Method of production of human insulin methyl ester

1. A method of obtaining a human insulin methyl ester, the method comprising contacting, in the presence of trypsin, insulin desB30 with L-threonine methyl ester in a solvent, the solvent comprising water, dimethyl sulfoxide and dimethylformamide in a ratio of water/dimethyl sulfoxide from about 1:1 (v/v) to about 1:0.5 (v/v), and a ratio of water/dimethylformamide from about 1:0.6 (v/v) to about 1:0.3 (v/v); and wherein the solvent further comprises dioxane, wherein dioxane is present in the solvent in a ratio of water/dioxane of about 1:0.2 (v/v) or less.

2. The method of claim 1 , wherein the ratio of water/dimethyl sulfoxide in the solvent is from about 1:0.8 (v/v) to about 1:0.7 (v/v), and the ratio of water/dimethylformamide is from about 1:0.45 (v/v) to about 1:0.35 (v/v).

3. The method of claim 1 , wherein the ratio of water/dimethyl sulfoxide in the solvent is about 1:0.74 (v/v), the ratio of water/dimethylformamide is about 1:0.4 (v/v), and the ratio of water/dioxane is about 1:0.19 (v/v).

4. The method of claim 1 , wherein the solvent contains tris(hydroxymethyl)aminomethane (Tris).

5. The method of claim 1 , wherein the solvent comprises in an amount from about 0.03 to about 0.3 M at least one selected from the group consisting of carbonate salt, sodium carbonate, potassium carbonate, bicarbonate salt, sodium hydrogen carbonate, and potassium hydrogen carbonate, or a hydrate thereof.

6. The method of claim 1 , wherein desB30 is obtained from at least one selected from the group consisting of (a) a precursor insulin, and (b) human preproinsulin.

7. The method of claim 6 , wherein desB30 is obtained by contacting the precursor insulin with trypsin in a buffer of about 0.05M to 0.125M histidine comprising at least one selected from the group of (a) water and dioxane in a ratio of water/dioxane from about 1:0.5 (v/v) to about 1:0.3 (v/v); and (b) water and dimethylformamide in a ratio of water/dimethylformamide from about 1:0.9 (v/v) to about 1:0.3 (v/v).

8. The method of claim 7 , wherein the solvent in obtaining desB30 further comprises a carbonate and/or a bicarbonate salt in an amount of about 0.05 to about 0.3 M.

9. The method of claim 7 , wherein the solvent in obtaining desB30 does not contain tris(hydroxymethyl)aminomethane (Tris).

10. The method of claim 1 , wherein contacting desB30 with L-threonine methyl ester is carried out in the presence of trypsin at an amount selected from the group consisting of about 20 U or more trypsin per mg of desB30, and about 50 U or more trypsin per mg of desB30.

11. The method of claim 1 , wherein contacting desB30 with L-threonine methyl ester is carried out at a pH in the range from about 6.0 to about 7.5.

12. The method of claim 1 , further comprising collecting the produced human insulin methyl ester after a period of 7 hours or more at a temperature from about 18° C. to about 30° C.

13. The method of claim 12 , comprising crystallising the produced human insulin methyl ester by adding citrate buffer and zinc chloride, and maintaining the solution at about 12° C. or less.

14. The method of claim 13 , wherein crystallising the produced human insulin methyl ester comprises, after adding citrate and zinc chloride, diluting the solvent with water to an organic solvent concentration of about 13% or less.

15. The method of claim 7 , wherein the buffer in obtaining desB30 has a pH in the range from about 8.5 to about 10.0.

16. The method of claim 7 , wherein contacting desB30 with L-threonine methyl ester is carried out in the presence of trypsin at an amount selected from the group consisting of about 10 U or more trypsin per mg desB30, and about 20 U or more trypsin per mg of the precursor insulin.

17. The method of claim 12 , wherein the temperature is about 25° C.

18. The method of claim 13 , wherein the amount of zinc chloride is selected from the group consisting of about 0.5 mmol to about 5 mmol per gram of human insulin methyl ester, and about 1 mmol to about 2 mmol zinc chloride per gram of human insulin methyl ester.

19. The method of claim 13 , wherein the citrate buffer comprises 0.02 to 0.06 molar citric acid and 0.2 to 0.6 molar disodium hydrogen orthophosphate.