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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.

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

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SDS-PAGE of rFVIII molecules before and after thrombin cleavage. From Kristensen et al. 26.
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fig03: SDS-PAGE of rFVIII molecules before and after thrombin cleavage. From Kristensen et al. 26.

Mentions: During secretion, some rFVIII molecules are cleaved in the A2 domain leaving part of the FVIII molecules with a heavy chain with C-terminus at amino acid 720 or 729 20,21. As the C-terminal end of the A2 domain (amino acids 720–740) contains residues required for optimal interaction with thrombin 22, the purification process for turoctocog alfa was optimised in order to secure isolation of molecules with intact A2 domain. This was obtained by selecting an antibody (F25) for immunoaffinity chromatography that binds selectively to molecules with intact A2 domain. The antibody binds to the C-terminus of the A2 domain (Fig.1), enabling removal of FVIII degraded within the A2 domain 13. The gene encoding the antibody was transferred to CHO cells for production and therefore no additional murine antigens or host cell proteins are introduced 23. The removal of degraded heavy chain is clearly seen by SDS–PAGE analysis after thrombin cleavage, where more than one band represents the A2 domain for ReFacto AF® (Fig.3, lane 8). For turoctocog alfa (Fig.3, lane 2) or rFVIII derived from the full-length genes (Fig.3, lanes 4 and 6), no C-terminal degradation in the A2 domain was observed.


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

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

SDS-PAGE of rFVIII molecules before and after thrombin cleavage. From Kristensen et al. 26.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: SDS-PAGE of rFVIII molecules before and after thrombin cleavage. From Kristensen et al. 26.
Mentions: During secretion, some rFVIII molecules are cleaved in the A2 domain leaving part of the FVIII molecules with a heavy chain with C-terminus at amino acid 720 or 729 20,21. As the C-terminal end of the A2 domain (amino acids 720–740) contains residues required for optimal interaction with thrombin 22, the purification process for turoctocog alfa was optimised in order to secure isolation of molecules with intact A2 domain. This was obtained by selecting an antibody (F25) for immunoaffinity chromatography that binds selectively to molecules with intact A2 domain. The antibody binds to the C-terminus of the A2 domain (Fig.1), enabling removal of FVIII degraded within the A2 domain 13. The gene encoding the antibody was transferred to CHO cells for production and therefore no additional murine antigens or host cell proteins are introduced 23. The removal of degraded heavy chain is clearly seen by SDS–PAGE analysis after thrombin cleavage, where more than one band represents the A2 domain for ReFacto AF® (Fig.3, lane 8). For turoctocog alfa (Fig.3, lane 2) or rFVIII derived from the full-length genes (Fig.3, lanes 4 and 6), no C-terminal degradation in the A2 domain was observed.

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