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Title:
SOLID STATE FERMENTATION AND FED BATCH FOR THE PRODUCTION OF AN IMMUNOSUPPRESSANT
Document Type and Number:
WIPO Patent Application WO/2004/003214
Kind Code:
A1
Abstract:
The present invention provides a novel method for producing compound of Formula (I) or its any salt form, by solid state fermentation, optionally with fed-batch technique by culturing microorganisms capable of producing the compound of Formula (I).

Inventors:
Khedkar, Anand Prakash (20th Km Hosur Road, Hebbagodi Bangalore 9, Karnataka, 561 22, IN)
Patil, Nitin Sopanrao (20th Km Hosur Road, Hebbagodi Bangalore 9, Karnataka, 561 22, IN)
Ul-ainn, Nazhath (20th Km Hosur Road, Hebbagodi Bangalore 9, Karnataka, 561 22, IN)
Neelakantan, Bhasker (20th Km Hosur Road, Hebbagodi Bangalore 9, Karnataka, 561 22, IN)
Gururaja, Ramavana (20th Km Hosur Road, Hebbagodi Bangalore 9, Karnataka, 561 22, IN)
Melarkode, Ramakrishnan (20th Km Hosur Road, Hebbagodi Bangalore 9, Karnataka, 561 22, IN)
Suryanarayan, Shrikumar (20th Km Hosur Road, Hebbagodi Bangalore 9, Karnataka, 561 22, IN)
Application Number:
PCT/IN2002/000141
Publication Date:
January 08, 2004
Filing Date:
June 28, 2002
Export Citation:
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Assignee:
BIOCON LIMITED (20th Km Hosur Road, Hebbagodi Bangalore 9, Karnataka, 561 22, IN)
Khedkar, Anand Prakash (20th Km Hosur Road, Hebbagodi Bangalore 9, Karnataka, 561 22, IN)
Patil, Nitin Sopanrao (20th Km Hosur Road, Hebbagodi Bangalore 9, Karnataka, 561 22, IN)
Ul-ainn, Nazhath (20th Km Hosur Road, Hebbagodi Bangalore 9, Karnataka, 561 22, IN)
Neelakantan, Bhasker (20th Km Hosur Road, Hebbagodi Bangalore 9, Karnataka, 561 22, IN)
Gururaja, Ramavana (20th Km Hosur Road, Hebbagodi Bangalore 9, Karnataka, 561 22, IN)
Melarkode, Ramakrishnan (20th Km Hosur Road, Hebbagodi Bangalore 9, Karnataka, 561 22, IN)
Suryanarayan, Shrikumar (20th Km Hosur Road, Hebbagodi Bangalore 9, Karnataka, 561 22, IN)
International Classes:
C12P17/18; (IPC1-7): C12P17/18
Attorney, Agent or Firm:
Anand, Pravin (Anand & Anand, B-41 Nizamuddin East, New Delhi 3, 110 01, IN)
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Claims:
We claim:
1. A process for the manufacture of compound of Formula I and its salts by solid substrate fermentation comprising the steps of Formula I preparing an inoculum of the microorganism of the genus Streptomyces, inoculating a solid substrate matrix with the inoculum prepared incubating the inoculated solid substrate matrix for 47 days at 2530 deg. C and extracting the incubated solid substrate matrix to obtain the said product.
2. A process as claimed in claim 1 wherein further comprising the step of purifying the said extract.
3. A process as claimed in claim 1, wherein said microorganism is Streptomyces tsukubaensis.
4. A process as claimed in claim 1, wherein the solid substrate for fermentation is selected from wheat bran, wheat rava, oat meal, broken wheat, boiled rice, rice bran, rice rava, beaten rice, maize bran, maize grits, oat bran, bagasse, tapioca residue, soy grits, soy flakes, rice flakes, ceramic beads, glass beads, sponge or a mixture of two or more of these.
5. A process as claimed in claim 1, wherein the solid substrate fermentation is a fedbatch fermentation.
6. A process as claimed in claim 5, wherein the feeding for fed batch fermentation is done at the beginning of the fermentation or at intervals throughout the fermentation.
