FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
PROVISIONAL SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION:
"Isolation and purification process for mycophenolic acid or its derivatives"


2. APPLICANT:
(a) NAME: IPCA LABORATORIES LTD.
(b) NATIONALITY: Indian Company incorporated under the Indian Companies
ACT, 1956
(c) ADDRESS: 48, Kandivli Industrial Estate, Charkop, Kandivli (West),
Mumbai-400067, Maharashtra, India.
3. PREAMBLE TO THE DESCRIPTION
The following specification describes the invention.


Field of invention.
The present invention relates to an isolation and purification process for mycophenolic acid obtained from the fermentation process.
Background of the invention.
Mycophenolic acid, chemically known as 6-[4-hydroxy-6-methoxy-7-methyl-3-oxo-5-phthalanyl]-4-methyl-4-hexanoic acid (figure I), was discovered & isolated in 1893, as a secondary metabolite of fungus of penicillium genus. Later several species of Penicillium were identified to produce Mycophenolic acid including P. brevi-compactum, P. scabrum, P. nagemi, P. roqueforti, P. patris-mei and P. viridictum. Among these strains Penicillium Brevicompactum is widely used for the industrial production of mycophenolic acid.

Figure I Mycophenolic acid possesses various pharmaceutically valuable activities such as antibiotic, antiviral and anti-tumor properties and is a powerful immunosuppressant. Two forms of mycophenolic acid are approved for clinical use, which are its sodium salt and prodrug form- mycophenolate mofetil, when administered is metabolized in the liver to the active moiety mycophenolic acid. They are clinically approved for treatment in psoriasis and as an immunosuppressant agent for the prevention of rejection in organ transplantation.
Various prior arts disclose process for preparation and isolation of mycophenolic acid, using wild or mutated strain of Penicillium species.
Submerged culture process and isolation from alkaline culture medium is disclosed in GB 1158387 which results into a yield of 363mg/litre with a purity of 77% and 70 mg/litre with a purity of 94-95%.
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An almost similar process is reported in GB 1157099, wherein the Mycophenolic acid
can be isolated from an acidic culture medium by extraction of the fermentation
broth/medium.
Some other processes report for use of a mutated strain such as US 4115197 and US
4452891.The reagent used for mutation is N-methyl-N'-nitrosoguanidine,
ethylmethylsulfonate, nitrous acid, ultra-violet light with a series of treatments with each
reagent. The mutated strains of the prior art yields mycophenolic acid in the range of
1.85mg/ml and 1.3 g/ml respectively.
Similar mutating agent are disclosed in WO2008/002665, however, higher production
rate of mycophenolic acid is claimed to be achieved based on maintaining the oxygen
level at a specified rate during the fermentation process.
EPl 624070 discloses multilevel fermentation and chromatographic separation of
mycophenolic acid from the broth by using adsorption resin.
Solid state fermentation is reported in Sadhukhan etal, J.ind. Micro. Biotechnology,
resulting into a yield of 3286mg MPA/Kg.
WO01/21607 discloses use of mutated strain of P.walksmeni which provide the product
in range of 2000-3000mg/liter. The mutation is done by using chemicals such as N-
methyl-N-nitro-N'nitrosoguanidine or UV radiation.
Purification of mycophenolic acid by using pH adjustment and extraction of whole broth
or medium with water immiscible solvents is reported in WO2005/105768, wherein
119.4 Kg concentrated mycophenolic acid suspension provides only 553 gm of
mycophenolic acid.
