PRABHU SUREKHA K (IN)
SHIVAKUMAR M C (IN)
SENGUPTA PRIJYAJIT (IN)
TIWARI SANJAY (IN)
MENDHE RAKESH (IN)
PATIL NITIN (IN)
ADHIKARY LAXMI (IN)
KHEDKAR ANAND (IN)
MELARKODE RAMAKRISHNAN (IN)
GURURAJA RAMAVANA (IN)
SURYANARAYAN SHRIKUMAR (IN)
KULKARNI MADHAV (IN)
PRABHU SUREKHA K (IN)
SHIVAKUMAR M C (IN)
SENGUPTA PRIJYAJIT (IN)
TIWARI SANJAY (IN)
MENDHE RAKESH (IN)
PATIL NITIN (IN)
ADHIKARY LAXMI (IN)
KHEDKAR ANAND (IN)
MELARKODE RAMAKRISHNAN (IN)
GURURAJA RAMAVANA (IN)
SURYANARAYAN SHRIKUMAR (IN)
WO2003068980A2 | 2003-08-21 |
EP0184162B1 | 1994-04-27 |
See also references of EP 1756290A4
1. | A new strain of Streptomycessp. BICC 7522. 2. A new strain of Streptomycessp. BICC 7522, a variant or mutant thereof. 3. Use of the new strain of claim 1 or 2, for the production of macrolides. 4. A use of the new strain as in claim 3, comprising a) fermentation of a new strain of Streptomyces BICC 7522 and b) isolation and purification of the macrolide. 5. A process of claim 4, wherein the macrolide is isolated from broth or aqueous phase by extraction with water immiscible organic solvent. 6. A process of claim 5, wherein the organic solvent is ethyl acetate. 7. A process of claim 5, wherein the organic phase containing product is concentrated. 8. A process of claim 5 or 7, wherein the organic phase containing the product is treated with ammonia gas. 9. A process of claim 8, wherein the precipitated impurities are separated from the organic phase. 10. A process of claim 9, wherein the separation of the precipitated impurities is effected by either filtration or centrifugation. 11. A process of claim 9, wherein the organic phase containing macrolide is optionally concentrated. 12. A process of claim 4, 5, 7, 9 or 11 wherein the organic phase or the concentrate containing product is subjected to chromatography. 13. A process of claim 12, wherein the chromatography stationary phase is silica. 14. A process of claim 13, wherein the silica is optionally pretreated with silver ion. 15. A process of claim 14, wherein the source of silver ion is silver nitrate. 16. A process of claim 12, wherein the chromatography stationary phase is reversed phase silica. 17. A process of claim 12, wherein the macrolide is eluted with organic solvent or mixture of solvents. 18. A process of claim 14, wherein the solvent or mixture of solvents selected are one or more among ethyl acetate, propyl acetate, butyl acetate, alkyl alcohols, chloroform, dichloromethane, hexane, heptane, isooctane, petroleum ether. 19. A process of any preceding claim, wherein the product is in solution with organic solvent. 20. A process as in claim 19, wherein the organic solvent is selected from solvents in which the product is in dissolved state at room temperature or at elevated temperature. 21. A process as in claim 18, wherein the organic solvent is selected from one or more among methyl acetate, ethyl acetate, propyl acetate, butyl acetate, acetonitrile, diethyl ether, methanol, ethanol, propyl alcohol, butanol, tbutyl methyl ether, acetone, chloroform and dichloromethane. 22. A process as in claim 18, wherein the organic solvent is ethyl acetate. 23. A process as in claim 18, wherein the product is crystallized or precipitated from organic solvent by addition of antisolvent. 24. A process as in claim 19, wherein the antisolvent is selected from one or more among water, hexane, heptane, isooctane and petroleum ether. 25. A process as in claim 23, wherein the antisolvent is hexane. 26. Substantially pure macrolide afforded by any preceding claim. 27. A process of crystallization or precipitation of macrolides from ethyl acetate by addition of hexane. 28. A process of crystallization or precipitation of Tacrolimus from ethyl acetate by addition of hexane. 29. A process of purification of macrolide by treating a solution of the compound with gaseous ammonia. 30. A process of purification of Tacrolimus by treating a solution of the compound with gaseous ammonia. 31. A process of purification of macrolide by silica chromatography treated with silver. 