WO2001081611A1 - A novel process for the manufacture and purification of compactin - Google Patents

A novel process for the manufacture and purification of compactin Download PDF

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Publication number
WO2001081611A1
WO2001081611A1 PCT/IN2000/000049 IN0000049W WO0181611A1 WO 2001081611 A1 WO2001081611 A1 WO 2001081611A1 IN 0000049 W IN0000049 W IN 0000049W WO 0181611 A1 WO0181611 A1 WO 0181611A1
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WIPO (PCT)
Prior art keywords
improved method
compactin
solid state
fermentation
ethyl acetate
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PCT/IN2000/000049
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French (fr)
Inventor
Anindya Sircar
Anand Prakash Khedkar
Pampapathy Subramaniyam
Shreehas Pradeep Tambe
Konayakanahalli Nanjunda Swamy Anand
Melarkode Ramkrishna
Gururaja Ramavana
Shrikumar Suryanarayan
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Biocon India Limited
Priority date (The priority date 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 date listed.)
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Publication date
Application filed by Biocon India Limited filed Critical Biocon India Limited
Priority to AU2000255633A priority Critical patent/AU2000255633A1/en
Priority to PCT/IN2000/000049 priority patent/WO2001081611A1/en
Publication of WO2001081611A1 publication Critical patent/WO2001081611A1/en

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    • 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
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein

