WO2013164834A1

WO2013164834A1 - Cost-effective process for commercial production of paclitaxel

Info

Publication number: WO2013164834A1
Application number: PCT/IN2012/000533
Authority: WO
Inventors: Jayabaskaran Chelliah; Pahan Jayeeta
Original assignee: Phyto Biotech Pvt. Ltd.
Priority date: 2012-05-04
Filing date: 2012-07-30
Publication date: 2013-11-07

Abstract

Disclosed herein a cost effective, improved, continuous process for production of Paclitaxel from endophytic fungi, Fusarium solani, with substantially high yield. In an aspect, the process for the production of paclitaxel includes preparing the autoclaved culture media, preparing the inoculum followed by inoculating the inoculum on culture media and placed the culture media in the fermenter. The cells from the fermenter are harvested after 15 days. Mycelia are separated using filtration method. The mycelia is kept for drying at about 40°C and after drying crushed in Liq. Nitrogen. The solvent is added to the filtrate in the ratio of 1 :3 and kept under shaking conditions. Separating the organic layer using centrifugation and evaporating the organic solvent using rotary evaporator to obtain the dried residue of the product Paclitaxel. The dried residue is dissolved in solvent and kept overnight for evaporation until the solvent is completely evaporated.

Description

"COST-EFFECTIVE PROCESS FOR COMMERCIAL PRODUCTION OF

PACLITAXEL"

TECHNICAL FIELD OF THE INVENTION:

The present invention relates to a cost effective, improved, continuous process for commercial production of Paclitaxel throughout the year with substantially high yield of Paclitaxel involving endophytic fungus such as Fusarium solani.

BACKGROUND AND PRIOR ART:

Paclitaxel, is a mitotic inhibitor that is used in cancer chemotherapy. Since 1967 this drug is well-known and initially it was used to be extracted from the bark of Taxus trees (Wall and Wani). In spite of offering substantial improvement in cancer patients, it soon became a controversial drug mainly because of its environmental impact of its original sourcing. Quite a large number of 100 year old Yew trees had to be felled to extract a few kgs of Paclitaxel. The mainly novelty of the present invention lies in the extraction of good amounts of Paclitaxel without the felling of trees or even cutting down needles of these trees.

Paclitaxel is now used to treat patients with lung, ovarian, breast, head and neck cancer, and advanced forms of Kaposi's sarcoma. Paclitaxel is also used for the prevention of restenosis. According to market reports, the demand of paclitaxel is expected to reach 1040 kgs by 2012. Strong demand for anti-cancer drugs is expected to sustain the demand for bulk Paclitaxel as an effective and powerful pharmaceutical ingredient.

However, recent reports in the year 201 1 reveal the difficulty faced by breast cancer patients due to the non-availability of the drug in the market during summer being dependent on depleting natural source. There have been continuous and persistent reports on inability to meet the market demand due to unavailability of natural raw material of plant origin throughout the year, especially during summer. An endophytic fungi, Fusarium solani is a filamentous fungus in the genus Fusarium, and the anamorph of Haematonectria haematococca is commonly isolated from soil and plant debris and has a worldwide distribution. The fungi can be also obtained from Taxus trees which are capable of producing Paclitaxel.

Production of Paclitaxel from Fusarium Solani is known in the art.

An article titled "Production of paclitaxel by Fusarium solani isolated from Taxus celebica" by Chakravarthi B V S K et al., published in J. Biosci. 33(2), June 2008, 259- 267 discloses production of paclitaxel by Fusarium solani. However, the yield has been poor such as 1.6 μg/L.

The reported process remains nonviable and devoid of potential of commercial production due to poor yield.

An article titled, "Fusarium solani, Tax-3, a new endophytic taxol-producing fungus from Taxus chinensis" by Bai Wan Deng et al., published in World Journal of Microbiology and Biotechnology Volume 25, Number 1, 139-143, discloses isolation of an endophytic fungus, Tax-3, from barks of Taxus chinensis grown in the Qinba mountains, China. The article further discloses that High performance liquid chromatography (HPLC) assay showed F. solani, Tax-3, produced taxol with a higher yield of 163.35 μg L in the reformative potato dextrose liquid medium, revealing its potential applications for Taxol production.

An article titled "Endophytic fungi for producing bioactive compounds originally from their host plants" by J. Zhao et. al., published in Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology. The said article mainly describes the research progress on the different types of endophytic fungi for producing bioactive compounds such as paclitaxel, podophyllotoxin, camptothecine, vinblastine, hypericin and diosgenin, which were also produced by their host plants.

An article titled "Identification and characterization of antimicrobial metabolite from an endophytic fungus, Fusarium solani isolated from bark of Himalayan yew" by Tayung K et al. published in Mycosphere Journals Volume 2 Issue 3, page 203-213 reports an endophytic F. solani isolated from T. baccata producing volatile hydrocarbons including Taxols with antimicrobial activity.

However, all the above-mentioned reported processes suffer from poor yield which make the reported process non-viable for commercial production. The alternative methods for paclitaxel production known in the art, such as chemical synthesis, tissue and cell cultures of the Taxus species, are expensive and give low yields.

Keeping in view, the potential commercial viability of endophytic fungus, Fusarium Solani obtained from Taxus tree which are capable of producing Paclitaxel and the disadvantages in the prior art process, the present inventors felt a need to develop an improved process for production of Paclitaxel from Fusarium Solanii to a level where the yield is good enough for commercial production.

The other object of the invention is to provide an optimized culture media containing readily available nutrient content which can scale-up the yield of fungi, Fusarium Solanii and consequently results in higher production of paclitaxel at low cost.

SUMMARY OF THE INVENTION:

In an aspect, the present invention provides an economically viable and commercially attractive process design to produce higher yield of Paclitaxel from endophytic fungus, Fusarium solani.

In another aspect, the present invention provides a culture media containing readily available nutrient content which scales-up the growth of Fusarium solani useful for subsequent production of Paclitaxel at low cost.

In yet another aspect, the culture media used for growth of Fusarium solani, comprises carbohydrates, proteins, vitamins and inorganic salts. In yet another aspect, the process for the production of paclitaxel includes preparing the autoclaved culture media, preparing the inoculum followed by inoculating the inoculum on culture media and placed the culture media in the fermenter. The cells from the fermenter are harvested after 15 days. Mycelia are separated using filtration method. The mycelia is kept for drying at about 40°C and after drying crushed in Liq. Nitrogen. The solvent is added to the filtrate in the ratio of 1:3 and kept under shaking conditions. Separating the organic layer using centrifugation and evaporating the organic solvent using rotary evaporator to obtain the dried residue of the product Paclitaxel. The dried residue is dissolved in solvent and kept overnight for evaporation until the solvent is completely evaporated. The dried product (Paclitaxel) so obtained is further dissolved in methanol for HPLC Analysis.