32. A process of purification of Tacrolimus by silica chromatography treated with silver. 33. A process of purification of macrolide by reversed phase chromatography. 34. A process of purification of Tacrolimus by reversed phase chromatography. 35. Substantially pure macrolide obtained by reversed phase chromatography. 36. Substantially pure macrolide obtained by purification on silica chromatography treated with silver. 37. Substantially pure macrolide containing less than 0.5% total impurities obtained by reversed phase chromatography. 38. Substantially pure Tacrolimus containing less than 0.5% total impurities obtained by reversed phase chromatography. 39. Substantially pure macrolide containing less than 0.5% total impurities obtained by silica chromatography treated with silver. 40. Substantially pure Tacrolimus containing less than 0.5% total impurities obtained by silica chromatography treated with silver. 41. Substantially pure macrolide containing total impurities less than 1%. 42. Substantially pure Tacrolimus containing total impurities less than 1%. 43. Substantially pure Tacrolimus containing less than 0.5% of 17 ethyl analog. 44. Substantially pure Tacrolimus containing less than 0.5% of 17 ethyl analog obtained by a process of any preceding claim. 45. Substantially pure Tacrolimus containing less than 0.2% of 17 ethyl analog. 46. Substantially pure Tacrolimus containing less than 0.2% of 17 ethyl analog obtained by a process of any preceding claim. 47. Tacrolimus substantially free of its 17ethyl analog. 48. Substantially pure Tacrolimus. 49. A process of any preceding claim, wherein the macrolide is Tacrolimus. 50. A process of any preceding claim, wherein the macrolide is Immunomycin. 51. A process of any preceding claim, wherein the macrolide is Sirolimus. AMENDED CLAIMS [received by the International Bureau on 13 September 2004 (13.09.04); Original claims 151 replaced by new claims 142 (4 pages).] 1 A new strain of Streptomycessp. BICC 7522. 2. A new strain of Streptomyces sp. BICC 7522, a variant or mutant thereof. 3. Use of the new strain of claim 1 or 2, for the production of macrolides. 4. A use of the new strain as in claim 3, comprising a) fermentation of a new strain of Streptomyces BICC 7522 and b) isolation and purification of the macrolide. 5. A process of claim 4, wherein the macrolide is isolated from broth or aqueous phase by extraction with water immiscible organic solvent. 6. A process of claim 5, wherein the organic solvent is ethyl acetate. 7. A process of claim 5, wherein the organic phase containing product is concentrated* 8» A process of claim 5 or 7, wherein the organic phase containing the product is treated with ammonia gas. 9, A process of claim 8, wherein the precipitated impurities are separated from the organic phase. 10, A process of claim 9, wherein the separation of the precipitated impurities is effected by either filtration or centrifugation. 11, A process of claim 9, wherein the organic phase containing macrolide is optionally concentrated. |
2. | 22 12. A process of claim 4, 5, 7, 9 or 11 wherein the organic phase or the u concentrate containing product is subjected to chromatography. 13» A process of claim 12, wherein the chromatography stationary phase is silica. 14, A process of claim 13, wherein the silica is optionally pretreated with silver ion. 15. A process of claim 14, wherein the source of silver ion is silver nitrate. 16» A process of claim 12, wherein the chromatography stationary phase is reversed phase silica. 17. A process of claim 12, wherein the macrolide is eluted with organic solvent or mixture of solvents. IS, A process of claim 14, wherein the solvent or mixture of solvents selected are one or more among ethyl acetate, propyl acetate, butyl acetate, alkyl alcohols, chloroform, dichloromethane, hexane, heptane, isooctane, petroleum ether. 19. A process of any preceding claim, wherein the product is in solution with organic solvent. 20. A process as in claim 19, wherein the organic solvent is selected from solvents in which the product is in dissolved state at room temperature or at elevated temperature. 