Definitions

  • the present invention relates to an improved method for the manufacture and purification of Compactin.
  • Compactin (also known as mevastatin, ML-236B) is an antihypercholesterolemic and antiartereosclerotic agent from the statin family. Compactin is also known to exhibit anti-fungal properties.
  • the biosynthetic pathway of compactin formation is similar to lovastatin biosynthetic pathway. It is a condensation product of polyketide synthetic pathway where a nonaketide and a diketide are formed separately and then condensed to form mevastatin.
  • Aspergillus terreus which is primarily used for production of lovastatin is also reported to produce mevastatin along with lovastatin at 24°C (Manzoni, M.
  • Compactin derivatives are produced by fermenting ML-236B with Mucor, Rhizopus, Actinomucor, Rhizoctonia, etc.
  • M. hiemalis is shake-cultured at 26°C for 4 days on a medium containing glucose 1, peptone 0.2, meat extract 0.1, yeast extract 0.1, and corn steep liquor 0.3%.
  • 0.05% compactin is added and the broth shake-cultured for 6 days.
  • the filtrate of the culture is extracted with ethyl acetate.
  • the ethyl acetate extract is washed with saturated sodium chloride, mixed with diazomethane in Ethyl acetate, and evaporated to dryness under reduced pressure
  • the residue obtained from this is then worked upon column chromatography (Japanease Patent No. JP 57155995). All in all 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 manufacture of compactin by solid substrate fermentation in a novel bioreactor PLAFRACTO TM and its subsequent purification .
  • this invention provides an improved method for the manufacture and purification of Compactin comprising: loading a contained bioreactor with solid state nutritive matrix and sterilizing it, mixing the said sterilized solid state nutritive matrix with Penicillium citrinum, incubating the said inoculated solid state nutritive matrix for 4
  • Penicillium citrinum used is in the form of a macroconidial suspension.
  • 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 novel bioreactor is a PLAFRACTORTM
  • the solid state nutritive matrix is selected from wheat bran, rice bran, soya grits, rice grits, millet flour or a mixture of these.
  • the organic solvent used for extraction is selected from acetone, methanol, toluene, benzene or ethyl acetate.
  • the aqueous buffer used for extraction is at pH 7.0 to 9.5.
  • the filtration is carried out with the help of a filter aid selected from celite, perlite or alumina.
  • the water miscible solvent used to dissolve the crude crystals is selected from acetone, methanol, ethanol or acetronitrile.
  • Compaction obtained may be further processed to get pravastatin or its salts.
  • the bioreactor used for the solid state fermentation in our invention has been described in our PCT Appln No. PCT/99IB/01688 dated October 15, 1999.
  • the modular construction of this bioreactor has 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 state 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 micfobial metabolites, which are cytotoxic in nature.
  • the base plate of the bioreactor has multiple channels called noncommunicatmg 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 said novel contained bioreactor, 'PLAFRACTOR' is capable of sterilizing the solid state fermentation media, cooling it to the required temperature, fermenting at the desired set conditions, m situ extraction of the end product, recovery of the solvents and post harvest sterilization.
  • Example 1 Heavily sporulated slants of Penicillium citrinum was taken and 5 mL of sterile distilled water was added to it. It was shaken thoroughly and 500 L of the spore suspension was spread on PGA (Potato Glycerol Agar) plates and incubated at 25°C for 7 days. After 7 days the macroconida lodged as a slime is scraped by a sterile loop in sterile distilled water. This microconidal suspension, devoid of mycelial bits is used as the inoculum. 15 Kg of wheat bran was loaded on the contained bioreactor of approximately 22600 cm ⁇ 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 1-2 hours.
  • 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 10 4 macroconidia/ml suspension of Penicillium citrinum in 14 L of sterilized distilled water. This was used to inoculate the sterilized wheat bran so that the final moisture after inoculation was 65 %.
  • the inoculum was mixed thoroughly with the sterilized bran.
  • the contained bioreactor was sterilized and inoculated as in Example 1. In this experiment, the temperature was maintained at 25°C for all 5 days. The Compactin production titres were assayed following extraction using the HPLC.
  • the contained bioreactor was sterilized and inoculated as in Example 1.
  • rice bran was used instead of wheat bran and the temperature was maintained at 25°C for all 5 days.
  • the Compactin production titres were assayed following extraction using the HPLC.
  • Example 4 5 Kg. fermented wheat bran obtained from Example 3 was then extracted by using acetone. 10 L of acetone extract was collected, analyzed and taken for further processing. The extraction efficiency of acetone was found to be greater than 98%, as quantitated by HPLC.
  • the extract obtained from Example 2 was concentrated by azeotropic distillation to remove acetone, leaving behind 1.5 L of aqueous residue.
  • the pH of the aqueous residue obtained was adjusted to 4.0.
  • 1.5 L of ethyl acetate was added and the mixture was stirred for 1 hr.
  • the two layers were allowed to separate and ethyl acetate layer was collected.
  • This ethyl acetate layer was washed with saturated solution of NaCl.
  • the ratio of NaCl solution to ethyl acetate was maintained at 0.075: 1.
  • Ethyl acetate layer was concentrated by azeotropic distillation at a temperature of 55°C. The residue left behind was chilled to about 5°C for 8 hr.
  • the crystals of compactin formed were filtered and washed with hexane. Crystals obtained from ethyl acetate were dissolved in 1 :10 (w/v) times acetone, to this solution, 50 gm alumina per L of acetone solution was added and stirred for 1 hr. Alumina was removed by filtration. The filtrate was then concentrated by azeotropic distillation. The concentrate on crystallization gives crystals of pure compactin.

Abstract

The present invention provides an improved method for the manufacture of Compactin. Compactin is produced by Penicillium citrinum, using solid state fermentation in a bioreactor under optimal fermentation parameters. The product is extracted and further purified to get pharmaceutical acceptable purity.

