Recombinant elastase proteins and methods of manufacturing and use thereof

Abstract

The present invention relates to methods for the manufacture, purification, formulation, and use of biologically active recombinant elastase proteins. Described are recombinant methods for producing therapeutically useful elastase proteins, as are pharmaceutical compositions comprising said elastase proteins. Novel recombinant elastase proteins and protein preparations are also disclosed. Methods are described for treating and preventing diseases of biological conduits using pharmaceutical compositions containing the elastase proteins of the invention.

What is claimed is: 
     
         1 . A protein comprising the amino acid sequence of SEQ ID NO:64 or SEQ ID NO:69. 
     
     
         2 . The protein of  claim 1  which is isolated. 
     
     
         3 . A nucleic acid molecule comprising a nucleotide sequence encoding a protein of  claim 1 . 
     
     
         4 . The nucleic acid molecule of  claim 3  wherein the protein comprises a signal sequence operably linked to said amino acid sequence of SEQ ID NO:64 or SEQ ID NO:69. 
     
     
         5 . The nucleic acid molecule of  claim 4 , wherein the signal sequence is operable in  Pichia pastoris.    
     
     
         6 . The nucleic acid molecule of  claim 5 , wherein the signal sequence is a yeast α-factor signal peptide. 
     
     
         7 . A vector comprising the nucleic acid molecule of  claim 4 . 
     
     
         8 . The vector of  claim 7  in which the nucleotide sequence is multimerized. 
     
     
         9 . A host cell comprising the vector of  claim 7 . 
     
     
         10 . The host cell of  claim 9  in which at least one copy of said vector is integrated into the host cell genome. 
     
     
         11 . The host cell of  claim 10  in which two to five copies of said vector are integrated into the host cell genome. 
     
     
         12 . The host cell of  claim 9  in which the nucleotide sequence is multimerized. 
     
     
         13 . The host cell of  claim 12  in which the vector comprises two to five copies of said nucleotide sequence. 
     
     
         14 . A cell genetically engineered to express the nucleic acid molecule of  claim 3 . 
     
     
         15 . The cell of  claim 14 , which is a  Pichia pastoris  cell. 
     
     
         16 . The cell of  claim 15 , wherein the nucleotide sequence is operably linked to a methanol-inducible promoter. 
     
     
         17 . A cell culture supernatant comprising the protein of  claim 1 . 
     
     
         18 . A method of producing an elastase protein, comprising culturing the cell of  claim 15  under conditions in which the protein of SEQ ID NO:64 or SEQ ID NO:69 is expressed. 
     
     
         19 . The method of  claim 18 , wherein said conditions include one, two, three or all four of: (i) a period of growth or induction at a pH of 2 to 6; (ii) a period of growth or induction at a temperature of 22° C. to 28° C.; (iii) culturing in complex medium; or (iv) culturing in the presence of a citrate, succinate or acetate compound. 
     
     
         20 . The method of  claim 18  which further comprises recovering the protein. 
     
     
         21 . The method of  claim 18 , which further comprises exposing said protein of SEQ ID NO:64 or SEQ ID NO:69 to activation conditions to produce a mature elastase protein. 
     
     
         22 . The method of  claim 21 , wherein said protein is purified prior to said exposure to activating conditions. 
     
     
         23 . The method of  claim 22 , wherein said protein is purified in the presence of a citrate, succinate or acetate compound. 
     
     
         24 . The method of  claim 21 , further comprising purifying the mature elastase protein. 
     
     
         25 . The method of  claim 24 , further comprising lyophilizing the purified mature elastase protein. 
     
     
         26 . A method of making a mature elastase protein, comprising subjecting a cell culture supernatant according to  claim 17  to autoactivation conditions, thereby producing a mature elastase protein. 
     
     
         27 . The method of  claim 26 , further comprising purifying the mature elastase protein. 
     
     
         28 . The method of  claim 27 , further comprising lyophilizing the purified mature elastase protein. 
     
     
         29 . A method of making a pharmaceutical composition comprising a mature elastase protein, reconstituting a lyophilisate comprising the lyophilized proelastase protein produced by the method of  claim 25  or  claim 28 . 
     
     
         30 . The method of  claim 29 , wherein the lyophilisate (a) comprises one or more buffer ingredients or (b) does not comprise buffer ingredients. 
     
     
         31 . The method of  claim 30 , wherein the lyophilisate is reconstituted with water or buffer. 
     
     
         32 . The method of  claim 31 , wherein upon reconstitution a solution of mature elastase protein in full strength buffer, greater than full strength buffer, or less than full strength buffer is produced. 
     
     
         33 . The method of  claim 32 , wherein the buffer is phosphate buffered saline. 
     
     
         34 . The method of  claim 29 , wherein the mature elastase protein is reconstituted to a concentration of 0.001 mg/ml to 50 mg/ml. 
     
     
         35 . The method of  claim 29 , wherein the mature elastase protein has a specific activity of 1 to 40 U/mg protein. 
     
     
         36 . A pharmaceutical composition produced by the method of  claim 29 . 
     