BRIEF DESCRIPTION OF THE DRAWING:

Figure 1 depicts a flow diagram of the process of the present invention

DETAILED DESCRIPTION OF THE INVENTION:

The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

Isolation and identification of fungus, Fusarium solani:

Inner bark of the Taxus tree is stripped out and plated on PDA (Potato dextrose agar) medium. The cut end of the inner bark eventually shows growth of fungus. Repeated culturing and sub-culturing of the colonies are done in order to get a pure culture. ITS (Internal transcribed spacer) sequencing of the fungal strain is carried for species identification.

The present invention discloses economically viable and commercially attractive process for production of Paclitaxel from fungus Fusarium solani in high yield. In an embodiment, the present invention discloses carbohydrate rich culture media containing other readily available nutrient contents that scales-up the growth of Fusarium solani useful for subsequent production of Paclitaxel at low cost.

The carbohydrate rich culture media of the present invention consisting of carbohydrates in the range of 10-60g/L; along with proteins in the range of 0.002-20g/L, vitamins in the range of 0.002-0.006g/L, and inorganic salts in the range of 0.001 -5g/L.

In the process of the present invention, production of Paclitaxel have been scaled up manifold from 126 micrograms/lt. to 50-500 mg/lt., or more which is 100 fold or more.

Accordingly, in a preferred embodiment, the present invention discloses cost effective, improved, continuous process for the production of Paclitaxel from Fusarium solani in high yield including the steps of;

1. Preparing autoclaved carbohydrate rich culture media consisting of carbohydrates in the range of 10-60g/L; along with proteins in the range of 0.002-20g/L, vitamins in the range of 0.002-0.006g/L, and inorganic salts in the range of 0.001- 5g/L at optimum pH between 5 to 8 to scale - up the growth of fungi;

2. preparing the inoculum in shaker flask condition using 10% inoculum for 200 ml culture media;

3. inoculating the said culture media of step (1) with the inoculum of step (2) in the shaker flask at 120 rpm at 23°C, transferring the culture to the fermenter at 150- 300 rpm at 23°C and maintaining the culture under same condition for about 21 days;

4. harvesting the cells from the fermenter after 21 days;

5. separating the mycelia and the filtrate using filtration;

6. drying the mycelia, separated in step (5), at 40°C followed by crushing in liquid nitrogen into fine powder;

7. dissolving the crushed mycelia of step (6) in organic solvent, placing on a shaker overnight, filtering, evaporating and purifying to obtain paclitaxel; 8. adding organic solvent to the filtrate of step (5), placing on a shaker overnight, separating the solvent and collecting the lower layer, filtering, evaporating and purifying to obtain paclitaxel.

Paclitaxel so obtained from the above process is transferred into preweighed homeophile vials.

The process flow diagram is. depicted in Figure 1.

Accordingly, in one preferred embodiment, the present invention provides autoclaved carbohydrate rich culture media i.e. GAMP culture media consisting of nutrient ingredients such as 50g/L of glucose, 6g/L of ammonium nitrate, 0.3g/L of MgS0₄.7H₂0, 0.5g/L of KH₂P0₄, 0.005g/L of Vitamin B l , 3mg/L of phenylalanine, 40mg/L of tryptophan and 30mg/L of sodium acetate at pH 7.

In another preferred embodiment, the present invention provides autoclaved carbohydrate rich culture media i.e. MGP culture media consisting of the nutrient ingredients such as 20g/L of malt extract, 20g/L of Glucose and lg/L of Peptone.

In yet another preferred embodiment, the present invention provides autoclaved carbohydrate rich culture media i.e. WCS (B+C) culture media consisting of nutrient ingredients such as 40g/L of wheat bran, 2g/L of corn steep liquor, 20g/L of soya flour, 5g/L of KH₂P0₄, 3g/L of MgS0₄.7H20, 3g/L of (NH^SC , lmg/L of ZnS0₄, lmg/L of Cu(N0₃)₂ and 5mg/L of FeCl₃.

The above-mention culture media are autoclaved at 121°C for 15 min at 15 lb pressure.

In another embodiment, the present invention discloses the process for inoculation of endophytic fungus viz. Fusarium solani on the above mentioned cultural media and further the process for extraction of paclitaxel, includes preparing 20 ml of culture media in two falcon tubes inoculated with 10% spores of Fusarium solani i.e., 2 ml of spores of Fusarium solani from glycerol stock; (wherein the Glycerol stock preservation is a way of preserving fungal strains in glycerol and saline at -80 degree centigrade). This is used as the inoculum for 200 ml culture media. The autoclaved culture media is transferred to the flask and inoculated with said inoculum. The flask is placed on a Shaker at 120 rpm and the temperature is set to about 23°C. This culture is maintained at 150-300 rpm at 23°C in a fermenter for a period of 21 days. The culture is monitored daily for any contamination. The cells are harvested from the fermenter after 21 days. The cells of the fungi obtained after harvesting are filtered to separate mycelia and filtrate. The separated mycelia is placed inside the preheated hot air oven and after drying crushed into fine powder using liquid nitrogen, adding organic solvent selected from halogenated hydrocarbons such as Dichloromethane, alcohols such as ethyl alcohol, ethers etc. and placed the same on a shaker for overnight. The solvent is filtered, evaporated using rotary vacuum evaporator at 45°C; and the product paclitaxel is further dissolved in polar solvent such as dimethyl sulfoxide (DMSO) or lower alcoholic solvent such as methanol, which further purified and transferred into preweighed homeophile vials:

Further to the filtrate, after separating mycelia, is added organic solvent selected from halogenated hydrocarbons, alcohols, ethers etc. in the ratio of 1 :3 and placed on a shaker for overnight. The solvent is filtered, evaporated using rotary vacuum evaporator at 45°C; and the residue (paclitaxel) obtained is further dissolved in lower alcoholic solvent, purified and transferred for further HPLC analysis. The purified product is transferred into preweighed vials.

The vials are then stored in a freezer at a temperature of about -80°C to freeze the sample completely followed by lyophilization till the sample turned into powder form.

In another embodiment, the present invention provides cost effective, improved, continuous process for production of Paclitaxel from Fusarium solani in high yield using autoclaved carbohydrate rich culture media selected from GAMP culture media consisting of nutrient ingredients such as 50g/L of glucose, 6g/L of ammonium nitrate, 0.3g/L of MgS0₄.7H₂0, 0.5g/L of KH₂P0₄, 0.005g/L of Vitamin B l, 3mg/L of phenylalanine, 40mg/L of tryptophan and 30mg/L of sodium acetate at pH7 and isolating paclitaxel by the process as described above.

In another embodiment, the present invention provides cost effective, improved, continuous process for production of Paclitaxel from Fusarium solani in high yield using autoclaved carbohydrate rich culture media selected from MGP culture media consisting of the nutrient ingredients such as 20g/L of malt extract, 20g/L of Glucose and lg/L of Peptone and isolating paclitaxel by the process as described above.

