Methods for the preparation, isolation and purification of epothilone B, and X-ray crystal structures of epothilone B

1. A process for isolation of epothilone B from an epothilone-producing microorganism comprising:

(a) fermenting a strain of epothilone-producing microorganism in the presence of a resin that adsorbs epothilone B by hydrophobic interaction;

(b) collecting the resin in a water-based medium;

(c) extracting the resin with a solvent selected to extract epothilone B and to separate it from the water-based medium; and

(d) crystallizing epothilone B from the extraction phase; wherein said fermentation step further comprises feeding an additive capable of improving the amount of epothilone B produced as compared with the amount of epothilone A produced that increases the ratio of epothilone B to epothilone A produced from the fermentation, as compared with when the fermentation is performed without feeding the additive.

2. The process of claim 1 wherein the crystallized epothilone B from step (d) is substantially pure.

3. The process of claim 1 wherein the resin is extracted with a polar solvent.

4. The process of claim 1 wherein said fermentation step further comprises fermenting said epothilone-producing microorganism in the presence of skim milk, soy flour, yeast extract, maltrin starch, and/or glycerol.

5. The process of claim 1 wherein said fermentation step comprises continuously feeding said additive capable of improving the ratio of epothilone B to epothilone A.

6. The process of claim 1 wherein said additive is a propionic acid salt or ester.

7. The process of claim 6 wherein said additive is sodium propionate, propionic acid methyl ester or propionic acid ethyl ester.

8. The process of claim 1 wherein the crystallization is conducted to reduce the amount of epothilone A to about 55% or less of the amount of epothilone A present after extraction step (c).

9. The process of claim 8 further comprising

(e) at least a second crystallization step effective to reduce the amount of epothilone A to about 55% or less of the amount of epothilone A present after crystallization step (d).

10. The process of claim 1 wherein the epothilone-producing microorganism is a strain of Sorangium cellulosum.

11. The process of claim 10 1 , wherein said epothilone- producing microorganism is Sorangium cellulosum strain ATCC No. PTA 3880.

12. The process of claim 10 1 , wherein said epothilone- producing microorganism is Sorangium cellulosum strain ATCC No. PTA 3881.

13. The process of claim 1 wherein the resin is a styrene/divinylbenzene-based polymer.

14. The process of claim 13 wherein the resin is present in a range of from about 0.2 w/v % to about 5.0 w/v %.

15. The process of claim 1 wherein said step (d) comprises:

(i) adding a second solvent in which epothilone B is either not soluble or sparingly soluble;

(ii) removing at least a portion of the extraction solvent; and

(iii) transitioning the resultant solvent or solvent mixture to a temperature at which epothilone B crystallizes.

16. The process of claim 15 wherein the extraction solvent is ethyl acetate or MTBE, and the second solvent is toluene.

17. The process of claim 1 further comprising:

(f) prior to step (c), washing the resin with aqueous acetonitrile, or aqueous methanol, or an aqueous medium comprising a detergent and an amine reagent added in base form, the aqueous medium selected to not elute epothilone B.

18. The process of claim 1 , wherein step (c) further comprises polish filtering the epothilone B containing solvent.

19. The process of claim 1 , wherein epothilone B and epothilone A are produced in an epothilone B/A ratio of at least one.

20. The process of claim 1 , wherein epothilone B and epothilone A are produced in an epothilone B/A ratio of at least 1.5.

21. The process of claim 1 , wherein epothilone B and epothilone A are produced in an epothilone B/A ratio in the range of 1.5 to 4.0.