High pH protein refolding methods

Patent metadata

Field	Value
Publication number	US-10065987-B2
Application number	US-201414766848-A
Country	US
Kind code	B2
Filing date	Feb 11, 2014
Priority date	Feb 12, 2013
Publication date	Sep 4, 2018
Grant date	Sep 4, 2018

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

The invention claimed is:

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 11.5 to 12.8 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 10 to 10.9 to thereby obtain a composition comprising refolded proteins;
wherein the method does not include the use of a denaturing agent.

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

3. 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.

4. 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.

5. 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.

6. 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.

7. 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.

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

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

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

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

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

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

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

15. The method of claim 14 , 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.

16. The method of claim 15 , wherein the alternative scaffold binding domain is a fibronectin based scaffold domain.

Assignees

• Bristol Myers Squibb Co

Inventors

• Hollander Cristopher
• Blum Benjamin C

Classifications

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C07K1/1136 Primary

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

• C07K1/1133

  by redox-reactions involving cystein/cystin side chains · 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

• C07K2319/70

  containing domain for protein-protein interaction · CPC title