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 (i) an elastase activation sequence comprising an elastase recognition sequence operably linked to (ii) the amino acid sequence of a mature elastase. 
     
     
         2 - 39 . (canceled) 
     
     
         40 . A nucleic acid encoding a protein of  claim 1 . 
     
     
         41 - 69 . (canceled) 
     
     
         70 . A vector comprising the nucleic acid molecule of  claim 40 . 
     
     
         71 - 72 . (canceled) 
     
     
         73 . A host cell comprising the vector of  claim 70 . 
     
     
         74 - 78 . (canceled) 
     
     
         79 . A host cell comprising at least one copy of the nucleic acid molecule of  claim 40  integrated into its genome. 
     
     
         80 - 83 . (canceled) 
     
     
         84 . A cell genetically engineered to express the nucleic acid molecule of any one of  claim 40 . 
     
     
         85 - 87 . (canceled) 
     
     
         88 . A cell culture supernatant comprising the protein of  claim 1 . 
     
     
         89 . A method of producing an elastase protein, comprising culturing the host cell of  claim 84  under conditions in which the protein is produced. 
     
     
         90 - 110 . (canceled) 
     
     
         111 . A method of producing an elastase protein, comprising:
 (a) culturing a host cell capable of expressing a recombinant proelastase protein in the presence of a first citrate, succinate or acetate compound;   (b) recovering the recombinant proelastase protein from said host cell culture; and   (c) optionally, exposing the recombinant proelastase protein to activation conditions to produce a mature elastase protein,   thereby producing an elastase protein.   
     
     
         112 - 131 . (canceled) 
     
     
         132 . A method of producing a mature elastase protein, comprising:
 (a) lyophilizing a proelastase protein;   (b) storing the lyophilized proelastase protein;   (c) reconstituting the lyophilized proelastase protein; and   (d) activating the reconstituted proelastase protein,   thereby producing a mature elastase protein.   
     
     
         133 - 150 . (canceled) 
     
     
         151 . A method of producing a mature type I elastase protein comprising subjecting a recombinant autoactivated type I proelastase protein to autoactivation conditions, contacting a recombinant autoactivated type I proelastase protein with a catalytic amount of elastase, or both, thereby producing a mature type I elastase protein. 
     
     
         152 - 189 . (canceled) 
     
     
         190 . A method of producing a mature type I elastase protein comprising:
 (a) culturing the host cell of  claim 84  under conditions in which the protein is expressed;   (b) recovering the expressed protein;   (c) purifying the recovered protein;   (d) raising the pH of a solution containing the protein or contacting the recovered protein with a catalytic amount of elastase to produce a mature type I elastase protein,   thereby producing a mature type I elastase protein.   
     
     
         191 - 224 . (canceled) 
     
     
         225 . A method of producing a formulation of mature elastase protein, comprising:
 (a) subjecting an autoactivated proelastase protein to autoactivating conditions to produce mature elastase protein; and   (b) formulating the mature elastase protein,   thereby producing a formulation of mature elastase protein.   
     
     
         226 - 260 . (canceled) 
     
     
         261 . A method of producing a lyophilized mature type I elastase comprising:
 (a) producing a mature type I elastase according to the method of  claim 151 ;   (b) isolating mature type I elastase; and   (c) lyophilizing said isolated mature type I elastase,   thereby producing a lyophilized mature type I elastase.   
     
     
         262 - 313 . (canceled) 
     
     
         314 . A lyophilized formulation of a protein consisting essentially of SEQ ID NO: 5, 84, or 87 which upon reconstitution to a concentration of said protein of 1 mg/ml the trypsin activity corresponds to less than 2 ng/ml trypsin. 
     
     
         315 - 324 . (canceled) 
     
     
         325 . A liquid formulation comprising a solution of a least 0.1 mg/ml of a protein consisting essentially of SEQ ID NO: 5, 84, or 87 in which the trypsin activity corresponds to less than 2 ng/ml trypsin. 
     
     
         326 - 339 . (canceled) 
     
     
         340 . A formulation made by or obtainable by the method of  claim 225 . 
     
     
         341 - 346 . (canceled) 
     
     
         347 . 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.   
     
     
         348 - 373 . (canceled) 
     
     
         374 . A method of producing a pharmaceutical composition comprising a mature type I elastase, said method comprising (i) producing a lyophilized mature type I elastase by the method of  claim 261 ; and (ii) reconstituting the lyophilized mature type I elastase in water or a pharmaceutically acceptable carrier, thereby producing a pharmaceutical composition comprising a mature human type I elastase. 
     
     
         375 - 424 . (canceled)