Capture purification processes for proteins expressed in a non-mammalian system

Patent metadata

Field	Value
Publication number	US-10577392-B2
Application number	US-201715476691-A
Country	US
Kind code	B2
Filing date	Mar 31, 2017
Priority date	Jun 25, 2009
Publication date	Mar 3, 2020
Grant date	Mar 3, 2020

Abstract

Methods of purifying proteins expressed in non-mammalian expression systems in a non-native soluble form directly from cell lysate are disclosed. Methods of purifying proteins expressed in non-mammalian expression systems in a non-native limited solubility form directly from a refold solution are also disclosed. Resin regeneration methods are also provided.

First claim

What is claimed is:

1. A method of purifying a protein expressed in a non-native soluble form in a non-mammalian expression system comprising:
(a) expressing a protein in a non-native soluble form in E. coli;
(b) lysing the E. coli to generate a cell lysate;
(c) contacting the cell lysate with a separation matrix under conditions suitable for the protein to associate with the separation matrix;
(d) washing the separation matrix; and
(e) eluting the protein from the separation matrix.

2. The method of claim 1 , wherein the protein is a complex protein.

3. The method of claim 2 , wherein the complex protein is selected from the group consisting of: a multimeric protein, an antibody, and an Fc fusion protein.

4. The method of claim 1 , wherein the separation matrix is an affinity resin selected from the group consisting of: Protein A, Protein G, and a synthetic mimetic affinity resin.

5. The method of claim 1 , wherein the separation matrix is a non-affinity resin selected from the group consisting of: ion exchange, mixed mode, and a hydrophobic interaction resin.

6. The method of claim 1 , wherein the cell lysate is filtered before it is contacted with the separation matrix.

7. The method of claim 1 , further comprising:
(f) refolding the protein to its native form.

8. A method of purifying a protein expressed in a non-native limited solubility form in a non-mammalian expression system comprising:
(a) expressing a protein in a non-native limited solubility form in a non-mammalian cell;
(b) lysing the non-mammalian cell;
(c) solubilizing the expressed protein in a solubilization solution comprising one or more of the following:
(i) a denaturant;
(ii) a reductant; and
(iii) a surfactant;

(d) forming a refold solution comprising the solubilized protein and a refold buffer, the refold buffer comprising one or more of the following:
(i) a denaturant;
(ii) an aggregation suppressor;
(iii) a protein stabilizer; and
(iv) a redox component;

(e) applying the refold solution to a separation matrix under conditions suitable for the protein to associate with the separation matrix;
(f) washing the separation matrix; and
(g) eluting the protein from the separation matrix to yield a purified protein.

9. A method of purifying a protein expressed in a non-native soluble form in non-mammalian expression system comprising:
(a) expressing a protein in a non-native soluble form in a non-mammalian cell;
(b) lysing the non-mammalian cell to generate a cell lysate;
(c) contacting the cell lysate with a separation matrix under conditions suitable for the protein to associate with the separation matrix without diluting the protein prior to the contacting;
(d) washing the separation matrix; and
(e) eluting the protein from the separation matrix.

10. A method of purifying a protein expressed in a non-native limited solubility form in E. coli comprising:
(a) expressing a protein in a non-native limited solubility form in E. coli;
(b) lysing the E. coli;
(c) solubilizing the expressed protein in a solubilization solution comprising one or more of the following:
(i) a denaturant;
(ii) a reductant; and
(iii) a surfactant;

(d) forming a refold solution comprising the solubilized protein and a refold buffer, the refold buffer comprising one or more of the following:
(i) a denaturant;
(ii) an aggregation suppressor;
(iii) a protein stabilizer; and
(iv) a redox component;

(e) applying the refold solution to a separation matrix under conditions suitable for the protein to associate with the separation matrix and obtaining a purified protein.

11. The method of claim 8 or 10 , wherein the non-native limited solubility form is a component of an inclusion body.

12. The method of claim 8 or 10 , wherein the protein is a complex protein.

13. The method of claim 12 , wherein the complex protein is selected from the group consisting of: a multimeric protein, an antibody, a peptibody, and an Fc fusion protein.

14. The method of claim 8 or 10 , wherein the non-native limited solubility form is a component of an inclusion body.

15. The method of claim 8 or 10 , wherein the denaturant in the solubilization solution comprises one or more of: urea, guanidinium salts, dimethyl urea, methylurea, and ethylurea.

16. The method of claim 8 or 10 , wherein the reductant comprises one or more of: cysteine, dithiothreitol (DTT), beta-mercaptoethanol, and glutathione.

17. The method of claim 8 or 10 , wherein the surfactant comprises one or more of: sarcosyl and sodium dodecylsulfate.

18. The method of claim 8 or 10 , wherein the aggregation suppressor is selected from the group consisting of: arginine, proline, polyethylene glycols, nonionic surfactants, ionic surfactants, polyhydric alcohols, glycerol, sucrose, sorbitol, glucose, Tris, sodium sulfate, potassium sulfate, and osmolytes.

19. The method of claim 8 or 10 , wherein the protein stabilizer comprises one or more of: arginine, proline, polyethylene glycols, non-ionic surfactants, ionic surfactants, polyhydric alcohols, glycerol, sucrose, sorbitol, glucose, tris, sodium sulfate, potassium sulfate, and osmolytes.

20. The method of claim 8 or 10 , wherein the redox component comprises one or more of: glutathione-reduced, glutathione-oxidized, cysteine, cystine, cysteamine, cystamine, and beta-mercaptoethanol.

21. The method of claim 8 or 10 , wherein the separation matrix is:
an affinity resin selected from the group consisting of: Protein A, Protein G, and synthetic mimetic affinity resin.

22. The method of claim 8 or 10 , wherein the separation matrix is: a non-affinity resin selected from the group consisting of: ion exchange, mixed mode, and a hydrophobic interaction resin.

23. The method of claim 8 or 10 , wherein the refold solution is directly applied to the separation matrix.

Assignees

• Amgen Inc

Inventors

• Shultz Joseph Edward
• Hart Roger

Classifications

• C07K1/22 Primary

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• C07K1/32

  as complexes · CPC title

• C07K1/145

  by extraction or solubilisation · CPC title

• C07K1/18

  Ion-exchange chromatography · CPC title

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title