Although there are synthetic methods reported for its production, however, a biological
fermentation method is most commonly used. There are two common type of
fermentation methods used for the production of mycophenolic acid; they are submerged
or solid state fermentation method in either batch mode or fed-batch mode. Better
conversions were reported in fed batch mode of fermentation. Submerged culturing is a
preferred method as uniform mixing and feeding or aeration can be easily achieved. The
yield of mycophenolic acid in the fermentation broth was reported to be very low in the
range of 70mg/liter to 363 mg/liter. The yield of mycophenolic acid reportedly depends
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on several parameters like fermentation conditions; feed character and fed rate, and ultimately the type of the strain used. Although all other parameters are critical, the type of strain is the most crucial for getting higher production. It is also known that wild strain gives only small yields of mycophenolic acid. There are approaches used for the mutation of the wild strain to increase the production capability of wild strain. GB1593208 discloses a mutated strain of P. brevicompactum to increase the productivity of the strain, in which the strain is mutated by treatment with a chemical like N-methyl-N'-nitro-N-nitroso-guanidine, ethylmethylsulfonate, nitrous acid and ultraviolet light. However, the production yield in the fermentation medium is reported to be in the range of 1.85mg/litre- 2.0mg/liter.
A major technical difficulty is the isolation of mycophenolic acid from the fermentation broth containing such a low concentration of the active compound -mycophenolic acid. Generally the isolation comprises pH adjustment of the fermentation broth to either acidic or alkaline and extraction of the whole broth with a water immiscible organic solvent. Typically, according to the literature data, the isolation from the fermentation media comprises extraction of the medium with an organic solvent with or without elimination of mycelia from the broth. Extraction is also reported with adjustment of pH of broth to either acidic or basic range, but the whole fermentation medium was extracted to recover very small amount of mycophenolic acid from the broth. A considerable amount of organic solvents are required to extract most of the mycophenolic acid contained in the medium, and then recovery from the organic solution is effected by energy intensive distillation of large mass of organic solvent, which takes up considerable energy and resources.
For the economy of the process, the mycophenolic acid should be recovered completely from the fermentation broth, which is almost impractical in reported aqueous processes because of poor efficiency of extraction from large volume of aqueous solutions. Being soluble in water, the recovery of mycophenolic acid from aqueous fermentation solution is very difficult and in most cases requires very large volume of organic solvent for recovering a small amount of mycophenolic acid, which is not economically viable.
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Thus, the search for a manufacturing process for the preparation of mycophenolic acid resulting in a satisfactory production yield in the fermentation process and its effective recovery from fermentation broth and final purity of product remains undoubtedly of interest.
It is also an objective of the present invention to provide high mycophenolic acid producing mutant strains and an efficient process to recover the mycophenolic acid from the fermentation broth, apart from simplifying the process for purification of mycophenolic acid. The present invention ameliorates above problems in the prior art and also reduced a number of unit operations of pH assisted extraction/back extractions.
Summary of the invention.
Accordingly, the present invention provides a process for the producing mycophenolic acid and its recovery from the fermentation broth.
According to one aspect of the present invention, a process for mycophenolic acid is provided comprising the steps of:
a) culturing a mycophenolic acid producing Penicillium strain under suitable conditions;
b) adjusting the pH of the fermentation culture obtained in step a) to acidic;
c) recovering the mycelia by filtration or other means;
d) leaching the mycelia with a solvent or alkaline aqueous solution; and
e) isolating the mycophenolic acid from the solution obtained in step (d) ; and optionally
f) purifying the mycophenolic acid obtained.
The microorganism is selected from a penicillium species of P. brevi-compactum, P. scabrum, P. nagemi, P. roqueforti, P. patris-mei and P. viridictum, preferably is Penicillium brevi-compactum. Mutated version of the high producing Penicillium species is used, and mutated penicillium bervi-compactum is especially preferred. The pH of the fermentation broth is adjusted to 4.5 or lower, preferably below 3, before recovering the mycelia from the fermentation broth by filtration or other means.
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In a second aspect, the present invention provides a mutated high producing strain of P. brevicompactum for the production of mycophenolic acid. The mutated strain is produced by treatment of wild P. Brevicompactum with N-methyl-N'-nitro-N-nitroso-guanidine, ethyl methyl sulfonate, clofibrate and methyl violgen.