Description

A Novel Process for the manufacture and purification of Compactin.
The present invention relates to an improved method for the manufacture and purification of Compactin.
BACKGROUND
Compactin (also known as mevastatin, ML-236B) is an antihypercholesterolemic and antiartereosclerotic agent from the statin family. Compactin is also known to exhibit anti-fungal properties. The biosynthetic pathway of compactin formation is similar to lovastatin biosynthetic pathway. It is a condensation product of polyketide synthetic pathway where a nonaketide and a diketide are formed separately and then condensed to form mevastatin. Aspergillus terreus which is primarily used for production of lovastatin is also reported to produce mevastatin along with lovastatin at 24°C (Manzoni, M. Qt al, Biotechnol.Lett.; (1999) 21, 3, 253-57). There is a constant requirement for improving the productivity's and in a shorter time. Submerged fermentation of compactin reportedly gives yields of 1.275 gm/1 at 23°C using P. Adamet∑ioides in 7 days and 1.33 gm/1 in 9 days. Spores of Penicillium cyclopium when immobilized on loofah sponge and used for continuous culture, produces 23.04mg/l.h of compactin (Bazaraa W.A., J.Ind.Microbiol.Biotechnol; (1998) 21, 4-5, 192-202). Compactin derivatives like pravastatin Na+are more potent and tissue specific than compactin itself. Compactin derivatives are produced by fermenting ML-236B with Mucor, Rhizopus, Actinomucor, Rhizoctonia, etc. Thus, M. hiemalis is shake-cultured at 26°C for 4 days on a medium containing glucose 1, peptone 0.2, meat extract 0.1, yeast extract 0.1, and corn steep liquor 0.3%. Then, 0.05% compactin is added and the broth shake-cultured for 6 days. The filtrate of the culture is extracted with ethyl acetate. The ethyl acetate extract is washed with saturated sodium chloride, mixed with diazomethane in Ethyl acetate, and evaporated to dryness under reduced pressure The residue obtained from this is then worked upon column chromatography (Japanease Patent No. JP 57155995). All in all 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 manufacture of compactin by solid substrate fermentation in a novel bioreactor PLAFRACTO ™ and its subsequent purification .
To achieve the said objective, this invention provides an improved method for the manufacture and purification of Compactin comprising: loading a contained bioreactor with solid state nutritive matrix and sterilizing it, mixing the said sterilized solid state nutritive matrix with Penicillium citrinum, incubating the said inoculated solid state nutritive matrix for 4
6 days at 25 - 30°C, extracting the fermented matrix with an organic solvent or an aqueous buffer; - concentrating the extract obtained, and re-extracting in ethyl acetate; optionally giving the ethyl acetate extract a wash with saturated NaCl solution; concentrating the ethyl acetate extract and cooling the slurry obtained at 0 to 10 °C for 4 to 12 hours; filtering to get crude crystals, optionally giving a hexane wash; dissolving the crude crystals in a water miscible solvent followed by the addition of filter aid and agitating for 0.5 to 3 hour;, filtering to get a clear solution which is concentrated; and crystallizing of the concentrate to get pure compactin.
The Penicillium citrinum used is in the form of a macroconidial suspension.
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 novel bioreactor is a PLAFRACTOR™
The solid state nutritive matrix is selected from wheat bran, rice bran, soya grits, rice grits, millet flour or a mixture of these.
The organic solvent used for extraction is selected from acetone, methanol, toluene, benzene or ethyl acetate.
The aqueous buffer used for extraction is at pH 7.0 to 9.5.
The filtration is carried out with the help of a filter aid selected from celite, perlite or alumina.
The water miscible solvent used to dissolve the crude crystals is selected from acetone, methanol, ethanol or acetronitrile.
Compaction obtained may be further processed to get pravastatin or its salts. The bioreactor used for the solid state fermentation in our invention has been described in our PCT Appln No. PCT/99IB/01688 dated October 15, 1999. The modular construction of this bioreactor has 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 state 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 micfobial metabolites, which are cytotoxic in nature.