21. A process as in claim 18, wherein the organic solvent is selected from one or more among methyl acetate, ethyl acetate, propyl acetate, butyl acetate, acetonitrile, diethyl ether, methanol, ethanol, propyl alcohol, butanol, tbutyl methyl ether, acetone, chloroform and dichloromethane. 22. A process as in claim 18, wherein the organic solvent is ethyl acetate. 23. A process as in claim 18, wherein the product is crystallized or precipitated from organic solvent by addition of antisolvent. 24. A process as in claim 19, wherein the antisolvent is selected from one or more among water, hexane, heptane, isooctane and petroleum ether. 25. A process as in claim 23, wherein the antisolvent is hexane. 26. Substantially pure macrolide afforded by any preceding claim. 27. A process of any preceding claim, wherein the macrolϊde is Tacrolimus. 28. A process of any preceding claim, wherein the macrolide is Immunomycini 29. A process of any preceding claim, wherein the macrolide is Sirolimus. 30. Substantially pure Tacrolimus obtained by any preceding claim. 31. Substantially pure Tacrolimus containing less than 0.5% of 17 ethyl analog obtained by a process of any preceding claim. 32. Substantially pure Tacrolimus containing less than 0.2% of 17 ethyl analog obtained by a process of any preceding claim. 33. A process of purification of macrolide as in claim 4(b) by treating a solution of the compound with gaseous ammonia. |
3. | 24 34. A process of purification of macrolide as in claim 4(b) by silica chromatography treated with silver* 35. A process of purification of Tacrolimus by treating a solution of the compound with gaseous ammonia. 36. A process of purification of Tacrolimus by silica chromatography treated with silver, 37. Substantially pure macrolide obtained by a process of purification by reversed phase chromatography wherein the elution is performed with a mixture comprising acetonitrilet n butanol: buffer. 38. A process of claim 36 wherein the ratio of acetonltrile: n butanol: buffer is 12.5: 10: 77.5. 39. A process of claim 36 wherein the buffer comprises KH2PO4, triethyl amine, phosphoric acid. 40. Substantially pure Tacrolimus obtained by a process of purification by reversed phase chromatography wherein the elution is performed with a mixture comprising acetonitrile: n butanol: buffer. 41. A process of claim 36 wherein the ratio of acetonitrile: n butanol: buffer is 12.5: 10: 77,5, 42. A process of claim 36 wherein the buffer comprises KH2PO4, triethyl amine, phosphoric acid, 25. |
Sufficient seed for running a 20 L fermentor is generated by sub-culturing above seed in to a 2 L flask containing 350 ml of above mentioned seed medium. After a growth for 48 log hours, 170 ml of inoculum was transferred to 17 L of production medium sterilized at 121 ° C for 1 h holding period in a 20 L fermentor. Following production medium was employed for the experiment Ingredients Concentration g/L) Soluble Starch 90 Corn Steep Liquor 10 Dried Yeast 20 CaCO3 1 pH adjusted to 6.8 +/- 0.1 with NaOH before sterilization. Throughout the fermentation, pH was controlled at 6.8 +/- 0.2 with sodium hydroxide or orthophosphoric acid. Incubation temperature was controlled at 23+/-10C throughout the batch. Aginations, airflow, head pressure of fermentor were manipulated in order to control dissolved oxygen above 25 %. Samples were withdrawn every day for analysis of product content. Batch was continued till the activity stabilized in the broth. Final yield was 194 mg/L in 180 h with 15 mg/L of Ascomycln. Example 2 Production of Tacrolimus by fermentation of mutant of new Streptomyces sp. The mutant was obtained by classical UV mutation technique. Inoculum was developed in the same way as in the previous example. Production medium was used as given below. Ingredients Concentration( g/L) Soluble Starch 99 Corn Steep Liquor 11 Cotton Seed Flour 2.5 Dried Yeast 20 CaCO3 1 pH adjusted to 6.8 +/- 0.1 with NaOH before sterilization. Throughout the batch, pH was controlled at 6.8 +Λ 0.2 with sodium hydroxide or orthophosphoric acid. Incubation temperature was controlled at 23+/-1 ° C throughout the batch. Agitations, airflow, head pressure of fermentor were manipulated in order to control dissolved oxygen above 25 %. On sixth day, a feed of following medium composition is prepared and added in such a way that volume of the fermentor broth increases by 10 %. Ingredients Concentration g/L) Dried yeast 44 Cotton seed flour 27.5 Starch 200 CaCO3 1
