WO2001064931A1 - Manufacture and purification of mycophenolic acid - Google Patents
Manufacture and purification of mycophenolic acid Download PDFInfo
- Publication number
- WO2001064931A1 WO2001064931A1 PCT/IN2000/000017 IN0000017W WO0164931A1 WO 2001064931 A1 WO2001064931 A1 WO 2001064931A1 IN 0000017 W IN0000017 W IN 0000017W WO 0164931 A1 WO0164931 A1 WO 0164931A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- improved method
- flour
- organic solvent
- solid substrate
- rice
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
Definitions
- the present invention relates to an improved method for the manufacture and purification of Mycophenohc acid (MPA) BACKGROUND OF THE INVENTION:
- Mycophenohc acid was initially isolated from a culture of Pemcillium (B. Gosio, Riv. Igiene Sanita Pub. Ann, 7, 825-869, 1896). Its value as an immunomodulator was realized much later. Morpholino ester of mycophenolic acid is used as a prodrug in pharmaceutical composition for treatment of rheumatoid arthritis, psoriasis and in prevention of tissue rejection in organ transplant patients.
- Mycophenolic acid is produced by aerobic fermentation of several Pemcillium species. It has a broad spectrum of activity like antitumor activity, antiviral, antipsoriatic, immunosuppressive and anti-inflammatory activity. It also exhibits antibacterial and antifungal activities. It is tolerable in large doses and has minimal side effects. It inhibits inosine monophosphate dehydrogenase which is an important enzyme in de novo synthesis of inosine monophosphate, a precursor of purines. MPA also inhibits proliferation of lymphocytes that are responsible for immune response. This immuno repressory effect of mycophenolic acid has been important in treatment of organ rejection after organ transplant surgery.
- Penicillium brevi-compactum strain has been used in submerged fermentation where it produces 2.4 mg/ml at 27°C in 6 days on shaking and gives 3.6 mg/ml at 27°C in 14 days without shaking (US patent No.
- the object of the present invention is to provide an improved method for the production of mycophenolic acid by solid substrate fermentation in a novel bioreactor 'PLAFRACTOR' and its subsequent purification.
- this invention provides an improved method for the manufacture of Mycophenolic acid comprising:
- Pemcillium brevi-compactum used is in the form of spore suspension or in mycelial form.
- the solid substrate matrix is selected from wheat bran, rice bran, ragi flour, soya flour, cotton seed flour, wheat flour, rice flour, rice husk.
- the solid substrate matrix is a mixture of two or more solid substances selected from wheat bran, rice bran, ragi flour, soya flour, cotton seed flour, wheat flour, rice flour, rice husk.
- the said contained bioreactor allows solid state fermentation to be carried out in a manner such that the fermentation micro-organisms and the fermentation products produced are kept isolated from the outside environment during the course of fermentation.
- the said contained bioreactor is "PLAFRACTOR".
- the organic solvent used for extraction is selected from acetone, methanol, toluene, benzene or ethyl acetate.
- the filter aid is selected from celite, perlite or alumina.
- the solvent used to dissolve the filter aid cake is selected from cyclohexane, toluene, benzene, ethyl acetate or butyl acetate.
- the alcohol used for crystallization is selected from methanol, ethanol or iso-propanol.
- the inorganic acid used for adjusting pH is sulphuric acid and the concentration of the organic layer is carried out by azeotropic distillation.
- the present invention uses Penicillium brevi-compactum .
- the colony of this isolate was comparatively fast growing and the aerial mycelium was cottony and white.
- the microbial culture sporulated showing green coloration on third day.
- the bioreactor used for the solid substrate fermentation is our invention and is described in our PCT Appln No. PCT/99IB/01688 dated October 15, 1999.
- the said bioreactor is modular in nature and carries out all of the processes of solid substrate fermentation in a single contained environment.
- the modular construction of the bioreactor provides multiple modules stacked on top of one another, each with a base connected to frame for holding the solid medium in isolation from the exterior environment.
- the construction of the bioreactor allows solid substrate fermentation to be carried out in a manner such that the fermenting microorganisms and the fermentation products it produces are kept isolated from the outside environment during the course of the fermentation. This containment of the fermentation process is of significant importance when working with microbial metabolites, which are cytotoxic in nature e.g. Gyclosporin, mycophenolic acid.
- the said bioreactor operates in a contained manner and is capable of sterilizing the solid state fermentation media, cooling it to the required temperature, fermenting at the desired set conditions, in situ extraction of the end product, recovery of the solvents and post harvest sterilization.
- An important aspect of the bioreactor is a mechanism of heat removal resulting in stringent temperature control of the fermentation process. In comparison, maintaining a constant temperature of growth in solid substrate fermentation using tray cultures is not efficient.
