CLIAMS:
1. The process for the purification of Pneumocandin B0 from fermentation broth comprising.
a) Extracting the Pneumocandin B0 from whole broth or by separated biomass from fermentation broth by using suitable solvent;
b) Concentration of solvent containing product;
c) Liquid –Liquid extraction by suitable solvent;
d) Carbon treatment;
e) Precipitation.

2. The process of Claim1, where in the solvent used for extraction from whole broth or filtered biomass is n-butanol, isobutanol and methanol.

3. The process of Claim 2, where in the solvent is n-butanol.

4. The process of Claim 1, where in the impurities were separated by liquid –liquid extraction by suitable solvent.

5. The process of Claim 4, where in the suitable solvents are (cyclohexane or methylcyclohexane, methanol and water).

6. The Process of Claim 4, where in the suitable solvents are methylcyclohexane, methanol and water.

7. The process of Claim 4, where in the ratio of the solvent is more important for the formation of phases.

8. The process of Claim 7, where in the suitable solvent ratios are 1:0.8:0.6:1.3 (n-butanol: methanol: water: methylcyclohexane)

9. The process of Claim 1, where in the separated product n-butanol layer is treated with activated carbons.

10. The process of Claim 9, where in the suitable carbons are Norit SA plus, CN1 and CGP super activated carbons.

11. The process of Claim 1, where in the precipitation is carried out by using antisolvent

12. The process of Claim 11, where in the antisolvent is acetonitrile.

13. The process of Claim 12, where in the cooling temperature for precipitation is 0 to 10 deg C.

14. The process of Claim 13, where in the suitable temperature is about 5 deg C.

15. The process of Claim 1, where in the purity of the dried product is 50-72%

16. The process of Claim 1, where in over all yield of product 60-71%
,TagSPECI:TITLE:PROCESS FOR THE PURIFICATION OF PNEUMOCANDIN B0 FROM FERMENTATION BROTH

TECHNICAL FIELD OF INVENTION:
The present invention is related to the process for purification of Pneumocandin B0 from fermentation broth. The process involves Pneumocandin B0 extraction from whole broth or filtered biomass by using suitable solvents, impurities removal by liquid –liquid separation, carbon treatment and precipitation by suitable anti solvent.

BACKGROUND ART:
The invention is related to the process of purifying echinocandin type product from fermentation broth. The Pneumocandin, a type of echinocandin derived from the fermentation of the fungus Glarea lozoyensis, has been successfully used to develop an antifungal drug. Pneumocandin is similar to the echinocandin and aculeacin class of natural products; the amino acids that compose the polar, hexapeptide nucleus possess one or more hydroxyl groups. Similarly, the Pneumocandin also possess a nonpolar, amide-linked acyl side chain. Pneumocandin B0 is produced as a secondary metabolite by fermentation of the fungus Glarea lozoyensis (U.S. Pat. Nos. 5,194,377 and 5,202,309) which is precursor for Caspofungin acetate (CANCIDAS), a semi synthetic lipopeptide Echinocandin B derivative currently being sold in US as an antifungal agent for intravenous administration.

US Patent No 6,610,822 B2 describes a process for purifying a natural product after initial isolation by whole broth extraction using a two phase, multi-solvent system. Recovery of the product from the back extraction steps involves additional vacuum concentration and a two stage precipitation step. Typically three or more solvents are employed since the multi-solvent system can impact the relative polarity balance of the solution in which the compound is being purified. The process of the invention can be used to purify lipophilic natural products such as echinocandin-type cyclic peptides.

World Patent No WO 2011/121599 A1 describes a process for purifying Pneumocandin having one or more polar impurities and one or more non-polar impurities comprising extraction of product from fermentation broth using suitable solvent and partially concentrated, washing with immiscible solvent, charcoalization, concentration and filtration, loading the solids obtained in a column with an adsorbent, eluting with suitable solvents, concentration of product rich fraction and crystallization.

DISCLOSURE OF THE INVENTION:
The patent is related to process for the purification of Pneumocandin B0, a secondary metabolite obtained from fermentation of anamorphic fungi Glarea lozoyensis. The present invention for the process of purifying Pneumocandin B0 comprises.
A. Extracting the Pneumocandin B0 from whole broth or by separated biomass from fermentation broth by using suitable solvent;
B. Concentration of solvent containing product;
C. Liquid –Liquid Extraction by suitable solvent;
D. Carbon treatment;
E. Precipitation.
In one embodiment, the present invention is directed to a process for purifying Pneumocandin B0 a natural product, initial isolation by whole broth extraction or biomass separated using suitable solvent .The suitable solvent for extraction of product from fermentation broth or from separated biomass comprises n-butanol, isobutanol and methanol. Preferably n-butanol is used.

