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Last Updated: March 29, 2024

Claims for Patent: 7,592,432


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Summary for Patent: 7,592,432
Title:Polypeptide cleavage process
Abstract: The invention provides a palladium-promoted hydrolytic polypeptide cleavage process which selectively cleaves the polypeptide at a Cys-His cleavage site comprising solubilizing the polypeptide in a reaction mixture comprised of a palladium promoter dissolved in a high-concentration formic acid, wherein the concentration of the formic acid in the reaction mixture is between about 1 to about 22 molar.
Inventor(s): Holmquist; Barton (Eagle, NE), Strydom; Daniel (Lincoln, NE), Seo; Jin Seog (Brampton, CA), Cryer; Kirk (Madison, WI)
Assignee: Restoragen, Inc. (Lincoln, NE)
Application Number:10/997,822
Patent Claims:1. A palladium-promoted hydrolytic polypeptide cleavage process which selectively cleaves the polypeptide at a Cys-His cleavage site comprising solubilizing the polypeptide in a reaction mixture comprised of a palladium promotor dissolved in a formic acid solvent having a concentration of 50% to 80%, wherein the concentration of the formic acid in the reaction mixture is between about 13 to about 22 molar, wherein the cleavage occurs at the carboxamide bond between the histidine residue and the residue immediately adjacent to the histidine on the C-terminal side.

2. The process of claim 1, wherein the polypeptide is a recombinantly expressed chimeric protein in the form of an inclusion body and the molar ratio of palladium promotor to inclusion body in the reaction mixture is from about 2:1 to about 20:1.

3. The process of claim 2, wherein the molar ratio of palladium promotor to inclusion body in the reaction mixture is from about 2:1 to about 20:1 and the concentration of formic acid is 50 to 80%.

4. The process of claims 1, 2 or 3, wherein the reaction mixture temperature is maintained at about 50.degree. C. to about 70.degree. C.

5. The process of claims 1, 2 or 3, wherein the cleavage reaction proceeds for 0.5 to 6 hours.

6. The process of claim 1, wherein the cleavage site includes a linker, which is N-terminally adjacent to Cys.

7. The process of claim 6, wherein the linker is selected from the group consisting of acidic residues, hydrophobic residues and basic residues inserted in front of a Cys-His sequence.

8. The process of claims 1 or 2, wherein the palladium promoter is a Palladium(II) complex selected from the group consisting of Na.sub.2PdCl.sub.4; cis-[Pd(en)Cl.sub.2]; cis-[Pd(bp)Cl.sub.2]; cis-[Pd(phen)Cl.sub.2]; cis-[Pd(pn)Cl.sub.2]; cis-[Pd(pic)Cl.sub.2]; cis-[Pd(dtco-OH)Cl.sub.2; [Pd(en)(OH.sub.2).sub.2].sup.2+, cis-[Pd(pn)(OH.sub.2).sub.2].sup.2+, cis-[Pd(Pic)(OH.sub.2).sub.2].sup.2+-, cis-[Pd(bp)(OH.sub.2).sub.2].sup.2+; cis-[Pd(phen)(OH.sub.2).sub.2].sup.2+, cis-[Pd(dtco-OH)(OH.sub.2).sub.2].sup.2+; and [Pd(OH.sub.2).sub.3(OH)](NO.sub.3).

9. The process of claim 1, wherein the chimeric protein was expressed recombinantly in host cells and was recovered from the host cells in the form of an inclusion body.

10. The process of claim 9, wherein the peptide is hGRF, the host cells are E. coli and the chimeric protein is T7tag-Vg-D.sub.4-CH-GRF (1-44)-Xaa (SEQ ID NO:9), where Xaa is any amino acid.

11. The process of claim 9, wherein the peptide is rPTH, the host cells are E. coli and the chimeric protein is T7tag-Vg-G.sub.5PR-CH-PTH(1-34) (SEQ ID NO:4).

12. The process of claims 1 or 2, wherein the formic acid is combined with an inorganic acid selected from the group consisting of HCl, H.sub.3PO.sub.4, H.sub.2SO.sub.4, or HClO.sub.4.

