Process for obtaining insulin with correctly formed disulfide bonds

1. A process for obtaining a precursor molecule of insulin or insulin analog having correctly bonded cysteine bridges, which comprises:

(a) providing a suspension of inclusion bodies obtained from E. coli host cells, wherein the inclusion bodies comprise precursor molecules of insulin or insulin analog having incorrectly bonded cysteine bridges;

(b) solubilizing the inclusion bodies by adding urea, dithiothreitol (DTT), and ethanolamine to the suspension to provide a solubilization solution, wherein the solubilization solution comprises about 12 to 18 g/L of the precursor molecules of insulin or insulin analog having incorrectly bonded cysteine bridges, about 3.8 to 4.3 M urea, about 2.2 to 2.8 mM DTT, and about 265 to 365 mM ethanolamine, and has a pH of about 10.2 to 11.0;

(c) diluting the solubilization solution about 10-fold with a refold diluent solution comprising ethanolamine and cystamine to provide a refold solution having about 1.2 to 1.8 g/L of the precursor molecules of insulin or insulin analog having incorrectly bonded cysteine bridges, 0.38 to 0.43 M urea, 345 to 404 μM cystamine, about 0.22 to 0.28 mM DTT, and about 26.5 to 36.5 mM ethanolamine and has a pH of about 10.2 to 11.0; and

(d) incubating the refold solution with stirring to provide aeration for 6 to 12 hours to provide the precursor of insulin or insulin analog having correctly bonded cysteine bridges.

2. A process for obtaining an insulin or insulin analog having correctly bonded cysteine bridges, which comprises:

(a) providing a suspension of inclusion bodies obtained from E. coli host cells microbiological host comprising a precursor of insulin or insulin analog having incorrectly bonded cysteine bridges in an aqueous solution comprising urea, dithiothreitol (DTT), and ethanolamine to provide a solubilization solution, wherein the solubilization solution comprises about 12 to 18 g/L of the precursor molecules of insulin or insulin analog having incorrectly bonded cysteine bridges, about 3.8 to 4.3 M urea, about 2.2 to 2.8 mM DTT, and about 265 to 365 mM ethanolamine, and has a pH of about 10.2 to 11.0;

(b) diluting the solubilization solution about 10-fold with a refold diluent solution comprising ethanolamine and cystamine to provide a refold solution having about 1.2 to 1.8 g/L of the precursor molecules of insulin or insulin analog having incorrectly bonded cysteine bridges, 0.38 to 0.43 M urea, 345 to 404 μM cystamine, about 0.22 to 0.28 mM DTT, and about 26.5 to 36.5 mM ethanolamine and has a pH of about 10.2 to 11.0;

(c) incubating the refold solution with stirring to provide aeration for 6 to 12 hours to provide a refold solution comprising a precursor of insulin or insulin analog having correctly bonded cysteine bridges;

(d) reducing the pH of the refold solution from step (c) to about 9.0 to 9.4;

(e) reacting the precursor of insulin or insulin analog having correctly bonded cysteine bridges from step (d) with trypsin or a trypsin-like enzyme and optionally additionally with carboxypeptidase B or mixture of the enzymes to provide the insulin or insulin analog having correctly bonded cysteine bridges; and

(f) purifying the insulin or insulin analog having correctly bonded cysteine bridges to provide insulin or insulin analog having correctly bonded cysteine bridges.

3. The process of claim 2 , wherein step (e) comprises reacting the precursor insulin or insulin analog having correctly bonded cysteine bridges from step (d) with citraconic anhydride to produce a citraconylated precursor insulin or insulin analog having correctly bonded cysteine bridges and then reacting the citraconylated precursor insulin or insulin analog having correctly bonded cysteine bridges with trypsin or a trypsin-like enzyme and optionally additionally with the carboxypeptidase B or mixture of the enzymes to provide a citraconylated insulin or insulin analog having correctly bonded cysteine bridges.

4. The process of claim 3 , wherein step (f) comprises reacting the citraconylated insulin or insulin analog having correctly bonded cysteine bridges with acetic acid and then purifying the insulin or insulin analog having correctly bonded cysteine bridges to provide insulin or insulin analog having correctly bonded cysteine bridges.

