Methods for producing Factor VIII proteins
Methods are provided for purification of Factor VIII polypeptides by affinity chromatography and ion exchange chromatography, in which the eluate from the affinity column is diluted with a solution comprising higher salt concentration, or lower non-polar agent concentration than that of the elution solution, prior to passing the diluted solution through the ion exchange column. The affinity matrix may comprise a monoclonal antibody or a peptide ligand. The methods result in improved purification without significant yield loss.
1. A method for purification of a Factor VIII polypeptide comprising:
(a) adding a mixture containing a Factor VIII polypeptide to be purified to an affinity matrix which binds the Factor VIII polypeptide by hydrophobic and/or electrostatic and/or van der Waals attractions;
(b) eluting the Factor VIII polypeptide from the affinity matrix with an elution solution which desorbs the Factor VIII polypeptide;
(c) diluting the elution solution about 3-fold to about 5-fold with a solution comprising higher salt concentration than that of the elution solution, resulting in a diluted Factor VIII solution comprising about 7 to about 20 mM of a salt;
(d) passing the diluted Factor VIII solution through an ion exchange column which binds the Factor VIII polypeptide, thereby allowing contaminants to pass through the ion exchange column; and
(e) eluting the purified factor VIII polypeptide from the ion exchange column.
2. The method of claim 1 , wherein the affinity matrix comprises a monoclonal antibody or peptide ligand.
3. The method of claim 2 , wherein the affinity matrix comprises a monoclonal antibody.
4. The method of claim 2 , wherein the affinity matrix comprises a peptide ligand.
5. The method of claim 4 , wherein the peptide ligand is TN 8.2.
6. The method of claim 1 , wherein a salt is added to the elution solution of step (b).
7. The method of claim 6 , wherein the salt is NaCl.
8. The method of claim 1 , wherein a salt is added to the diluted Factor VIII solution in step (c).
9. The method of claim 8 , wherein the salt is NaCl.
10. The method of claim 8 , wherein the elution solution of step (b) comprises no NaCl salt, and the diluted Factor VIII solution in step (c) comprises about 15 mM NaCl.
11. The method of claim 1 , wherein the elution solution of step (b) comprises NaCl salt, and the diluted Factor VIII solution in step (c) comprises about 15 mM NaCl.
12. The method of claim 1 , wherein the elution solution is diluted about 3-fold.
13. The method of claim 1 , wherein the affinity matrix comprises monoclonal antibody, peptide ligand or triazine dye.
14. The method of claim 1 , wherein the elution solution comprises ethylene glycol.
15. The method of claim 14 , wherein the solution used to dilute the elution solution in step (c) comprises ethylene glycol.
16. A method for purification of a Factor VIII polypeptide comprising:
(a) adding a mixture containing a Factor VIII polypeptide to be purified to an affinity matrix which binds the Factor VIII polypeptide by hydrophobic and/or electrostatic and/or van der Waals attractions;
(b) eluting the Factor VIII polypeptide from the affinity matrix with an elution solution which desorbs the Factor VIII polypeptide, wherein the elution solution comprises at least one of ethylene glycol, dioxane, propylene glycol and polyethylene glycol;
(c) diluting the elution solution about 1.5-fold to about 3-fold with a solution comprising lower concentration of the ethylene glycol, dioxane, propylene glycol and/or polyethylene glycol than that of the elution solution, resulting in a diluted Factor VIII solution comprising about 17% to about 33% (v/v) ethylene glycol, dioxane, propylene glycol and/or polyethylene glycol;
(d) passing the diluted Factor VIII solution through an ion exchange column which binds the Factor VIII polypeptide, thereby allowing contaminants to pass through the ion exchange column; and
(e) eluting the purified Factor VIII polypeptide from the ion exchange column.
17. The method of claim 16 , wherein the affinity matrix comprises a monoclonal antibody or peptide ligand.
18. The method of claim 17 , wherein the affinity matrix comprises a monoclonal antibody.
19. The method of claim 17 , wherein the affinity matrix comprises a peptide ligand.
20. The method of claim 19 , wherein the peptide ligand is TN 8.2.
21. The method of claim 16 , wherein the elution solution of step (b) comprises 50% (v/v) ethylene glycol, and the dilution of step (c) is performed using a solution comprising less than 50% (v/v) ethylene glycol, such that the final concentration of ethylene glycol in the diluted factor VIII solution is from about 17% to about 33% (v/v).
22. The method of claim 16 , wherein the elution solution of step (b) contains 50% (v/v) ethylene glycol, and the dilution of step (c) is performed using a solution comprising no ethylene glycol, such that the final concentration of ethylene glycol in the diluted factor VIII solution is from about 17% to about 33% (v/v).
23. The method of claim 16 , wherein the elution solution is diluted about 1.5-fold.
24. The method of claim 16 , wherein a salt is added to the elution solution of step (b).
25. The method of claim 24 , wherein the salt is NaCl.
26. The method of claim 16 , wherein a salt is added to the diluted Factor VIII solution in step (c).
27. The method of claim 26 , wherein the salt is NaCl.
28. The method of claim 16 , wherein the elution solution of step (b) comprises no NaCl salt, and the diluted Factor VIII solution in step (c) comprises about 15 mM NaCl.
29. The method of claim 16 , wherein the elution solution of step (b) comprises NaCl salt, and the diluted Factor VIII solution in step (c) comprises about 15 mM NaCl.
30. The method of claim 16 , wherein the affinity matrix comprises monoclonal antibody, peptide ligand or triazine dye.
31. The method of claim 16 , wherein the elution solution comprises ethylene glycol.
32. The method of claim 31 , wherein the solution used to dilute the elution solution in step (c) comprises ethylene glycol.
