Claims for Patent: 5,416,007

Summary for Patent: 5,416,007

Title:	Enhanced proteolytic cleavage of recombinant fusion proteins
Abstract:	Disclosed are methods for increasing the yield of intact target proteins by cleaving fused polypeptides made by recombinant DNA techniques. The fused polypeptides are designed at the DNA level to have a preselected primary cleavage site in a pendant polypeptide fused to a protein of interest. Structural features of the fused polypeptide and cleavage reaction environment are controlled to favor cleavage by a preselected cleavage agent at the primary cleavage site over a second cleavage agent-sensitive amino acid sequence in the target protein. The cleavage reaction is terminated before completion when the ratio of intact target protein to truncated, cleaved target protein is optimized, and the remaining reaction mixture comprising uncleaved fused polypeptide is resubjected to the cleavage agent. The presence of charged organic molecules in the cleavage reaction mixture favors cleavage at the primary cleavage site. The endopeptidase used for cleavage may be immobilized on an insoluble support matrix.
Inventor(s):	Charette; Marc F. (West Roxbury, MA), Crea; Roberto (Boston, MA), Cohen; Charles M. (Medway, MA)
Assignee:	Creative BioMolecules, Inc. (Hopkinton, MA)
Application Number:	08/273,870
Patent Claims:	1. In a process for producing an intact, recombinant, target protein by cleaving a recombinant fused polypeptide, said fused polypeptide comprising a continuous sequence of amino acids defining said target protein, a pendant polypeptide, and a primary cleavage site disposed adjacent to the target protein in the pendant polypeptide, said target protein comprising an amino acid sequence defining a secondary cleavage site, each of said primary and secondary cleavage sites being hydrolyzable by a single selected cleavage agent such that treatment with said selected cleavage agent under cleavage conditions induces hydrolytic cleavage of said fused polypeptide at both said primary and secondary cleavage sites, the improvement wherein the fused polypeptide is treated to yield enhanced quantities of an intact target protein by the steps of: A) subjecting the fused polypeptide to said cleavage agent in a reaction mixture under conditions in which the cleavage agent cleaves the fused polypeptide at both the primary and secondary cleavage sites, the rate of cleavage at the primary cleavage site being initially greater than the rate of cleavage at the secondary cleavage site, and the rate of cleavage at the secondary cleavage site approaching the rate of cleavage at the primary cleavage site as the cleavage reaction progresses; B) separating cleaved, intact target protein from the reaction mixture of step A prior to completion of the cleavage reaction at said primary cleavage site and prior to the establishment of equilibrium between the rates of cleavage at the primary and secondary cleavage sites, thereby producing intact target protein and a remaining reaction mixture comprising an uncleaved amount of said fused polypeptide; and C) subjecting the uncleaved amount of said fused polypeptide in said remaining reaction mixture to the cleavage agent to produce an additional amount of cleaved intact target protein, and a second remaining reaction mixture. 2. The process of claim 1 comprising the additional steps of separating said additional amount of cleaved intact target protein from said second reaction mixture and subjecting said second reaction mixture of step C to the cleavage agent to produce a further amount of cleaved, intact protein. 3. The process of claim 1 comprising the step of renaturing said fused polypeptide prior to step A, and conducting step A under conditions in which the target protein remains in its tertiary conformation during cleavage. 4. The process of claim 3 wherein the target protein comprises multiple Cys residues, and wherein said fused polypeptide is oxidized prior to step A so that the target protein is held in its tertiary conformation during cleavage. 5. The process of claim 1 wherein the cleavage agent and the primary and secondary cleavage sites are selected from the group consisting of the cleavage agents: trypsin, chymotrypsin, elastase, pepsin, papain, subtilisin, thermolysin, V-8 protease, endoproteinase, Arg C, clostripain, thrombim, collagenase, lysobacter, enzymogenese, mysobacter A1-1, protease, armillaria mellea, flavobacterium, meringosepticum, factor Xa, CNBr, BNPS-skatole, N-bromosuccinidine, 0-iodosobenzoic acid, HBr/DMSO, NTCB, sodium metal in liquid ammonia hydroxylamino, and dilute acid. 6. The process of claim 1 wherein said selective cleavage agent is an endopeptidase. 7. The process of claim 6 wherein step A is conducted by immobilizing the endopeptidase on an insoluble support matrix and passing a solution of the fused polypeptide over the support matrix. 