Process for purifying proteins in a hydrophobic interaction chromatography flow-through fraction

• US 20050136521A1
• Filed: 10/22/2004
• Published: 06/23/2005
• Est. Priority Date: 10/24/2003
• Status: Active Grant

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Ghose, Sanchayita, Shukla, Abhinav A.
• Granted Patent: US 7,427,659 B2
• Time in Patent Office: Days
• US Class Current: 435/183
• CPC Class Codes: C07K 1/20 Partition-, reverse-phase or hydrophobic interaction chromatography
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

The present invention is a process for separating a target protein (such as a recombinant protein produced in a cell culture) from a mixture containing the target protein and contaminants (such as cell culture contaminants), by contacting the mixture with a hydrophobic adsorbent comprising branched hydrocarbon functional groups in an aqueous salt solution and collecting the unbound flow-through fraction containing the target protein. In one embodiment, the hydrophobic adsorbent may be a branched alkyl functional group. In another embodiment, the branched alkyl functional group has from 3 to 8 carbon atoms. In another embodiment, the branched alkyl functional group is a tertiary carbon atom, such as tert-butyl.

Claims

1. A process for separating a target protein from a mixture containing the target protein and contaminants, which comprises:

a) contacting the mixture with a hydrophobic adsorbent comprising branched hydrocarbon functional groups in an aqueous salt solution; and

b) collecting the portion of the mixture that does not bind to the hydrophobic adsorbent, which contains the target protein.

2. The method of claim 1, wherein the hydrophobic adsorbent comprises a branched alkyl functional group.

3. The method of claim 2, wherein the branched alkyl functional group has from 3 to 8 carbon atoms.

4. The method of claim 3, wherein the branched alkyl functional group comprises a tertiary carbon atom.

5. The method of claim 4, wherein the branched alkyl functional group is tert-butyl.

38. A protein purified by the process of any one of claims 1-37.

6. A process for separating a recombinant target protein, produced as a product of cell culture expression in a host cell, from a mixture containing the target protein and contaminants, which comprises:

a) contacting the mixture with a hydrophobic adsorbent comprising branched hydrocarbon functional groups in an aqueous salt solution; and

b) collecting the portion of the mixture that does not bind to the hydrophobic adsorbent, which contains the target protein.

7. The method of claim 6, wherein the hydrophobic adsorbent comprises a branched alkyl functional group.

8. The method of claim 7, wherein the branched alkyl functional group has from 3 to 8 carbon atoms.

9. The method of claim 8, wherein the branched alkyl functional group comprises a tertiary carbon atom.

10. The method of claim 9, wherein the branched alkyl functional group is tert-butyl.

11. A process for separating a recombinant F_c fusion target protein, produced as a product of cell culture expression in a host cell, from a mixture containing the target protein and contaminants, which comprises:

a) contacting the mixture with a hydrophobic adsorbent comprising branched hydrocarbon functional groups in an aqueous salt solution; and

b) collecting the portion of the mixture that does not bind to the hydrophobic adsorbent, which contains the target protein.

12. The method of claim 11, wherein the hydrophobic adsorbent comprises a branched alkyl functional group.

13. The method of claim 12, wherein the branched alkyl functional group has from 3 to 8 carbon atoms.

14. The method of claim 13, wherein the branched alkyl functional group comprises a tertiary carbon atom.

15. The method of claim 14, wherein the branched alkyl functional group is tert-butyl.

16. A process for separating a recombinant target protein, produced as a product of cell culture expression in a host cell, from a mixture containing the target protein and contaminants, which comprises:

a) preparing a chromatography column having a support comprising hydrophobic branched alkyl functional groups, wherein the branched alkyl functional groups have from 4 to 8 carbon atoms, at least one of which is a tertiary carbon atom;

b) preparing the mixture in an aqueous solution having a salt concentration such that the contaminants bind to the column while the target protein in the mixture does not bind to the column;

c) contacting the mixture with the column; and

d) collecting from the column the portion of the mixture that does not bind to the column, which contains the target protein.

17. The method of claim 16, wherein the branched alkyl functional group is tert-butyl.

18. A process for removing Protein A from a mixture containing a target protein and Protein A, which comprises:

a) contacting the mixture with a hydrophobic adsorbent comprising branched hydrocarbon functional groups in an aqueous salt solution; and

b) collecting the portion of the mixture that does not bind to the hydrophobic adsorbent, which contains the target protein.

19. The method of claim 18, wherein the hydrophobic adsorbent comprises a branched alkyl functional group.

20. The method of claim 19, wherein the branched alkyl functional group has from 3 to 8 carbon atoms.

21. The method of claim 20, wherein the branched alkyl functional group comprises a tertiary carbon atom.

22. The method of claim 21, wherein the branched alkyl functional group is tert-butyl.

23. A process for removing a misfolded variant of a recombinant target protein from a mixture containing correctly folded variants and misfolded variants of the target protein, which comprises:

a) contacting the mixture with a hydrophobic adsorbent comprising branched hydrocarbon functional groups in an aqueous salt solution; and

b) collecting the portion of the mixture that does not bind to the hydrophobic adsorbent, which contains the correctly folded variant of the target protein.

24. The method of claim 23, wherein the hydrophobic adsorbent comprises a branched alkyl functional group.

25. The method of claim 24, wherein the branched alkyl functional group has from 3 to 8 carbon atoms.

26. The method of claim 25, wherein the branched alkyl functional group comprises a tertiary carbon atom.

27. The method of claim 26, wherein the branched alkyl functional group is tert-butyl.

28. A process for removing aggregated forms of a recombinant target protein from a mixture containing individual forms and aggregated forms of the target protein, which comprises:

a) contacting the mixture with a hydrophobic adsorbent comprising branched hydrocarbon functional groups in an aqueous salt solution; and

b) collecting the portion of the mixture that does not bind to the hydrophobic adsorbent, which contains the individual form of the target protein.

29. The method of claim 28, wherein the hydrophobic adsorbent comprises a branched alkyl functional group.

30. The method of claim 29, wherein the branched alkyl functional group has from 3 to 8 carbon atoms.

31. The method of claim 30, wherein the branched alkyl functional group comprises a tertiary carbon atom.

32. The method of claim 31, wherein the branched alkyl functional group is tert-butyl.

34. The method of claim 28, wherein the hydrophobic adsorbent comprises a branched alkyl functional group.

35. The method of claim 29, wherein the branched alkyl functional group has from 3 to 8 carbon atoms.

36. The method of claim 30, wherein the branched alkyl functional group comprises a tertiary carbon atom.

33. A process for separating a recombinant target protein from a mixture containing the target protein and cell culture contaminants produced by cell culture expression of the recombinant protein in a Chinese Hamster Ovary host cell, which comprises:

a) contacting the mixture with a hydrophobic adsorbent comprising branched hydrocarbon functional groups in an aqueous salt solution; and

b) collecting the portion of the mixture that does not bind to the hydrophobic adsorbent, which contains the target protein.

37. The method of claim 37, wherein the branched alkyl functional group is tert-butyl.