- the base plate of the bioreactor has multiple channels called noncommunicating channels that carry heating and cooling fluids sandwiched between two sheets. Heat is transferred to and from the modules by conduction. In this way the temperature of the module is precisely maintained to meet the specific requirement of different microorganisms.
- the base of the module contains a second set of channels, the communicating channels to deliver sterile air as supply of oxygen into the solid substrate bed for optimum growth of organism.
- Moisture loss because of passage of sterile air is significantly reduced by regularly reversing the direction of airflow every few hours. Using this, homogeneity in moisture content is maintained throughout the bioreactor.
- a single spore isolate of Penicillium brevi-compactum was used.
- the organism was subcultured on a fresh MEA (Malt Extract Agar) slant and incubated at 24°C. After 5 days, the sporulated slant was suspended in 10ml of water containing 0.01% tween 80. 500 ⁇ l of this spore suspension were spread on a fresh plate containing MEA. The plate was allowed Co grow for 5 days. After 5 days the spores were scraped from the plate with a sterile loop and suspended in sterile distilled water. This spore suspension, devoid of mycelial fragments was used as the inoculum.
- the master seed for inoculation of culture was a 10 6 spores/ml suspension of Penicillium brevi-compactum in 14 L of sterilized distilled water containing 20% glycerol. This was used to inoculate the sterilized wheat bran so that the final moisture after inoculation was 60 %. The inoculum was mixed thoroughly with the sterilized bran. Sterile airflow at a rate of 20 Lpm on the first day, 40 Lpm on second and third day and 20 Lpm on fourth and fifth day were sent into the bioreactor continuously. The temperature was controlled at 25°C for all 5 days by conductive heating and cooling. The Mycophenolic acid production titres were assayed following extraction using the HPLC.
- Example 2 The Mycophenolic acid production titres were assayed following extraction using the HPLC.
- the Bioreactor was sterilized and inoculated as in Example 1. In this experiment, the temperature was maintained at 30°C for all 5 days. The Mycophenolic acid production titres were assayed following extraction using the HPLC. Example 3
- the extract obtained from Example 3 was concentrated by azeotropic distillation to remove acetone, leaving behind 1.5 L of aqueous residue.
- the pH of this aqueous residue was adjusted to 2.0 with concentrated H 2 S0 4 and allowed to stand at 10°C.
- large needle shaped crystals of Mycophenolic acid were found floating at the surface of the liquid. These crystals were separated by filtration through a celite bed. Recovery of mycophenolic acid by this crystallization was found to be 100%. Crystals trapped on the celite bed were re-dissolved completely in 2 L of ethyl acetate. Ethyl acetate layer was separated and treated with alumina to remove colour.
- Alumina was removed by filtration and ethyl acetate layer was concentrated by distillation to leave behind light brown coloured crystals of mycophenolic acid. These crystals were further dissolved in methanol and dispersing the methanolic solution in water to obtain white crystals of mycophenolic acid. These crystals obtained from aqueous methanol were dissolved in 10 parts of acetone. To this acetone solution equivalent amount of hexane was added and the mixture was chilled to 10°C. The crystals of mycophenolic acid were separated by filtration and dried. The crystals thus obtained were of acceptable pharmaceutical grade.
- the present invention has the following advantages over the other reported methods: (i) Fermentation in a bioreactor, which is fully contained as a result assuring full safety for the cytotoxic fermentation products like mycophenolic acid.
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Saccharide Compounds (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides an improved method for the manufacture of Mycophenolic acid by solid substrate fermentation of Penicillium brevi-compactum in a contained bioreactor under optimal fermentation parameters with its subsequent purification steps.
Description
TITLE OF THE INVENTION:
'Manufacture and Purification of Mycophenohc Acid9
FIELD OF THE INVENTION: The present invention relates to an improved method for the manufacture and purification of Mycophenohc acid (MPA) BACKGROUND OF THE INVENTION:
Mycophenohc acid (MPA) was initially isolated from a culture of Pemcillium (B. Gosio, Riv. Igiene Sanita Pub. Ann, 7, 825-869, 1896). Its value as an immunomodulator was realized much later. Morpholino ester of mycophenolic acid is used as a prodrug in pharmaceutical composition for treatment of rheumatoid arthritis, psoriasis and in prevention of tissue rejection in organ transplant patients.