7. A process as claimed in claims 5 and 6, wherein the carbon source for feeding is selected from glucose, sucrose, starch (maize, wheat, tapioca, potato), modified starch, maltose, maltodextrin, soybean oil, acetate or a mixture of two or more of these.
8. A process as claimed in claim 5 and 6, wherein the nitrogen source for feeding is selected from ammonium sulphate, dried yeast, ammonium nitrate, sodium nitrate, bacteriological peptone, yeast extract, casein hydrolyzate, soy peptone, soy flour, cotton seed flour, corn steep liquor or a mixture of two or more of these.
9. A process as claimed in claim 1 wherein in the step of purification is carried by filtration, centrifugation, chromatography, extraction, distillation, concentration, precipitation, crystallization and drying. AMENDED CLAIMS [received by the International Bureau on 16 December 2002 (16.12. 02); original claim 1 amended; claim 5 deleted; claims 69 amended and renumbered; remaining claims unchanged; (3 pages)] 1. A process for the manufacture of compound of Formula I and its salts by solid substrate fermentation with feeding comprising the steps of Formula I preparing an inoculum of the microorganism of the genus Streptomyces, inoculating a solid substrate matrix with the inoculum prepared incubating the inoculated solid substrate matrix for 47 days at 2530 deg. C and extracting the incubated solid substrate matrix to obtain the product. 2. A process as claimed in claim 1 further comprising the step of purifying the said extract.
10. 3 A process as claimed in claim 1, wherein the microorganism is Streptomyces tsukubaensis.
11. 4 A process as claimed in claim 1, wherein the solid substrate for fermentation is selected from wheat bran, wheat rava, oat meal, broken wheat, boiled rice, rice bran, rice rava, beaten rice, maize bran, maize grits, oat bran, bagasse, tapioca residue, soy grits, soy flakes, rice flakes, ceramic beads, glass beads, sponge or a mixture of two or more of these.
12. 5 A process as claimed in claim 1, wherein the feeding for solid state fermentation is done at the beginning of the fermentation or at intervals throughout the fermentation.
13. 6 A process as claimed in claim 1 or 5, wherein the carbon source for feeding is selected from glucose, sucrose, starch (maize, wheat, tapioca, potato), modified starch, maltose, maltodextrin, soybean oil, acetate or a mixture of two or more of these.
14. A process as claimed in claim 1 or 5, wherein the nitrogen source for feeding is selected from ammonium sulphate, dried yeast, ammonium nitrate, sodium nitrate, bacteriological peptone, yeast extract, casein hydrolyzate, soy peptone, soy flour, cotton seed flour, corn steep liquor or a mixture of two or more of these.
15. A process as claimed in claim 2 wherein the step of purification is carried by filtration, centrifugation, chromatography, extraction, distillation, concentration, precipitation, crystallization and drying.
Description:
TITLE OF INVENTION SOLID STATE FERMENTATION AND FED BATCH FOR THE PRODUCTION OF AN IMMUNOSUPPRESSANT FIELD OF THE INVENTION The present invention provides a novel method for producing compound of Formula I or its any salt form by solid state fermentation, optionally with fed-batch technique by culturing microorganisms capable of producing the compound of Formula I.

