Tangential flow filtration based protein refolding methods

Patent metadata

Field	Value
Publication number	US-11053278-B2
Application number	US-201816211094-A
Country	US
Kind code	B2
Filing date	Dec 5, 2018
Priority date	Feb 12, 2013
Publication date	Jul 6, 2021
Grant date	Jul 6, 2021

Abstract

Provided herein are methods for refolding proteins that are denatured. Exemplary methods comprise solubilizing the denatured protein with a denaturing agent, e.g., a chaotropic agent, and renaturing the protein using a buffer exchanging system, e.g., tangential flow filtration (TFF).

First claim

The invention claimed is:

1. A method for refolding a denatured protein, comprising
(i) combining the denatured protein with a solubilization buffer that comprises a denaturing agent and has a pH in the range of 7 to 10, wherein the denaturing agent is guanidine or guanidinium, to obtain a first protein composition comprising solubilized denatured protein, wherein the denatured protein comprises a tenth fibronectin type III ( 10 Fn3) domain;
(ii) diafiltering the first protein composition comprising solubilized denatured protein with 2-4 diavolumes of a refold buffer that comprises arginine and has a pH in the range of 9 to 11 to obtain a second protein composition comprising partially refolded protein; and
(iii) incubating the second protein composition comprising partially refolded protein with a refold/oxidizing buffer that comprises arginine and glutathione and has a pH in the range of 9 to 11 to obtain a third protein composition comprising the protein in a refolded state,
wherein the protein in a refolded state comprises at least one disulfide bond.

2. The method of claim 1 , wherein incubating the second protein composition comprising partially refolded protein with a refold/oxidizing buffer comprises diafiltering the second protein composition with 2-6 diavolumes of a refold/oxidizing buffer.

3. The method of claim 1 , wherein diafiltering with the refold buffer takes between 0.5 to 3 hours.

4. The method of claim 1 , wherein the diafiltering with the refold/oxidizing buffer takes between 0.5 to 3 hours.

5. The method of claim 1 , wherein the first composition comprising solubilized denatured protein is not diluted prior to diafiltering.

6. The method of claim 1 , wherein the first composition comprising solubilized denatured protein is filtered prior to diafiltering.

7. The method of claim 1 , wherein the third protein composition comprises 1-10 mg/ml of protein.

8. The method of claim 1 , wherein the protein in a refolded state has an efficiency of recovery of at least about 70%.

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

10. The method of claim 1 , wherein the solubilization buffer, refold buffer, and/or refold/oxidizing buffer comprises Tris.

Assignees

• Bristol Myers Squibb Co

Inventors

• Blum Benjamin C
• Hollander Christopher

Classifications

• C07K2319/30

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

• C07K1/1136 Primary

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

• C07K14/78

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

• C07K2317/14

  Specific host cells or culture conditions, e.g. components, pH or temperature · CPC title

• C07K1/34

  by filtration, ultrafiltration or reverse osmosis · CPC title