Purification of glucagon-like peptide 1 analogs

Abstract

The present invention refers to a method of purifying a glucagon-like peptide 1 analogs, the method comprising a two dimensional reversed phase high performance liquid chromatography protocol, wherein the first step is carried out at a pH value between 7.0 to 7.8 using a mobile phase comprising a phosphate buffer and acetonitrile, and the second step is carried out at a pH value below 3.0 using a mobile phase comprising trifluoroacetic acid and acetonitrile.

1 - 15 . (canceled) 
     
     
         16 . A method for the purification of Liraglutide, comprising:
 a) providing a liquid composition C comprising Liraglutide and at least one unwanted component;   b) subjecting the composition C to a first reversed phase high performance liquid chromatograph (RP-HPLC) purification at a pH between 7.0 and 7.8, wherein a hydrocarbon bonded silica is used as a stationary phase, a mobile phase comprising an aqueous phosphate buffer AB1 and acetonitrile is used, and elution is effected by gradually increasing the acetonitrile concentration within the mobile phase while collecting Liraglutide containing fractions; and   c) subjecting the pooled Liraglutide containing fractions obtained in step b) to a second reversed phase HPLC purification at a pH below 3.0, wherein a hydrocarbon bonded silica is used as a stationary phase, a mobile phase comprising trifluoroacetic acid and acetonitrile is used, and elution is effected by gradually increasing the acetonitrile concentration within the mobile phase while collecting fractions containing purified Liraglutide.   
     
     
         17 . The method according to  claim 16 , wherein the aqueous phosphate buffer AB1 in step b) is ammonium phosphate buffer, preferably at a concentration of 5 mM to 50 mM. 
     
     
         18 . The method according to  claim 16 , wherein the gradient in step b) is selected from the range of 19 to 67% (v/v) acetonitrile and/or wherein the gradient in step c) is selected from the range of 31 to 100% (v/v) acetonitrile. 
     
     
         19 . The method according to  claim 16 , wherein the trifluoroacetic acid concentration within the mobile phase used in step c) is selected from the range of 0.05 to 0.5% (v/v), preferably 0.05 to 0.1% (v/v). 
     
     
         20 . The method according to  claim 16 , further comprising the step of:
 d) subjecting the Liraglutide obtained in step c) to a third reversed phase HPLC purification at a pH between 7.0 and 7.8, wherein a hydrocarbon bonded silica is used as a stationary phase, a mixture of an aqueous buffer AB2 with acetonitrile is used as a mobile phase, and elution is effected by gradually increasing the acetonitrile concentration within the mobile phase while collecting fractions containing purified Liraglutide.   
     
     
         21 . The method according to  claim 20 , wherein said aqueous buffer AB2 is selected from the group consisting of:
 a mixture of sodium dihydrogen phosphate and disodium hydrogen phosphate,   a mixture of potassium dihydrogen phosphate and dipotassium hydrogen phosphate,   potassium acetate, and   sodium acetate.   
     
     
         22 . The method according to  claim 16 , wherein all or parts of step b) and/or step c) and/or step d), if present, is/are carried out at a temperature selected from the range of 4° C. to 25° C., preferably 4° C. to 10° C. 
     
     
         23 . The method according to  claim 16 , wherein the stationary phase used in steps b) and c) and step d), if present, is C8 bonded silica or C18 bonded silica. 
     
     
         24 . The method according to  claim 16 , further comprising a step e) of size exclusion chromatography. 
     
     
         25 . The method according to  claim 16 , further comprising a step f) of desalting the peptide, preferably wherein desalting is performed by ion exchange chromatography, by size exclusion chromatography, or by ultrafiltration. 
     
     
         26 . The method according to  claim 24 , wherein all or parts of the respective step is/are carried out at a temperature selected from the range of 4° C. to 20° C., preferably 4° C. to 10° C. 
     
     
         27 . The method according to  claim 16 , wherein step a) comprises dissolving a dried crude Liraglutide peptide in an aqueous phosphate buffer AB0 at a pH selected from the range of 7.0 to 7.5. 
     
     
         28 . The method according to  claim 16 , wherein the crude Liraglutide peptide is obtained by solid phase peptide synthesis, followed by trifluoroacetic acid mediated cleavage and peptide precipitation from the cleavage composition. 
     
     
         29 . The method according to  claim 16 , wherein a purified Liraglutide is lyophilized, preferably at a pH selected from the range of 6.6 to 7.9, preferably 7.0 to 7.8, and most preferably 7.0 to 7.5. 
     
     
         30 . A composition LC comprising Liraglutide obtainable from a method according to  claim 16 , characterized in that said composition contains Liraglutide at a purity above 99%, and does contain detectable levels, but not more than 0.5%, preferably not more than 0.3%, more preferably not more than 0.2%, and most preferably not more than 0.1% of each of i) any Liraglutide derivative, where the indole moiety in the side chain of Trp at position 25 is oxidized by incorporation of a single oxygen atom, and/or of ii) any Liraglutide derivative, where the indole moiety in the side chain of Trp at position 25 is oxidized by incorporation of two oxygen atoms, and/or of iii) a Liraglutide derivative comprising kynurenine instead of Trp at position 25, and/or of iv) a Liraglutide deletion variant lacking Gly31.