BACKGROUND OF THE INVENTION The compound of Formula I or its any salt form produced by the inventive method is used as an immunosuppressant therapeutically.

This agent inhibits the proliferative response of lymphocytes to alloantigen stimulation, and a variety of T cell associated immune reaction. The compound suppresses immune responses in vivo as well as in vitro and is more highly potent than cyclosporin. The immunosuppressive action of FK506 is applicable in organ transplantation.

Formula I EP 0 184162 disclosed production of FK-506 by submerged fermentation from Streptomyces tsukubaensis.

Tanaka, Hirokazu et al. discussed isolation of the compound of Formula I, known as FK506 or Tacrolimus hydrate from Streptomyces tsukubaensis by submerged fermentation (Yakugaku Zasshi (1997), 17 (8), 542-554).

Kino, Toru et al. discussed isolation of FK-506 from submerged fermentationbrothof Streptomyces tsukobaensís a Antibiot. (1987), 40 (9), 1249-55).

EP 0 497 515 disclosed manufacture of immunosuppressant FK- 506 with cultures of Streptomyces sp. ATCC 55098.

Yoon, Yeo Joon et al. discussed nutrient effect on FK-506 production by Streptomyces sp. in a defined medium for submerged fermentation a. Ferment. Bioeng. (1997), 83 (6), 599-603).

Soeda, Shinsuke et al. also discussed studies on development of tacrolimus production by submerged fermentation.

The prior art literature does not disclose production of compound of Formula I or its any salt form, by solid substrate fermentation or solid state fermentation as well as with fed-batch technique.

SUMMARY OF THE INVENTION The present invention provides a novel method for production of compound of Formula I or its salts.

In preferred embodiments, the invention provides a fermentation process in which the compound of Formula I or it's any salt form, is produced on solid nutritious matrix. The fermentation is also carried out in fed-batch mode to increase the productivity/yields of the final product in a contained bio-reactor.

To achieve the said object, the present invention provides a process for the manufacture of compound of Formula I and its salts by solid substrate fermentation comprising the steps of

Formula I preparing an inoculum of the microorganism of the genus Streptomyces, inoculating a solid substrate matrix with the inoculum prepared, incubating the inoculated solid substrate matrix for 4-7 days at 25-30 deg. C and extracting the incubated solid substrate matrix to obtain the said product.

The extract if desired is subjected to further purification step by conventional techniques, such as filtration, centrifugation, chromatography, extraction, distillation, concentration, precipitation, crystallization and drying.

The micro-organism is Streptomyces tsukubaensis. The solid substrate for fermentation is selected from wheat bran, wheat rava, oat

meal, broken wheat, boiled rice, rice bran, rice rava, beaten rice, maize bran, maize grits, oat bran, bagasse, tapioca residue, soy grits, soy flakes, rice flakes, ceramic beads, glass beads, sponge or a mixture of two or more of these.

The solid substrate fermentation is a fed-batch fermentation. The feeding for fed-batch fermentation is done at the beginning of the fermentation or at intervals throughout the fermentation.

The carbon source for feeding is selected from glucose, sucrose, starch (maize, wheat, tapioca, potato), modified starch, maltose, malto- dextrin, soybean oil, acetate or a mixture of two or more of these.

The nitrogen source for feeding is selected from ammonium sulphate, dried yeast, ammonium nitrate, sodium nitrate, bacteriological peptone, yeast extract, casein hydrolyzate, soy peptone, soy flour, cotton seed flour, corn steep liquor or a mixture of two or more of these.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Definitions "Solid state fermentation"or"solid state cultivation" : The term "solid state fermentation"or"solid state cultivation", sometimes referred to as"semi-solid state fermentation"as used herein, means the process of fermenting microorganisms on a solid medium that provides anchorage points for the microorganisms in the absence of any freely flowing substance. The amount of water in the solid medium can be any amount of water. For example, the solid medium could be almost dry, or it could be slushy. A person skilled in the art

knows that the terms"solid state fermentation"and"semi-solid state fermentation"are interchangeable.

"Fed-batch fermentation"or"fed-batch technique" : The term fed-batch fermentation as used herein, means a fermentation process carried out where substrate or nutrients are added in small increments as the fermentation progresses. The substrate or nutrient is added in small increment that would encourage the production of secondary metabolites, because some secondary metabolite production is inhibited by high concentrations of substrate or substrates, so this method would encourage the production of such metabolites.

Supplement of nutrients at a time when the initially fed nutrient are consumed by the microorganisms or culture also help in providing more energy to the microorganism which in turn increases the overall production of the secondary metabolites.

"Bioreactor" : The term"bioreactor"as used herein, means a device capable of holding fermentation media inoculated with microorganism and carrying out the process of solid state fermentation in a contained manner. A bioreactor can be used to grow any microorganism capable of growing under specified conditions in a contained environment. Some examples of microorganisms capable of growing in a bioreactor are fungi, yeast and bacteria.

The present invention discloses a process for production of compound of Formula I or its any salt form, by culturing a microorganism capable of producing such compound on solid nutrient matrix wherein, optionally the nutrients are fed in adequate quantities

during the growth of the culture so that the production of the product increases significantly.