     
         37 . The pharmaceutical composition of  claim 36  which is characterized by at least one, at least two, at least three, at least four, at least five, at least six or at least seven of the following properties:
 (a) the composition is free of trypsin; 
 (b) the composition is substantially free of trypsin; 
 (c) the composition is free of any protein consisting of SEQ ID NOS:70 and 71; 
 (d) the composition is substantially free of any protein consisting of SEQ ID NOS:2 and 3; 
 (e) the composition is free of bacterial proteins; 
 (f) the composition is substantially free of bacterial proteins; 
 (g) the composition is free of mammalian proteins other than said mature elastase protein; 
 (h) the composition is substantially free of mammalian proteins other than said mature elastase protein; 
 (i) the composition is free or substantially free of one, two, three or all four proteins consisting of SEQ ID NO:85, 86, 94 and 95; 
 (j) the composition is free or substantially free of one, two, or all three proteins consisting of SEQ ID NO:106, 107 and 108; 
 (k) the composition contains pharmaceutically acceptable levels of endotoxins; 
 (l) the mature elastase protein in the composition is characterized by a specific activity of 1 to 40 U/mg of protein; 
 (m) the trypsin activity in said composition corresponds to less than 4 ng per 1 mg of mature elastase protein; 
 (n) the composition comprises polysorbate-80; 
 (o) the composition comprises dextran; 
 (p) the composition comprises sodium ions, potassium ions, phosphate ions, chloride ions and polysorbate-80; 
 (q) the composition comprises sodium ions, potassium ions, phosphate ions, chloride ions and dextran; 
 (r) the composition comprises sodium ions, potassium ions, phosphate ions, chloride ions, polysorbate-80, and dextran; 
 (s) the mature elastase protein in said composition maintains 60% to 100% of its specific activity after at least a week of storage at 4° C., after at least a month of storage at 4° C., after at least two months of storage at 4° C., after at least three months of storage at 4° C., or after at least month six months of storage at 4° C.; and 
 (t) the composition comprises a unit dosage of 0.0033 mg to 200 mg of said mature elastase protein. 
 
     
     
         38 . The pharmaceutical composition of  claim 37 , wherein the pharmaceutical composition is characterized by at least three characteristics, at least four characteristics or five characteristics independently selected from the following groups (i) through (v):
 (i) (a), (b) or (m)   (ii) (e) or (f)   (iii) (g) or (h)   (iv) (k)   (v) (l)   
     
     
         39 . The pharmaceutical composition of  38 , wherein two of said at least three or at least said four characteristics are selected from groups (i) and (iv) or (v). 
     
     
         40 . The pharmaceutical composition of  38 , wherein three of at least said four characteristics are selected from groups (i), (iv) and (v). 
     
     
         41 . A method of removing one or more incorrectly processed mature elastase proteins from a mixture of correctly and incorrectly processed mature elastase proteins, said method comprising:
 (a) subjecting a composition comprising a mixture of correctly and incorrectly processed mature elastase proteins to a pH at which the correctly processed mature enzyme is active;   (b) maintaining such pH until such time that one or more incorrectly processed mature elastase proteins are degraded,   thereby removing said one or more incorrectly processed mature elastase proteins from a mixture of correctly and incorrectly processed mature elastase proteins.   
     
     
         42 . The method of  claim 41 , wherein said one or more incorrectly processed mature elastase proteins contain at least one additional or fewer amino acid at the N-terminus relative to correctly processed mature elastase proteins. 
     
     
         43 . The method of  claim 41  wherein said pH is between 5 and 12. 
     
     
         44 . The method of any one of  claim 41 , wherein said one or more incorrectly processed mature elastase proteins are degraded by 50% to 100%. 
     
     
         45 . A method for therapeutically increasing the diameter of an artery or vein in a human subject in need thereof, the method comprising: locally administering to the wall of the artery or vein in the human subject the pharmaceutical composition of  claim 36  in a dose sufficient to increase the diameter of the artery or vein. 
     
     
         46 . The method of  claim 45 , wherein the diameter of the vessel, the lumenal diameter of the vessel, or both, are increased. 
     
     
         47 . A method for preventing or treating vasospasm of an artery or vein in a human subject in need thereof, the method comprising: locally administering to the wall of the artery or vein in the human subject the pharmaceutical composition of  claim 36  in a dose sufficient to prevent or treat vasospasm of the artery or vein. 
     
     
         48 . A method for treating an obstructed artery or vein in a human subject in need of such treatment, the method comprising: locally administering to the wall of the artery or vein in the human subject the pharmaceutical composition of  claim 36 , wherein said administration results in proteolysis of elastin in the wall of the artery or vein leading to enlargement of the diameter of the artery or vein. 
     
     
         49 . A method for treating an artery or vein connected to an arteriovenous hemodialysis graft or arteriovenous fistula in a human subject in need of such treatment, the method comprising: locally administering to the wall of the artery or vein in the human subject the pharmaceutical composition of  claim 36 , wherein said administration results in proteolysis of elastin in the wall of the artery or vein leading to enlargement of the diameter of the artery or vein. 
     
     
         50 . A method for treating a vein in a human subject for use in hemodialysis, the method comprising: locally administering to the wall of the vein in the human subject the pharmaceutical composition of  claim 36 , wherein said administration results in proteolysis of elastin in the wall of the vein leading to enlargement of the diameter of the vein. 
     
     
         51 . The kit comprising the pharmaceutical composition of  claim 36 . 
     
     
         52 . The kit of  claim 51  wherein the pharmaceutical composition is in a container, pack, dispenser, or catheter.