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

A method for treating diabetes in a patient in need thereof comprising administering a single chain insulin (SCI) compound of formula (I):

• B chain -C′-A chain (Formula (I))
• having the properties of higher affinity for the insulin receptor and lower affinity for the IGF-1 receptor as compared to those of native proinsulin with chemical and thermodynamic degradation profiles such that the SCI can be formulated and stored for extended periods of time without refrigeration;
• wherein B chain and A chain are modified human insulin chains;
• and
• wherein C′ covalently links the C-terminus of the B chain to the N-terminus of the A chain, and is a peptide of 5 amino acids comprising the following sequence:
• Y-P-G-D-X (SEQ ID NO:
• 1);
• wherein X is any amino acid;
• wherein the B chain is modified from a native human insulin B chain (SEQ ID NO:11), and A chain is modified from a native human insulin A chain (SEQ ID NO:10), wherein the modifications comprise one or more mutations at (1) Gln5, Gln15, Asn18, or Asn21 of SEQ ID NO:10, or (2) Asn3 or Gln5 of SEQ ID NO:11;
• resulting in enhanced resistance to deamidation.

A method for producing single chain insulin (SCI) comprising introducing a recombinant vector into an expression system, expressing a protein comprising a fusion tag and single chain insulin, cleaving said fusion tag from said single chain insulin, and isolating said single chain insulin, wherein the recombinant vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the SCI of formula (I):

• B chain -C′-A chain (Formula (I))
• having the properties of higher affinity for the insulin receptor and lower affinity for the IGF-1receptor as compared to those of native proinsulin with chemical and thermodynamic degradation profiles such that the SCI can be formulated and stored for extended periods of time without refrigeration;
• wherein B chain and A chain are modified human insulin chains;
• and
• wherein C′ covalently links the C-terminus of the B chain to the N-terminus of the A chain, and is a peptide of 5 amino acids comprising the following sequence:
• Y-P-G-D-X (SEQ ID NO:
• 1);
• wherein X is any amino acid;
• wherein the B chain is modified from a native human insulin B chain (SEQ ID NO:11), and A chain is modified from a native human insulin A chain (SEQ ID NO:10), wherein the modifications comprise one or more mutations at (1) Gln5, Gln15, Asn18, or Asn21 of SEQ ID NO:10, or (2) Asn3 or Gln5 of SEQ ID NO:11;
• resulting in enhanced resistance to deamidation.

A single chain insulin (SCI) compound of formula (I):

• B chain -C′-A chain (Formula I));
• having the properties of higher affinity for the insulin receptor and lower affinity for the IGF-1 receptor as compared to those of native proinsulin with chemical and thermodynamic degradation profiles such that the SCI can be formulated and stored for extended periods of time without refrigeration;
• wherein the B chain and A chain are native or modified human insulin chains;
• and
• wherein C′ covalently links the C-terminus of the B chain to the N-terminus of the A chain, and is a peptide of 5 amino acids comprising the following sequence:
• Y-P-G-D-X (SEQ ID NO:
• 1);
• wherein X is any amino acid.