Mycophenolic acid is produced by aerobic fermentation of several Pemcillium species. It has a broad spectrum of activity like antitumor activity, antiviral, antipsoriatic, immunosuppressive and anti-inflammatory activity. It also exhibits antibacterial and antifungal activities. It is tolerable in large doses and has minimal side effects. It inhibits inosine monophosphate dehydrogenase which is an important enzyme in de novo synthesis of inosine monophosphate, a precursor of purines. MPA also inhibits proliferation of lymphocytes that are responsible for immune response. This immuno repressory effect of mycophenolic acid has been important in treatment of organ rejection after organ transplant surgery.
There is>a continuous need to find improved process for production of mycophenolic acid that will be commercially viable. It has been shown that mutants Periicillium brevi-compactum resistant to polyene antibiotics, HMG CoA reductase inhibitors, methyl viologen and surfactants produce more MPA than the parent strain (US patent No. 4,452,891).
The proposed biosynthetic pathway of mycophenolic acid production involves steroid biosynthetic pathway as well as polyketide biosynthetic pathway. It is a condensation product of a tetraketide and geranyl pyrophosphate, wherein geranyl moiety is cleaved after condensation and subsequent o-methylation in the tetraketide ring yields mycophenolic acid (Muth and Nash, Antimicrobial Agents and Chemotherapy, 8, 321-327, 1896). The biosynthetic pathway was studied in P. stoloniferum.
Penicillium brevi-compactum strain has been used in submerged fermentation where it produces 2.4 mg/ml at 27°C in 6 days on shaking and gives 3.6 mg/ml at 27°C in 14 days without shaking (US patent No.
4,452,891). In solid substrate fermentation (SSF) it is reported to produce 3286 mg per Kg of wheat bran (Sadhukhan et al, J. Ind. Microbiol. Biotechnol. 22, 33-38, (1999). Regarding the economics of using a producing strain, the described volume-time-yields as an industrial process are not economically attractive.
The object of the present invention is to provide an improved method for the production of mycophenolic acid by solid substrate fermentation in a novel bioreactor 'PLAFRACTOR' and its subsequent purification.
To achieve the said objective this invention provides an improved method for the manufacture of Mycophenolic acid comprising:
- loading a contained bioreactor with solid substrate matrix and sterilizing it,
- mixing the said sterilized solid substrate matrix with Penicillium brevi-compactum, - adding 5 - 20 % of glycerol, if desired,
- incubating the said inoculated solid substrate matrix for 4 - 7 days at 20 - 35°C,
- extracting the fermented solid substrate matrix with an organic solvent,
- concentrating the organic solvent extract,
- crystallizing mycophenolic acid by adjusting the pH to about 2.0 with an inorganic acid and allowing to stand for about 3 hours, followed by filtration using a filter aid, - dissolving the filter aid cake in a water immiscible organic solvent and treating with alumina,
- filtering the water immiscible organic solvent followed by concentration of the organic layer by distillation,
- dissolving the concentrate obtained in an alcohol,, - dispersing the alcoholic solution in water and filtering to get the crude crystals,
- dissolving the crude crystals thus obtained in an organic solvent,
- adding another organic solvent to the previous step solution and chilling to 4 to -20°C to get pure mycophenolic acid crystals.
The Pemcillium brevi-compactum used is in the form of spore suspension or in mycelial form.
The solid substrate matrix is selected from wheat bran, rice bran, ragi flour, soya flour, cotton seed flour, wheat flour, rice flour, rice husk. The solid substrate matrix is a mixture of two or more solid substances selected from wheat bran, rice bran, ragi flour, soya flour, cotton seed flour, wheat flour, rice flour, rice husk.
The said contained bioreactor allows solid state fermentation to be carried out in a manner such that the fermentation micro-organisms and the fermentation products produced are kept isolated from the outside environment during the course of fermentation. The said contained bioreactor is "PLAFRACTOR".
The organic solvent used for extraction is selected from acetone, methanol, toluene, benzene or ethyl acetate.
The filter aid is selected from celite, perlite or alumina.
The solvent used to dissolve the filter aid cake is selected from cyclohexane, toluene, benzene, ethyl acetate or butyl acetate.
The alcohol used for crystallization is selected from methanol, ethanol or iso-propanol.
The inorganic acid used for adjusting pH is sulphuric acid and the concentration of the organic layer is carried out by azeotropic distillation.
The present invention uses Penicillium brevi-compactum . The colony of this isolate was comparatively fast growing and the aerial mycelium was cottony and white. The microbial culture sporulated showing green coloration on third day.
The bioreactor used for the solid substrate fermentation is our invention and is described in our PCT Appln No. PCT/99IB/01688 dated October 15, 1999. The said bioreactor is modular in nature and carries out all of the processes of solid substrate fermentation in a single contained environment. The modular construction of the bioreactor provides multiple modules stacked on top of one another, each with a base connected to frame for holding the solid medium in isolation from the exterior environment. The construction of the bioreactor allows solid substrate fermentation to be carried out in a manner such that the fermenting microorganisms and the fermentation products it produces are kept isolated from the outside environment during the course of the fermentation. This containment of the fermentation process is of significant importance when working with microbial metabolites, which are cytotoxic in nature e.g. Gyclosporin, mycophenolic acid.