In another embodiment, the present invention provides cost effective, improved, continuous process for production of Paclitaxel from Fusarium solani in high yield using autoclaved carbohydrate rich culture media selected from WCS(B+C) culture media consisting of nutrient ingredients such as 40g/L of wheat bran, 2g/L of corn steep liquor, 20g/L of soya flour, 5g/L of KH₂P0₄, 3g/L of MgS0₄.7H20, 3g/L of (NH₄)₂S0₄, lmg/L of ZnS0₄, lmg/L of Cu(N0₃)₂ and 5mg/L of FeCl₃ and isolating paclitaxel by the process as described above.

In further embodiment, the present invention describes a pharmaceutical composition consisting of Paclitaxel prepared according to the invention together other pharmaceutical acceptable excipients, used to treat patients with lung, ovarian, breast, head and neck cancer, advanced forms of Kaposi's sarcoma and restenosis.

In further embodiment, the present invention describes a method of treating different types of cancers such as lung, ovarian, breast, head and neck cancer, advanced forms of Kaposi's sarcoma and restenosis by administrating pharmaceutical composition of paclitaxel of the present invention.

In further, embodiment, the present invention describes use of paclitaxel prepared according to the invention for the treatment of different types of cancers such as lung, ovarian, breast, head and neck cancer, advanced forms of Kaposi's sarcoma and restenosis.

The yield of paclitaxel from filtrate and mycelia of Fusarium solani, using different culture media of the present invention such as GAMP media, MGP media and WCS (B+C) media is given in Table 1 below:- Media GAMP Media MGP Media WCS (B+C) Media

Filtrate Mycelia Total Filtrate Mycelia Total Filtrate Mycelia Total

Batch 1 2.1208 0.248 mg/ 2.368 2.48 mg/ 1.637 mg/ 4.1 17 0.42 mg/ 0.0098 0.4298 mg/ 400 3.3 lg dry 8 mg 400 ml 4.15g dry mg 400 ml mg mg ml culture weight of culture weight of culture from

mycelia mycelia mycelia

Batch 2 3.267 mg/ 0.139 mg/ 3.406 2.545 1.362 mg/ 3.907 0.4939 .0.13053 0.6244

400 ml 3.3 l g dry mg mg/ 400 3.63g dry mg mg/ 400 mg 3 mg culture weight of ml weight of ml from

mycelia culture mycelia culture mycelia

Batch 3 2.243 mg/ 0.172 mg/ 2.415 3.841 0.0829 3.9239 0.802 0.15645 0.9584

400 ml 2.92g dry mg mg/ 400 mg/ 4.15g mg mg/ 400 mg 5 mg culture weight of ml dry ml from

mycelia culture weight of culture mycelia

mycelia

Table 1

EXPERIMENTAL DETAILS;

GAMP MEDIA (BATCH 1) Preparation of Media:-

GAMP media, the medium contained per litre:- glucose- 50g, ammonium nitrate- 6g,

MgS04.7H20- 0.3g, KH2P04 -0.5g, vitamin Bl - 5* 10-3g, phenylalanine-3mg, tryptophan-40mg, sodium acetate-30mg, the chemicals were individually weighed into a clean conical flask. The components were dissolved in 200 ml of distilled water. The media was prepared in two flasks each containing 200 ml. The pH was adjusted to 7.0 by using IN NaOH (base).The mouth of the flask was closed with a cotton plug packed and autoclaved at 121°C for 15 mins at 15 lb pressure.

Inoculum Preparation:-

10% inoculum of Fusarium solani is used for inoculation. 20 ml of GAMP media was prepared in two falcon tubes and was inoculated with 10% spores of Fusarium solani i.e., 2 ml of spores from glycerol stock. This was used as the inoculum for 200 ml GAMP media.

Inoculation Method:-

The laminar air flow hood was wiped with alcohol and the U.V light was turned on for 20 minutes. The flask containing the autoclaved media was opened inside the hood and the neck was heated properly. In the proximity of flame the falcon tubes were opened and the inoculum was transferred to the flask. The flask were packed and placed on shaker.

Maintenance of Culture:-

The flasks containing the inoculated media were placed on a shaker at 120 rpm and the temperature was set to 23°C. This culture was maintained in the above condition for a period of 21 days. The culture was monitored daily for any contamination.

Cell Separation :-

The 21 days old culture was passed through double layered cheese cloth to separate the mycelia and the filtrate. The separated mycelia was squeezed to remove excess filtrate and weighed to obtain the fresh mycelial biomass which was found to be 11.92g and 10.20g. This was placed inside the preheated hot air oven at 45°C for 24-48 hrs. to dry.

Product Extraction from Filtrate:-

The volume of filtrate obtained after filtration from each flask was 190ml.To the filtrate obtained after separation of mycelia l /3^rd volume that is 63.3 ml of dichloromethane was added and the flasks were foiled and placed on a shaker at 120rpm for overnight. The following day the filtrate containing DCM was transferred to a separating funnel. As soon as the separation of different layers were observed, the lower layer was collected in a preweighed round bottom flask by passing it through Whattman filter paper (3mm).The sample collected in the round bottom flask was evaporated using rotary vacuum evaporator at 45°C.After evaporation the round bottom flask was weighed again to. obtain the dry weight of the sample. Then the evaporated sample was dissolved in 4ml dichloromethane and allowed to evaporate overnight at room temperature. The following day the residue was dissolved in 2 ml of methanol and transferred into preweighed vials.

Product Extraction from Mycelia:-

The dried mycelia was removed from the hot air oven and weighed to obtain the dry weight. The dry weight of the mycelia was found to be 1.76g and 1.54g. This was crushed in a sterile mortar and pestle using liquid nitrogen into fine powder, and then it was transferred into a flask, to this 20ml of dichloromethane was added, packed and placed on a shaker at 120rmp for overnight. After 24 hr it was taken out from shaker and filtered by passing it through Whatmann filter paper (3mm) into a clean preweighed round bottom flask .The sample is evaporated in rotary vacuum evaporator. After evaporation the round bottom flask was weighed again to obtain the dry weight of the sample. This was dissolved in 3ml of dichloromethane and allowed to evaporate overnight at room temperature. The following day the residue was dissolved in 2 ml methanol and transferred into preweighed homeophile vials.

Product Purification:-

The sample dissolved in methanol was passed through 0.2 μηι filter. Fraction collection was done during HPLC.

Sample Preparation for HPLC:-

The sample was filtered using 0.2μηι Α1ίεΓ.40μ1 of the filtered sample was transferred to labeled HPLC vials containing the inserts.

Filling:-

The filtered samples were transferred to cryovials and stored in -80° freezer to freeze the sample completely. Lyophilization:-

The lyophilizer was turned on and the shelf temperature was set to -30°C. The sample was frozen in a slanting position inside the vial. As the shelf temperature reaches -30°C the cryovials were placed on the rack with the lids open. The samples were placed in the lyophilizer till it turned into a powdered form.

