Methods for refolding G-CSF from inclusion bodies

• US 9,458,207 B2
• Filed: 03/18/2013
• Issued: 10/04/2016
• Est. Priority Date: 03/19/2012
• Status: Active Grant

• Current Assignee: Richter Gedeon Rt.
• Original Assignee: Richter Gedeon Rt.
• Inventors: Felfldi, Ferenc, Ballagi, Andras, Bcsi, Jnos
• Primary Examiner(s): STOICA, ELLY GERALD
• Application Number:
  US14/386,128
• Publication Number:
  US 20150057439A1
• Time in Patent Office: 1,296 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C07K 14/535 Granulocyte CSF; Granulocyte-macrophage CSF
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

New methods for the refolding of recombinant granulocyte colony stimulating factor (G-CSF) from inclusion bodies are disclosed. The methods comprise two refolding steps. In particular, the methods comprise the solubilizing of G-CSF with a solubilizing agent, the oxidative refolding (first refolding step) of G-CSF in the presence of the solubilizing agent and an oxidizing agent, the efficient removal of the solubilizing agent, and a second refolding step to complete the folding of G-CSF in the absence of the solubilizing agent. Various methods are described for the efficient removal of the solubilizing agent from partially refolded G-CSF.

Claims

1. A method for refolding granulocyte colony stimulating factor (G-CSF) from inclusion bodies, comprising:

a) solubilising G-CSF in the presence of a solubilising agent selected from the group consisting of a detergent, a tenside and a surfactant;

b) performing an oxidation and first refolding step, comprising incubating the solubilised G-CSF in the presence of an oxidizing agent and the solubilising agent;

c) removing the solubilising agent by ion exchange resin adsorption and/or ion exchange chromatography, and optionally performing an acid precipitation; and

d) performing a second refolding step at a pH value above pH 7, comprising diluting the G-CSF of step (c) with a low conductivity buffer or water and incubating the diluted G-CSF at a conductivity below 2 mS/cm for more than 12 hours, wherein the solubilizing agent after step c) is below 0.01 mg/ml.

2. The method according to claim 1, wherein the inclusion bodies are from a microorganism, preferably from E. coli.

3. The method according to claim 1, wherein the G-CSF is recombinant bovine or human methionyl-G-CSF.

4. The method according to claim 1, wherein the solubilising agent is N-Lauroylsarcosine.

5. The method according to claim 1, wherein the oxidizing agent is CuSO₄.

6. The method according to claim 1, wherein the solubilisation of G-CSF is performed at a pH value greater than pH 7.

7. The method according to claim 1, wherein the solubilising agent is N-Lauroylsarcosine at a concentration of 0.5% to 1.5%.

8. The method according to claim 1, wherein the oxidation and first refolding step is performed for at least two hours.

9. The method according to claim 1, wherein the oxidation and first refolding step is performed under airflow and without cooling.

10. The method according to claim 1, wherein the oxidation and first refolding step is performed at a pH value of 7-9 and/or at a temperature of 20-28°

C. and/or for 15-25 hours.

11. The method according to claim 1, wherein the removal of the solubilising agent in step (c) comprises:

anion exchange chromatography (AEX) and cation exchange chromatography (CEX), optionally in this order.

12. The method according to claim 1 wherein the removal of the solubilising agent in step (c) comprises:

a) binding to an anion exchange resin material by mixing the G-CSF solution with the suspended resin material and removal of the resin material by filtration, and/or

b) ion exchange chromatography under conditions where the solubilising agent binds to the resin and G-CSF remains in the flow through and/or,

c) ion exchange chromatography under conditions where G-CSF binds to the resin and the solubilising agent remains in the flow through.

13. The method according to claim 1 wherein the solubilising agent and other impurities are removed by the sequential application of the following steps:

(i) anion exchange chromatography (AEX),

(ii) acid precipitation,

(iii) anion exchange chromatography (AEX), and

(iv) cation exchange chromatography (CEX).

14. The method according to claim 1 wherein the solubilising agent and other impurities are removed by the sequential application of the following steps:

a) binding of the solubilising agent to an anion exchange resin material by mixing the G-CSF solution with the suspended resin material and removal of the resin material by filtration;

b) precipitation of impurities by lowering the pH below pH 5 and by removal of the precipitate by filtration;

c) anion exchange chromatography conducted under conditions wherein the residual solubilising agent binds to the resin and G-CSF remains in the flow through;

d) cation exchange chromatography conducted under conditions wherein G-CSF binds to the resin and the residual solubilising agent remains in the flow through; and

e) elution of bound G-CSF from the cation exchange resin by step or gradient elution using an elution buffer with increased pH or salt concentration.

15. The method according to claim 1, wherein the second refolding step is performed under cooled conditions.

16. The method according to claim 1, wherein the second refolding step is performed at a temperature of 2-8°

C. and/or for at least 24 hours.

17. The method according to claim 1, wherein the method further comprises a polishing step, which comprises one or more ion exchange chromatographies.

18. The method according to claim 17, wherein the one or more ion exchange chromatographies in the polishing step comprise(s) an anion exchange chromatography followed by a cation exchange chromatography.

19. The method of claim 17, wherein the one or more ion exchange chromatographies comprise(s) the following steps:

a) anion exchange chromatography conducted under conditions where G-CSF binds to the resin;

b) elution of bound G-CSF by step or gradient elution using an elution buffer with decreased pH or increased salt concentration;

c) cation exchange chromatography conducted under conditions where G-CSF binds to the resin;

d) elution of bound G-CSF by step or gradient elution using an elution buffer with increased pH or salt concentration;

characterised in that the backbone polymers of the anion and cation exchange resins both comprise methacrylate derivatives.