N-Terminal Polysialylation

Abstract

The present invention relates to a composition comprising a population of polysaccharide derivatives of a protein, wherein the protein is insulin or an insulin-like protein and the polysaccharide is anionic and comprises between 2 and 125 saccharide units, and wherein the population consists of substantially only N-terminal derivatives of the protein. Typically, the polysaccharide is PSA. The present invention also relates to pharmaceutical compositions comprising the novel compounds, and methods for making the novel compounds.

1 - 10 . (canceled) 
     
     
         11 . A method for producing a population of amino-terminal polysialic acid derivatives of insulin the method comprising:
 performing a derivatization reaction in a first aqueous solution having a pH in the range of 4.0 to 6.5 which couples a polysialic acid comprising 2-125 saccharide units to only the amino-terminal amine of the B-chain of the insulin, thereby producing the population of amino-terminal polysialic acid derivatives of insulin; and   purifying the population of amino-terminal polysialic acid derivatives of insulin using a chromatographical method and eluting in a second aqueous solution having a pH in the range of 6.5 to 9.0,   wherein at least 85% of the population of amino-terminal polysialic acid derivatives of insulin consists of a polysialic acid coupled to only at the amino-terminal amine of the B-chain of the insulin.   
     
     
         12 . The method according to  claim 11 , wherein the polysialic acid has a reactive aldehyde group which reacts with the insulin and the coupling reaction is carried out under reducing conditions. 
     
     
         13 . The method according to  claim 12 , wherein the reactive aldehyde group is at the non-reducing end of the polysialic acid. 
     
     
         14 . The method according to  claim 12 , wherein the reactive aldehyde is at the reducing end of the polysialic acid and the non-reducing end has been passivated such that it does not react with the insulin. 
     
     
         15 - 17 . (canceled) 
     
     
         18 . The method according to  claim 11 , wherein the polysialic acid has a reactive aldehyde group and the method comprises:
 (a) converting said aldehyde group to an amine;   (b) reacting said amine with a bifunctional reagent comprising at least one functional group selected from N-maleimide, vinylsulphone, N-iodoacetamide, orthopyridyl group and N-hydroxysuccinimide, to form a reaction intermediate; and   (c) reacting the reaction intermediate with the insulin.   
     
     
         19 . (canceled) 
     
     
         20 . The method according to  claim 11  wherein the pH of the first aqueous solution is in the range 4.0-6.0. 
     
     
         21 . The method according to  claim 11 , which is carried out in the presence of a formulation additive. 
     
     
         22 . The method according to  claim 21 , wherein the formulation additive is selected from the group consisting of one or more buffers, stabilizers, surfactants, salts, polymers, metal ions, sugars, polyols and amino acids. 
     
     
         23 - 33 . (canceled) 
     
     
         34 . The method according to  claim 11 , wherein the derivatization reaction includes a bifunctional reagent with at least one functional group comprising N-maleimide, vinylsulphone, N-iodoacetamide, orthopyridyl group or N-hydroxysuccinimide. 
     
     
         35 . The method according to  claim 11 , wherein the derivatization reaction is performed under reducing conditions. 
     
     
         36 . The method according to  claim 35 , wherein the derivatization reaction includes hydrogen with catalysts, a hydride, L-ascorbic acid, sodium metabisulphite or L-selectride. 
     
     
         37 . The method according to  claim 36 , wherein the hydride is an alkali metal hydride. 
     
     
         38 . The method according to  claim 11 , wherein the polysialic acid is α-2,8 linked polysialic acid. 
     
     
         39 . The method according to  claim 11 , wherein the polysialic acid comprises 10-80 sialic acid units. 
     
     
         40 . The method according to  claim 39 , wherein the polysialic acid comprises 20-60 sialic acid units. 
     
     
         41 . The method according to  claim 11 , wherein the polysialic acid has a weight average molecular weight in the range about 1 kDa to about 35 kDa. 
     
     
         42 . The method according to  claim 41 , wherein the polysialic acid has a weight average molecular weight in the range about 10 kDa to about 20 kDa. 
     
     
         43 . The method according to  claim 11 , wherein the insulin is derivatised by the polysialic acid at the reducing terminal unit of the polysialic acid. 
     
     
         44 . The method according to  claim 11 , wherein the population of amino-terminal polysialic acid derivatives of insulin has a polydispersity that is less than 1.3. 
     
     
         45 . The method according to  claim 11 , wherein the chromatographical method comprises hydrophobic interaction chromotography, size exclusion chromotography, high performance liquid chromatography or ion exchange chromatography. 
     
     
         46 . The method according to  claim 11 , wherein each amino-terminal polysialic acid derivative of insulin has the general formula (I): 
       
         
           
           
               
               
           
         
         wherein m is any integer from 2 to 125; 
         HN is derived from NH 2  which is the amino terminus of the B-chain of the insulin; 
         B is insulin; 
         L is a bond, or a linking group; 
         GlyO is a sialic acid unit; 
         wherein the linking group is of the formula: —Y—C(O)—R 1 —C(O)—, in which R 1  is a bifunctional organic radical selected from the group consisting of alkanediyl, arylene, alkarylene, heteroarylene and alkylheteroarylene, any of which may be substituted, and any of which may be interrupted by carbonyl, ester, sulfide, ether, amide and/or amine linkages; and Y is NR 2  or NR 2 —NR 2 , wherein R 2  is H or C 1-6  alkyl. 
       
     
     
         47 . The method according to  claim 46 , wherein L is a bond or is: 
       
         
           
           
               
               
           
         
       
     
     
         48 . The method according to  claim 46 , wherein m is an integer from 10 to 80. 
     
     
         49 . The method according to  claim 11 , wherein the pH of the second aqueous solution is in the range of 6.5 to 8.5. 
     
     
         50 . The method according to  claim 49 , wherein the pH of the second aqueous solution is in the range of 6.5 to 8.0. 
     
     
         51 . The method according to  claim 11 , wherein the population of amino-terminal polysialic acid derivatives of insulin is mixed with one or more pharmaceutically acceptable excipients to produce a pharmaceutical composition.