Chemically and thermodynamically stable insulin analogues and improved methods for their production

Abstract

The subject matter of this invention is directed towards chemically and thermodynamically stable single-chain insulin (SCI) analogues that are resistant to deamidation and fibrillation. The invention further discloses improved methods for the recombinant expression, purification and refolding of SCI.

What is claimed is: 
     
         1 . A compound comprising a B chain peptide, an A chain peptide and a C′ linker peptide; wherein
 the C′ linker peptide covalently links a C-terminus of the B chain peptide to an N-terminus of the A chain peptide, and consists of an amino acid sequence Y 1 Y 2 Y 3 Y 4 Y 5 Y 6 Y 7 ; wherein Y 1  is R or absent, Y 2  is R or absent, Y 3  is Y or V, Y 4  is P or absent, Y 5  is G or D, Y 6  is D or G, and Y 7  is any amino acid; 
 the B chain peptide and the A chain peptide are each independently an insulin peptide or chemostable analogue thereof; and 
 the B chain peptide and the A chain peptide are linked together by a disulfide bond. 
 
     
     
         2 . The compound of  claim 1 , wherein the compound has a Formula (I):
   B chain-C′-A chain  Formula (I).
   
     
     
         3 . The compound of  claim 1 , wherein the B chain peptide and the A chain peptide are linked together by two disulfide bonds. 
     
     
         4 . The compound of  claim 1 , wherein the B chain peptide is a peptide of 50 or fewer amino acids. 
     
     
         5 . The compound of  claim 1 , wherein the A chain peptide is a peptide of 50 or fewer amino acids. 
     
     
         6 . The compound of  claim 1 , wherein the B chain peptide is a peptide of 27-30 amino acids. 
     
     
         7 . The compound of  claim 1 , wherein the A chain peptide is a peptide of 21 amino acids. 
     
     
         8 . The compound of  claim 1 , wherein Y 7  is V or K. 
     
     
         9 . The compound of  claim 1 , wherein the compound has higher affinity for an insulin receptor and lower affinity for an insulin growth factor-1 (IGF-1) receptor as compared to native insulin. 
     
     
         10 . The compound of  claim 1 , wherein the compound is capable of being stored for extended periods of time without refrigeration. 
     
     
         11 . The compound of  claim 1 , wherein the compound displays less than one percent degradation with incubation at 40° C. for at least 28 days. 
     
     
         12 . The compound of  claim 1 , wherein the compound displays less than 5 percent denaturation with incubation at 40° C. for at least 90 days. 
     
     
         13 . The compound of  claim 1 , wherein the compound has an affinity for an insulin receptor greater than or equal to that of natural insulin, as measured in vitro, and having an affinity for Insulin-like Growth Factor Receptor no greater than that of natural insulin as measured in vitro. 
     
     
         14 . The compound of  claim 1 , wherein the B chain is native human insulin, B chain polypeptide, or human insulin B chain with chemical degradation resistance mutation. 
     
     
         15 . The compound of  claim 1 , wherein the A chain is native human insulin, A chain polypeptide, or human insulin A chain with chemical degradation resistance mutation.