Final Packaging:-

The powdered samples obtained after lyophilization were packed and sealed. Result:-

The extracts obtained from the above experiment were analyzed by HPLC. The concentration of paclitaxel obtained from the analysis of the sample was found to be:- a) Filtrate: - 2.1208mg/400ml culture.

b) Mycelia: - 0.248mg/3.31 g dry weight of mycelia.

GAMP MEDIA (BATCH 2) Preparation of Media:-

GAMP media, the medium contained per litre:- glucose- 50g, ammonium nitrate- 6g, MgS04.7H20- 0.3g, KH2P04 -0.5g, vitamin B l-5* 10-3g, phenylalanine-3mg, tryptophan-40mg, sodium acetate-30mg, the chemicals were individually weighed into a clean conical flask. The components were dissolved in 200 ml of distilled water .The media was prepared in two flasks each containing 200 ml .The pH was adjusted to 7.0 by using IN NaOH (base).The mouth of the flask was closed with a cotton plug packed and autoclaved at 121°C for 15 mins at 15 lb pressure.

Inoculum Preparation:-

10% inoculum of Fusarium solani is used for inoculation. 20 ml of GAMP media was prepared in two falcon tubes and was inoculated with 10% spores of Fusarium solani i.e., 2 ml of spores from glycerol stock. This was used as the inoculum for 200 ml GAMP media. Inoculation Method:-

The laminar air flow hood was wiped with alcohol and the U.V light was turned on for 20 minutes. The flask containing the autoclaved media was opened inside the hood and the neck was heated properly .In the proximity of flame the falcon tubes were opened and the inoculum was transferred to the flask. The flask were packed and placed on shaker- Maintenance of Culture:-

Cell Separation:-

The 21 days old culture was passed through double layered cheese cloth to separate the mycelia and the filtrate. The separated mycelia was squeezed to remove excess filtrate and weighed to obtain the fresh mycelial biomass which was found to be 12.66g and lli06g. This was placed inside the preheated hot air oven at 45°C for 24-48 hrs. to dry.

Product Extraction from Filtrate:-

The volume of filtrate obtained after filtration from each flask was 190ml. To the filtrate obtained after separation of mycelia l/3^rd volume that is 63.3 ml of dichloromethane was added and the flasks were foiled and placed on a shaker at 120rpm for overnight. The following day the filtrate containing DCM was transferred to a separating funnel. As soon as the separation of different layers were observed, the lower layer was collected in a preweighed round bottom flask by passing it through Whattman filter paper (3mm).The sample collected in the round bottom flask was evaporated using rotary vacuum evaporator at 45°C.After evaporation the round bottom flask was weighed again to obtain the dry weight of the sample. Then the evaporated sample was dissolved in 4ml dichloromethane and allowed to evaporate overnight at room temperature. The following day the residue was dissolved in 2 ml of methanol and transferred into preweighed vials.

Product Extraction from Mycelia:-

The dried mycelia was removed from the hot air oven and weighed to obtain the dry weight. The dry weight of the mycelia was found to be 1.82g and 1.49g. This was crushed in a sterile mortar and pestle using liquid nitrogen into fine powder, and then it was transferred into a flask, to this 20ml of dichloromethane was added, packed and placed on a shaker at 120rmp for overnight. After 24 hr. it was taken out from shaker and filtered by passing it through Whatmann filter paper (3mm) into a clean preweighed round bottom flask .The sample is evaporated in rotary vacuum evaporator. After evaporation the round bottom flask was weighed again to obtain the dry weight of the sample. This was dissolved in 3ml of dichloromethane and allowed to evaporate overnight at room temperature. The following day the residue was dissolved in 2 ml methanol and transferred into preweighed homeophile vials.

Product Purification:-

The sample dissolved in methanol was passed through 0.2 μιη filter .Fraction collection was done during HPLC.

Sample Preparation for HPLC:-

The sample was filtered using 0.2μηι filter.40 l of the filtered sample was transferred to labeled HPLC vials containing the inserts.

Filling:-

The filtered samples were transferred to cryovials and stored in -80° freezer to freeze the sample completely.

Lyophilization:-

The lyophilizer was turned on and the shelf temperature was set to -30°C.The sample was frozen in a slanting position inside the vial. As the shelf temperature reaches -30°C the cryovials were placed on the rack with the lids open. The samples were placed in the lyophilizer till it turned into a powdered form.

Final Packaging:-

The extracts obtained from the above experiment were analyzed by HPLC. The concentration of paclitaxel obtained from the analysis of the sample was found to be:- a) Filtrate: - 3.267mg/400ml culture.

b) Mycelia: - 0.139mg/3.31 g dry weight of mycelia.

GAMP MEDIA (BATCH 3) Preparation of Media:-

GAMP media, the medium contained per litre:- glucose- 50g, ammonium nitrate- 6g, MgS04.7H20- 0.3g, KH2P04 -0.5g, vitamin B l- 5* 10-3g, phenylalanine-3mg, tryptophan-40mg, sodium acetate-30mg, the chemicals were individually weighed into a clean conical flask. The components were dissolved in 200 ml of distilled water .The media was prepared in two flasks each containing 200 ml .The pH was adjusted to 7.0 by using IN NaOH (base).The mouth of the flask was closed with a cotton plug packed and autoclaved at 121°C for 15 mins at 15 lb pressure.

Inoculum Preparation:-

Inoculation Method:-

The laminar air flow hood was wiped with alcohol and the U.V light was turned on for 20 minutes. The flask containing the autoclaved media was opened inside the hood and the neck was heated properly .In the proximity of flame the falcon tubes were opened and the inoculum was transferred to the flask. The flask were packed and placed on shaker.

Maintenance of Culture:-

The flasks containing the inoculated media were placed on a shaker at 120rpm and the temperature was set to 23°C. This culture was maintained in the above condition for a period of 21 days. The culture was monitored daily for any contamination. Cell Separation :-

The 21 days old culture was passed through double layered cheese cloth to separate the mycelia and the filtrate. The separated mycelia was squeezed to remove excess filtrate and weighed to obtain the fresh mycelial biomass which was found to be 12.66g and 11.06g. This was placed inside the preheated hot air oven at 45°C for 24-48 hrs. to dry.

Product Extraction from Filtrate:-

The volume of filtrate obtained after filtration from each flask was 190ml.To the filtrate obtained after separation of mycelia l/3^rd volume that is 63.3 ml of dichloromethane was added and the flasks were foiled and placed on a shaker at 120rpm for overnight. The following day the filtrate containing DCM was transferred to a separating funnel. As soon as the separation of different layers were observed, the lower layer was collected in a preweighed round bottom flask by passing it through Whattman filter paper (3mm).The sample collected in the round bottom flask was evaporated using rotary vacuum evaporator at 45°C.After evaporation the round bottom flask was weighed again to obtain the dry weight of the sample. Then the evaporated sample was dissolved in 4ml dichloromethane and allowed to evaporate overnight at room temperature. The following day the residue was dissolved in 2 ml of methanol and transferred into preweighed vials.

