High ph protein refolding methods

Patent metadata

Field	Value
Publication number	US-2016002287-A1
Application number	US-201414766848-A
Country	US
Kind code	A1
Filing date	Feb 11, 2014
Priority date	Feb 12, 2013
Publication date	Jan 7, 2016
Grant date	—

Abstract

Provided herein are methods for refolding denatured protein (e.g., from inclusion bodies) that do not require the use of a denaturing agent. Exemplary methods use a high pH for solubilizing denatured protein, followed by a decrease in pH for refolding the proteins.

First claim

1 . A method for refolding a denatured protein, comprising
suspending a denatured protein in a suspension solution to obtain a composition comprising suspended denatured proteins; combining the composition comprising suspended denatured proteins with a solubilization buffer having a pH in the range of 10.5 to 13 to thereby obtain a composition comprising solubilized denatured proteins; and combining the composition comprising solubilized denatured proteins with a refold buffer having a pH in the range of 9 to 11 to thereby obtain a composition comprising refolded proteins; wherein the method does not include the use of a significant amount of denaturing agent.

2 . (canceled)

3 . (canceled)

4 . (canceled)

5 . (canceled)

6 . The method of claim 1 , wherein the suspension solution consists of water.

7 . (canceled)

8 . (canceled)

9 . (canceled)

10 . (canceled)

11 . (canceled)

12 . (canceled)

13 . The method of claim 1 , wherein the denatured proteins are suspended in suspension solution at a ratio of weight (g) of denatured proteins:volume (ml) of suspension solution of 1:1-3.

14 . (canceled)

15 . (canceled)

16 . The method of claim 1 , wherein the composition comprising suspended denatured proteins is combined with solubilization buffer at a ratio of weight (g) of denatured proteins: volume (ml) of solubilization buffer of 1:10-30.

17 . (canceled)

18 . The method of claim 1 , wherein the composition comprising solubilized denatured proteins is combined with refold buffer at a ratio of volume of solubilization buffer:volume of refold buffer of 1:1-5.

19 . (canceled)

20 . The method of claim 1 , wherein the denatured proteins are suspended in suspension solution at a ratio of weight (g) of denatured proteins:volume (ml) of suspension solution of 1:1-3;
the composition comprising suspended denatured proteins is combined with solubilization buffer at a ratio of weight (g) of denatured proteins:volume (ml) of solubilization buffer of 1:10-30; and the composition comprising solubilized denatured proteins is combined with refold buffer at a ratio of volume of solubilization buffer:volume of refold buffer of 1:1-5.

21 . The method of claim 20 , wherein the solubilization buffer has a pH in the range of 11.5 to 12.8 and the refold buffer has a pH in the range of 10 to 10.9.

22 . (canceled)

23 . (canceled)

24 . The method of claim 1 , wherein the suspended denatured proteins and the solubilization buffer are combined for 1-10 minutes prior to being combined with the refold buffer.

25 . (canceled)

26 . The method of claim 1 , wherein the composition comprising the solubilized denatured proteins is combined with the refold buffer for 5-60 minutes.

27 . (canceled)

28 . (canceled)

29 . The method of claim 1 , wherein the solubilization buffer comprises Arginine.

30 . The method of claim 1 , wherein the refold buffer comprises Arginine.

31 . The method of claim 1 , wherein the refold buffer comprises an oxidizing agent.

32 . (canceled)

33 . (canceled)

34 . The method of claim 1 , wherein the method does not comprise first suspending the denatured protein in a suspension solution.

35 . The method of claim 1 , wherein the method does not include the use of a denaturing agent.

36 . The method of claim 1 , wherein the denatured proteins are in the form of inclusion bodies (IBs).

37 . (canceled)

38 . (canceled)

39 . The method of claim 1 , wherein the protein comprises an Fc region.

40 . (canceled)

41 . (canceled)

42 . The method of claim 39 , wherein the protein comprises a binding domain that specifically binds to a target protein, and wherein the binding domain is an alternative scaffold binding domain.

43 . The method of claim 42 , wherein the alternative scaffold binding domain is a fibronectin based scaffold domain.

44 . (canceled)

45 . (canceled)

46 . A composition comprising a protein comprising at least two cysteines that form a disulfide bond under appropriate conditions, and water, wherein the composition does not comprise a buffer or a denaturing agent.

47 . (canceled)

48 . A composition comprising a protein comprising at least two cysteines that form a disulfide bond under appropriate conditions, and a solubilization buffer having a pH in the range of pH 10 to 13, wherein the composition does not comprise a denaturing agent.

49 . (canceled)

50 . (canceled)

51 . (canceled)

52 . (canceled)

53 . (canceled)

54 . (canceled)

55 . (canceled)

56 . (canceled)

Assignees

• Bristol Myers Squibb Co

Inventors

• Hollander Cristopher
• Blum Benjamin C

Classifications

• C07K2319/70

  containing domain for protein-protein interaction · CPC title

• C07K1/1133

  by redox-reactions involving cystein/cystin side chains · CPC title

• C07K1/1136 Primary

  by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents · CPC title

• C07K2319/30

  Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto · CPC title

• C07K14/78

  Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG] · CPC title