Process and intermediate products for preparing cardiodilatin fragments, and highly purified cardiodilatin fragments

Abstract

The invention relates to a process for the preparation of cardiodilatin fragments, to highly purified cardiodilatin fragments, and to appropriate intermediates for the preparation of said fragments. Furthermore, the invention relates to highly purified cardiodilatin fragments which are free of peptide impurities and exhibit a single migration peak in capillary electrophoresis, as well as to appropriate processes for the preparation of same.

1 - 20 . (canceled) 
     
     
         21 . A process for purifying a cardiodilatin fragment having the formula R 1 -ANP(105-121)-R 2  and a total chain length of 17-37 amino acids, wherein
 ANP(105-121) is the amino acid sequence of SEQ ID NO:1,   R 1  is absent or the amino acid sequence ANP(90-104) (SEQ ID NO:2) or a fragment thereof,   R 2  is absent or the amino acid sequence ANP(122-126) (SEQ ID NO:3) or a fragment thereof, wherein the process comprises the steps of   loading a crude product containing the cardiodilatin fragment on a reversed-phase HPLC column, and   eluting the cardiodilatin fragment with a buffer system containing either triethylammonium phosphate and acetonitrile, or trifluoroacetic acid and acetonitrile.   
     
     
         22 . The process of  claim 21 , wherein R 1  is a fragment selected from the group consisting of ANP(95-104), ANP(99-104), and ANP(102-104). 
     
     
         23 . The process of  claim 21 , wherein R 2  is a fragment selected from the group consisting of ANP(122-125) and ANP(122-126). 
     
     
         24 . The process of  claim 21 , wherein the HPLC column is a C 18  column. 
     
     
         25 . The process of  claim 21 , wherein the buffer system contains triethylammonium phosphate and acetonitrile. 
     
     
         26 . The process of  claim 21 , wherein the buffer system contains trifluoroacetic acid and acetonitrile. 
     
     
         27 . The process of  claim 21 , wherein the pH of the elution buffer is 2-5. 
     
     
         28 . The process of  claim 27 , wherein the pH of the elution buffer is 2-3. 
     
     
         29 . The process of  claim 28 , wherein the pH of the elution buffer is 2.25. 
     
     
         30 . The process of  claim 21 , wherein the process further comprises equilibrating the reversed-phase HPLC column with a triethylammonium phosphate buffer prior to loading the crude product containing the cardiodilatin fragment. 
     
     
         31 . The process of  claim 30 , wherein the buffer has a concentration of 10-200 mM. 
     
     
         32 . The process of  claim 31 , wherein the buffer has a concentration of 50 mM. 
     
     
         33 . The process of  claim 30 , wherein the cardiodilatin fragment is eluted by continuous charging of a buffer mixture of triethylammonium phosphate in water and acetonitrile in a continuous gradient. 
     
     
         34 . The process of  claim 33 , wherein the concentration of triethylammonium phosphate in the buffer mixture is 10-200 mM. 
     
     
         35 . The process of  claim 34 , wherein the concentration is 50 mM. 
     
     
         36 . The process of  claim 33 , wherein the ratio of triethylammonium phosphate in water to acetonitrile is 2:3 v/v. 
     
     
         37 . The process of  claim 33 , wherein the pH of the elution buffer is 2-5. 
     
     
         38 . The process of  claim 33 , wherein the pH of the elution buffer is 2-3. 
     
     
         39 . The process of  claim 33 , wherein the pH of the elution buffer is 2.25. 
     
     
         40 . The process of  claim 21 , wherein the process produces a cardiodilatin fragment selected from the group consisting of ANP(95-126), ANP(99-126), ANP(102-106) and ANP(103-126). 
     
     
         41 . The process of  claim 21 , wherein the process produces a cardiodilatin fragment having a purity of 96-99.9%.