Tandem folding methods to improve protein folding yield

Patent metadata

Field	Value
Publication number	US-10633414-B2
Application number	US-201615748012-A
Country	US
Kind code	B2
Filing date	Jul 27, 2016
Priority date	Jul 27, 2015
Publication date	Apr 28, 2020
Grant date	Apr 28, 2020

Abstract

Methods of refolding proteins are provided, especially cysteine-containing proteins such as insulin, proinsulin, and analogues thereof. The methods make used of tandem folding via the addition, at two different time, of two different reducing agents. A first reversible reducing agent can be added to induce folding and, at a later time, a second irreversible reducing agent can be added to prevent and/or reverse the formation of aggregates via intermolecular sulfide bonds. The methods can be used to reform a variety of cysteine-containing proteins with high yield of the native form, e.g. about 60 mol %, 70 mol %, or more.

First claim

We claim:

1. A method of refolding a solubilized cysteine-containing protein to its native form, the method comprising
adding a reversible reducing agent to a solution containing the solubilized cysteine-containing protein to initiate folding of the cysteine-containing protein, wherein the reversible reducing agent is added at a ratio of [reversible reducing agent]:[thiols in the cysteine-containing protein] of 3:1 to 4:1; and
adding an irreversible reducing agent to the solution at a time after the initiation of folding to breakdown aggregates of the cysteine-containing protein.

2. The method of claim 1 , wherein the time after the initiation of folding is when the concentration of the dimer in the solution is 5 mol % to 12 mol % based upon the concentration of the cysteine-containing protein.

3. The method of claim 1 , wherein the time after the initiation of folding is when the concentration of the dimer in the solution is 1.5 to 3.0 times the concentration of the dimer in the solution prior to the addition of the reversible reducing agent.

4. The method of claim 1 , wherein the time after the initiation of folding is 5 minutes to 15 minutes.

5. The method of claim 1 , wherein the irreversible reducing agent has a redox potential of −0.3 V to −0.35 V at a pH above 7.0, and wherein the irreversible reducing agent has a redox equilibrium constant of 10 3 to 10 5 .

6. The method of claim 1 , wherein the irreversible reducing agent is added at a ratio of [irreversible reducing agent]:[thiols in the cysteine-containing protein] of 0.5:1 to 0.8:1.

7. The method of claim 1 , wherein the cysteine-containing protein is a recombinant protein selected from the group consisting of an enzyme, an antibody, an antigen, a hormones, and a cytokine.

8. The method of claim 7 , wherein the recombinant protein is an inclusion body isolated from an Escherichia coli expression system.

9. The method of of claim 1 , wherein the cysteine-containing proteins is insulin, an insulin analogue, or a proinsulin.

10. The method of claim 1 , wherein the concentration of the cysteine-containing protein in the solution prior to addition of the reversible reducing agent is 0.3 g/L to 1 g/L.

11. The method of claim 1 , further comprising dissolution of the cysteine-containing protein at an alkaline pH to form the solubilized cysteine-containing protein.

12. The method of claim 11 , where the alkalinepH is 11.5-12.5.

13. The method of claim 11 , where the temperature is 4° C. to 8° C.

14. The method of claim 1 , wherein the pH of the solution after the addition of the reversible reducing agent is 10.75 to 10.85.

15. The method of claim 1 , wherein the reversible reducing agent is cysteine-HCl dissolved to a 200 mM to 900 mM at 4° C. to 8° C. and acidic pH.

Assignees

• Purdue Research Foundation

Inventors

• Wang Nien-Hwa Linda
• Yuan Chongli
• Crawford Morgan H

Classifications

• C07K1/14

  Extraction; Separation; Purification · CPC title

• A61K47/42

  Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein (oligopeptides having up to five amino acids {A61K47/183}; polyamino acids A61K47/34) · CPC title

• C07K1/1133 Primary

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

• C12R1/19

  Chemistry & Metallurgy · mapped topic

• C12R2001/19

  Escherichia coli · CPC title