In a further aspect of the present invention, a process for recovery of mycophenolic acid from the fermentation broth is provided comprising the steps of:
a) adjusting the pH of the whole fermentation broth containing mycophenolic acid to acidic
b) filtering out the mycelia;
c) leaching the mycelia with an organic solvent; and
d) Isolating mycophenolic acid from the organic solvent.
The organic solvents used for leaching includes solvents such as, but not limited to, hydrocarbons, esters, ketones, alcohols, protic solvents, aprotic dipolar solvents, ether and their mixtures thereof. The pH is adjusted to be lower than 4.5.
In a third aspect of the present invention, there is provided a purification method for mycofenolic acid comprising the steps of:
a) forming a solution of mycophenolic acid in an alkaline aqueous solution;
b) optionally, filtering the obtained solution; and
c) precipitating mycophenolic acid from the aqueous solution of step a) or b) by mixing with an acid.
The details of one or more embodiments of the inventions are set forth in the description below. Other features, objects and advantages of the inventions will be apparent from the appended examples and claims.
Detailed description of the invention.
Unless specified otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the
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preferred methods and materials are described. To describe the invention, certain terms
are defined herein specifically as follows.
Unless stated to the contrary, any of the words "including," "includes," "comprising," and
"comprises" mean "including without limitation" and shall not be construed to limit any
general statement that it follows to the specific or similar items or matters immediately
following it. Embodiments of the invention are not mutually exclusive, but may be
implemented in various combinations. The described embodiments of the invention and
the disclosed examples are given for the purpose of illustration rather than limitation of
the invention as set forth the appended claims.
The term "treating" means adding or combining or mixing the stated reagent or materials
to the thing being treated.
The term "aqueous medium" means a solvent medium that does contain water in
significant amounts. The term does not exclude water containing insignificant amounts of
water miscible organic solvent, which may be less than 10%, more preferably less than
5%.
The term "leaching" includes without limitation any means of recovering the product
from a solid mass with a solvent that commonly understood in the art, and includes
extraction, infusion or steeping.
It has now been found that a new process makes it possible to obtain mycophenolic acid
in high yield and purity, ensuring the near complete recovery of mycophenolic acid from
dilute fermentation broth media. The various aspects of the invention are described in
detail with the specific embodiments/conditions hereafter.
Accordingly, in one aspect of the present invention, mycophenolic acid is prepared by a process broadly comprising the steps of:
a) culturing a mycophenolic acid producing microorganism under suitable fermentation conditions;
b) adjusting the pH of the fermentation culture obtained in step a) to acidic;
c) isolating the mycelia from the fermentation broth/medium by filtration or other suitable means;
d) leaching the mycelia with an organic solvent to recover mycophenolic acid;
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e) isolating the mycophenolic acid from the organic solvent; and optionally g) purifying the resulting mycophenolic acid.
The microorganism for the production of mycophenolic acid is selected from any penicillium species capable of producing mycophenolic acid, the known strains includes P. brevi-compactum, P. scabrum, P. nagemi, P. roqueforti, P. patris-mei and P. viridictum. The most preferred strain is Penicillium brevi-compactum. The pH of the fermentation broth is adjusted to 4.5 or lower, preferably below 3, before recovering the mycelia from the fermentation broth by filtration or other means.
The yield of mycophenolic acid in the fermentation medium with the wild strain is very poor. Thus the present invention provides a mutated version of the high producing Penicillium species, especially P. brevi-compactum for increasing the production yield of mycophenolic acid in the broth. With the use of the mutated form of penicillium bervicompactum strain the production yield reaches more than 4.5gm/litre in the fermentation medium compared to 1.3gm/l with the use of wild strain. The high producing mutated strain of Penicillium brevi-compactum is obtained by treating the wild strain with chemical agents to increase the potency for mycophenolic acid. The chemical agent used in the present invention is N-methyl-N'-nitro-N-nitroso-guanidine, ethyl methyl sulfonate, clofibrate, and methyl viologen.