In another embodiment, the solvent is concentrated by vacuum distillation and the impurities in the concentrated solvent are then removed by two phase liquid – liquid separation. The liquid –liquid separation process comprising, the product concentrated n-butanol layer is combined in different volumetric ratios of 3 different solvents (cyclohexane or methylcyclohexane, methanol and water).

In the present embodiment, the first step comprises of two phases the upper phase Methylcyclohexane and lower phase n-butanol, methanol and water. The upper phase is separated and the lower aqueous phase is vacuum concentrated to form two layers upper phase n-butanol and lower phase water.

In another embodiment, the purification of Pneumocandin B0 impurities separation is done by using suitable solvents (methylcyclohexane, methanol and water).

In another embodiment, in the present invention the ratio of the solvent is more important for the formation of two phases, preferably 1:0.8:0.6:1.3 (n-butanol: methanol: water: methylcyclohexane)

In another embodiment of the present invention, second step upper n-butanol layer is treated with activated carbon. Preferably with Norit SA Plus and CN1 activated carbon.

In another embodiment, the purification of the carbon treated n-butanol layer is done by using an antisolvent for precipitation. In another embodiment the suitable antisolvent is acetone, isopropyl acetate, and acetonitrile, preferably 1:3 ratio of acetonitrile.

In another embodiment of the present invention, the crystallized dry product is amorphous powder.

EXAMPLES
Example-1: Whole broth extraction with carbon treatment.
Step A -Whole broth extraction
1 Liter of fermentation broth containing 1.51gms/l of Pneumocandin B0 was extracted with 1:2 ratio of n-butanol. The two phases were separated. The separated n-butanol phase (1.7Liter) contains 1.25gms of Pneumocandin B0 and the spent biomass layer was rextracted with additional 1liter of n-butanol, the two phases were separated. The separated n-butanol phase (1100ml) contains 0.15gms product. Two n-butanol layers were mixed and mixed n-butanol layers (2.8liter) contains 1.4gms of product at a yield of 93% and purity of the product is 11%. The spent biomass layer contains 0.016gm of product concentration.

Step B –Liquid Liquid extraction
2.8liter of n-butanol layer is concentrated to 225ml. The concentrated n-butanol layer was mixed with methanol, water and methylcyclohexane in the ratio of 1:0.8:0.6:1.3 (225:180:135:292ml). The solvents are mixed by agitation and the two phase separated by settling. The upper methylcyclohexane phase 300ml with product concentration of 0.01gms, the lower phase contains methanol, water and n-butanol is again washed with methylcyclohexane, the solvents are mixed by agitation and two phase separated by settling. The upper methylcyclohexane phase 295ml with 0.005gms product, the lower phase contains methanol, water and n-butanol is further concentrated under vacuum by maintaining temperature 40-45 deg C, the concentration is carried out till the two-layer formation.

The upper phase n-butanol layer is further washed with 1:0.5 ratio of water, the separated n-butanol layer 120ml contains 1.28gms of product at a step yield of 91.4% and the purity increased from 11 to 46%. The lower phase water layer contains 0.01gm of product concentration.

Step C –Precipitation
To the concentrated n-butanol layer 25gms/l concentration of Norit activated carbons (CN1, SA Plus and CGP Super) are added and mixed for 1 hour. After mixing the n-butanol layer is filtered under vacuum. The clear carbon free filtered n-butanol layer contains 1.119gms of product and purity of the product is 57%. The n-butanol layer is transferred to a glass beaker and maintained at a temperature not exceeding 5 deg C. To this chilled n-butanol layer under stirring, acetonitrile 1:3 ratio is added over the period of 3 hours. The precipitate is filtered and kept for drying in VTD at temperature not exceeding 40deg C. After drying 1.56gms of dried product was obtained at a step yield of 84% and the purity of the product is 69%. The acetonitrile layer contained 0.07gms of product. The overall active product recovery is 71.3%.

Example-2 Whole broth extraction without carbon treatment
Step A -Whole broth extraction
600ml of fermentation broth containing 1.176gms of Pneumocandin B0 was extracted with 1:2 ratio of n-butanol. The two phases were separated. The separated n-butanol phase (1005ml) contains 0.896 gms of Pneumocandin B0 and the spent biomass layer was rextracted with additional 1liter of n-butanol, the two phases were separated. The separated n-butanol phase (1050ml) contains 0.126gms product. The two n-butanol layers were mixed and mixed n-butanol layer (2.055 liters) contained 1.022gms of product at a yield of 87% and purity of the product is 13.3%. The spent biomass layer contains 0.024gm of product concentration.

