Refolding proteins using a chemically controlled redox state

• US 9,856,287 B2
• Filed: 02/01/2017
• Issued: 01/02/2018
• Est. Priority Date: 06/22/2009
• Status: Active Grant

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Shultz, Joseph Edward, Hart, Roger, Keener, III, Ronald Nixon
• Primary Examiner(s): Kim, Yunsoo
• Application Number:
  US15/422,327
• Publication Number:
  US 20170145049A1
• Time in Patent Office: 335 Days
• Field of Search: None
• CPC Class Codes: C07K 1/1133 by redox-reactions involving cystein/cystin side chains
• CPC Class Codes: C07K 1/1136 by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
• CPC Class Codes: C07K 1/14 Extraction; Separation; Purification
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

A method of refolding proteins expressed in non-mammalian cells present in concentrations of 2.0 g/L or higher is disclosed. The method comprises identifying the thiol pair ratio and the redox buffer strength to achieve conditions under which efficient folding at concentrations of 2.0 g/L or higher is achieved and can be employed over a range of volumes, including commercial scale.

Claims

1. A method of refolding proteins expressed in a non-mammalian expression system, the method comprising:

contacting the proteins with a preparation that supports the renaturation of at least one of the proteins to a biologically active form, to form a refold mixture, the preparation comprising;

at least one ingredient selected from the group consisting of a denaturant, an aggregation suppressor and a protein stabilizer;

an amount of oxidant; and

an amount of reductant,

wherein the amounts of the oxidant and the reductant are related through a thiol-pair ratio and a thiol-pair buffer strength,

wherein the thiol-pair ratio is in the range of 0.001-100; and

wherein the thiol-pair buffer strength maintains the solubility of the preparation; and

incubating the refold mixture so that at least about 25% of the proteins are properly refolded.

2. The method of claim 1, wherein the refold mixture has a protein concentration in a range of 1-40 g/L.

3. The method of claim 1, wherein the refold mixture has a protein concentration of 2.0 g/L or greater.

4. The method of claim 1, wherein the thiol-pair buffer strength is 2 mM or greater.

5. The method of claim 1, wherein the thiol-pair buffer strength is increased proportionally to an increase in a total protein concentration in the refold mixture.

6. The method of claim 1, wherein the thiol-pair buffer strength is decreased proportionally to a decrease in a total protein concentration in the refold mixture.

7. The method of claim 1, wherein the at least one of the proteins is a complex protein.

8. The method of claim 1, wherein the thiol-pair ratio is calculated according to the following equation:

9. The method of claim 1, wherein the thiol-pair buffer strength is calculated according to the following equation:

2[the oxidant]+[the reductant].

14. The method of claim 1, wherein:

the thiol-pair ratio is calculated according to the following equation;

10. A method of refolding proteins expressed in a non-mammalian expression system, the method comprising:

contacting the proteins with a preparation that supports the renaturation of at least one of the proteins to a biologically active form, to form a refold mixture, the preparation comprising;

at least one ingredient selected from the group consisting of a denaturant, an aggregation suppressor and a protein stabilizer;

an amount of oxidant; and

an amount of reductant,

wherein the amounts of the oxidant and the reductant are related through a thiol-pair ratio and a thiol-pair buffer strength,

wherein the thiol-pair ratio is in the range of 0.001-100; and

wherein the thiol-pair buffer strength maintains the solubility of the preparation; and

incubating the refold mixture so that about 30-80% of the proteins are properly refolded.

11. The method of claim 10, wherein the refold mixture has a protein concentration in a range of 1-40 g/L.

12. The method of claim 10, wherein the thiol-pair buffer strength is 2 mM or greater.

13. The method of claim 10, wherein the refold mixture has a protein concentration of 2.0 g/L or greater.

15. The method of claim 10, wherein:

the thiol-pair ratio is calculated according to the following equation;

16. A method of refolding proteins expressed in a non-mammalian expression system, the method comprising:

preparing a solution comprising;

the proteins;

at least one ingredient selected from the group consisting of a denaturant, an aggregation suppressor and a protein stabilizer;

an amount of oxidant; and

an amount of reductant,

wherein the amounts of the oxidant and the reductant are related through a thiol-pair ratio and a thiol-pair buffer strength,

wherein the thiol-pair ratio is in the range of 0.001-100, and

wherein the thiol-pair buffer strength maintains the solubility of the solution; and

incubating the solution so that at least about 25% of the proteins are properly refolded.

17. The method of claim 16, wherein the solution has a protein concentration in a range of 1-40 g/L.

18. The method of claim 16, wherein the solution has a protein concentration of 2.0 g/L or greater.

19. The method of claim 16, wherein the thiol-pair buffer strength is 2 mM or greater.

20. The method of claim 16, wherein the thiol-pair buffer strength is increased proportionally to an increase in a total protein concentration in the solution.

21. The method of claim 16, wherein the thiol-pair buffer strength is decreased proportionally to a decrease in a total protein concentration in the solution.

22. The method of claim 16, wherein the at least one of the proteins is a complex protein.

23. The method of claim 16, wherein the thiol-pair ratio is calculated according to the following equation:

24. The method of claim 16, wherein the thiol-pair buffer strength is calculated according to the following equation:

2[the oxidant]+[the reductant].

25. The method of claim 16, wherein:

the thiol-pair ratio is calculated according to the following equation;

26. A method of refolding proteins expressed in a non-mammalian expression system, the method comprising:

preparing a solution comprising;

the proteins;

at least one ingredient selected from the group consisting of a denaturant, an aggregation suppressor and a protein stabilizer;

an amount of oxidant; and

an amount of reductant,

wherein the amounts of the oxidant and the reductant are related through a thiol-pair ratio and a thiol-pair buffer strength,

wherein the thiol-pair ratio is in the range of 0.001-100, and

wherein the thiol-pair buffer strength maintains the solubility of the solution; and

incubating the solution so that about 30-80% of the proteins are properly refolded.

27. The method of claim 26, wherein the solution has a protein concentration in a range of 1-40 g/L.

28. The method of claim 26, wherein the solution has a protein concentration of 2.0 g/L or greater.

29. The method of claim 26, wherein the thiol-pair buffer strength is 2 mM or greater.

30. The method of claim 26, wherein:

the thiol-pair ratio is calculated according to the following equation;