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 state fermentation using tray cultures is not efficient. The base plate of the bioreactor has multiple channels called noncommunicatmg 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 said novel contained bioreactor, 'PLAFRACTOR' is capable of sterilizing the solid state fermentation media, cooling it to the required temperature, fermenting at the desired set conditions, m situ extraction of the end product, recovery of the solvents and post harvest sterilization. The invention will now be described with reference to the following examples:
Example 1: Heavily sporulated slants of Penicillium citrinum was taken and 5 mL of sterile distilled water was added to it. It was shaken thoroughly and 500 L of the spore suspension was spread on PGA (Potato Glycerol Agar) plates and incubated at 25°C for 7 days. After 7 days the macroconida lodged as a slime is scraped by a sterile loop in sterile distilled water. This microconidal suspension, devoid of mycelial bits is used as the inoculum. 15 Kg of wheat bran was loaded on the contained bioreactor of approximately 22600 cm^ 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 1-2 hours. 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 104macroconidia/ml suspension of Penicillium citrinum in 14 L of sterilized distilled water. This was used to inoculate the sterilized wheat bran so that the final moisture after inoculation was 65 %. 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 was sent into the bioreactor continuously. The temperature was controlled at 25°C for all 5 days by conductive heating and cooling. The Compactin production litres was assayed following extraction using the HPLC. Example 2:
The contained bioreactor was sterilized and inoculated as in Example 1. In this experiment, the temperature was maintained at 25°C for all 5 days. The Compactin production titres were assayed following extraction using the HPLC.
Example 3:
The contained bioreactor was sterilized and inoculated as in Example 1. In this experiment, rice bran was used instead of wheat bran and the temperature was maintained at 25°C for all 5 days. The Compactin production titres were assayed following extraction using the HPLC.
Example 4: 5 Kg. fermented wheat bran obtained from Example 3 was then extracted by using acetone. 10 L of acetone extract was collected, analyzed and taken for further processing. The extraction efficiency of acetone was found to be greater than 98%, as quantitated by HPLC.
Example 5:
The extract obtained from Example 2 was concentrated by azeotropic distillation to remove acetone, leaving behind 1.5 L of aqueous residue. The pH of the aqueous residue obtained was adjusted to 4.0. After adjusting the pH, 1.5 L of ethyl acetate was added and the mixture was stirred for 1 hr. The two layers were allowed to separate and ethyl acetate layer was collected. This ethyl acetate layer was washed with saturated solution of NaCl. The ratio of NaCl solution to ethyl acetate was maintained at 0.075: 1. Ethyl acetate layer was concentrated by azeotropic distillation at a temperature of 55°C. The residue left behind was chilled to about 5°C for 8 hr. The crystals of compactin formed were filtered and washed with hexane. Crystals obtained from ethyl acetate were dissolved in 1 :10 (w/v) times acetone, to this solution, 50 gm alumina per L of acetone solution was added and stirred for 1 hr. Alumina was removed by filtration. The filtrate was then concentrated by azeotropic distillation. The concentrate on crystallization gives crystals of pure compactin.
The examples given above are not exhaustive.
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 fermentation product - Compactin. (ii) Less fermentation time and easier control of temperature making the process economically attractive.
(iii) Fewer steps for the isolation and purification to get the pure product, thus saving processing time and additional expenses, (iv) High efficiency of the purification process results in higher yields.

