Method for extracting a macrolide from biomatter

1. A method for obtaining a macrolide selected from the group consisting of tacrolimus, ascomycin, pimecrolimus, sirolimus, and everolimus comprising the step of extracting macrolide-containing biomatter that is whole fermentation broth with a hydrophobic extraction solvent to obtain a solution of the macrolide in the hydrophobic extraction solvent, wherein the pH of the macrolide-containing biomatter being extracted is between 8 and about 13 and recovering said macrolide from said solution, wherein the macrolide-containing biomaffer is tacrolimus-containing biomatter when tacrolimus is to be obtained, ascomycin-containing biomatter when ascomycin is to be obtained, pimecrolimus-containing biomatter when primecrolimus is to be obtained, sirolimus-containing biomatter when sirolimus is to be obtained, and everolimus-contaming biomatter when everolimus is to be obtained.

2. The method of claim 1 wherein the hydrophobic extraction solvent is selected from the group consisting of n-butanol, iso-butanol, C 2 -C 6 linear and branched esters of acetic acid or formic acid, C 3 -C 6 linear or branched aliphatic ketones, halogenated methanes, halogenated ethanes, and aromatic hydrocarbons that are liquid at 25° C. and that have a boiling point at atmospheric pressure less than about 150° C.

3. The method of claim 2 wherein the hydrophobic extraction solvent is selected from the group consisting of n-butanol, iso-butanol, n-butyl acetate, iso-butyl acetate, t-butyl acetate, ethyl acetate, propyl acetate, ethyl formate, butyl methyl ketone, methyl iso-butyl ketone, dichloromethane, chloroform, tetrackloromethane, dichioroethane, and toluene.

4. The method of claim 3 wherein the hydrophobic extraction solvent is ethyl acetate, iso-butyl acetate, or a mixture of these.

5. The method of claim 4 wherein the hydrophobic extraction solvent is iso-butyl acetate.

6. The method of claim 1 wherein the extraction is at a temperature between about 2° C. and about 70° C.

7. The method of claim 6 wherein the extraction is at a temperature between about 15° C. and about 35° C.

8. The method of claim 1 wherein the macrolide-containing biomatter is obtained from a microorganism selected from Streptomyces tsukubaensis, Streptomyces hygroscopicus, Streptomyces lividans, Streptomyces hygroscopicus hygroscopicus, and Streptomyces hygroscopicus ascomyceticus.

9. The method of claim 1 wherein the macrolide is ascomycin.

10. The method of claim 1 wherein the macrolide is pimecrolimus.

11. The method of claim 1 wherein the macrolide is sirolimus.

12. The method of claim 1 wherein the macrolide is everolimus.

13. The method of claim 1 further comprising the steps of, after extraction:

separating the solution contaming the macrolide from the extracted macrolide-containing biomatter to obtain a separated macrolide-containing solution,

concentrating the separated macrolide-contaming solution,

loading the concentrated macrolide-containing solution onto a silica gel column and eluting with an eluent to obtain at least one macrolide-containing fraction that is a macrolide-containing solution,

and isolating the macrolide.

14. A method for obtaining a macrolide selected from the group consisting of tacrolimus, ascomycin, pimecrolimus, sirolimus, and everolimus from macrolide-containing biomatter, wherein the macrolide-containing biomatter is tacrolimus-containing biomatter when tacrolimus is to be obtained, ascomycin-containing biomatter when ascomycin is to be obtained, pimecrolimus-containing biomatter when pimecrolimus is to be obtained, sirolimus-containing biomatter when sirolimus is to be obtained, and everolimus-containing biomatter when everolimus is to be obtained, comprising the steps of:

a) extracting macrolide-containing biomaffer that is whole fermentation broth obtained by fermentation of a microorganism selected from the group consisting of Streptomyces tsukubaensis, Streptomyces hygroscopicus, Streptomyces lividans, Streptomyces hygroscopicus hygroscopicus , and Streptomyces hygroscopicus ascomyceticus with a hydrophobic extraction solvent selected form the group consisting of n-butanol, iso-butanol, n-butyl acetate, iso-butyl acetate, t-butyl acetate, ethyl acetate, propyl acetate, ethyl formate, butyl methyl ketone, dichloromethane, chloroform, tetrachioromethane, dichloroethane and toluene to obtain a solution of macrolide in hydrophobic extraction solvent at a temperature between about 20° C. and about 70° C. and a pH of between 8 and about 13,

b) separating the macrolide-containing solution from the extracted macrolide-containing biomatter,

c) concentrating the macrolide-contaiing solution,

d) loading the concentrated macrolide-containing solution onto a silica gel chromatography column and eluting the column with an eluent to obtain at least one macrolide-containing fraction that is a macrolide-containing solution, and

e) separating the macrolide.

