Process for preparing human insulin or B-30 esters thereof

• US 4,601,979 A
• Filed: 04/11/1983
• Issued: 07/22/1986
• Est. Priority Date: 06/07/1982
• Status: Expired due to Term

• Current Assignee: Nordisk Insulinlaboratorium
• Original Assignee: Nordisk Insulinlaboratorium
• Inventors: Balschmidt, Per, Andresen, Finn H., Hejnos, Kim R., Kofod, Hans
• Primary Examiner(s): Wiseman, Thomas G.
• Assistant Examiner(s): Huleatt, Jayme A.
• Application Number:
  US06/491,325
• Time in Patent Office: 1,198 Days
• Field of Search: 435/70, 435/71, 435/212, 435/213, 435/68, 435/69, 435/262
• US Class Current: 435/68.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/62 Insulins
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

Human insulin or B-30 esters thereof are prepared by reacting an insulin derivative of the formula ##STR1## wherein R is hydroxyl or an amino acid radical being different from threonine, and -A- and -B- represent the A- and B-chains with the same amino acid sequence as in human insulin, with a carboxyl protected and optionally hydroxyl protected L-threonine derivative in a concentration of from 2 to about 6 moles per liter in the presence of trypsin or a trypsin-like enzyme in a reaction medium containing water and optionally also an organic cosolvent at a pH value of from 5 to 9 and at a temperature below 50° C., followed, if desired, by removal of any protecting groups present.

Human insulin is prepared in an easy and simple way in high yield and in high purity.

Claims

1. A process for the preparation of a B-30 ester of human insulin by reacting a carboxyl protected L-threonine derivative with an insulin derivative of the formula ##STR3## wherein R is hydroxyl or an amino acid radical being different from threonine, and --A-- and --B-- represent the A- and B-chains with the same amino acid sequence as in human insulin, in the presence of a proteolytic enzyme, capable of splitting lysine carbonyl peptide bonds, in an aqueous reaction medium substantially free of organic cosolvent, at a pH value of from 5 to 9 and at a temperature below 50°

C., and for a time sufficient to accumulate the human insulin ester, wherein the L-threonine derivative is used in the reaction mixture in a concentration of from 2 to about 6 moles/liter.

2. A process of claim 1 wherein the L-threonine derivative is present in the reaction mixture in a concentration of from 3 to 5 moles/liter.

3. A process of claim 1, wherein the insulin derivative used is desalanine-B30 insulin (DAI) derived from porcine insulin.

4. A process of claim 1, wherein the insulin derivative is porcine insulin, R representing alanine.

5. A process of claim 1, wherein the L-threonine derivative is a methyl, ethyl, or t-butyl ester of L-threonine.

6. A process of claim 1, wherein the proteolytic enzyme is trypsin.

7. A process of claim 1, wherein the carboxyl protected L-threonine derivative is also hydroxyl protected.

8. A process of claim 1, wherein any protecting groups present following the reaction of said L-threonine derivative with the said insulin derivative are removed.

9. A process of claim 2, wherein the insulin derivative used is desalanine-B30 insulin (DAI) derived from porcine insulin.

10. A process of claim 2, wherein the insulin derivative is porcine insulin, R representing alanine.

11. A process of claim 2 wherein the L-threonine derivative is a methyl, ethyl or t-butyl ester of L-threonine.

12. A process of claim 2 wherein the proteolytic enzyme is trypsin.

13. A process of claim 3, wherein the L-threonine derivative is a methyl, ethyl, or t-butyl ester of L-threonine.

14. A process of claim 3, wherein the proteolytic enzyme is trypsin.

15. A process of claim 4, wherein the L-threonine derivative is a methyl, ethyl, or t-butyl ester of L-threonine.

16. A process of claim 4, wherein the proteolytic enzyme is trypsin.

17. A process of claim 5, wherein the proteolytic enzyme is trypsin.