The culturing of the organism may be done according to any known method including either submerged or solid state with or without a fed-batch mode of feeding the organism. The most preferred method is submerged culturing with a fed-batch mode of feeding the organism in the production fermentation stage. The nutrient feed may contain the materials known per se for good growth of the organism, including carbon and nitrogen sources, buffers, and growth enzymes.
Exemplarily carbon source includes, but not limited to, glucose, sucrose, starch, maltose, malto-dextrin, malt, cotton seal meal, soyabean oil meal etc. Exemplarily nitrogen source material may be used in the fermentation process includes, known amino acids, ammonium salts like ammonium nitrate & ammonium nitrate, nitrates like sodium nitrate, bacteriological peptone, yeast extract, casein hydrolyzate, soya peptone, glycine,
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seed meals, for example cottonseed meal, and corn steep liquor, peptone, urea, yeast extract and meat extract. For the maintenance of pH of the fermentation medium, known buffers are added which are customarily used for this application, including phosphate buffer, the source of phosphorous, sulphur and potassium may be for example potassium dihydrogen phosphate, a soluble suphate and potassium chloride respectively. The pH of the medium is maintained during the course of fermentation in the range between 6.0 to 7.5. The fermentation medium usually comprised of aqueous solvent, especially water. Sufficient aeration may be given for maintenance of the oxygen level
The fermentation is carried out usually at a temperature not lower than ambient temperature and not higher than the temperature for the survival of the strain. Preferably the fermentation is carried out at a temperature from ambient temperature to 37 degrees. Especially preferred fermentation temperature is between 25 -30 degree Celsius. The fermentation time is usually range from about 100 hours to about 4-16 days preferably 8-10 days.
The present inventors have found that the isolation of the mycophenolic acid from the fermentation medium according to the prior art processes present substantial difficulties due to the requirement of large volume of the organic solvents for recovering it from the medium and solubility of mycophenolic acid in both water and water immiscible solvents makes it even worse. The efficiency of the extraction is also limited due to very limited choices of organic solvents which has good sosolubility differences between aqueous and water immiscible solvents for better recovery of mycophenolic acid. The present inventors had recognized that in using water immiscible solvents for extraction of large volume of fermentation broth containing 1 gm/litre- 5 gm/litre mycophenolic acid is not practical for the industrial application, owing to the economy of the process. Apart from this, repeated extraction of the fermentation medium might lead to contamination of the product with various organic impurities from the fermentation medium. The invention, therefore, aims to provide an improved method for quantitative recovery of mycophenolic
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acid from the fermentation broth/medium, and even makes it possible to use water miscible solvents for recovery of mycophenolic acid.
Thus the process for recovery of mycophenolic acid from the fermentation broth, according to the present invention, includes the steps of:
e) adjusting the pH of the whole fermentation broth containing mycophenolic acid to acidic
f) filtering out the mycelia;
g) leaching the mycelia with an organic solvent; and
h) isolating mycophenolic acid from the organic solvent. The pH of the fermentation broth is adjusted to 4.5 or lower, preferably below 3, before recovering the mycelia from the fermentation broth by filtration or other means. The acid can be of organic or inorganic acid customarily used for acidification including, but not limited to, hydrochloric acid, sulfuric acid, phosphoric acid, methane sulfonic acid, acetic acid, formic acid etc.
In the process, after acidification of the fermentation broth, the mycelia are separated from the fermentation medium by any conventional means such as filtration or centrifugation. It has been found that the isolation & separation of mycelia leads to quantitative recovery of mycophenolic acid from the medium and better utilization of solvent for the recovery of mycophenolic acid as the mycelia (containing mycophenolic acid) quantity is only about l/6th of the total fermentation medium. The mycelia are leached with an organic solvent to recover the mycophenolic acid.