Step B –Liquid Liquid Extraction
2.055 liter of n-butanol layer is concentrated to 140ml. The concentrated n-butanol layer was mixed with methanol, water and methylcyclohexane in the ratio of 1:0.8:0.6:1.3 (140:112:84:182ml). The solvents are mixed by agitation and the two phases separated by settling. The upper methylcyclohexane phase 195ml with product concentration of 0.009gms, the lower phase contains methanol, water and n-butanol is again washed with methylcyclohexane, the solvents are mixed by agitation and two phases separated by settling. The upper methylcyclohexane phase 185ml with 0.004gms product, the lower phase contains methanol, water and n-butanol is further concentrated under vacuum by maintaining temperature 40-45 deg C. The concentration is carried out till the two layer formation.

The upper phase n-butanol layer is further washed with 1:0.5 ratio of water, the separated n-butanol layer 100ml contained 0.84gms of product at a step yield of 82% and the purity increased from 13.3 to 44%. The lower phase water layer contains 0.019gm of product concentration.

Step C – Precipitation
The concentrated n-butanol layer contains 10gm/l of product. Without carbon treatment, the n-butanol layer was transferred to a glass beaker and maintained at a temperature not exceeding 5 deg C. To this chilled n-butanol layer under stirring, acetonitrile 1:3 ratio is added over the period of 3 hours. The precipitate is filtered and kept for drying in VTD at temperature not exceeding 40deg C. After drying 1.4gms of dried product was obtained at a step yield of 88% and the purity of the product is 53%. The acetonitrile layer contained 0.09gms of product. The overall active product recovery is 63%.

Example-3 Filtered biomass extraction
Step A -Biomass extraction
600ml of fermentation broth containing 1.176gms of Pneumocandin B0 was filtered to separate the biomass and obtained140gms wet biomass, and then the biomass is extracted with 1:2.5 ratio of n-butanol (350ml). The biomass and n-butanol were separated by filtration. The separated n-butanol layer (330ml) contains 0.98 gms of Pneumocandin B0 and the spent biomass was rextracted with an additional 350ml of n-butanol. The biomass and n-butanol were separated by filtration. The separated n-butanol layer (320ml) contain 0.118gms product. The two n-butanol layers were mixed and mixed n-butanol layers (650ml) contains 1.098gms of product at a yield of 93% and purity of the product is 13.3%. The spent biomass contains 0.0034gm of product concentration.

Step B –Liquid Liquid extraction
650ml of n-butanol layer is concentrated to 100ml. The concentrated n-butanol layer was mixed with methanol, water and methylcyclohexane in the ratio of 1:0.8:0.6:1.3 (100:80:60:130ml). The solvents are mixed by agitation and the two phases separated by settling. The upper methylcyclohexane phase 140ml with product concentration of 0.008gms, the lower phase contains methanol, water and n-butanol is again washed with methylcyclohexane, the solvents are mixed by agitation and two phases separated by settling. The upper methylcyclohexane phase 135ml with 0.001gms product, the lower phase contains methanol, water and n-butanol is further concentrated under vacuum by maintaining temperature 40-45 deg C. The concentration is carried out till the two layer formation.

The upper phase n-butanol layer is further washed with 1:0.5 ratio of water, the separated n-butanol layer 55ml contain 0.98gms of product at a step yield of 89% and the purity increased from 13.3 to 44.5%. The lower phase water layer contains 0.034gm of product concentration.

Step C – Precipitation
To the concentrated n-butanol layer 25gms/l concentration of Norit activated carbons (CN1, SA plus and CGP Super) are added and mixed for 1 hour. After mixing the n-butanol layer is filtered under vacuum. The clear carbon free filtered n-butanol layer contains 0.9gms of product and purity of the product is 59%. The n-butanol layer is transferred to a glass beaker and maintained at a temperature not exceeding 5 deg C. To this chilled n-butanol layer under stirring, acetonitrile1:3 ratio is added over a period of 3 hours. The precipitate is filtered and kept for drying in VTD at temperature not exceeding 40deg C. After drying 1.1gms of dried product was obtained at a step yield of 80% and the purity of the product is 71%. The acetonitrile layer contains 0.092gms of product. The overall active product recovery is 66%.