Insulin analogues and method of preparing the same

Claim 1

A method for the preparation of human insulin analogues of the formula I wherein the X's are the amino acid residues of human insulin or the same or different amino acid residue substitutions, the net function of which is to impart to the molecule the same charge or a greater negative charge at neutral pH than that of human insulin, with the proviso that at least one X is different from the amino acid residues of human insulin at the respective position in the insulin molecule and that when X in position A(8) is His or Phe, X in position A(2l) is Asp, X in position B(5) is Ala, X in position B(9) is Leu, X in position B(l0) is Asn or Leu, X in position B(l2) is Asn or X in position B(26) is Ala, then at least one of the remaining X's are different from the amino acid residues of human insulin at the respective position in the insulin molecule and with the further proviso that one or more amino acid residues may have been removed from the N- and/or C-­terminal ends of the A- and/or B-chain, wherein a yeast strain containing a replicable expression vehicle comprising a DNA-sequence encoding a precursor of the insulin analogue is cultured in a suitable nutrient medium, and the precursor is recovered from the culture medium and converted into the novel insulin analogue by enzymatic and chemical in vitro conversion.

Claim 2

A method for the preparation of human insulin analogues of the formula I wherein the X's are the amino acid residues of human insulin or the same or different amino acid residue substitutions, the net function of which is to impart to the molecule the same charge or a greater negative charge at neutral pH than that of human insulin, with the proviso that at least one X is different from the amino acid residues of human insulin at the respective position in the insulin molecule and that when X in position A(8) is His or Phe, X in position A(2l) is Asp, X in position B(5) is Ala, X in position B(9) is Leu, X in position B(l0) is Asn or Leu, X in position B(l2) is Asn or X in position B(26) is Ala, then at least one of the remaining X's are different from the amino acid residues of human insulin at the respective position in the insulin molecule and with the further proviso that one or more amino acid residues may have been removed from the N- and/or C-­terminal ends of the A- and/or B-chain, wherein a yeast strain containing a replicable expression vehicle comprising a DNA-sequence encoding a precursor of the insulin analogue is cultured in a suitable nutrient medium, and the precursor is recovered from the culture medium and converted into the novel insulin analogue by enzymatic and chemical in vitro conversion.

Claim 3

A method for the preparation of human insulin analogues of the formula I wherein the X's are the amino acid residues of human insulin or the same or different amino acid residue substitutions, the net function of which is to impart to the molecule the same charge or a greater negative charge at neutral pH than that of human insulin, with the proviso that at least one X is different from the amino acid residues of human insulin at the respective position in the insulin molecule and that when X in position A(8) is His or Phe, X in position A(2l) is Asp, X in position B(5) is Ala, X in position B(9) is Leu, X in position B(l0) is Asn or Leu, X in position B(l2) is Asn or X in position B(26) is Ala, then at least one of the remaining X's are different from the amino acid residues of human insulin at the respective position in the insulin molecule and with the further proviso that one or more amino acid residues may have been removed from the N- and/or C-­terminal ends of the A- and/or B-chain, wherein a biosynthetic precursor of the general formula II wherein Q n is a peptide chain with n naturally occuring amino acid residues, R is Lys or Arg, n is an integer from 0 to 33, m is 0 or l, and the X's are defined as above with the proviso that the peptide chain -Q n -R- does not contain two adjacent basic amino acid residues is reacted with an L-threonine ester in the presence of trypsin or a trypsin derivative followed by conversion of the obtained threonine ester of the human insulin analogue into the human insulin analogue.

Claim 4

A method for the preparation of human insulin analogues of the formula I wherein the X's are the amino acid residues of human insulin or the same or different amino acid residue substitutions, the net function of which is to impart to the molecule the same charge or a greater negative charge at neutral pH than that of human insulin, with the proviso that at least one X is different from the amino acid residues of human insulin at the respective position in the insulin molecule and that when X in position A(8) is His or Phe, X in position A(2l) is Asp, X in position B(5) is Ala, X in position B(9) is Leu, X in position B(l0) is Asn or Leu, X in position B(l2) is Asn or X in position B(26) is Ala, then at least one of the remaining X's are different from the amino acid residues of human insulin at the respective position in the insulin molecule and with the further proviso that one or more amino acid residues may have been removed from the N- and/or C-­terminal ends of the A- and/or B-chain, wherein a biosynthetic precursor of the general formula II wherein Q n is a peptide chain with n naturally occuring amino acid residues, R is Lys or Arg, n is an integer from 0 to 33, m is 0 or l, and the X's are defined as above with the proviso that the peptide chain -Q n -R- does not contain two adjacent basic amino acid residues is reacted with an L-threonine ester in the presence of trypsin or a trypsin derivative followed by conversion of the obtained threonine ester of the human insulin analogue into the human insulin analogue.