The said bioreactor operates in a contained manner and is capable of sterilizing the solid state fermentation media, cooling it to the required temperature, fermenting at the desired set conditions, in situ extraction of the end product, recovery of the solvents and post harvest sterilization.
An important aspect of the bioreactor is a mechanism of heat removal resulting in stringent temperature control of the fermentation process. In comparison, maintaining a constant temperature of growth in solid substrate fermentation using tray cultures is not efficient. The base plate of the bioreactor has multiple channels called noncommunicating channels that carry heating and cooling fluids sandwiched between two sheets. Heat is transferred to and from the modules by conduction. In this way the temperature of the module is precisely maintained to meet the specific requirement of different microorganisms. The base of the module contains a second set of channels, the communicating channels to deliver sterile air as supply of oxygen into the solid substrate bed for optimum growth of organism. Moisture loss because of passage of sterile air is significantly reduced by regularly reversing the direction of airflow every few hours. Using this, homogeneity in moisture content is maintained throughout the bioreactor. These aspects provide ample convenience over previous SSF methodologies that require multiple manipulations at each step of the fermentation process.
The invention will now be described with reference to the following examples: Example 1
A single spore isolate of Penicillium brevi-compactum was used. The organism was subcultured on a fresh MEA (Malt Extract Agar) slant and incubated at 24°C. After 5 days, the sporulated slant was suspended in 10ml of water containing 0.01% tween 80. 500μl of this spore suspension were spread on a fresh plate containing MEA. The plate was allowed Co grow for 5 days. After 5 days the spores were scraped from the plate with a sterile loop and suspended in sterile distilled water. This spore suspension, devoid of mycelial fragments was used as the inoculum. 15 Kg of wheat bran was loaded on the bioreactor of approximately 22600 cm2 of plate area. The
bioreactor was sterilized by sending steam simultaneously into the communicating and the noncommunicating channels to heat the bioreactor and its contents to a temperature of 121°C for 90 minutes. The steam pressure was released and simultaneously sterile air was sent into the communicating channels while cooling water at approximately 25°C was sent into the noncommunicating channels.
The master seed for inoculation of culture was a 106 spores/ml suspension of Penicillium brevi-compactum in 14 L of sterilized distilled water containing 20% glycerol. This was used to inoculate the sterilized wheat bran so that the final moisture after inoculation was 60 %. The inoculum was mixed thoroughly with the sterilized bran. Sterile airflow at a rate of 20 Lpm on the first day, 40 Lpm on second and third day and 20 Lpm on fourth and fifth day were sent into the bioreactor continuously. The temperature was controlled at 25°C for all 5 days by conductive heating and cooling. The Mycophenolic acid production titres were assayed following extraction using the HPLC. Example 2:
The Bioreactor was sterilized and inoculated as in Example 1. In this experiment, the temperature was maintained at 30°C for all 5 days. The Mycophenolic acid production titres were assayed following extraction using the HPLC. Example 3
5 Kg. fermented wheafbran obtained from example 1 was then extracted by using 10 L of acetone and the extract was collected, analyzed and taken for further processing. The extraction efficiency of acetone was found to be 98%, as quantitated by HPLC. Example 4
The extract obtained from Example 3 was concentrated by azeotropic distillation to remove acetone, leaving behind 1.5 L of aqueous residue. The
pH of this aqueous residue was adjusted to 2.0 with concentrated H2S04 and allowed to stand at 10°C. After 4 hours large needle shaped crystals of Mycophenolic acid were found floating at the surface of the liquid. These crystals were separated by filtration through a celite bed. Recovery of mycophenolic acid by this crystallization was found to be 100%. Crystals trapped on the celite bed were re-dissolved completely in 2 L of ethyl acetate. Ethyl acetate layer was separated and treated with alumina to remove colour. Alumina was removed by filtration and ethyl acetate layer was concentrated by distillation to leave behind light brown coloured crystals of mycophenolic acid. These crystals were further dissolved in methanol and dispersing the methanolic solution in water to obtain white crystals of mycophenolic acid. These crystals obtained from aqueous methanol were dissolved in 10 parts of acetone. To this acetone solution equivalent amount of hexane was added and the mixture was chilled to 10°C. The crystals of mycophenolic acid were separated by filtration and dried. The crystals thus obtained were of acceptable pharmaceutical grade.