Product Extraction from Mycelia:-

The dried mycelia was removed from the hot air oven and weighed to obtain the dry weight. The dry weight of the mycelia was found to be 1.81g and l.llg. This was crushed in a sterile mortar and pestle using liquid nitrogen into fine powder, and then it was transferred into a flask, to this 20ml of dichloromethane was added, packed and placed on a shaker at 120rmp for overnight. After 24 hr. it was taken out from shaker and filtered by passing it through Whatmann filter paper (3mm) into a clean preweighed round bottom flask .The sample is evaporated in rotary vacuum evaporator. After evaporation the round bottom flask was weighed again to obtain the dry weight of the sample. This was dissolved in . 3ml of dichloromethane and allowed to evaporate overnight at room temperature. The following day the residue was dissolved in 2 ml methanol and transferred into preweighed homeophile vials. Product Purification:-

The sample dissolved in methanol was passed through 0.2 μπι filter .Fraction collection was done during HPLC.

Sample Preparation for HPLC:-

The sample was filtered using 0.2μιη ί!1ΐβΓ.40μ1 of the filtered sample was transferred to labeled HPLC vials containing the inserts.

Filling:-

Lyophilization:-

Final Packaging:-

The extracts obtained from the above experiment were analyzed by HPLC. The concentration of paclitaxel obtained from the analysis of the sample was found to be:- a) Filtrate:-2.243mg/400ml culture.

b) Mycelia:-0.172 mg/2.92g dry weight of mycelia

MGP MEDIA (BATCH 1) Preparation of Media:-

The MGP media contained per litre: - Malt extract- 20g, Glucose- 20g, Peptone- lg. Individual components were weighed accurately into a clean conical flask. The components were dissolved in 200 ml of distilled water and the flask was closed and packed. The prepared media was autoclaved at 121 °C for 15 minutes at 15 lb pressure. 2 flasks containing 200 ml of MGP media was prepared.

Inoculum Preparation:-

10% inoculum of Fusarium solani is used for inoculation. 20 ml of MGP media was prepared in two falcon tubes and was inoculated with 10% spores of Fusarium solani i.e., 2 ml of spores from glycerol stock. This was used as the inoculum for 200 ml MGP media.

Inoculation Method:-

Maintenance of Culture:-

The flasks containing the inoculated media were placed on a shaker at 120rpm and the temperature was set to 23°C. This culture was maintained in the above condition for a period of 21 days. The culture was monitored daily for any contamination.

Cell Separation :-

The 21 days old culture was passed through double layered cheese cloth to separate the mycelia and the filtrate. The separated mycelia was squeezed to remove excess filtrate and weighed to obtain the fresh mycelial biomass which was found to be 9.21g and 6.70g. This was placed inside the preheated hot air oven at 45°C for 24-48 hrs to dry.

Product Extraction from Filtrate:-

The volume of filtrate obtained after filtration from each flask was 185ml. To the filtrate obtained after separation of mycelia l/3^rd volume that is 61.6 ml of dichloromethane was added and the flasks were foiled and placed on a shaker at 120rpm for overnight. The following day the filtrate containing DCM was transferred to a separating funnel. As soon as the separation of different layers were observed, the lower layer was collected in a preweighed round bottom flask by passing it through Whattman filter paper (3mm).The sample collected in the round bottom flask was evaporated using rotary vacuum evaporator at 45°C .After evaporation the round bottom flask was weighed again to. obtain the dry weight of the sample. Then the evaporated sample was dissolved in 4ml dichloromethane -and allowed to evaporate overnight at room temperature. The following day the residue was dissolved in 2 ml of methanol and transferred into preweighed vials.

Product Extraction from Mycelia:-

The dried mycelia was removed from the hot air oven and weighed to obtain the dry weight. The dry weight of the mycelia was found to be 2.45g and 1.70g. This was crushed in a sterile mortar and pestle using liquid nitrogen into fine powder, and then it was transferred into a flask, to this 20ml of dichloromethane was added, packed and placed on a shaker at 120rpm for overnight. After 24 hr it was taken out from shaker and filtered by passing it through Whatmann filter paper (3mm) into a clean preweighed round bottom flask .The sample is evaporated in rotary vacuum evaporator. After evaporation the round bottom flask was weighed again to obtain the dry weight of the sample. This was dissolved in 3ml of dichloromethane and allowed to evaporate overnight at room temperature. The following day the residue was dissolved in 2 ml methanol and transferred into preweighed homeophile vials.

Product Purification :-

The sample dissolved in methanol was passed through 0.2 μηι filter .Fraction collection was done during HPLC.

Sample Preparation for HPLC:-

The sample was filtered using 0.2μιη ί 1ΐεΓ.40μ1 of the filtered sample was transferred to labeled HPLC vials containing the inserts.

Filling:-

Final Packaging:-

The extracts obtained from the above experiment were analyzed by HPLC. The concentration of paclitaxel obtained from the analysis of the sample was found to be:- a) Filtrate:-2.48mg/400ml culture.

b) Mycelia: - 1.637mg/4.15g dry weight of mycelia

MGP MEDIA (BATCH 2) Preparation of Media:-

The MGP media contained per litre: - Malt extract- 20g, Glucose- 20g, Peptone- lg. Individual components were weighed accurately into a clean conical flask. The components were dissolved in 200 ml of distilled water and the flask was closed and packed. The prepared media was autoclaved at 121°C for 15 minutes at 15 lb pressure. 2 flasks containing 200 ml of MGP media was prepared.

Inoculum Preparation:-

Inoculation Method :-

Maintenance of Culture:-

Cell Separation:-

The 21 days old culture was passed through double layered cheese cloth to separate the mycelia and the filtrate. The separated mycelia was squeezed to remove excess filtrate and weighed to obtain the fresh mycelial biomass which was found to be 9.14g and 6.50g. This was placed inside the preheated hot air oven at 45°C for 24-48 hrs. to dry.

Product Extraction from Filtrate:-

The volume of filtrate obtained after filtration from each flask was 185ml.To the filtrate obtained after separation of mycelia l/3^rd volume that is 61 .6 ml of dichloromethane was added and the flasks were foiled and placed on a shaker at 120rpm for overnight. The following day the filtrate containing DCM was transferred to a separating funnel. As soon as the separation of different layers were observed, the lower layer was collected in a preweighed round bottom flask by passing it through Whattman filter paper (3mm).The sample collected in the round bottom flask was evaporated using rotary vacuum evaporator at 45°C.After evaporation the round bottom flask was weighed again to obtain the dry weight of the sample. Then the evaporated sample was dissolved in 4ml dichloromethane and allowed to evaporate overnight at room temperature. The following day the residue was dissolved in 2 ml of methanol and transferred into preweighed vials.

