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.

1 . A protein comprising the amino acid sequence of SEQ ID NO:64 or SEQ ID NO:69. 
     
     
         2 . (canceled) 
     
     
         3 . A nucleic acid molecule comprising a nucleotide sequence encoding a protein of  claim 1 . 
     
     
         4 - 6 . (canceled) 
     
     
         7 . A vector comprising the nucleic acid molecule of  claim 3 . 
     
     
         8 . (canceled) 
     
     
         9 . A host cell comprising the vector of  claim 7 . 
     
     
         10 - 13 . (canceled) 
     
     
         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 . (canceled) 
     
     
         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 - 25 . (canceled) 
     
     
         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 28 . 
     
     
         30 - 35 . (canceled) 
     
     
         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 - 40 . (canceled) 
     
     
         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 - 44 . (canceled) 
     
     
         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 . (canceled) 
     
     
         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 - 52 . (canceled)