8. The process of claim 1 wherein step A is conducted in an aqueous solution having a pH which favors cleavage at the primary cleavage site. 9. The process of claim 1 wherein the primary and secondary cleavage sites comprise the same amino acid or amino acid sequence. 10. The process of claim 1 wherein the target protein is EGF. 11. The process of claim 1 wherein the primary and secondary cleavage sites comprise a glutamic acid residue, and the cleavage agent is S. aureus V-8 protease. 12. The process of claim 11 wherein the target protein is EGF. 13. The process of claim 11 or 12 wherein step A is conducted in the presence of a charged organic molecule. 14. The process of claim 11 or 12 wherein step A is conducted in the presence of an organic molecule having 12 carbon atoms attached to a charged moiety. 15. The process of claim 14 wherein said charged organic molecule is selected from the group consisting of dodecyl sulfate salt, a lauric acid salt, and an N-dodecyl, N-lower alkyl sulfonic acid quaternary ammonium zwitterion. 16. The process of claim 1 wherein the target protein is human EGF, the primary and secondary cleavage sites comprise glutamic acid residues, the cleavage agent comprises S. aureus V-8 protease, and step A is conducted in the presence of a charged organic molecule which inhibits cleavage of a glutamic acid residue in said EGF. 17. The process of claim 16 wherein the S. aureus V-8 protease is immobilized on a solid support during step A. 18. The process of claim 16 wherein step A is conducted under conditions during cleavage in which the EGF is disposed in its tertiary conformation. 19. The process of claim 1 wherein said separation step is conducted by altering the properties of the reaction mixture to insolubilize one of said fused protein and said target polypeptide, and separating a supernatant from a precipitate. 20. A process for cleaving a fused, recombinant polypeptide to obtain intact EGF, said fused polypeptide comprising EGF having a Glu residue at position 51 and a pendant polypeptide having a Glu residue adjacent said EGF, said process comprising the step of treating said fused polypeptide with S. aureus V-8 protease in a reaction mixture containing a charged organic molecule under conditions in which the S. aureus V-8 protease cleaves said fused polypeptide at the Glu residue adjacent the EGF preferentially to the Glu residue at EGF position 51. 21. The process of claim 20 wherein said charged organic molecule is selected from the group consisting of dodecyl sulfate salt, a lauric acid salt, and an N-dodecyl, N-lower alkyl sulfonic acid quaternary ammonium zwitterion. 22. The process of claim 20 wherein said charged organic molecule comprises 12 carbon atoms and an anionic moiety. 23. The process of claim 20 wherein the treatment step is conducted while the fused polypeptide is disposed in its tertiary conformation. 24. A method of producing an intact, recombinant target protein by cleaving a recombinant fused polypeptide, said fused polypeptide comprising a continuous sequence of amino acids defining said target protein, a pendant polypeptide, and a primary cleavage site disposed adjacent the target protein hydrolyzable by a selected endopeptidase, said target protein comprising a secondary cleavage site each of said primary and secondary cleavage sites being hydrolyzable by said selected endopeptidase, the initial rate of cleavage at said primary site exceeding the initial rate of cleavage at said secondary site under cleavage conditions, said method comprising the steps of: A. immobilizing said selected endopeptidase on the exposed surface of an insoluble support matrix; B. passing an aqueous solution of said fused polypeptide over said matrix in reactive contact with the immobilized endopeptidase; C. controlling the time the fused polypeptide solution is in reactive contact with said immobilized cleavage agent so as to prevent the establishment of equilibrium between the rates of cleavage at said primary and secondary sites, such that said fused polypeptide is cleaved preferentially at said primary cleavage site, producing intact target protein and a remaining reaction mixture; and D. separating said target protein from the remaining reaction mixture. 25. The method of claim 24 wherein said fused polypeptide is renatured prior to step B, and step B is conducted under conditions in which said target protein remains in its tertiary conformation during cleavage. 26. The method of claim 24 wherein said primary cleavage site is a Glu residue and said endopeptidase is S. aureus V-8 protease. 27. The method of claim 24 or 26 wherein said target protein is EGF. 28. The method of claim 24 wherein said insoluble support matrix comprises cross-linked polyglucose. 29. The method of claim 24 wherein said insoluble support matrix comprises a polyvinylidine fluoride surface.

Details for Patent 5,416,007

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2007-03-20