The present invention has the following advantages over the other reported methods: (i) Fermentation in a bioreactor, which is fully contained as a result assuring full safety for the cytotoxic fermentation products like mycophenolic acid.
(ii) In situ extraction of the fermented end product.
(iii) Less fermentation time making the process economically attractive.
(iv) Fewer steps for the isolation and purification to get the pure product, thus saving processing time and additional expenses.
Claims
1. An improved method for the manufacture of Mycophenolic acid comprising: - loading a contained bioreactor with solid substrate matrix and sterilizing it, mixing the said sterilized solid substrate matrix with Penicillium brevi-compactum, adding 5 - 20 % of glycerol, if desired, - incubating the said inoculated solid substrate matrix for 4 - 7 days at 20 - 35°C, extracting the fermented solid substrate matrix with an organic solvent, concentrating the organic solvent extract, - crystallizing mycophenolic acid by adjusting the pH to 2.0 to 5.0 with an inorganic acid and allowing to stand for 3 to 10 hours, followed by filtration using a filter aid, dissolving the filter aid cake in a water immiscible organic solvent and treating with alumina, - filtering the water immiscible organic solvent followed by concentration of the organic layer by distillation, dissolving the concentrate obtained in an alcohol, dispersing the alcoholic solution in water and filtering to get the crude crystals, „ - dissolving the crude crystals thus obtained in an organic solvent, adding another organic solvent to the previous step solution and chilling to 4 to -20°C to get pure mycophenolic acid crystals. 2. An improved method as claimed in claim 1 wherein Penicillium brevi- compactum used is in the form of spore suspension or in mycelial form.
3. An improved method as claimed in claim 1 wherein the solid substrate matrix is selected from wheat bran, rice bran, ragi flour, soya flour, cotton seed flour, wheat flour, rice flour, rice husk.
4. An improved method as claimed in claim 1 wherein the solid substrate matrix is a mixture of two or more solid substances selected from wheat bran, rice bran, ragi flour, soya flour, cotton seed flour, wheat flour, rice flour, rice husk.
5. An improved method as claimed in claim 1 wherein the said contained bioreactor allows solid state fermentation to be carried out in a manner such that the fermentation micro-organisms and the fermentation products produced are kept isolated from the outside environment during the course of fermentation.
6. An improved method as claimed in claim 5 wherein the said contained bioreactor is "PLAFRACTOR". 7. An improved method as claimed in claim 1 wherein the organic solvent used for extraction is selected from acetone, methanol, toluene, benzene or ethyl acetate.
8. An improved method as claimed in claim 1 wherein the filter aid is selected from celite, perlite or alumina. 9. An improved method as claimed in claim 1 wherein the solvent used to dissolve the filter aid cake is selected from cyclohexane, toluene, benzene, ethyl acetate or butyl acetate.
10. An improved method as claimed in claim 1 wherein the alcohol used for crystallization is selected from methanol, ethanol or iso-propanol. 11. An improved method as claimed in claim 1 wherein the inorganic acid used for adjusting pH is sulphuric acid
12. An improved method as claimed in claim 1 wherein the concentration of the organic layer is carried out by azeotropic distillation. AMENDED CLAIMS
[received bv the International Bureau on 9 Januai 2001 (090101) original claims 1-12 replaced bv new claims 1-10 (2 pages)]
Claims :
1 An ιmpro\ed method tor the manufacture of Ivhcophenolic acid comprising loading a contained bioreactor "PLAFRACTOR"' with solid substrate matrix and sterilizing it. mixing the said sterilized solid substrate matπx with Penicillium brevi-compactum adding 5 - 20% of ghcerol. if desired. incubating the said inoculated solid substrate matrix tor 4-7 
at 20 -35 °C. extracting the fermented solid substrate matrix with an organic solvent. concentrating the organic solvent extract. crvstalhzing l copheno c acid b adjusting the pH to 20 to 50 with an inorganic acid and allowing to stand for 3 to 10 Hours, followed b> filtration using a filter aid. - dissohing the filter aid cake in a water immiscible organic solvent and treating ith alumina. filtering the water immiscible organic solvent followed 
concentration of the organic la\er b> distillation. disso ing the concentrate obtained in a alcohol - dispersing the alcoholic solution in water and filtering to get the crude cn stals. dissolving the crude crystals thus obtained in an organic solvent, adding another organic solvent to the previous step solution and chilling to 4 to -20°C to get pure mycophenolic acid crystals.
2. An improved method as claimed in claim 1 wherein Penicillium brevi- compactum used in the form of spore suspension or in mycelial form.
3. An improved method as claimed in claim 1 wherein the solid substrate matrix is selected from wheat bran, rice bran, ragi flour, soya flour, cotton seed flour, wheat flour, rice flour, rice husk.