Claim 5

A method for the production of human insulin analogues of the formula I wherein the X's are the amino acid residues of human insulin or the same or different amino acid residue substitutions, the net function of which is to impart to the molecule the same charge or a greater negative charge at neutral pH than that of human insulin, with the proviso that at least one X is different from the amino acid residues of human insulin at the respective position in the insulin molecule and that when X in position A(8) is His or Phe, X in position A(2l) is Asp, X in position B(5) is Ala, X in position B(9) is Leu, X in position B(l0) is Asn or Leu, X in position B(l2) is Asn or X in position B(26) is Ala, then at least one of the remaining X's are different from the amino acid residues of human insulin at the respective position in the insulin molecule and with the further proviso that one or more amino acid residues may have been removed from the N- and/or C-­terminal ends of the A- and/or B-chain, wherein a biosynthetic precursor of the general formula III wherein S and T are each Lys or Arg and the X's are defined as above, are reacted with trypsin and carboxypeptidase B in aqueous solution and the human insulin analogue is recovered from the reaction mixture.

Claim 6

A method for the production of human insulin analogues of the formula I wherein the X's are the amino acid residues of human insulin or the same or different amino acid residue substitutions, the net function of which is to impart to the molecule the same charge or a greater negative charge at neutral pH than that of human insulin, with the proviso that at least one X is different from the amino acid residues of human insulin at the respective position in the insulin molecule and that when X in position A(8) is His or Phe, X in position A(2l) is Asp, X in position B(5) is Ala, X in position B(9) is Leu, X in position B(l0) is Asn or Leu, X in position B(l2) is Asn or X in position B(26) is Ala, then at least one of the remaining X's are different from the amino acid residues of human insulin at the respective position in the insulin molecule and with the further proviso that one or more amino acid residues may have been removed from the N- and/or C-­terminal ends of the A- and/or B-chain, wherein a biosynthetic precursor of the general formula III wherein S and T are each Lys or Arg and the X's are defined as above, are reacted with trypsin and carboxypeptidase B in aqueous solution and the human insulin analogue is recovered from the reaction mixture.

Claim 9

A method according to any one of Claims l to 4, wherein the amino acid residue substitutions in the insulin analogue prepared are more hydrophilic than the amino acid residue of human insulin at the respective position in the insulin molecule.

Claim 10

A method according to any one of Claims l to 5, wherein, in the insulin analogue prepared, not more than about 7 of the X's are different from the amino acid residue at the corresponding position in human insulin.

Claim 11

A method according to any one of Claims l to 6, wherein the amino acid substitutions in the insulin analogue prepared are selected from the group consisting of Asp, Glu, Ser, Thr, His, and Ile.

Claim 13

A method according to any one of Claims l to 8, wherein at least one X in position B(9), B(l0), B(l2), B(26), or B(27) of the insulin analogue prepared is different from the amino acid residue at the corresponding site in the molecule of human insulin.

Claim 15

A method according to any one of Claims l to 4, wherein, in the insulin analogue prepared, X in position B27 is Glu, X in position Bl2 is Ile, or Tyr, X in position A2l is Asp and position B27 is Glu, X in position B9 is Asp, X in position A2l and in position B9 is Asp and in position B27 is Glu, X in position A8 is His, in position B9 is Asp and in position B27 is Glu, X in position Bl0 is Asp, or X in position B9 is Asp and in position B27 is Glu, or X in position B28 is Asp.

Claim 16

A method according to any one of Claims l to 4, wherein the insulin analogue prepared lacks up to four amino acid residues at the N-terminal of the B-chain and/or up to five amino acid residues at the C-terminal end of the B-chain.