Claims

Claims:
1. An improved method for the manufacture and purification of Compactin comprising: loading a contained bioreactor with solid state nutritive matrix and sterilizing it, mixing the said sterilized solid state nutritive matrix with Penicillium citrinum, incubating the said inoculated solid state nutritive matrix for 4 - , 6 days at 25 - 30°C, - extracting the fermented matrix with an organic solvent or an aqueous buffer; concentrating the extract obtained, and re-extracting in ethyl acetate; optionally giving the ethyl acetate extract a wash with saturated NaCl solution; - concentrating the ethyl acetate extract and cooling the slurry obtained at 0 to 10 °C for 4 to 12 hours; filtering to get crude crystals, optionally giving a hexane wash;, dissolving the crude crystals in a water miscible solvent followed by the addition of filter aid and agitating for 0.5 to 3 hour;. - filtering to get a clear solution which is concentrated; and crystallizing of the concentrate to get pure compactin.
2. An improved method of claim 1 wherein the Penicillium citrinum used is in the form of a macroconidial suspension.
3. An improved method of 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.
4. An improved method of claim 3 wherein the said contained bioreactor is "PLAFRACTOR".
5. An improved method of claim 1 wherein the solid state nutritive matrix is selected from wheat bran, rice bran, soya grits, rice grits, millet flour or a mixture of these.
6. An improved method of claim 1 wherein the organic solvent used for extraction is selected from acetone, methanol, toluene, benzene or ethyl acetate.
7. An improved method of claim 1 wherein the aqueous buffer used for extraction is at pH 7.0 to 9.5.
8. An improved method of claim 1 wherein the filtration is carried out with the help of a filter aid selected from celite, perlite or alumina.
9. An improved method of claim 1 wherein the water miscible solvent used to dissolve the crude crystals is selected from acetone, methanol, ethanol or acetronitrile.
10. An improved method of 1 wherein the compactin obtained is further processed to get pravastatin or its salts.
PCT/IN2000/000049 2000-04-27 2000-04-27 A novel process for the manufacture and purification of compactin WO2001081611A1 (en)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100453452B1 (en) * 2002-10-23 2004-10-15 씨제이 주식회사 A Penicillium cyclopium sp. and a process for producing a compactin using the same
KR100458067B1 (en) * 2002-12-13 2004-11-18 씨제이 주식회사 A Penicillium sp. and a process for producing a compactin using the same
WO2005051372A2 (en) * 2003-11-24 2005-06-09 Teva Gyógyszergyár Zártköruen Muködo Részvénytársaság Method of purifying pravastatin
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
CN102827123A (en) * 2012-08-02 2012-12-19 丽珠集团新北江制药股份有限公司 Separation and extraction process for Mevastatin
CN108977469A (en) * 2018-01-29 2018-12-11 北大方正集团有限公司 Rice bran meal soaks application of the juice in production Pravastatin
CN115521944A (en) * 2022-08-29 2022-12-27 云南大学 Preparation method and application of penicillium fermentation product ethyl acetate extract
CN115521944B (en) * 2022-08-29 2024-04-19 云南大学 Preparation method and application of penicillium fermentation product ethyl acetate extract

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US5942423A (en) * 1995-06-07 1999-08-24 Massachusetts Institute Of Technology Conversion of compactin to pravastatin by actinomadura

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US5942423A (en) * 1995-06-07 1999-08-24 Massachusetts Institute Of Technology Conversion of compactin to pravastatin by actinomadura

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BARRIOS-GONZALEZ J ET AL: "PRODUCTION OF SECONDARY METABOLITES BY SOLID-STATE FERMENTATION", BIOTECHNOLOGY ANNUAL REVIEW,XX,XX, vol. 2, 1996, pages 85 - 121, XP000952872 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100453452B1 (en) * 2002-10-23 2004-10-15 씨제이 주식회사 A Penicillium cyclopium sp. and a process for producing a compactin using the same
KR100458067B1 (en) * 2002-12-13 2004-11-18 씨제이 주식회사 A Penicillium sp. and a process for producing a compactin using the same
WO2005051372A2 (en) * 2003-11-24 2005-06-09 Teva Gyógyszergyár Zártköruen Muködo Részvénytársaság Method of purifying pravastatin
WO2005051372A3 (en) * 2003-11-24 2005-08-11 Teva Gyogyszergyar Reszvenytar Method of purifying pravastatin
US7425644B2 (en) 2003-11-24 2008-09-16 TEVA Gyógyszergyár Zártkörűen Működő Részvénytársaság Method of purifying pravastatin
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
CN102827123A (en) * 2012-08-02 2012-12-19 丽珠集团新北江制药股份有限公司 Separation and extraction process for Mevastatin
CN102827123B (en) * 2012-08-02 2015-04-22 丽珠集团新北江制药股份有限公司 Separation and extraction process for Mevastatin
CN108977469A (en) * 2018-01-29 2018-12-11 北大方正集团有限公司 Rice bran meal soaks application of the juice in production Pravastatin
CN108977469B (en) * 2018-01-29 2022-04-08 北大方正集团有限公司 Application of rice bran meal immersion juice in pravastatin production
CN115521944A (en) * 2022-08-29 2022-12-27 云南大学 Preparation method and application of penicillium fermentation product ethyl acetate extract
CN115521944B (en) * 2022-08-29 2024-04-19 云南大学 Preparation method and application of penicillium fermentation product ethyl acetate extract

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