Refolding transforming growth factor beta family proteins

• US 8,722,862 B2
• Filed: 08/18/2005
• Issued: 05/13/2014
• Est. Priority Date: 08/19/2004
• Status: Expired due to Fees

• Current Assignee: Sinfonia Biotherapeutics AB
• Original Assignee: Biogen Idec MA Inc. (Biogen, Inc.)
• Inventors: Rossomando, Anthony, Pepinsky, R. Blake, Gong, BangJian
• Primary Examiner(s): Emch, Gregory S
• Application Number:
  US11/573,771
• Publication Number:
  US 20080249287A1
• Time in Patent Office: 3,190 Days
• Field of Search: None
• US Class Current: 530/427
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/495 Transforming growth factor [TGF]
• 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

Compositions and methods for folding proteins belonging to the transforming growth factor beta superfamily are disclosed. The compositions and methods allow for the folding of such proteins when produced in an expression system that does not yield a properly folded, biologically active product.

Claims

1. A composition comprising an amount of a refolding buffer effective to, when diluted by a factor of 1 to 10, induce folding of a neublastin polypeptide, wherein the refolding buffer comprises the following components at 1 to 10 times the stated concentrations:

(i) potassium phosphate or sodium phosphate at a concentration of 25 mM to 150 mM with a pH ranging from 5.8 to 8.0;

(ii) guanidine-HCl at a concentration of 0.3 M to 2 M;

(iii) L-Arginine at a concentration of 0.25 M to 1 M;

(iv) Tween-80 at a concentration of 0.05% to 1%; and

(v) oxidized glutathione at a concentration of 1 mM to 4 mM and reduced glutathione at a concentration of 0.05 mM to 0.8 mM, wherein the ratio of oxidized to reduced glutathione is from 5;

1 to 20;

1.

2. The composition of claim 1, wherein the refolding buffer comprises L-Arginine at 1 to 10 times a concentration of 0.30 M to 0.5M.

3. The composition of claim 1, wherein the refolding buffer comprises L-Arginine at 1 to 10 times a concentration of at least 0.30 M.

4. The composition of claim 1, wherein the refolding buffer comprises L-Arginine at 1 to 10 times a concentration of at least 0.35 M.

5. The composition of claim 1, wherein the refolding buffer comprises L-Arginine at 1 to 10 times a concentration of 0.35 M.

6. The composition of claim 1, wherein the refolding buffer comprises Tween-80 at 1 to 10 times a concentration of 0.1% to 1%.

7. The composition of claim 1, wherein the refolding buffer comprises Tween-80 at 1 to 10 times a concentration of 0.1% to 0.5%.

8. The composition of claim 1, wherein the refolding buffer comprises Tween-80 at 1 to 10 times a concentration of at least 0.1%.

9. The composition of claim 1, wherein the refolding buffer comprises Tween-80 at 1 to 10 times a concentration of 0.1%.

10. The composition of claim 1, wherein the refolding buffer comprises oxidized and reduced glutathione at a ratio of from 5:

1 to 10;

1.

11. The composition of claim 1, wherein the refolding buffer comprises oxidized and reduced glutathione at a ratio of 5:

1.

12. The composition of claim 1, wherein the refolding buffer comprises oxidized glutathione at 1 to 10 times a concentration of 1 mM to 2 mM.

13. The composition of claim 1, wherein the refolding buffer comprises oxidized glutathione at 1 to 10 times a concentration of 1 mM.

14. The composition of claim 1, wherein the refolding buffer comprises guanidine-HCl at 1 to 10 times a concentration of 0.5 M to 1.0 M.

15. The composition of claim 1, wherein the refolding buffer comprises guanidine-HCl at 1 to 10 times a concentration of at least 0.5 M.

16. The composition of claim 1, wherein the refolding buffer comprises guanidine-HCl at 1 to 10 times a concentration of 0.5 M.

17. The composition of claim 1, wherein the refolding buffer comprises potassium phosphate at 1 to 10 times a concentration of 25 mM to 100 mM.

18. The composition of claim 1, wherein the refolding buffer comprises potassium phosphate at 1 to 10 times a concentration of 25 mM to 75 mM.

19. The composition of claim 1, wherein the refolding buffer comprises potassium phosphate at 1 to 10 times a concentration of at least 50 mM.

20. The composition of claim 1, wherein the refolding buffer comprises potassium phosphate at 1 to 10 times a concentration of 50 mM.

21. The composition of claim 1, wherein the refolding buffer comprises potassium phosphate at a pH of 7.0 to 8.0.

22. The composition of claim 1, wherein the refolding buffer comprises potassium phosphate at a pH of 7.5 to 8.0.

23. The composition of claim 1, wherein the refolding buffer comprises potassium phosphate at a pH of about 7.8.

24. The composition of claim 1, wherein the refolding buffer does not consist of (i) potassium phosphate pH 7.8 at a concentration of 50 mM, (ii) guanidine-HCl at a concentration of 0.5 M, (iii) L-Arginine at a concentration of 0.35 M, (iv) Tween-80 at a concentration of 0.1%, (v) oxidized glutathione at a concentration of 1 mM, and (vi) reduced glutathione at a concentration of 0.2 mM.

25. The composition of claim 1, wherein the refolding buffer comprises the following components at 1 to 10 times the stated concentrations:

(i) potassium phosphate pH 7.8 at a concentration of 50 mM;

(ii) guanidine-HCl at a concentration of 0.5 M;

(iii) L-Arginine at a concentration of 0.35 M;

(iv) Tween-80 at a concentration of 0.1%;

(v) oxidized glutathione at a concentration of 1 mM; and

(vi) reduced glutathione at a concentration of 0.2 mM.

26. The composition of claim 1, wherein the refolding buffer consists of the following components at 1 to 10 times the stated concentrations:

(i) potassium phosphate pH 7.8 at a concentration of 50 mM;

(ii) guanidine-HCl at a concentration of 0.5 M;

(iii) L-Arginine at a concentration of 0.35 M;

(iv) Tween-80 at a concentration of 0.1%;

(v) oxidized glutathione at a concentration of 1 mM; and

(vi) reduced glutathione at a concentration of 0.2 mM.

27. The composition of claim 1, wherein the refolding buffer lacks urea.

28. The composition of claim 1, wherein the refolding buffer lacks glycine.