4. An improved method as claimed in claim 1 wherein the sold substrate matrix is a mixture of two or more solid substances selected from wheat bran, rice bran, ragi flour, soya flour, cotton seed flour, wheat flour, rice flour, rice hust.
5. An improved method as claimed in claim 1 wherein the organic solvent used for extraction is selected from acetone, methanol. toluene, benzene or ethyl acetate.
6. An improved method as claimed in claim 1 wherein the filter aid is selected from celite. perlite or alumina.
7. An improved method as claimed in claim 1 wherein the solvent used to dissolve the filter aid cake is selected from cyclohexane. toluene, benzene, ethyle acetate or butyl acetate.
8. An improved method as claimed in claim 1 w herein the alcohol used for crystallization is selected from methanol. ethanol or iso-propanυl.
9. An improved method as claimed in claim 1 wherein the inorganic acid used for adjusting pH is sulphuric acid.
10. An improved method as claimed in claim 1 wherein the concentration of the organic layer is carried out by azeotropic distillation.
Priority Applications (9)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002401699A CA2401699A1 (en) | 2000-02-29 | 2000-02-29 | Manufacture and purification of mycophenolic acid |
| EP00911239A EP1259631B1 (en) | 2000-02-29 | 2000-02-29 | Manufacture and purification of mycophenolic acid |
| CZ20022913A CZ20022913A3 (en) | 2000-02-29 | 2000-02-29 | Preparation and purification process of mycophenolic acid |
| PCT/IN2000/000017 WO2001064931A1 (en) | 2000-02-29 | 2000-02-29 | Manufacture and purification of mycophenolic acid |
| DE60023187T DE60023187D1 (en) | 2000-02-29 | 2000-02-29 | PREPARATION AND PURIFICATION OF MYCOPHENOLIC ACID |
| US10/220,225 US6927047B1 (en) | 2000-02-29 | 2000-02-29 | Manufacture and purification of mycophenolic acid |
| AT00911239T ATE306557T1 (en) | 2000-02-29 | 2000-02-29 | PRODUCTION AND PURIFICATION OF MYCOPHENOLIC ACID |
| AU2000233225A AU2000233225A1 (en) | 2000-02-29 | 2000-02-29 | Manufacture and purification of mycophenolic acid |
| JP2001563619A JP2003525052A (en) | 2000-02-29 | 2000-02-29 | Production and purification of mycophenolic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/IN2000/000017 WO2001064931A1 (en) | 2000-02-29 | 2000-02-29 | Manufacture and purification of mycophenolic acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2001064931A1 true WO2001064931A1 (en) | 2001-09-07 |
Family
ID=11076227
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/IN2000/000017 Ceased WO2001064931A1 (en) | 2000-02-29 | 2000-02-29 | Manufacture and purification of mycophenolic acid |
Country Status (9)
| Country | Link |
|---|---|
| US (1) | US6927047B1 (en) |
| EP (1) | EP1259631B1 (en) |
| JP (1) | JP2003525052A (en) |
| AT (1) | ATE306557T1 (en) |
| AU (1) | AU2000233225A1 (en) |
| CA (1) | CA2401699A1 (en) |
| CZ (1) | CZ20022913A3 (en) |
| DE (1) | DE60023187D1 (en) |
| WO (1) | WO2001064931A1 (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005508959A (en) * | 2001-10-17 | 2005-04-07 | ノバルティス アクチエンゲゼルシャフト | Pharmaceutical composition comprising mycophenolic acid or mycophenolate |
| WO2005105768A3 (en) * | 2004-04-26 | 2006-03-16 | Teva Gyogyszergyar Reszvenytar | Process for preparation of mycophenolic acid and ester derivatives thereof |
| WO2006031665A1 (en) * | 2004-09-10 | 2006-03-23 | Ivax Pharmaceuticals S.R.O. | Process for isolation of mycophenolic acid |
| EP1873235A1 (en) * | 2006-06-26 | 2008-01-02 | Universita' degli Studi di Milano | A method for the preparation of Penicillium spores and the use of the latter in the food field |
| US7358247B2 (en) | 2004-04-27 | 2008-04-15 | TEVA Gyógyszergyár Zártköruen Muködö Részvénytársaság | Mycophenolate mofetil impurity |
| US7439373B2 (en) | 2004-07-20 | 2008-10-21 | TEVA Gyógyszergyár Zártkörúen Múködö Részvénytársaság | Crystalline mycophenolate sodium |
| WO2008125616A3 (en) * | 2007-04-12 | 2009-01-22 | Dsm Ip Assets Bv | Method for the purification of bio-molecules |
