Regeneration of Chromatographic Stationary Phases

• US 20080312131A1
• Filed: 04/07/2008
• Published: 12/18/2008
• Est. Priority Date: 04/08/2003
• Status: Active Grant

• Current Assignee: Novo Nordisk A/S
• Original Assignee: Novo Nordisk A/S
• Inventors: Schou, Ole, Larsen, Per, Rasmussen, Eigil Schroder
• Granted Patent: US 9,364,772 B2
• Time in Patent Office: Days
• US Class Current: 514/2
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: B01D 15/20 relating to the conditioning of the sorbent material
• CPC Class Codes: B01D 15/203 Equilibration or regeneration
• CPC Class Codes: B01J 20/283 based on silica
• CPC Class Codes: B01J 20/285 based on polymers
• CPC Class Codes: B01J 20/34 Regenerating or reactivating
• CPC Class Codes: B01J 20/3425 of sorbents or filter aids comprising organic materials
• CPC Class Codes: B01J 20/3433 of sorbents or filter aids other than those covered by B01J20/3408 - B01J20/3425
• CPC Class Codes: B01J 20/3475 in the liquid phase
• CPC Class Codes: C07K 1/20 Partition-, reverse-phase or hydrophobic interaction chromatography

Abstract

Process for regenerating a chromatographic stationary phase

Claims

1. A process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one organic acid and less than about 75% w/w water.

2. The process according to claim 1, wherein the regeneration solution comprises at least one organic acid and less than about 1% w/w water.

3. The process according to claim 1, wherein the concentration of said organic acid is at least about 25% w/w.

4. The process according to claim 1, wherein said organic acid is formic acid.

5. The process according to claim 1, wherein said organic acid is acetic acid.

6. The process according to claim 1, wherein said regeneration solution comprises an organic solvent.

7. The process according to claim 6, wherein said organic solvent is ethanol.

8. The process according to claim 6, wherein said organic solvent is 2-propanol.

9. The process according to claim 6, wherein said organic solvent is acetonitrile.

10. The process according to claim 6, wherein said organic solvent is selected from the group consisting of methanol, 1-propanol, and hexylene glycol.

11. The process according to claim 1, wherein said regeneration solution contains less than 0.5% water.

13. The process according to claim 1, wherein said chromatographic stationary phase is contacted with said regeneration solution inside the chromatographic column.

14. The process according to claim 13, wherein said chromatographic stationary phase is regenerated without repacking the column.

15. The process according to claim 13, wherein said chromatographic stationary phase is fluidized during said regeneration.

16. The process according to claim 13, wherein the chromatographic eluent or equilibrium buffer is displaced by a water miscible organic solvent before said chromatographic stationary phase is contacted with said regeneration solution.

17. The process according to claim 16, wherein said organic solvent is also present in the chromatographic eluent or equilibrium buffer.

18. The process according to claim 16, wherein said water miscible organic solvent is also present in the regeneration solution.

19. The process according to claim 1, wherein said chromatographic stationary phase is contacted with said regeneration solution outside the chromatographic column.

20. The process according to claim 1, wherein said chromatographic stationary phase is a RP-HPLC matrix.

21. The process according to claim 1, wherein said chromatographic stationary phase is a silica or substituted silica material.

22. The process according to claim 21, wherein said chromatographic stationary phase is C16 or C18 substituted silica.

23. The process according to claim 21, wherein said chromatographic stationary phase is C4, C8 or phenyl-substituted silica.

24. The process according to claim 21, wherein said chromatographic stationary phase is a polymeric material.

25. The process according to claim 1, wherein said chromatographic stationary phase is contacted with said regeneration solution for at least 1 minute.

26. The process according to claim 1, wherein said chromatographic stationary phase is contacted with said regeneration solution until the pressure drop over the length of the chromatographic column at normal flow rate decreases by at least 10%.

27. The process according to claim 1, wherein contacting of said chromatographic stationary phase with said regeneration solution is performed at a temperature in the range from about 5°

C. to 50°

C.

