Tangential flow filtration based protein refolding methods

Patent metadata

Field	Value
Publication number	US-10183967-B2
Application number	US-201414766849-A
Country	US
Kind code	B2
Filing date	Feb 11, 2014
Priority date	Feb 12, 2013
Publication date	Jan 22, 2019
Grant date	Jan 22, 2019

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, e.g., that is present in inclusion bodies (IBs), comprising
(i) providing the denatured protein that is present in IBs, wherein the protein comprises a tenth fibronectin type III ( 10 Fn3) domain and an Fc region;
(ii) suspending denatured protein in a suspension solution, to obtain a composition comprising suspended denatured protein;
(iii) combining the composition comprising suspended 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;
(iv) 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
(v) 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.

2. The method of claim 1 , wherein step (v) comprises diafiltering the second protein composition with 2-6 diavolumes of a refold/oxidizing buffer.

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

4. The method of claim 2 , wherein the diafiltering with the refold/oxidizing buffer takes between 0.5 to 3 hours.

5. The method of claim 1 , wherein the denatured protein is suspended in a suspension solution at a ratio of weight (grams) of denatured protein:volume (ml) of suspension solution of 1:1-10.

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

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

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

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

10. The method of claim 1 , wherein the denatured protein comprises at least one disulfide bond.

11. 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 Cristopher

Classifications

• C07K2319/30

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

• C07K1/34

  by filtration, ultrafiltration or reverse osmosis · 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

• C07K2317/52

  Constant or Fc region; Isotype · CPC title