Product Extraction from Mycelia:-

The dried mycelia was removed from the hot. air oven and weighed to obtain the dry weight. The dry weight of the mycelia was found to be 2.04g and 1.59-g. This was crushed in a sterile mortar and pestle using liquid nitrogen into fine powder, and then it was transferred into a flask, to this 20ml of dichloromethane was added, packed and placed on a shaker at 120rpm for overnight. After 24 hrs. it was taken out from shaker and filtered by passing it through Whatmann filter paper (3mm) into a clean preweighed round bottom flask. The sample is evaporated in rotary vacuum evaporator. After evaporation the round bottom flask was weighed again to obtain the dry weight of the sample. This was dissolved in 3ml of dichloromethane and allowed to evaporate overnight at room temperature. The following day the residue was dissolved in 2 ml methanol and transferred into preweighed homeophile vials.

Product Purification:-

The sample dissolved in methanol was passed through 0.2 μπι filter. Fraction collection was done during HPLC.

Sample Preparation for HPLC:-

The sample was filtered using 0.2μιη filter. 40μ1 of the filtered sample was transferred to labeled HPLC vials containing the inserts.

Filling:-

Lyophilization:-

Final Packaging:-

The extracts obtained from the above experiment were analyzed by HPLC. The concentration of paclitaxel obtained from the analysis of the sample was found to be:- a) Filtrate:-2.545mg/400ml culture.

b) Mycelia: - 1.362mg/3.63g dry weight of mycelia MGP MEDIA (BATCH 3)

Preparation of Media:-

The MGP media contained per litre: - Malt extract- 20g, Glucose- 20g, Peptone-lg. Individual components were weighed accurately into a clean conical flask. The components were dissolved in 200 ml of distilled water and the flask was closed and packed. The prepared media was autoclaved at 121°C for 15 minutes at 15 lb pressure. 2 flasks containing 200 ml of MGP media was prepared.

Inoculum Preparation:-

Inoculation Method:-

Maintenance of Culture:-

Cell Separation :-

The 21 days old culture was passed through double layered cheese cloth to separate the mycelia and the filtrate. The separated mycelia was squeezed to remove excess filtrate and weighed to obtain the fresh mycelial biomass which was found to be 9.66g and 6.51g. This was placed inside the preheated hot air oven at 45°C for 24-48 hrs. to dry. Product Extraction from Filtrate:-

The volume of filtrate obtained after filtration from each flask was 185ml.To the filtrate obtained after separation of mycelia l/3^rd volume that is 61.6 ml of dichloromethane was added and the flasks were foiled and placed on a shaker at 120rpm for overnight. The following day the filtrate containing DCM was transferred to a separating funnel. As soon as the separation of different layers were observed, the lower layer was collected in a preweighed round bottom flask by passing it through Whattman filter paper (3mm).The sample collected in the round bottom flask was evaporated using rotary vacuum evaporator at 45°C. After evaporation the round bottom flask was weighed again to obtain the dry weight of the sample. Then the evaporated sample was dissolved in 4ml dichloromethane and allowed to evaporate overnight at room temperature. The following day the residue was dissolved in 2 ml of methanol and transferred into preweighed vials.

Product Extraction from Mycelia:-

The dried mycelia was removed from the hot air oven and weighed to obtain the dry weight. The dry weight of the mycelia was found to be 2.49g and 1.60g. This was crushed in a sterile mortar and pestle using liquid nitrogen into fine powder, and then it was transferred into a flask, to this 20ml of dichloromethane was added, packed and placed on a shaker at 120rpm for overnight. After 24 hr. it was taken out from shaker and filtered by passing it through Whatmann filter paper (3mm) into a clean preweighed round bottom flask .The sample is evaporated in rotary vacuum evaporator.^' After evaporation the round bottom flask was weighed again to obtain the dry weight of the sample. This was dissolved in 3ml of dichloromethane and allowed to evaporate overnight at room temperature. The following day the residue was dissolved in 2 ml methanol and transferred into preweighed homeophile vials.

Product Purification :-

Sample Preparation for HPLC:-

The sample was filtered using 0.2μπι Α1ΐεΓ.40μΙ of the filtered sample was transferred to labeled HPLC vials containing the inserts. Filling:-

Lyophilization:-

The lyophilizer was turned on and the shelf temperature was set to -30°C.The sample was frozen in a slanting position inside the vial.As the shelf temperature reaches -30°C the cryovials were placed on the rack with the lids open. The samples were placed in the lyophilizer till it turned into a powdered form.

Final Packaging:-

The extracts obtained from the above experiment were analyzed by HPLC. The concentration of paclitaxel obtained from the analysis of the sample was found to be:- a) Filtrate:-3.84 lmg/400ml culture.

b) Mycelia: - 0.0829mg/4.15g dry weight of mycelia

WCS (B+C) BATCH 1 Preparation of Media:-

WCS(B+C) media, the medium contained per litre :- Wheat bran- 40g, Cornsteep liquor- 2g, Soya flour-20g, KH2P04-5g, MgS04.7H20-3g, (NH4)2S04-3g, ZnS04-lmg, Cu(N03)2, FeC13-5mg. lO g of wheat bran was weighed into a clean conical flask. The wheat bran was treated with 13 ml of 0.2 N HC1 and autoclaved at 121°C for 15 mins at 15 lb pressure. The remaining constituents were weighed accurately and the flask was closed and autoclaved at 121°C for 15 min at 15 lb pressure.

Inoculum Preparation:-

10% inoculum of Fusarium solani is used for inoculation. 20 ml of WCS(B+C) media was prepared in two falcon tubes and was inoculated with 10% spores of Fusarium solani i.e., 2 ml of spores from glycerol stock. This was used as the inoculum for 200 ml WCS(B+C) media.

Inoculation Method :-

Maintenance of Culture:-

Cell Separation :-

The 21 days old culture was passed through double layered cheese cloth to separate the mycelia and the filtrate. The separated mycelium was squeezed to remove excess filtrate. This was placed inside the preheated hot air oven at 45°C for 24-48 hrs. to dry.

Product Extraction from Filtrate:-

The volume of filtrate obtained after filtration from each flask was 180ml.To the filtrate obtained after separation of mycelia l/3^rd volume that is 60ml of dichloromethane was added and the flasks were foiled and placed on a shaker at 120rpm for overnight. The following day the filtrate containing DCM was transferred to a separating funnel. As soon as the separation of different layers were observed, the lower layer was collected in a preweighed round bottom flask by passing it through Whattman filter paper (3mm).The sample collected in the round bottom flask was evaporated using rotary vacuum evaporator at 45°C.After evaporation the round bottom flask was weighed again to obtain the dry weight of the sample. Then the evaporated sample was dissolved in 4ml dichloromethane and allowed to evaporate overnight at room temperature. The following day the residue was dissolved in 2 ml of methanol and transferred into preweighed vials. Product Extraction from Mycelia:-

The dried mycelia was removed from the hot air oven. This was crushed in a sterile mortar and pestle using liquid nitrogen into fine powder, and then it was transferred into a flask, to this 20ml of dichloromethane was added, packed and placed on a shaker at I20rmp for overnight. After 24 hr it was taken out from shaker and filtered by passing it through Whatmann filter paper (3mm) into a clean preweighed round bottom flask .The sample is evaporated in rotary vacuum evaporator. After evaporation the round bottom flask was weighed again to obtain the dry weight of the sample. This was dissolved in 3ml of dichloromethane and allowed to evaporate overnight at room temperature. The following day the residue was dissolved in 2 ml methanol and transferred into preweighed homeophile vials.

