Separation of VWF and VWF propeptide by chromatographic methods
The present invention relates to a method for separating a mature von Willebrand Factor (mat-VWF) from von Willebrand Factor pro-peptide (VWF-PP) by incubating a composition comprising inducing dissociation of mat-VWF and VWF-PP by disruption of the non-covalently associated mat-VWF and VWF-PP, wherein said dissociation is induced by: (i) addition of at least one chelating agent, or (ii) increasing the pH to a pH of at least 7, and then collecting said mat-VWF to obtain a high purity, propeptide depleted mature VWF (mat-VWF).
1. A method for obtaining a composition comprising a high purity, propeptide depleted mature recombinant rVWF (mat-rVWF), said method comprising the steps of:
a) loading a solution comprising mat-rVWF/rVWF-PP complex, mat-rVWF, and rVWF propeptide (rVWF-PP) onto a size exclusion column;
b) washing said size exclusion column with a buffer, thereby dissociating said mat-rVWF/rVWF-PP complex in said solution in a) into mat-rVWF and rVWF-PP, wherein said dissociation occurs by disruption of the non-covalently associated mat-rVWF and rVWF-PP, wherein said buffer comprises at least one chelating agent and exhibits a pH of at least 7; and
c) collecting said mat-rVWF to obtain a high purity, mat-rVWF composition, wherein said high purity, mat-rVWF composition comprises at least 95% mature rVWF and less than 5% rVWF-PP.
2. The method of claim 1 , wherein said high purity, mat-rVWF composition comprises at least 96% mat-rVWF and less than 4% rVWF-PP, at least 97% mat-rVWF and less than 3% rVWF-PP, at least 98% mat-rVWF and less than 2% rVWF-PP, at least 99% mat-rVWF and less than 1% rVWF-PP, or at least 99.5% mat-rVWF and less than 0.5% rVWF-PP, or 99.9% mat-rVWF and less than 0.1% rVWF-PP.
3. The method according to claim 1 , wherein said solution is selected from the group consisting of a cell culture medium, an antibody column flow-through solution, and a buffered solution.
4. The method according to claim 1 , wherein said solution has been treated with furin prior to step a).
5. The method according to claim 1 , wherein said solution is an antibody column flow-through solution.
6. The method according to claim 1 , wherein said at least one chelating agent is a divalent cation chelating agent.
7. The method according to claim 6 , wherein said divalent cation chelating agent is selected from the group consisting of EDTA, EGTA, CDTA, and citrate.
8. The method according to claim 1 , wherein said pH is at least 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0.
9. The method according to claim 1 , wherein said pH is at least about 7.2 to about 7.8.
10. The method according to claim 9 , wherein said pH is at least about 7.6.
11. The method according to claim 1 , wherein said pH is increased by the addition of basic amino acids, Tris, NaOH, Tricine, or ethanolamine.
12. The method according to claim 1 , wherein said buffer comprises a buffering agent selected from the group consisting of glycine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TrisHCl (Tris(hydroxymethyl)-aminomethane), histidine, imidazole, acetate citrate, MES, and 2-(N-morpholino)ethanesulfonic acid.
13. The method according to claim 1 , wherein said buffer further comprises one or more monovalent cations.
14. The method according to claim 13 , wherein said one or more monovalent cations are selected from the group consisting of Na+, K+, Li+, and Cs+.
15. The method according to claim 13 , wherein said monovalent cation is Na+.
16. The method according to claim 1 , wherein said buffer further comprises one or more monovalent, divalent and/or trivalent anions.
17. The method according to claim 16 , wherein said one or more monovalent, divalent and/or trivalent anions are selected from the group consisting of Cl“, acetate”, SO 4 2− , Br − , and citrate 3− .
18. The method according to claim 1 , wherein said buffer comprises at least one buffer exhibiting a conductivity of >0.5 mS/cm at 25° C.
19. The method according to claim 1 , wherein said high purity mat-rVWF composition comprises a host cell (HC) impurity level of <2.0%.
20. The method according to claim 1 , wherein said high purity, mat-rVWF composition comprises a host cell (HC) impurity level of <0.6%.
21. The method according to claim 1 , wherein said solution comprising mat-rVWF/rVWF-PP complex, mat-rVWF, and rVWF-PP is derived from a capture step for rVWF.
22. The method according to claim 1 , wherein said solution comprising mat-rVWF/rVWF-PP complex, mat-rVWF, and rVWF-PP is derived from a method comprising a FVIII immunoaffinity step and anion exchange chromatography step.
23. The method of claim 12 , wherein said buffering agent is HEPES.
24. The method of claim 1 , wherein said buffer comprises Na citrate.
25. The method of claim 1 , wherein said buffer comprises NaCl.
26. The method of claim 1 , wherein said buffer comprises HEPES, Na citrate, and NaCl.
27. The method of claim 1 , wherein said buffer comprises 20 mM HEPES, 15 mM Na citrate, and 150 mM NaCl.
28. The method of claim 1 , further comprising lyophilizing said high purity, mat-rVWF composition after step (c).