Another aspect of invention is production of compound of Formula I or its any salt form. The compound is afforded by culturing Streptomyces sp. on solid nutrient matrix e. g. wheat bran, oatmeal, soybean meal, wheat flour, soybean flakes, maize bran etc. The culture is then fed with nutrients to increase production of the final product The product is purified by conventional techniques comprising filtration, centrifugation, chromatography, extraction, distillation, concentration, precipitation, crystallization and drying.

The advantages of the present invention over the other reported methods are: (i) cost effective process (ii) higher productivities of compound of Formula I with feeding (iii) self-contained bioreactor decreases hazardous exposure and risk of contamination.

The following Examples further illustrate the invention, it being understood that the invention is not intended to be limited by the details disclosed therein.

EXAMPLE 1 A well grown slant of Streptomyces tsukubaensis was taken and 5ml of distilled water was added. It was shaken thoroughly and 4ml of this spore suspension was used for the inoculation of 400ml seed medium taken in 2000 ml conical flask. The composition of seed medium is as follows:

Glycerol lOg/L Soluble starch = 10g/L Glucose = 5g/L Cottonseed meal = 5g/L Dried yeast = 5g/L Corn steep liquor = 5g/L Calcium carbonate = 2g/L pH of this medium is adjusted to 6. 5-after making up the volume with water. The seed flasks were grown at 28°C for 4 days and used as an inoculum for solid state fermentation.

Solid state fermentation: l0gm each of wheat bran, maize flakes, wheat rawa, rice rawa, oat meal, maize grits, maize bran, soy grits, rice flake, were taken in separate petri plates. Adequate. amount of water was added and sterilized at 121 deg C for 30 minutes. 10 ml inoculum from 4 day old seed medium was added. The entire substrate was mixed properly with the inoculum and incubated at 28 deg C for 7 days. Following results were obtained. Substrates FK506 mg (kg substrate Wheat bran 6 soy flakes 52 Wheat rawa 47 rice rawa 39 oat meal 58 Maize grits 16 soy grits 4 Maize bran 26 rice flake 32

EXAMPLE 2 Solid state fermentation was conducted as in Example 1 using 75 g ceramic beads as the solid support in a petri-plate, 15 mL of Streptomyces tsukubaensis inoculum grown in seed medium was added.

The result obtained is given in the table below. Solid substrate FK506 mg/kg substrate Ceramic beads 7

EXAMPLE 3 Solid state fermentation was conducted as in Example 1 using different solid supports in combination. l0gm of this substrate is taken in petri plate and 10ml of Streptomyces tsukubaensis inoculum grown in seed medium was added. The results obtained are given in the table below. Solid substrates FK506 mg/kg substrate Wheat bran + oat meal 69 Wheat bran + oat meal + soy flakes 75 Wheat bran + oatmeal + wheat rava 86 Wheat bran + maize bran 39

EXAMPLE 4 Solid state fermentation was conducted as in Example 3 using a mixture of wheat bran, wheat rawa and rice flakes. 10gm of this substrate is taken in petri plate and 10ml of Streptomyces tsukubaensis inoculum grown in seed medium was added along with a liquid nutrient feed consisting of glucose and dried yeast. The feed was added every alternate day up to 4 day.

Solid substrates FK506 mg/kg substrate Without feed 86 With feed 107 EXAMPLE 5 Seed inoculum of Streptomyces tsukubaensis is obtained as explained in Example 1.3. 5L of this inoculum was used for inoculating 35 L of the same seed medium taken in a 50L fermenter.

This is grown for 48 hr at 28°C. This is used as an inoculum for solid state fermentation. 15 kg of substrate mixture consisting of wheat bran, oatmeal and rice flake was loaded into a bioreactor having 22600 cm2 surface area. The bioreactor was sterilised at 121 deg C for 1 to 2 hours using steam. After the sterilization the temperature of the solid substrate was brought down to 28 deg C. 15 L of the above inoculum was added to the solid substrate along with 2 L of glucose and dried yeast feed and mixed. This was incubated at 27 to 29 deg C. On 2ndand 4th day 2 L of the above feed was added to the solid substrate matrix and mixed well. The entire biomass along with the solid substrate was harvested on 7th day and processed to get pharmaceutically acceptable grade of FK506.