Method for producing mature VWF from VWF pro-peptide
View Patent ↗The present invention relates to a method for producing a mature von Willebrand Factor (VWF) from von Willebrand Factor pro-peptide comprising the steps: immobilizing VWF pro-peptide on an ion exchange resin, incubating the immobilized VWF pro-peptide with furin to obtain immobilized mature VWF, and isolating mature VWF from the ion exchange resin by elution.
1. A method for producing mature von Willebrand Factor (VWF) from von Willebrand Factor pro-peptide comprising the steps of:
(a) immobilizing VWF pro-peptide on an ion exchange resin,
(b) incubating the immobilized VWF pro-peptide with furin comprising activity of at least 0.2 Units furin/Unit VWF antigen (Ag) to obtain immobilized mature VWF, and
(c) isolating at least 90% mature VWF from the ion exchange resin by elution.
2. The method according to claim 1 , wherein said VWF pro-peptide is of recombinant origin.
3. The method according to claim 1 , wherein said ion exchange resin comprises trimethylaminoethyl-groups (TMAE).
4. The method according to claim 1 , wherein said ion exchange resin is packed in a chromatographic column.
5. The method according to claim 3 , wherein said VWF pro-peptide is immobilized on the ion exchange resin and incubated with furin at a conductivity measured at 25° C. below 25 mS/cm.
6. The method according to claim 5 , wherein said conductivity is below 20 mS/cm.
7. The method according to claim 5 , wherein said conductivity is below 16 mS/cm.
8. The method according to claim 3 , wherein said mature VWF is eluted from the ion exchange resin at a conductivity measured at 25° C. of at least 40 mS/cm.
9. The method according to claim 8 wherein said conductivity is at least 60 mS/cm.
10. The method according to claim 8 wherein said conductivity is at least 80 mS/cm.
11. The method according to claim 1 , wherein said furin is comprised in a solution, which comprises further CaCl 2 at a concentration of 0.01 to 10 mM.
12. The method according to claim 11 , wherein said CaCl 2 is at a concentration of 0.1 to 5 mM.
13. The method according to claim 11 , wherein said CaCl 2 is at a concentration of 0.2 to 2 mM.
14. The method according to claim 1 , wherein the incubation is performed for 1 min to 48 hours.
15. The method according to claim 14 , wherein said incubation is performed for 10 min to 42 hours.
16. The method according to claim 15 , wherein said incubation is performed for 20 min to 36 hours.
17. The method according to claim 16 , wherein said incubation is performed for 30 min to 24 hours.
18. The method according to claim 1 , wherein the incubation is performed at a temperature of 2 to 40° C.
19. The method according to claim 18 , wherein said incubation is performed at a temperature of 4 to 37° C.
20. The method according to claim 1 , wherein said furin is of recombinant origin.
21. The method according to claim 1 wherein the furin comprises activity of at least 0.5 Units furin/Unit VWF Ag.
22. The method according to claim 21 wherein the furin comprises activity between 0.5-4.0 Units furin/Unit VWF Ag.
23. The method according to claim 21 wherein mature VWF is at least 95% mature VWF.
24. The method according to claim 21 wherein mature VWF is at least 98% mature VWF.
25. The method according to claim 21 wherein mature VWF is at least 99% mature VWF.
26. The method according to claim 22 wherein incubating is carried out for at least 1 hour.
27. The method according to claim 26 wherein incubating is carried out at 2° C. to 8° C.
28. The method according to claim 26 wherein incubating is carried out at 37° C.
29. The method according to claim 24 wherein a mean of 0.9 Units furin/Unit VWF Ag is incubated for at least 4 hours.
30. The method according to claim 1 wherein the furin comprises activity between 0.2 to 2.0 Units furin/Unit VWF Ag for 4 hours.