| CN108727318A (en) * | 2017-04-25 | 2018-11-02 | 鲁南制药集团股份有限公司 | The crystal form object of Mycophenolic Acid |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20080069571A (en) * | 2006-06-29 | 2008-07-28 | 테바 파마슈티컬 인더스트리즈 리미티드 | Regulation of Acid Metabolite Production |
| US20080254520A1 (en) * | 2007-04-11 | 2008-10-16 | Eva Gulyas | Method for reducing impurity level in mycophenolic acid fermentation |
| CN101348810B (en) * | 2008-09-02 | 2011-12-21 | 天津北洋百川生物技术有限公司 | Solid-state fermentation method of mycophenolic acid |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4452891A (en) * | 1980-09-08 | 1984-06-05 | Ajinomoto Company Incorporated | Method for production of mycophenolic acid by fermentation |
| WO2000029544A1 (en) * | 1998-11-17 | 2000-05-25 | Biocon India Limited | Solid state fermentation |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CH469806A (en) * | 1966-12-01 | 1969-03-15 | Afico Sa | Fermentation process and fermenter for the implementation of this process |
| US4212949A (en) * | 1978-08-21 | 1980-07-15 | Golger Leonid I | Apparatus for cultivating microorganisms |
| TR200003179T2 (en) * | 1998-04-30 | 2001-03-21 | Prophyta Biologischer Pflanzenschutz Gmbh | Solid state fermentation device and solid state fermentation method. |
-
2000
- 2000-02-29 WO PCT/IN2000/000017 patent/WO2001064931A1/en not_active Ceased
- 2000-02-29 JP JP2001563619A patent/JP2003525052A/en active Pending
- 2000-02-29 AU AU2000233225A patent/AU2000233225A1/en not_active Abandoned
- 2000-02-29 US US10/220,225 patent/US6927047B1/en not_active Expired - Fee Related
- 2000-02-29 DE DE60023187T patent/DE60023187D1/en not_active Expired - Lifetime
- 2000-02-29 CZ CZ20022913A patent/CZ20022913A3/en unknown
- 2000-02-29 AT AT00911239T patent/ATE306557T1/en not_active IP Right Cessation
- 2000-02-29 EP EP00911239A patent/EP1259631B1/en not_active Expired - Lifetime
- 2000-02-29 CA CA002401699A patent/CA2401699A1/en not_active Abandoned
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4452891A (en) * | 1980-09-08 | 1984-06-05 | Ajinomoto Company Incorporated | Method for production of mycophenolic acid by fermentation |
| WO2000029544A1 (en) * | 1998-11-17 | 2000-05-25 | Biocon India Limited | Solid state fermentation |
Non-Patent Citations (4)
| Title |
|---|
| "Patent non infringing strategies for pharmaceutical industry", BIOPULSE, no. 10, 2000, XP002151454, Retrieved from the Internet <URL:http://www.biocon.com/html/biopulse/issue10/patent.html> [retrieved on 20001017] * |
| BARRIOS-GONZALEZ J ET AL.: "Production of secondary metabolites by solid-state fermentation", BIOTECHNOLOGY ANNUAL REVIEW, vol. 2, 1996, pages 85 - 121, XP000952872 * |
| ISHIKAWA H: "Mizoribine and mycophenolic acid", CURR. MED. CHEM., vol. 6, no. 7, July 1999 (1999-07-01), pages 575 - 597, XP000956409 * |
| SADHUKHAN A K ET AL.: "Optimization of mycophenolic acid production in solid state fermentation using response surface methodology", JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY, vol. 22, no. 1, January 1999 (1999-01-01), pages 33 - 38, XP000956451 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005508959A (en) * | 2001-10-17 | 2005-04-07 | ノバルティス アクチエンゲゼルシャフト | Pharmaceutical composition comprising mycophenolic acid or mycophenolate |
| WO2005105768A3 (en) * | 2004-04-26 | 2006-03-16 | Teva Gyogyszergyar Reszvenytar | Process for preparation of mycophenolic acid and ester derivatives thereof |
| US7683188B2 (en) | 2004-04-26 | 2010-03-23 | TEVA Gyógyszergyár Zártkōrūen Mūkōdō Részvénytársaság | Process for preparation of mycophenolic acid and ester derivatives thereof |
| US7358247B2 (en) | 2004-04-27 | 2008-04-15 | TEVA Gyógyszergyár Zártköruen Muködö Részvénytársaság | Mycophenolate mofetil impurity |
| US7439373B2 (en) | 2004-07-20 | 2008-10-21 | TEVA Gyógyszergyár Zártkörúen Múködö Részvénytársaság | Crystalline mycophenolate sodium |
| WO2006031665A1 (en) * | 2004-09-10 | 2006-03-23 | Ivax Pharmaceuticals S.R.O. | Process for isolation of mycophenolic acid |
| EP1873235A1 (en) * | 2006-06-26 | 2008-01-02 | Universita' degli Studi di Milano | A method for the preparation of Penicillium spores and the use of the latter in the food field |
| WO2008125616A3 (en) * | 2007-04-12 | 2009-01-22 | Dsm Ip Assets Bv | Method for the purification of bio-molecules |