Samples were withdrawn every day for analysis of product content. Batch was harvested at 184 h and it gave a final activity of 254 mg/L with 25 mg/L of Ascomycin. Example 3 Production of Tacrolimus by fermentation of new Streptomyces sp. Inoculum was developed in the same way as in the previous example. However, modified production medium was used as given below. Ingredients Conceπtration( g/L) Soluble Starch 100 (NH4)2SO4 3 Corn Steep Liquor 15 Soya Flour 5 Cotton Seed Fiour 10 Dried Yeast 45 CaCO3 1 pH adjusted to 6.8 +/- 0-1 with NaOH before sterilization. Throughout the batch, pH was controlled at 6.8 +/- 0.2 with sodium hydroxide or orthophosphoric acid. Incubation temperature was controlled at 23+/-1 ° C " throughout the batch. Agitations, airflow, head pressure of fermentor were manipulated in order to control dissolved oxygen above 25 %. Samples were withdrawn every day for analysis of product content. Batch was harvested at 274 h and it gave a final activity of 337 mg/L with 30 mg/L of Ascomycin. Example 4 Isolation and purification of the Tacrolimus The fermentation broth (30 Kg) containing 10 g Tacrolimus was extracted with 30 L of ethyl acetate. The ethyl acetate extract was partially concentrated to 2.4 L. The concentrate was chilled to 40C and ammonia was sparged through the concentrate for 30 minutes. The solution was filtered using celite as filter aid to separate the precipitated impurities. The filtrate was concentrated to obtain 82 g oily residue. The residue was applied on a silica gel column. The column was washed with 3 column volumes of 25% ethyl acetate in. hexane and 3 column volumes of 50% ethyl acetate in hexane. The product was eluted with 75% ethyl acetate in hexane. The product containing fractions were pooled and concentrated to obtain 26 g oily residue. The residue was dissolved in 200 mi ethyl acetate. 27 g of activated charcoal was added to it. The mixture was stirred for 20 minutes and then filtered. The filtrate was concentrated to obtain 18 g of oiiy residue. To the residue, 5 ml of ethyl acetate was added. The crude product was crystallized at 40C by slow addition of hexane. The crude product (6.2 g) was filtered and dried. Example 5 Isolation and purification of Tacrolimus 3.1 g of crude product obtained in example 4 was applied to a 3-L silica gel column. Silica gel (230-400 mesh) was initially treated with silver nitrate. The column was eluted with 75% ethy! acetate and 25% hexane. The product containing fractions with acceptable purity were pooled and concentrated. The product was crystallized as mentioned earlier from ethyl acetate and hexane. The crystals were filtered and dried. Substantially pure Tacrolimus was afforded by this method. The Ascomycin concentration in the final product was less than 0.2%. Example 6 Isolation and purification of the Tacrolimus l. 3.1 g of crude product obtained in example 4 was applied to a C-8 reverse phase column (d ~ 50 mm, L ~ 210 mm). The product was eluted with acetonitrile: n-butanol: buffer in the ratio of 12.5: 10: 77.5. The buffer contained 1.36 g/L of KH2PO4, 1 ml/L Methyl amine, 1 ml/L phosphoric acid. The product containing fractions with acceptable purity were pooled and extracted with equal volume of ethyl acetate. The extract was washed with water, dried with sodium sulfate and concentrated. The product was crystallized as mentioned earlier from ethyl acetate and hexane. The crystals were filtered and dried. Substantially pure Tacrolimus was afforded by this method. The Ascomycin concentration in the final product was less than 0.1%.