28. The process according to claim 1, wherein the life time of said chromatographic stationary phase is at least 500 chromatographic cycles.

29. The process according to claim 1, wherein said process is applied to said chromatographic stationary phase for every chromatographic cycle.

30. The process according to claim 1, wherein said process is applied to said chromatographic stationary phase at least once every 100 chromatographic cycles.

31. A process for the production of a therapeutic polypeptide or a precursor thereof comprising at least one chromatographic step wherein the chromatographic stationary phase is regenerated by a process according to claim 1.

32. The process according to claim 31, wherein said therapeutic polypeptide is a derivative comprising a lipophilic substituent.

33. The process according to claim 31, wherein said therapeutic polypeptide is selected from the group consisting of glucagon, glucagon-like peptide 1, glucagon-like peptide 2, exendin-4, TFF peptides, human insulin, analogues thereof and derivatives thereof.

34. The process according to claim 31, wherein said polypeptide or a precursor thereof is selected from the group consisting of Lys²⁶(N^ε

-(γ

-Glu(N^α

-hexadecanoyl)))-GLP-1(7-37), Arg³⁴-GLP-1(7-37), exendin-4, Lys¹⁷Arg³⁰-GLP-2(1-33), Arg³⁰Lys¹⁷N^ε

(β

-Ala(N^α

-hexadecanoyl)) GLP-2(1-33) and Gly²-GLP-2(1-33).

35. The process according to claim 31, wherein said polypeptide or a precursor thereof is selected from the group consisting of

threonine methyl ester^B30 human insulin,

threonine ethyl ester^B30 human insulin,

Asp^B23 human insulin,

threonine methyl ester^B30 Asp^B23 human insulin,

threonine ethyl ester^B30 Asp^B23 human insulin,

Lys^B23 Pro^B29 human insulin,

Met^B-1Arg^B0Lys^B23 Pro^B29 human proinsulin,

Lys^B3 Glu^B29 human insulin,

Gly^A21 Arg^B31 Arg^B32 human insulin,

des(B30) human insulin,

N^ε

B29-tetradecanoyl des(B30) human insulin,

N<sup>ε

B29</sup>-litocholoyl-γ

-glutamyl des(B30) human insulin,

N<sup>ε

B29</sup>-octanoyl des(B30) human insulin, and.

N<sup>ε

B29</sup>-octanoyl human insulin.

41. An automated chromatographic equipment comprising piping and control system for implementing the process according to claim 1.

42. A pharmaceutical composition prepared by a process comprising the steps of

a) first purifying a polypeptide or a precursor thereof using a chromatographic stationary phase regenerated by the process according to claim 1,

b) then drying said polypeptide, and

c) finally admixing with a pharmaceutically acceptable excipient.

43. A pharmaceutical composition prepared by a process comprising the steps of

a) first purifying a polypeptide or a precursor thereof using a chromatographic stationary phase regenerated by the process according to claim 11,

b) then drying said polypeptide, and

c) finally admixing with a pharmaceutically acceptable excipient.

36. A chromatographic stationary phase which has been regenerated by contacting said chromatographic stationary phase with a regeneration solution comprising at least one organic acid and less than about 75% w/w water.

37. The chromatographic stationary phase according to claim 36, wherein said chromatographic stationary phase has been regenerated by contacting said chromatographic stationary phase with a regeneration solution comprising at least one organic acid and less than about 1% w/w water.

38. The chromatographic stationary phase according to claim 36, wherein said chromatographic stationary phase is a silica or a substituted silica material.

39. A polypeptide product manufactured by a process comprising the steps of

a) purifying a polypeptide or a precursor thereof using the chromatographic stationary phase according to claim 36, and

b) isolating said polypeptide or a precursor thereof to give the resulting polypeptide product.

40. A polypeptide product manufactured by a process wherein is used a chromatographic stationary phase according to claim 36.