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US 10,745,457 B2Granted
Process for obtaining insulin with correctly formed disulfide bonds
AssigneeMerck Sharp & Dohme Corp.
InventorsAllison D. Ortigosa, Rebecca A. Chmielowski, Mark C. Sleevi
Life of this patent
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Abstract
A process for solubilization and refolding of precursor insulin or insulin analogs from inclusion body isolates for use in the production of insulin or insulin analog is described.
Claims16
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 more elements
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.
4 more elements
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.
5 more elements
Dependencies are inferred from the claim text.