13. The process of claim 9, wherein the palladium promotor is tetrachloropalladate.

14. The process of claims 1, 2, 9 or 13, wherein the palladium promotor is tetrachloropalladate and the molar ratio of tetrachloropalladate to chimeric protein is around 10:1.

15. The process of claim 14, wherein the chimeric protein is T7tag-Vg-D.sub.4KCH-GRF(1-44)-CH (SEQ ID NO:6).

16. The process of claim 1, wherein the polypeptide is GLP-1, GLP-2, GRF, PTH, parathyroid hormone related hormone, ACTH, enkephalins, endorphins, exendins, amylins, various opioid peptides, gaegurin 5, gaegurin 6, brevinin 1, a ranatuerin selected from the group consisting of ranatuerin 1 through 9, an esculetin, GIP, glucagon, motilin, a thymopoietin, a thymosin, ubiquitin, serum thymic factor, thymic humoral factor, neurotensin or tuftsin.

17. The process of claim 1, wherein the polypeptide is gastrin, calcitonin, luteinizing-hormone-releasing hormone, pancreatic polypeptide, endothelin, corticotropin releasing factor, neuropeptide Y, atrial naturetic peptide, amylin, galanin, somatostatins, vasoactive intestinal peptide or insulin.

18. The process of claims 16 or 17, wherein the polypeptide is made synthetically.

19. The process of claims 16 or 17, wherein the chimeric protein was expressed recombinantly in host cells and was recovered from the host cells in the form of an inclusion body.

20. The process of claim 19, wherein the host cells are E. coli.

21. The process of claims 1 or 9, wherein the cleavage reaction temperature is approximately 60.degree. C.

22. The process of claim 1, wherein the Cys-His cleavage site is present in the polypeptide between a leader sequence and a desired polypeptide sequence.

23. The process of claim 1, wherein the polypeptide comprises a chimeric protein having a molecular weight of between about 400 daltons and about 100,000 daltons.

24. The process of claim 23, wherein the polypeptide comprises repeating units of the chimeric protein.

25. A process for producing a cleaved, deformylated peptide comprising: (a) cleaving a chimeric protein comprised of a leader sequence joined by a Cys-His cleavage site to the N-terminus of the peptide by solubilizing the chimeric protein in a reaction mixture comprised of a palladium promotor dissolved in formic acid solvent having a concentration of 50 to 80%, wherein the cleavage occurs at the carboxamide bond between the histidine residue and the residue immediately adjacent to the histidine on the C-terminal side; and (b) deformylating the peptide cleaved in step (a) by removing the formic acid Solvent from the reaction product of step (a) and reacting the resultant reaction product containing cleaved peptide with a mixture of 0 to 2 M glycine and 0 to 100 mM hydroxylamine at a pH of 6 to 9 or by incubating the formylated peptide for 4 to 16 hours in a dilute acid.

26. The process of claim 25, wherein the chimeric protein was expressed recombinantly in host cells and was recovered from the host cells in the form of an inclusion body.

27. The process of claim 22, wherein the reaction mixture temperature is maintained at about 50.degree. C. to about 70.degree. C.

28. The process of claim 27, wherein the cleavage reaction proceeds for 0.5 to 6 hours.

29. The process of claim 27, wherein the cleavage site includes a linker, which is adjacent to Cys.

30. The process of claim 27, wherein the linker is selected from the group consisting of acidic residues, hydrophobic residues and basic residues may be inserted in front of a Cys-His sequence.

31. The process of claims 27, wherein the formic acid is combined with one or more palladium complexes with ligands selected from the group consisting of ethylenediamine; propylenediamine; 2-aminomethyl pyridine; 2(2-aminoethyl) pyridine; 2(2-methylaminoethyl)pyridine; 2,2'-bypyridyl; 1,10-phenanthroline: 3-hydroxy-2(dimethylaminomethyl)pyridine; 3-hydroxy-1, 5-dithiacyclooctane; 3,6-dithiaoctane-1,8-diol; 1,2-diphenylphosphineethane; triphenylphosphine; diphenylphosphineferrocene; and diethylenetriamine.