Product Purification:-

Sample Preparation for HPLC:-

The sample was filtered using 0.2μιη ιΊΚβΓ.40μ1 of the filtered sample was transferred to labeled HPLC vials containing the inserts.

FHIing:-

Lyophilization:-

Final Packaging:-

The extracts obtained from the above experiment were analyzed by HPLC. The concentration of paclitaxel obtained from the analysis of the sample was found to be:- a) Filtrate:-0.42mg/400ml culture.

b) Mycelia: - 0.0098mg from mycelia.

WCS (B+C) BATCH 2 Preparation of Media:-

WCS(B+C) media, the medium contained per litre :- Wheat bran- 40g, Cornsteep liquor- 2g, Soya flour-20g, KH2P04-5g, MgS04.7H20-3g, (NH4)2S04-3g, ZnS04-lmg, Cu(N03)2, FeC13-5mg. l0 g of wheat bran was weighed into a clean conical flask. The wheat bran was treated with 13 ml of 0.2 N HCl and autoclaved at 121°C for 15 mins at 15 lb pressure. The remaining constituents were weighed accurately and the flask was closed and autoclaved at 121°C for 15 min at 15 lb pressure.

Inoculum Preparation:-

Inoculation Method:-

Maintenance of Culture:-

Product Extraction from Filtrate:-

The volume of filtrate obtained after filtration from each flask was 180ml.To the filtrate obtained after separation of mycelia l/3^rd volume that is 60ml of dichloromethane was added and the flasks were foiled and placed on a shaker at 120rpm for overnight. The following day the filtrate containing DCM was transferred to a separating funnel. As soon as the separation of different layers were observed, the lower layer was collected in a preweighed round bottom flask by passing it through Whattman filter paper (3mm).The sample collected in the round bottom flask was evaporated using rotary vacuum evaporator at 45°C.After evaporation the round bottom flask was weighed again to obtain the dry weight of the sample. Then the evaporated sample was dissolved in 4ml dichloromethane and allowed to evaporate overnight at room temperature.The following day the residue was dissolved in 2 ml of methanol and transferred into preweighed vials.

Product Extraction from Myceiia:-

The dried mycelia was removed from the hot air oven. This was crushed in a sterile mortar and pestle using liquid nitrogen into fine powder, and then it was transferred into a flask, to this 20ml of dichloromethane was added, packed and placed on a shaker at 120rmp for overnight. After 24 hr. it was taken out from shaker and filtered by passing it through Whatmann filter paper (3mm) into a clean preweighed round bottom flask .The sample is evaporated in rotary vacuum evaporator. After evaporation the round bottom flask was weighed again to obtain the dry weight of the sample. This was dissolved in 3ml of dichloromethane and allowed to evaporate overnight at room temperature. The following day the residue was dissolved in 2 ml methanol and transferred into preweighed homeophile vials.

Product Purification:-

The sample dissolved in methanol was passed through 0.2 μηι filter .Fraction collection was done during HPLC. Sample Preparation for HPLC:-

The sample was filtered using 0.2μηι filter.4(^l of the filtered sample was transferred to labeled HPLC vials containing the inserts.

Filling:-

Lyophilization:-

Final Packaging:-

The extracts obtained from the above experiment were analyzed by HPLC. The concentration of paclitaxel obtained from the analysis of the sample was found to be:- a) .FiItrate:-0.4939mg 400ml culture.

b) Mycelia: - 0.13053 from mycelia.

WCS (B+C) BATCH 3 Preparation of Media:-

WCS(B+C) media, the medium contained per litre :- Wheat bran- 40g, Cornsteep liquor- 2g, Soya flour-20g, KH2P04-5g, MgS04.7H20-3g, (NH4)2S04-3g, ZnS04-lmg, Cu(N03)2, FeC13-5mg. l0 g of wheat bran was weighed into a clean conical flask. The wheat bran was treated with 13 ml of 0.2 N HCl and autoclaved at 121°C for 15 mins at 15 lb pressure. The remaining constituents were weighed accurately and the flask was closed and autoclaved at 121 °C for 15 min at 15 lb pressure. Inoculum Preparation:-

Inoculation Method:-

Maintenance of Culture:-

Cell Separation:-

Product Extraction from Filtrate:-

The volume of filtrate obtained after filtration from each flask was 180ml.To the filtrate obtained after separation of mycelia l/3^rd volume that is 60ml of dichloromethane was added and the flasks were foiled and placed on a shaker at 120rpm for overnight. The following day the filtrate containing DCM was transferred to a separating funnel. As soon as the separation of different layers were observed, the lower layer was collected in a preweighed round bottom flask by passing it through Whattman filter paper (3mm).The sample collected in the round bottom flask was evaporated using rotary vacuum evaporator at 45°C.After evaporation the round bottom flask was weighed again to obtain the dry weight of the sample. Then the evaporated sample was dissolved in 4ml dichloromethane and allowed to evaporate overnight at room temperature. The following day the residue was dissolved in 2 ml of methanol and transferred into preweighed vials.

Product Extraction from Mycelia:-

The dried mycelia was removed from the hot air oven. This was crushed in a sterile mortar and pestle using liquid nitrogen into fine powder, and then it was transferred into a flask, to this 20ml of dichloromethane was added, packed and placed on a shaker at 120rmp for overnight. After 24 hr it was taken out from shaker and filtered by passing it through Whatmann filter paper (3mm) into a clean preweighed round bottom flask .The sample is evaporated in rotary vacuum evaporator. After evaporation the round bottom flask was weighed again to obtain the dry weight of the sample. This was dissolved in 3ml of dichloromethane and allowed to evaporate overnight at room temperature. The following day the residue was dissolved in 2 ml methanol and transferred into preweighed homeophile vials.

Product Purification :-

Sample Preparation for HPLC:-

The sample was filtered using 0.2μηι Α1ίβΓ.40μ1 of the filtered sample was transferred to labeled HPLC vials containing the inserts.

Filling:-

Lyophilization:-

The lyophilizer was turned on and the shelf temperature was set to -30°C.The sample was frozen in a slanting position inside the vial. As the shelf temperature reaches -30°C the cryovials were placed on the rack with the lids open. The samples were placed in the lyophilizer till it turned into a powdered form. Final Packaging:-

The extracts obtained from the above experiment were analyzed by HPLC. The concentration of paclitaxel obtained from the analysis of the sample was found to be:- a) Filtrate:-0.802mg/400ml culture.

b) Mycelia: - 0.15645 from mycelia.