15. The method of claim 14 wherein the hydrophobic extraction solvent is iso-butyl acetate.

16. The method of claim 14 wherein the extraction is at a temperature between about 15° C. and about 30° C.

17. A method for obtaining a macrolide selected from the group consisting of tacrolimus, ascomycin, pimecrolimus, sirolimus, and everolimus from macrolide-containing biomatter, wherein the macrolide-containing biomatter is tacrolimus-containing biomatter when tacrolimus is to be obtained, ascomycin-containing biomatter when ascomycin is to be obtained, pimecrolimus-containing biomatter when pimecrolimus is to be obtained, sirolimus-containing biomatter when sirolimus is to be obtained, and everolimus-contaming biomafter when everolimus is to be obtained, the step of extracting macrolide-containing biomatter that is whole fermentation broth with a hydrophobic extraction solvent selected from the group consisting of n-butyl acetate, iso-butyl acetate, t-butyl acetate, ethyl acetate, propyl acetate, ethyl formate, butyl methyl ketone, dichioromethane, chloroform, tetrachloromethane, and toluene, wherein the pH of the macrolide-containing biomatter is between 8 and about 13 and recovering said macrolide from said solution.

18. A method of obtaining a macrolide selected from the group consisting of tacrolimus, ascomycin, pimecrolimus, sirolimus, and everolimus from macrolide-containing biomatter, wherein the macrolide-containing biomatter is tacrolimus-containing biomatter when tacrolimus is to be obtained, ascomycin-containing biomatter when ascomycin is to be obtained, pimecrolimus-containing biomafter when punecrolimus is to be obtained, sirolimus-containing biomatter when sirolimus is to be obtained, and everolimus-contaming biomaffer when everolimus is to be obtained, comprising the steps of:

a) extracting a macrolide-containing biomaffer that is whole fermentation broth obtained by fermentation of a microorganism selected from the group consisting of Streptomyces tsukubaensis, Streptomyces hygroscopicus, Streptomyces lividans, Streptomyces hygroscopicus hygroscopicus , and Streptomyces hygroscopicus ascomyceticus , wherein the pH of the macrolide-containing biomaffer is between 8 and about 13, with iso-butyl acetate to obtain a solution of

a macrolide in iso-butyl acetate at a temperature between about 2° C. and about 70° C.,

b) separating the macrolide-containing solution from the extracted macrolide-containing biomatter, to obtain a concentrated macrolide-containing solution,

c) concentrating the macrolide-containing solution,

d) loading the concentrated macrolide-containing solution onto a silica gel chromatography column and eluting with an eluent to obtain at least one macrolide-containing fraction that is a macrolide-containing solution,

e) concentrating at least one macrolide-containing fraction, and

f) separating the macrolide.

19. The method of claim 18 wherein the pH of the macrolide-containing biomatter is between about 10 and about 13.

20. The method of claim 18 wherein the temperature of extraction is between about 15° C. and about 35 C.

21. The method of claim 6 , wherein the extraction is at a temperature of from about 45° C. to about 70 C.

22. The method of claim 1 , wherein the macrolide is tacrolimus.

23. The method of claim 1 , wherein the pH of the biomaffer is between about 10 and about 13.

24. The method of claim 1 , wherein the pH of the biomatter is between 8 and about 10.

25. The method of claim 1 , wherein the yield of the macrolide obtained from macrolide-containing biomatter is about 66% to about 94%.

26. The method of claim 1 , wherein the yield of the macrolide obtained from macrolide-containing biomaffer is about 80% to about 94%.

27. The method of claim 14 , wherein the pH of the biomatter is between about 10 and about 13.

28. The method of claim 14 , wherein the pH of the biomaffer is between 8 and about 10.

29. The method of claim 17 , wherein the pH of the biomafter is between about 10 and about 13.

30. The method of claim 17 , wherein the pH of the biomatter is between 8 and about 10.

31. The method of claim 18 , wherein the pH of the biomaffer is between 8 and about 10.

32. The method of claim 1 , wherein the method is carried out using a single extraction step.

33. The method of claim 14 , wherein the method is carried out using a single extraction step.

34. The method of claim 17 , wherein the method is carried out using a single extraction step.

35. The method of claim 17 , wherein the method is carried out using a single extraction step.

36. The method of claim 1 , wherein the extracting step is carried out with only a hydrophobic extraction solvent.

37. The method of claim 14 , wherein the extracting step is carried out with only a hydrophobic extraction solvent.

38. The method of claim 17 , wherein the extracting step is carried out with only a hydrophobic extraction solvent.

39. The method of claim 18 , wherein the extracting step is carried out with only a hydrophobic extraction solvent.

40. The method of claim 14 , wherein the yield of the macrolide obtained from macrolide-containing biomatter is about 66% to about 94%.

41. The method of claim 14 , wherein the yield of the macrolide obtained from macrolide-containing biomatter is about 80% to about 94%.

42. The method of claim 17 , wherein the yield of the macrolide obtained from macrolide-containing biomatter is about 66% to about 94%.

43. The method of claim 17 , wherein the yield of the macrolide obtained from macrolide-containing biomatter is about 80% to about 94%.