32. The process of claim 27, wherein the palladium promoter is a Palladium(II) complex selected from the group consisting of Na.sub.2PdCl.sub.4; cis-[Pd(en)Cl.sub.2]; cis-[Pd(bp)Cl.sub.2]; cis-[Pd(phen)Cl.sub.2]; cis-[Pd(pn)Cl.sub.2]; cis-[Pd(pic)Cl.sub.2]; cis-[Pd(dtco-OH)Cl.sub.2]; cis-[Pd(en)(OH.sub.2).sub.2].sup.2+, cis-[Pd(Pn)(OH.sub.2).sub.2].sup.2+, cis-[Pd(pic)(OH.sub.2).sub.2].sup.2+, cis-[Pd(bp)(OH.sub.2).sub.2].sup.2+; cis-[Pd(phen)(OH.sub.2).sub.2].sup.2+; cis-[Pd(dtco-OH)(OH.sub.2).sub.2].sup.2+, And [Pd(OH.sub.2).sub.3(OH)](NO.sub.3) or a palladium (IV) complex including Na.sub.2PdCl..sub.6.

33. The process of claim 26, wherein the molar ratio of palladium promotor to inclusion body in the reaction mixture is from about 2:1 to about 20:1.

34. The process of claim 26, wherein the peptide is rGRF, the host cells are E. coli and the chimeric protein is T7tag-Vg-D.sub.4-CH-GRF(1-44)-CH (SEQ ID NO:6).

35. The process of claim 27, wherein the formic acid is combined with an inorganic acid selected from the group consisting of HCl, H.sub.3PO.sub.4, H.sub.2SO.sub.4, HClO.sub.4, or HClO.sub.4.

36. The process of claim 27, wherein the palladium promotor is tetrachloropalladate.

37. The process of claims 27 or 35, wherein the palladium promotor is tetrachloropalladate and the molar ratio of tetrachloropalladate to chimeric protein is around 10:1.

38. The process of claim 37, wherein the chimeric protein is T7tag-Vg-D.sub.44K-CH-GRF(1-44)-CH (SEQ ID NO:6).

39. The process of claim 27, wherein the peptide is GLP-1, GLP-2, GRF, PTH, parathyroid hormone releasing hormone, ACTH, enkephalins, endorphins, exendins, amylins, various opioid peptides, gaegurin 5, gaegurin 6, brevinin 1, a ranatuerin selected from the group consisting of ranatuerin 1 through 9, an esculetin, GIP, glucagon, motilin, a thymopoietin, a thymosin, ubiquitin, serum thymic factor, thymric humoral factor, neurotensin or tuftsin.

40. The process of claim 27, wherein the peptide is gastrin, calcitonin, luteinizing-hormone-releasing hormone, pancreatic polypeptide, endothelin, corticotropin releasing factor, neuropeptide Y, atrial naturetic peptide, amylin, galanin, somatostatins, vasoactive intestinal peptide or insulin.

41. The process of claims 39 or 40, wherein the chimeric protein is made synthetically.

42. The process of claims 39 or 40, wherein the chimeric protein was expressed recombinantly in host cells and was recovered from the host cells in the form of an inclusion body.

43. The process of claim 42, wherein the host cells are E. coli.

44. The process of claims 27 or 43, wherein the cleavage reaction temperature is approximately 60.degree. C.

45. The process of claims 27 or 43, wherein the cleaved, deformylated peptide produced in step (b) is recovered by ultrafiltration, filtration or ion-exchange chromatography or reverse phase chromatography or any means of chromatography.

46. The process of claim 1 wherein the Cys of the Cys-His cleavage site is in the oxidized disulfide state.

47. A process for producing a cleaved, deformylated peptide comprising: (a) cleaving a chimeric protein comprised of a leader sequence joined by a Cys-His cleavage site to the N-terminus of the peptide by solubilizing the chimeric protein in a reaction mixture comprised of a palladium promotor dissolved in formic acid solvent having a concentration of 50 to 80%; and (b) deformylating the peptide cleaved in step (a) by removing the formic acid solvent from the reaction product of step (a) and reacting the resultant reaction product containing cleaved peptide with a mixture of 0 to 2 M glycine and 0 to 100 mM hydroxylamine at a pH of 6 to 9 or by incubating the formylated peptide for 4 to 16 hours in a dilute acid.

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