| CN108727318A (en) * | 2017-04-25 | 2018-11-02 | 鲁南制药集团股份有限公司 | The crystal form object of Mycophenolic Acid |
Also Published As
| Publication number | Publication date |
|---|---|
| EP1259631A1 (en) | 2002-11-27 |
| US6927047B1 (en) | 2005-08-09 |
| DE60023187D1 (en) | 2006-02-23 |
| EP1259631B1 (en) | 2005-10-12 |
| CZ20022913A3 (en) | 2003-02-12 |
| JP2003525052A (en) | 2003-08-26 |
| AU2000233225A1 (en) | 2001-09-12 |
| ATE306557T1 (en) | 2005-10-15 |
| CA2401699A1 (en) | 2001-09-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4375464A (en) | Antibiotic AY24,668 and process of preparation | |
| JPH0764872B2 (en) | FR901228 substance and its manufacturing method | |
| EP1259631B1 (en) | Manufacture and purification of mycophenolic acid | |
| US20180187216A1 (en) | Use of microbacterium strains for the production of antibacterial agents | |
| CN101445784A (en) | Eupenicillium brefeldianum variety ZJB082702 and application thereof in preparation of Brefeldin A by fermentation | |
| US5563054A (en) | Process for preparation of benzo[B]thiophene glucuronides | |
| US4954641A (en) | Novel antitumor antibiotic substance and a method for production thereof | |
| WO2008092950A1 (en) | Fermentation process for preparing pravastatin | |
| WO2001081611A1 (en) | A novel process for the manufacture and purification of compactin | |
| KR100186758B1 (en) | Preparation of Pravastatin Precursor | |
| US12018305B2 (en) | Process for production of nigericin from Streptomyces sp. MCC-0151 | |
| EP1799808B1 (en) | Process for producing mycophenolic acid using penicillium arenicola bicc7673 | |
| CN110872338B (en) | Indole diterpenoid compound and preparation method and application thereof | |
| JP2695225B2 (en) | New substance UCT-1003 | |
| US7176001B1 (en) | Manufacture and purification of cyclosporin A | |
| JP2764759B2 (en) | Novel substance BT-38 substance, method for producing the same and antifungal agent containing the same as an active ingredient | |
| EP2032712A1 (en) | Regulation of acid metabolite production | |
| WO2004003214A1 (en) | Solid state fermentation and fed batch for the production of an immunosuppressant | |
| US6838268B2 (en) | Process for the preparation of β-keto aliphatic acid ester | |
| DE60116787T2 (en) | PROCESS FOR PREPARING PRAVASTATIN SODIUM SALT BY STREPTOMYCES FLAVIDOVIRENS DSM 14455 | |
| JPS6219599A (en) | Novel macrolide antibiotic m119 | |
| CN114807286A (en) | Method for refining prednisone acetate based on resting cell transformation | |
| JPH0515385A (en) | New antibiotic, alisamycin, and process for its production | |
| KR840000934B1 (en) | Process for preparing antibiotic sf-2080 a and sf-2080 b | |
| KR20010085642A (en) | Microbial process for preparation of optically active 3-hydroxypyrrolidine derivatives |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| AK | Designated states |
Kind code of ref document: A1 Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZW |
|
| AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
| DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
| WWE | Wipo information: entry into national phase |
Ref document number: PV2002-2913 Country of ref document: CZ |
|
| ENP | Entry into the national phase |
Ref country code: JP Ref document number: 2001 563619 Kind code of ref document: A Format of ref document f/p: F |
|
| WWE | Wipo information: entry into national phase |
Ref document number: 2401699 Country of ref document: CA |
|
| WWE | Wipo information: entry into national phase |
Ref document number: 2000911239 Country of ref document: EP |
|
| WWP | Wipo information: published in national office |
Ref document number: 2000911239 Country of ref document: EP |
|
| WWP | Wipo information: published in national office |
Ref document number: PV2002-2913 Country of ref document: CZ |
|
| REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
| WWE | Wipo information: entry into national phase |
Ref document number: 10220225 Country of ref document: US |
|
| WWG | Wipo information: grant in national office |
Ref document number: 2000911239 Country of ref document: EP |