33. A method for purification of a Factor VIII polypeptide comprising:
(a) adding a mixture containing a Factor VIII polypeptide to be purified to an affinity matrix which binds to the Factor VIII polypeptide;
(b) eluting the Factor VIII polypeptide from the affinity matrix with an elution solution which desorbs the Factor VIII polypeptide, wherein the elution solution comprises at least one of ethylene glycol, dioxane, propylene glycol and polyethylene glycol and a buffer;
(c) diluting the elution solution about 1.5-fold to about 3-fold with a solution comprising a lower concentration of the ethylene glycol, dioxane, propylene glycol and/or polyethylene glycol than that of the elution solution, resulting in a diluted Factor VIII solution comprising about 17% to about 33% (v/v) ethylene glycol, dioxane, propylene glycol and/or polyethylene glycol;
(d) passing the diluted Factor VIII solution through an ion exchange column which binds the Factor VIII polypeptide, thereby allowing contaminants to pass through the ion exchange column; and
(e) eluting the purified Factor VIII polypeptide from the ion exchange column.
34. The method of claim 33 , wherein the affinity matrix comprises a monoclonal antibody or peptide ligand.
35. The method of claim 34 , wherein the affinity matrix comprises a monoclonal antibody.
36. The method of claim 34 , wherein the affinity matrix comprises a peptide ligand.
37. The method of claim 36 , wherein the peptide ligand is TN 8.2.
38. The method of claim 33 , wherein the elution solution of step (b) comprises no NaCl salt, and the diluted Factor VIII solution in step (c) comprises about 15 mM NaCl.
39. The method of claim 33 , wherein the elution solution of step (b) comprises NaCl salt, and the diluted Factor VIII solution in step (c) comprises about 15 mM NaCl.
40. The method of claim 33 , wherein the elution solution is diluted about 1.5-fold.
41. The method of claim 33 , wherein a salt is added to the elution solution of step (b).
42. The method of claim 41 , wherein the salt is NaCl.
43. The method of claim 33 , wherein a salt is added to the diluted Factor VIII solution in step (c).
44. The method of claim 43 , wherein the salt is NaCl.
45. The method of claim 33 , wherein the affinity matrix comprises monoclonal antibody, peptide ligand or triazine dye.
46. The method of claim 33 , wherein the elution solution comprises ethylene glycol.
47. The method of claim 46 , wherein the solution used to dilute the elution solution in step (c) comprises ethylene glycol.
48. A method for purification of a Factor VIII polypeptide comprising:
(a) adding a mixture containing a Factor VIII polypeptide to be purified to an affinity matrix which binds the Factor VIII polypeptide by hydrophobic and/or electrostatic and/or van der Waals attractions;
(b) eluting the Factor VIII polypeptide from the affinity matrix with an elution solution which desorbs the Factor VIII polypeptide, wherein the elution solution comprises about 7 to about 20 mM of salt;
(c) passing the elution solution comprising Factor VIII solution through an ion exchange column which binds the Factor VIII polypeptide, thereby allowing contaminants to pass through the ion exchange column; and
(d) eluting the purified factor VIII polypeptide from the ion exchange column.
49. The method of claim 48 , wherein the elution solution of step (b) comprises about 15 mM NaCl.
50. The method of claim 48 , wherein the affinity matrix comprises monoclonal antibody, peptide ligand or triazine dye.
51. The method of claim 48 , wherein the elution solution comprises ethylene glycol.
52. A method for purification of a Factor VIII polypeptide comprising:
(a) adding a mixture containing a Factor VIII polypeptide to be purified to an affinity matrix which binds the Factor VIII polypeptide by hydrophobic and/or electrostatic and/or van der Waals attractions;
(b) eluting the Factor VIII polypeptide from the affinity matrix with an elution solution which desorbs the Factor VIII polypeptide, the elution solution comprising either a salt at a concentration of about 1 mM to about 25 mM or at least one of ethylene glycol, dioxane, propylene glycol and polyethylene glycol at a final concentration of about 50% (v/v);
(c) diluting the elution solution about 3-fold to about 5-fold with a solution of higher ionic strength than that of the elution solution;
(d) passing the diluted Factor VIII solution through an ion exchange column which binds the Factor VIII polypeptide, thereby allowing contaminants to pass through the ion exchange column; and
(e) eluting the purified factor VIII polypeptide from the ion exchange column.
53. The method of claim 52 , wherein the diluted Factor VIII solution comprises about 1 to about 50 mM of salt in step (c).
54. The method of claim 52 , wherein the diluted Factor VIII solution comprises about 7 to about 20 mM of salt in step (c).
55. The method of claim 52 , wherein the affinity matrix comprises monoclonal antibody, peptide ligand or triazine dye.
56. The method of claim 52 , wherein the affinity matrix comprises monoclonal antibody or peptide ligand.
57. The method of claim 56 , wherein the affinity matrix is a TN 8.2 peptide ligand.
58. The method of claim 52 , wherein the elution solution comprises ethylene glycol.
59. The method of claim 58 , wherein the solution used to dilute the elution solution in step (c) comprises ethylene glycol.
60. The method of claim 52 , wherein the elution solution comprises a salt at a concentration of about 5 mM to about 20 mM.
61. The method of claim 60 , wherein the elution solution comprises a salt at a concentration of about 5 mM, about 10 mM, about 15 mM, or about 20 mM.
62. The method of claim 52 , wherein the final concentration of at least one of ethylene glycol, dioxane, propylene glycol and polyethylene glycol is about 17% to about 33% (v/v) in step (c).