Claims for Patent: 8,507,221

Summary for Patent: 8,507,221

Title:	Process for the expression of peptides of interest using GCSF as a fusion partner
Abstract:	The present invention discloses an improved process for the production of desired recombinant peptides from bacterial cells by using G-CSF as a novel fusion partner for their high level expression in these cells. The invention further provides an expression system comprising the fusion protein wherein the G-CSF is operatively linked to the peptide of interest via an enzymatic or chemical cleavage site which can be used to separate the fusion partner from the said peptide.
Inventor(s):	Mendiretta; Sanjeev Kumar (Gujarat, IN), Patel; Pankaj R. (Gujarat, IN), Bandyopadhyay; Sanjay (Gujarat, IN), Saraswat; Vibhor (Gujarat, IN), Singh; Arun Kumar (Gujarat, IN)
Assignee:	Cadila Healthcare Limited (Ahmedabad, IN)
Application Number:	12/679,346
Patent Claims:	1. A process for producing a peptide using Granulocyte Colony Stimulating Factor (GCSF) as a fusion partner wherein the GCSF is fused with a peptide of interest, comprising the steps of: a) constructing a GCSF expression vector; b) inserting the gene encoding the peptide of interest into the GCSF expression vector; c) transforming a host cell with the GCSF expression vector which causes the host cell to express the peptide of interest; and d) purifying the peptide of interest. 2. The process as claimed in claim 1, wherein GCSF forms a fusion product construct with the peptide. 3. The process as claimed in claim 2, wherein the fusion product construct is represented by the formula: Fusion partner-CS-Fusion peptide wherein the fusion peptide is the peptide of interest, CS is a suitable cleavage site, and the fusion partner is GCSF. 4. The process as claimed in claim 3, wherein the fusion peptide is a physiologically active peptide. 5. The process as claimed in claim 3, wherein the CS is either present within the fusion partner or the fusion peptide amino acid sequences or is a suitable linker. 6. The process as claimed in claim 3, wherein the fusion partner is linked at its C terminal end with a peptide at its N terminal end via a cleavage site. 7. The process as claimed in claim 4, wherein the physiologically active peptide is selected from those polypeptides having a length from about 10 amino acids to about 90 amino acids. 8. The process as claimed in claim 4, wherein the physiologically active peptides is selected from the group consisting of caltrin, calcitonin, insulin, angiotensin, tissue plasminogen activator, growth hormone, growth factors, growth hormone releasing factors, cytokines, erythropoietin, interferons, interleukins, oxytocin, vasopressin, ACTH, collagen binding protein, parathyroid hormone, glucagon like peptide, glucagon, proinsulin, tumor necrosis factor, substance P, brain naturetic peptide, individual heavy and light antibody chains, peptide antibiotics, fuzeon, octreotide, and somatostatin. 9. The process as claimed in claim 3, wherein the fusion product construct is cloned in a suitable expression vector. 10. The process as claimed in claim 9, wherein the fusion product construct is encoded by an expression vector that is able to express the fusion product in E. coli. 11. The process as claimed in claim 4, wherein the physiologically active peptide is selected from parathyroid hormone (PTH) (1-34), angiotensin and proinsulin. 12. The process as claimed in claim 11, wherein the physiologically active peptide is PTH (1-34) of SEQ ID NO:6. 13. The process as claimed in claim 11, wherein the physiologically active peptide PTH (1-34) is expressed by expression vector pET27B-GCSF-PTH deposited under MTCC Accession Number 5425. 14. The process as claimed in claim 11, wherein the physiologically active peptide is proinsulin of SEQ ID NO:13. 15. The process as claimed in claim 11, wherein the physiologically active peptide proinsulin is expressed by expression vector pET27B-GCSF-Proinsulin deposited under MTCC Accession Number 5424. 16. The process as claimed in claim 11, wherein the physiologically active peptide is angiotensin of SEQ ID NO:17. 17. The process as claimed in claim 11, wherein the physiologically active peptide angiotensin is expressed by expression vector pET27B-GCS F-Angiotensin. 18. The process as claimed in claim 3, wherein the cleavage site is an enzymatic or a chemical cleavage site. 19. The process as claimed in claim 18, wherein the cleavage site is a chemical cleavage site. 20. The process as claimed in claim 18, wherein the cleavage site is an enzymatic cleavage site. 21. The process as claimed in claim 19, wherein the cleavage site is cleavable by a chemical selected from cyanogen bromide, 2-(2-nitrophenylsulphenyl)-methyl-3'-bromoindolenine, BNPS-skatole, N-bromosuccinamide, O-iodosobenzoic acid, HBr/DMSO, NTCB, Ssodium metal in liquid ammonia, hydroxylamine and dilute acids. 22. The process as claimed in claim 21, wherein the chemical is cyanogen bromide. 23. The process as claimed in claim 20, where the enzymatic cleavage site is cleavable by an enzyme selected from enterokinase, trypsin, chymotrypsin, elastase, pepsin, papain, subtilisin, thermolysin, V8 protease, endoproteinase Arg C (submaxillaris protease), clostripain, thrombin, collagenase, lysobacter enzymogenes (Lys C), Mysobacter Al-I Protease and Factor Xa. 24. The process as claimed in claim 23, where the enzyme is enterokinase. 25. The process as claimed in claim 1, wherein step c) further comprises the steps of: disrupting the said host cell[s] and collecting a fusion protein comprising GCSF and the peptide of interest as inclusion bodies; separating the fusion protein containing inclusion bodies from other host components; solubilizing the fusion protein with a suitable denaturing agent; and cleaving a cleavage site with a suitable enzyme or chemical to release the peptide of interest. 26. The process as claimed in claim 25, wherein the host cell for the expression of the fusion product is E. coli. 27. The process as claimed in claim 1, wherein the GCSF fusion partner is encoded by the nucleotide sequence set forth in SEQ ID NaI.

Details for Patent 8,507,221

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2027-09-21
Nps Pharmaceuticals, Inc.	NATPARA	parathyroid hormone	For Injection	125511	01/23/2015		2027-09-21