5. A process for obtaining an insulin or insulin analog having correctly bonded cysteine bridges, which comprises:

(a) providing a suspension of inclusion bodies obtained from E. coli host cells microbiological host comprising a precursor of insulin or insulin analog having incorrectly bonded cysteine bridges in an aqueous solution comprising urea, dithiothreitol (DTT), and ethanolamine to provide a solubilization solution, wherein the solubilization solution comprises about 12 to 18 g/L of the precursor molecules of insulin or insulin analog having incorrectly bonded cysteine bridges, about 3.8 to 4.3 M urea, about 2.2 to 2.8 mM DTT, and about 265 to 365 mM ethanolamine, and has a pH of about 10.2 to 11.0;

(b) diluting the solubilization solution about 10-fold with a refold diluent solution comprising ethanolamine and cystamine to provide a refold solution having about 1.2 to 1.8 g/L of the precursor molecules of insulin or insulin analog having incorrectly bonded cysteine bridges, 0.38 to 0.43 M urea, 345 to 404 μM cystamine, about 0.22 to 0.28 mM DTT, and about 26.5 to 36.5 mM ethanolamine and has a pH of about 10.2 to 11.0;

(c) incubating the refold solution with stirring to provide aeration for 6 to 12 hours to provide a refold solution comprising a precursor of insulin or insulin analog having correctly bonded cysteine bridges;

(d) reducing the pH of the refold solution from step (c) to about 9.0 to 9.4;

(e) isolating the precursor of insulin or insulin analog having correctly bonded cysteine bridges by anion exchange chromatography;

(f) cleaving the precursor of insulin or insulin analog having correctly bonded cysteine bridges from step (e) with trypsin or a trypsin-like enzyme and optionally additionally with carboxypeptidase B or mixture of the enzymes to provide the insulin or insulin analog having correctly bonded cysteine bridges; and

(g) purifying the insulin or insulin analog having correctly bonded cysteine bridges.

6. The process of claim 5 , wherein step (f) comprises reacting the precursor insulin or insulin analog having correctly bonded cysteine bridges from step (e) with citraconic anhydride to produce a citraconylated precursor insulin or insulin analog having correctly bonded cysteine bridges and then reacting the citraconylated precursor insulin or insulin analog having correctly bonded cysteine bridges with trypsin or a trypsin-like enzyme and optionally additionally with the carboxypeptidase B or mixture of the enzymes to provide a citraconylated insulin or insulin analog having correctly bonded cysteine bridges.

7. The process of claim 6 , wherein step (g) comprises reacting the citraconylated insulin or insulin analog having correctly bonded cysteine bridges with acetic acid and then purifying the insulin or insulin analog having correctly bonded cysteine bridges to provide insulin or insulin analog having correctly bonded cysteine bridges.

8. The process of claim 1 , wherein the insulin or insulin analog is insulin glargine or insulin lispro.

9. The process of claim 2 , wherein the insulin or insulin analog is insulin glargine or insulin lispro.

10. The process of claim 5 , wherein the insulin or insulin analog is insulin glargine or insulin lispro.

11. The process of claim 1 , wherein the solubilization solution comprises about 15 g/L of the precursor molecules of insulin or insulin analog having incorrectly bonded cysteine bridges bodies, about 4.0 M urea, about 2.5 mM DTT, and about 315 mM ethanolamine, and has a pH at about 10.5.

12. The process of claim 1 , wherein the refold solution comprises about 1.6 g/L of the precursor molecules of insulin or insulin analog having incorrectly bonded cysteine bridges, 0.38 to 0.43 M urea, 384 μM cystamine, about 0.22 to 0.28 mM DTT, and about 26.5 to 36.5 mM ethanolamine, and has a pH of about 10.5.

13. The process of claim 2 , wherein the solubilization solution comprises about 15 g/L of the precursor molecules of insulin or insulin analog having incorrectly bonded cysteine bridges bodies, about 4.0 M urea, about 2.5 mM DTT, and about 315 mM ethanolamine, and has a pH at about 10.5.

14. The process of claim 2 , wherein the refold solution comprises about 1.6 g/L of the precursor molecules of insulin or insulin analog having incorrectly bonded cysteine bridges, 0.38 to 0.43 M urea, 384 μM cystamine, about 0.22 to 0.28 mM DTT, and about 26.5 to 36.5 mM ethanolamine, and has a pH of about 10.5.

15. The process of claim 5 , wherein the solubilization solution comprises about 15 g/L of the precursor molecules of insulin or insulin analog having incorrectly bonded cysteine bridges bodies, about 4.0 M urea, about 2.5 mM DTT, and about 315 mM ethanolamine, and has a pH at about 10.5.

16. The process of claim 5 , wherein the refold solution comprises about 1.6 g/L of the precursor molecules of insulin or insulin analog having incorrectly bonded cysteine bridges, 0.38 to 0.43 M urea, 384 μM cystamine, about 0.22 to 0.28 mM DTT, and about 26.5 to 36.5 mM ethanolamine, and has a pH of about 10.5.