Suitable organic solvents for leaching the mycelia are those solvents possess reasonable solubility for mycophenolic acid, including, but not limited to, protic and aprotic polar solvent, and non-polar solvent. Exemplarily solvents include esters such as ethyl acetate, & butyl acetate, hydrocarbons such as toluene, cyclohexane, benzene etc., chlorinated hydrocarbons such as dichloromethane, ethylene dichloride, chloroform etc., ethers such as tetrahydrofuran, ketones such as acetone, methylethyl ketone, and alcohols such as isopropyl alcohol, butanol etc.. Especially preferred solvent for this application is toluene and ethyl acetate or their mixtures thereof. The process of the present invention is characterized by use of small volume of organic solvent compared to very large volume
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of solvent required for extraction of mycophenolic acid from the whole fermentation medium.
The extraction/leaching of the mycelia may be carried out at ambient temperature or under heating or with hot organic solvents. Typically the leaching is done at room temperature; however, leaching with hot solvents may lead to further reduction in the solvent volume.
Mycophenolic acid is isolated from the organic solvent after leaching of the mycelia by any conventional means such as crystallization, evaporation, chromatography or any other means known to a skilled artisan. The preferred method includes partial evaporation of the organic solvent and crystallization of the mycophenolic acid from the concentrated solution. Alternately, mycophenolic acid can be recovered from the solution by addition of an anti-solvent to the concentrated solution obtained after leaching the mycelia. The anti-solvent for precipitation of mycophenolic acid from the extraction solvent include non-polar solvents such as hexane. heptane etc.
In another embodiment of the present invention, the mycophenolic acid may be recovered from the mycelia with use of aqueous solvent alone comprising adjusting the pH to alkaline with a base in aqueous solvent and filtering out the mycelia free of mycophenolic acid, followed by precipitating the mycophenolic acid from the aqueous solvent by adjusting the pH to acidic. The aqueous solvent is water. This makes the process to partially eliminate the use of organic solvent.
The purity of the mycophenolic acid exceeds 96% by assay in the methods of the present invention, with a recovery yield of greater than 95% based on the overall titer value of mycophenolic acid in the whole fermentation broth.
The mycophenolic acid may further purified by a novel method. The method is characterized by use of primarily aqueous solvent, especially water for purification which provides a pharmaceutically acceptable purity mycophenolic acid without the need for extractive methods as described in the art. .
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Thus, according to the present invention, there is provided a purification method for mycofenolic acid comprising the steps of:
f) forming a solution of mycophenolic acid in an alkaline aqueous solution;
g) filtering the obtained solution
h) precipitating mycophenolic acid from the aqueous solution in step b) by acidification. The alkaline aqueous solution is made by dissolving appropriate quantity of alkali in water or else alkali may be added to a suspension of mycophenolic acid in aqueous solvent in order to form the solution of mycophenolic acid. The aqueous solvent is water. The alkali may be selected from any known base substance of organic or inorganic origin; however, inorganic bases are preferred for this application. Especially preferred inorganic base is sodium/potassium hydroxide or sodium/potassium carbonates/bicarbonates. The solution may be formed under heating or at room temperature, preferably under heating to about 30-50 degrees the alkaline aqueous solution of mycophenolic acid is optionally filtered to remove insolubles. The filtrate may be further subjected to active charcoal treatment to remove impurities,Mycophenolic acid can be precipitated by addition of acid to the filtrate. Typically the acid can be of organic or inorganic acid customarily used for acidification including, but not limited to, hydrochloric acid, sulfuric acid, phosphoric acid, methane sulfonic acid, acetic acid, formic acid etc. Preferably the acid is hydrochloric acid or sulfuric acid. The precipitation is carried out at ambient temperature or with appropriate cooling of the alkaline solution of mycophenolic acid.
Once the precipitation/crystallization is completed the pure product may be isolated by conventional methods such as filtration, centrifugation, or similar unit operations as exemplified in the accompanying illustrative examples.
The pure mycophenolic acid obtained according to the process of the present invention is then transformed into pharmaceutically acceptable salts, especially sodium salt or its prodrug form mycophenolate mofetil by any conventional method known in the art.
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The pure mycophenolic acid or its salt or its prodrug mofetil form obtained by the process of the present invention may be formulated into a dosage form, e.g., tablet, capsule, etc., by combining with one or more pharmaceutically acceptable excipients using known techniques. Further, the dosage form may be immediate release or extended release.
