 with permission from John Wiley and Sons.

Mentions: The human gene for factor VIII (FVIII) was cloned and expressed in 1984 1–5, making it possible to produce recombinant FVIII (rFVIII) for prevention and treatment of bleeds in patients with haemophilia A. The FVIII gene encodes a single chain of 2332 amino acid residues with the domain structure A1-A2-B-A3-C1-C2 (Fig.1) 1. During cellular processing, the molecule undergoes posttranslational modifications, including sulphation of specific tyrosine residues and glycosylation. Furthermore, the protein is processed into a heterodimer consisting of a heavy chain (HC) with the A1-A2-B domains and a light chain (LC) with the A3-C1-C2 domains. The two chains are held together by metal ions 6. While the B-domain plays a role in restricting the expression of endogenous FVIII, it is apparently not needed for the function of FVIII 7–9. Acidic regions C-terminal to the A1 and the A2 domains and N-terminal to the A3 domain (a1, a2 and a3, respectively) are important for interaction with thrombin and von Willebrand factor (VWF). Sulphation of Tyr1680 is essential for high affinity binding of FVIII to VWF 10,11, which is required for protection of FVIII from degradation and rapid clearance. During haemostasis, FVIII is activated by specific thrombin cleavages, thereby producing the A1, A2 and A3-C1-C2 fragments of activated FVIII (FVIIIa). This results in dissociation of VWF and assembly of the tenase complex (FVIIIa/FIXa) on the surface of activated platelets. FXa is generated, resulting in a thrombin burst, and ultimately leading to the formation of a stable haemostatic plug.


Turoctocog alfa (NovoEight®)--from design to clinical proof of concept.

Ezban M, Vad K, Kjalke M - Eur. J. Haematol. (2014)

Molecular structure of full-length FVIII and turoctocog alfa. The upper part of the figure illustrates full-length FVIII coding for 2332 amino acid residues. The middle part of the figure shows the structure of turoctocog alfa. The molecule consists of the heavy chain containing the A1-A2 domains, a truncated B-domain coding for 21 amino acids of the naturally occurring B-domain (amino acid 741–750 fused with 1638–1648) and the light chain (A3-C1-C2 domains). The lower part of the figure shows the sequence coding for the 21 amino acid residue truncated B-domain (light blue), which represents 10 amino residues from the N-terminal of the B-domain linked to 11 amino acid residues from the C-terminal of the B-domain. Cleavage sites, tyrosine sulphation sites and the epitope detected by F25 during purification are indicated in this region 13. Reproduced from Thim et al. 13 with permission from John Wiley and Sons.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232928&req=5

fig01: Molecular structure of full-length FVIII and turoctocog alfa. The upper part of the figure illustrates full-length FVIII coding for 2332 amino acid residues. The middle part of the figure shows the structure of turoctocog alfa. The molecule consists of the heavy chain containing the A1-A2 domains, a truncated B-domain coding for 21 amino acids of the naturally occurring B-domain (amino acid 741–750 fused with 1638–1648) and the light chain (A3-C1-C2 domains). The lower part of the figure shows the sequence coding for the 21 amino acid residue truncated B-domain (light blue), which represents 10 amino residues from the N-terminal of the B-domain linked to 11 amino acid residues from the C-terminal of the B-domain. Cleavage sites, tyrosine sulphation sites and the epitope detected by F25 during purification are indicated in this region 13. Reproduced from Thim et al. 13 with permission from John Wiley and Sons.
Mentions: The human gene for factor VIII (FVIII) was cloned and expressed in 1984 1–5, making it possible to produce recombinant FVIII (rFVIII) for prevention and treatment of bleeds in patients with haemophilia A. The FVIII gene encodes a single chain of 2332 amino acid residues with the domain structure A1-A2-B-A3-C1-C2 (Fig.1) 1. During cellular processing, the molecule undergoes posttranslational modifications, including sulphation of specific tyrosine residues and glycosylation. Furthermore, the protein is processed into a heterodimer consisting of a heavy chain (HC) with the A1-A2-B domains and a light chain (LC) with the A3-C1-C2 domains. The two chains are held together by metal ions 6. While the B-domain plays a role in restricting the expression of endogenous FVIII, it is apparently not needed for the function of FVIII 7–9. Acidic regions C-terminal to the A1 and the A2 domains and N-terminal to the A3 domain (a1, a2 and a3, respectively) are important for interaction with thrombin and von Willebrand factor (VWF). Sulphation of Tyr1680 is essential for high affinity binding of FVIII to VWF 10,11, which is required for protection of FVIII from degradation and rapid clearance. During haemostasis, FVIII is activated by specific thrombin cleavages, thereby producing the A1, A2 and A3-C1-C2 fragments of activated FVIII (FVIIIa). This results in dissociation of VWF and assembly of the tenase complex (FVIIIa/FIXa) on the surface of activated platelets. FXa is generated, resulting in a thrombin burst, and ultimately leading to the formation of a stable haemostatic plug.

Bottom Line: Viral inactivation is ensured by a detergent inactivation step as well as a 20-nm nano-filtration step.Tyr1680 was also fully sulphated in turoctocog alfa resulting in strong affinity (low nm Kd ) for binding to von Willebrand factor (VWF).The non-clinical data thus confirm the haemostatic effect of turoctocog alfa and, together with the comprehensive clinical evaluation, support the use as FVIII replacement therapy in patients with haemophilia A.

View Article: PubMed Central - PubMed

Affiliation: Novo Nordisk A/S, Maaloev, Denmark.

Show MeSH
Related in: MedlinePlus