EXAMPLES:

The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

Example 1: Production of Paclitaxel from fungal culture

Preparing the autoclaved culture media and preparing the inoculum in shaker flask. Inoculating the culture media with the inoculum and transferring the same to the fermenter. The cells from the fermenter are harvested after 15 days and separating the mycelia and filtrate using filtration method. The mycelia is kept for drying at 40°C and after drying the mycelia is crushed in Liq. Nitrogen and dissolve in organic solvent followed by placing the same on a shaker overnight, filtering, evaporating and purifying to obtain paclitaxel. Further to the filtrate, after separating mycelia, the solvent is added in the ratio of 1 :3 and kept under shaking conditions. Separating the solvent and collecting the lower layer, filtering, evaporating and purifying to obtain paclitaxel. The final product (Paclitaxel) so obtained is further dissolved in methanol for HPLC Analysis.

Example 2: GAMP Media

Components Amount per liter

Glucose 50g

Ammonium nitrate 6g MgS0₄.7H₂0 0.3g

KH₂P0₄ 0.5g

Vitamin B l 5* 10-3g

Phenylalanine 3mg

Tryptophan 40mg

Sodium acetate 30mg pH 7.0

Example 3: MGP Media

Example 4: WCS (B+C) Media

Components Amount per liter

Wheat bran 40g

Corn steep liquor 2g

Soya flour 20g

B

KH₂P0 5g

MgS0₄.7H₂0 3g

(NH₄)₂S0₄ 3g

c

ZnS0₄ lmg

Cu(N0₃)₂ lmg

FeCl₃ 5mg ADVANTAGES:

• The present invention resolves the long pending challenge to achieve enhanced sporulation in large scale bioreactor and subsequently achieves higher yield of paclitaxel.

• The present inventors have successfully concluded kinetics of the fermenter system and an apt downstream process mechanism by which the inventors have succeeded in extracting the maximum product output from culture, while cutting down the cost of the media of culturing of fungus to a base minimum.

• As the raw materials for large scale cultivation of fungus-carbohydrate from the malt industry and nitrogen compounds from dairy industry are available in bulk amounts, the process of drug production by fungus lends itself to lower production costs through efficient downstream processing.

• The cost effective process of the present invention allows the production of paclitaxel throughout the year thereby meeting the market demands for treating cancer affected patients.

• The process of the present invention provides the extraction of good amounts of Paclitaxel without the felling of trees or even cutting down needles of these trees.

Claims

We Claim,

1. A cost effective, improved, continuous process for production of Paclitaxel from Fusarium solani in high yield comprising;

a) preparing autoclaved carbohydrate rich culture media consisting of carbohydrate in the range of 10-60g/L; along with proteins in the range of 0.002-20g/L, vitamins in the range of 0.002-0.006g/L, and inorganic salts in the range of 0.001-5g/L at optimum pH to scale-up the growth of fungi; b) preparing the inoculum in shaker flask condition using 10% inoculum for 200ml culture media;

c) inoculating the said culture media of step (a) with the inoculum of step (b) in the shaker flask at 120 rpm at 23°C, transferring the culture to the fermenter at 150-300 rpm at 23°C and maintaining the culture under same condition for about 21 days;

d) harvesting the cells from the fermenter after 21 days;

e) separating the mycelia and the filtrate using filtration;

f) drying the mycelia, separated in step (e), at 40 °C followed by crushing in liquid nitrogen into fine powder;

g) dissolving the crushed mycelia of step (f) in organic solvent, placing on a shaker overnight, filtering, evaporating and purifying to obtain paclitaxel; h) adding organic solvent to the filtrate of step (e), placing on a shaker overnight, separating the solvent and collecting the lower layer, filtering, evaporating and purifying to obtain paclitaxel.

2. The cost effective, improved, continuous process for production of Paclitaxel according to claim 1, wherein the carbohydrate rich culture media selected from GAMP consisting of nutrient ingredients such as 50g/L of glucose, 6g/L of ammonium nitrate, 0.3g/L of MgS0₄.7H₂0, 0.5g/L of KH₂P0₄, 0.005g/L of vitamin B l , 3mg/L of phenylalanine, 40mg/L of tryptophan and 30mg/L of sodium acetate at pH7.

3. The cost effective, improved, continuous process for production of Paclitaxel according to claim 1 , wherein the carbohydrate rich culture media selected from MGP culture media consisting of the nutrient ingredients such as 20g/L of malt extract, 20g/L of Glucose and lg/L of Peptone.

4. The cost effective, improved, continuous process for production of Paclitaxel according to claim 1 , wherein the carbohydrate rich culture media selected from WCS (B+C) culture media consisting of nutrient ingredients such as 40g/L of wheat bran, 2g/L of corn steep liquor, 20g/L of soya flour, 5g L of KH₂P0₄, 3g/L of MgS0₄.7H₂0, 3g/L of (NH₄)₂S0₄, l mg/L of ZnS0₄, l mg/L of Cu(N0₃)₂ and 5mg/L of FeCl₃.

5. The cost effective, improved, continuous process for production of Paclitaxel from Fusarium solani in high yield using autoclaved carbohydrate rich culture media selected from GAMP culture media consisting of nutrient ingredients such as 50g/L of glucose, 6g L of ammonium nitrate, 0.3g/L of MgS0₄.7H₂0, 0.5g/L of KH₂P0₄, 0.005g/L of vitamin Bl, 3mg/L of phenylalanine, 40mg/L of tryptophan and 30mg/L of sodium acetate at pH7 and isolating paclitaxel by the process according to claim 1.

6. The cost effective, improved, continuous process for production of Paclitaxel from Fusarium solani in high yield using autoclaved carbohydrate rich culture media selected from MGP culture media consisting of the nutrient ingredients such as 20g/L of malt extract, 20g/L of Glucose and lg/L of Peptone and isolating paclitaxel by the process according to claim 1.

7. The cost effective, improved, continuous process for production of Paclitaxel from Fusarium solani in high yield using autoclaved carbohydrate rich culture media selected from WCS(B+C) culture media consisting of nutrient ingredients such as 40g/L of wheat bran, 2g/L of corn steep liquor, 20g L of soya flour, 5g L of KH₂P0₄, 3g/L of MgS0₄.7H₂0, 3g/L of (NH₄)₂S0₄, lmg/L of ZnS0₄, lmg/L of Cu(N0₃)₂ and 5mg/L of FeCl₃ and isolating paclitaxel by the process according to claim 1.

8. The cost effective, improved, continuous process for production of Paclitaxel according to any of the preceding claims, wherein the carbohydrate rich culture media is autoclaved at 121°C for 15 mins at 15 lb pressure.

9. The cost effective, improved, continuous process for production of Paclitaxel according to any of the preceding claims, wherein the yield of Paclitaxel ranging from 126 micrograms/lt. to 9-12 mg/lt.