The examples provided below are illustrative and are not intended to limit the scope of
the claimed invention.
Example 1.
0.5 ml of spore suspension of mutated/modified P. brevicompactum strain was inoculated
in 500 ml conical flask containing 60 ml of seed media.
Inoculated media was incubated at 25°C, for 36 hrs. The seed culture was loaded on to
the bioreactor containing production media.
Production Media

Sr.No Component Quantity (gm/lil)
1 Sucrose 48.0
2 Cotton seed meal 6.0
3 Casein enzymatic hydrolysate 9.0
4 KH2P04 2.0
5 MgS04 0.1
6 Polypropylene glycol 0.2
7 Distilled water 1000ml
8 pH 6.25
9 Glycerol
The pH was maintained at 6.0 with sucrose feeding. The temperature was controlled at 25°C for all 6 days, and 250 ml of soya oil and carbon source was added after completion of 146 hrs. Mycophenolic acid activity in the fermentation broth was 4.8 gm/liter.
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Example 2.
20 lit fermentation broth obtained by cultivation as per example 1, was acidified by addition of dil. HCl to pH 2.5 and the solution was stirred for 4-4.5 hrs at 30°C, and filtered. The residue 2-2.1 Kg (mycelia) was mixed with 6.6 liter of toluene and stirred for 2.0 hrs at 60-65°C. Solution was then filtered off; and the filtrate was concentrated to l/3r volume. The solution was cooled to room temperature and stirred at the same for 30 minutes, and further cooled to 5-10° C. The precipitated mycophenolic acid was collected by filtration and the product was dried under vacuum. Yield was 90gm and Purity was 90%by HPLCassay.
Example 3.
400 ml of water was added to the crude Mycophenolic acid (50 gm) obtained as per example 2, and aq. Solution of sodium carbonate (12.4 gm sodium carbonate dissolved in 100ml water) was added. The resultant mixture was stirred for 1.5 hrs at ambient temperature. The solution was filtered. PH of the clear solution was adjusted to 2.5 with dilute HCl and the precipitated crystals were filtered and washed with water; the product was dried at 70° C for 4-5 hrs to obtain 45 gm pure mycophenolic acid. Purity was 99%+ by HPLC Assay.
Dated this 10th Day of September, 2008

Dr.P.Aruna Sree Agent for the Applicant
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We claim -
1. A process for preparation of mycophenolic acid comprising:
a) culturing a mycophenolic acid producing microorganism;
b) adjusting the pH of the fermentation culture obtained in step (a) to acidic;
c) recovering the mycelia by filtration or other means;
d) leaching the mycelia with an organic solvent or an alkaline solution;
e) isolating mycophenolic acid from the solution of step (d).

2. A process for preparation of mycophenolic acid as claimed in claim 1, wherein the alkaline solution comprises base selected from alkali metal hydroxides, alkali metal carbonates or alkali metal bicarbonates.
3. A process for preparation of mycophenolic acid as claimed in claim 1, wherein the pH of the fermentation culture is adjusted to 4.5 or lower.
4. A process for preparation of mycophenolic acid as claimed in claim 1, wherein the pH of the fermentation culture is adjusted to below 3.
5. A process for preparation of mycophenolic acid as claimed in claim 1, further comprising purification of mycophenolic acid according to following steps:

a) forming a solution of mycophenolic acid in an alkaline aqueous solution;
b) optionally, filtering the obtained solution; and
c) precipitating mycophenolic acid from the aqueous solution of step a) or b.

6. A process as claimed in claim 5 wherein the purification of mycophenolic acid is carried out in presence of water.
7. A process for purification of mycophenolic acid comprising:

a) forming a solution of mycophenolic acid in an alkaline aqueous solution;
b) optionally, filtering the obtained solution; and
c) precipitating mycophenolic acid from the aqueous solution of step a) or b.
Dated this 10th Day of September, 2008

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