The present invention relates to an improved process for the preparation of the compound Lovastatin having the formula shown in Figure I of the accompanying drawings.
The compound Lovastatin has excellent properties of inhibiting cholesterol biosynthesis and is useful against hypercholesteremia, hyperlipemia and atherosclerosis.
Hypercholesterolemia is known to be one of the prime risk factors for ischemic cardiovascular diseases such as arteriosclerosis. Lovastatin is a known antihypercholesterolemic agent that functions by inhibiting the enzyme HMG-CoA reductase.
Lovastatin can be produced by fermentation using various strains of microorganisms for example, the strains belonging to the genus Aspergillus and Monascus such as Aspergillus terreus ATCC 20541 or 20542 (US Patent No.4231938, Nov. 4, 1980), A. oryaze (European Patent No.556699), A. obscurus (US Patent No.5403728, Apr, 1995), Monascus ruber (Ger. Patent No.3006216), Coniothyrium fuckleii (US Patent No.5409820, Apr. 25, 1995).
The recent commercial introduction of HMG-CoA reductase inhibitors like Lovastatin has provided a need for high yielding processes for their production. The techniques to improve processes include, but are not limited to improving the producer microorganism, scale up of the process, improving the culture medium or simplifying downstream recovery process. The present invention focuses on a method of increasing the process yield.
Methods of modification of the fermentation process have been used to increase the yield of the fermented product. For example British Patent No. 1,571,888 discloses that strict pH control in the range of 6.3 to 6.7 markedly increases the yield of the required fermented product. The pH control is done using acids or alkalies.
The improved process, according to the present invention, envisages :
(a) Providing a fermentation process for the preparation of Lovastatin to obtain higher yield
more readily and more economically than the hitherto known processes.
(b) Providing a modified extraction process to improve the yield of Mevinolinic acid
obtained which is mainly present in mycelium.
(c) Providing an improved process for the lactonisation of mevinolinic acid wherein the
lactonisation reaction is carried out on a solid support.
Accordingly the present invention provides an improved process for the preparation of Lovastatin having the Formula shown in Figure I of the accompanying drawings which process comprises fermentation of microfungus of genus Aspergillus in conventional culture media, adding assimilable carbon source, as herein described, continuously or in calculated batches during fermentation to maintain the pH of the fermentation broth between 5.5 to 7.5 and to maintain the residual sugar level in the fermentation broth between 1.0 to 2.8%. the fermentation broth is acidified, mixed with extraction solvent, such as herein described and re fluxed at 60°C to obtain mevinolinic acid having the Formula shown in Figure II in the accompanying drawings or its salt, the mevinolinic acid or its salt thus obtained is then subjected to the lactonisation reaction wherein during the lactonisation reaction, the mevinolinic acid or its salt is bound to a solid support such as resin as herein described and eluting by elutes containing catalyst, as herein described, to convert the mevinolinic acid or its salt to Lovastatin.
According to the process of the present invention, any assimilable carbon source can be employed, for example, mono-, di- or polysachharides, polyols but more specifically a chemically defined Carbon source. Examples of such compounds include dextrose, sucrose, lactose, maltose, glycerol, dextrin, starch or dextrinized starch.
In the process according to the present invention, the pH of the fermentation broth is regulated by controlled addition of assimilable carbon source to the fermentation broth and no acid or alkali is needed to be added to regulate the pH of the fermentaiton broth.
The reducing sugar level of the fermentation broth is kept preferably below 2%, correspondingly, it is preferred to add the assimilable carbon source in low amounts to the fermentation medium. The concentrated solution of the assimilable carbon source is normally used for addition purposes. The carbon source can be incorporated continuously or intermittently, preferably subject to the constraint that the level of the reducing sugar does not rise at any time above 2.8%. For instance, incorporation of assimilable carbon source can be continuous using constant metering of the amounts of carbon source alongwith constant metering of reducing sugar level of the fermentation medium. The continuous addition of carbon source is suitably effected at a rate of around 0.01 to 0.5% w/v per hour, more preferably about 0.1% w/v/h. Preferably the residual sugar level is maintained between 1.5 to
I U O7
1.5/0.
In an alternative mode, incorporation of assimilable carbon source is effected batchwise in accordance with a set protocol,. For example, intermittent addition can be spaced at intervals ranging normally every hour. Carbon source is preferably added in an amount of not more than 3% or better still not more than 1 %, relative to the medium in a single batch.
In another alternative mode, incorporation of the assimilable carbon source is effected in accordance with measured pH and carbon source addition is performed to prevent increase in pH and was stopped when pH started dropping. The addition can be used to maintain pH between 5.2 to 7.5 more specifically it can be controlled between pH 5.7 to 6.5, preferably betwen pH 5.8 to 6.2. The addition can be performed continuously or in batch mode. The rate of addition can be regulated not only by actual measured pH but also by rate of increase or decrease in pH values. The pH can be monitored on line or it can be measured by intermittent sampling.
Iii yet another alternative mode, the addition of assimilable carbon source can be regulated by combining both pH measurement and residual sugar level determination and hence maintaining the residual sugar between 10 to 28 g/L, more specifically between 15 to 20 g/L and maintaining pH between 5.2 to 7.5, more specifically 5.7 to 6.5.
The recovery process, according to the present invention, to improve extraction efficiency of Mevinolinic acid which is mainly present in mycelium, is performed by acidifying whole broth and mixing with extraction solvent and refluxing at 60°C. As extraction solvent, any organic solvent used for such purpose, may be used but preferably ethylacetate, butyl acetate, methylisobutylketone, chloroform may be used. The heating of the whole broth improves the extraction efficiency upto 99% and avoids multiple extractions. The extraction mixture is then padded through Westfalia whole broth extractor and separator to separate two layers. The organic layer is further extracted by aqueous alkali solution to get the compound in aqueous layer. The aqueous layer is acidified and extracted back in ethylacetate.
According.to one embodiment of the invention, the ethylacetate layer is mixed with acetone and ammonia is passed to get the ammonium salt of mevinolinic acid.
The process of lactonisation is to convert the mevinolinic acid or its salt to its lactone form i.e. Lovastatin wherein the reaction is carried out on a solid support. In this process, a column chromatography is performed to bind the mevinolinic acid or its salt to a stationary phase such as resins. The lactone is eluted using a mobile phase consisting of eluting agents alongwith acid catalysts. The process describes use of catalyst in a system where compound is present bound to a resin. The compound exists as a complex on the resin particles, either linked by absorption, ionic linkage or by adsorption. The resin could be hydrophilic, hydrophobic or ionexchange type. The examples of these resins include silica gel, ionexchange resin such as Amberlite CL-, DEAE Cl- or dianion or reverse phase resins such as XAD18, HP20.
The lactone is eluted using an agent which could be an aqueous or non-aqueous solution depending on the resin. The eluting agent is fortified by catalysts or an appropriate acidifying agent, organic or inorganic in catalytic amounts. The catalysts or acidifying agents are mainly organic or inorganic of catalysts such as trifluoro acetic acid, methylsulphonic acid, p-toluene sulphonic acid, sulphonic acid, perchloric acid, hydrochloric acid. Examples of eluting solution includes chloroform-ethylacetate gradient with 1% trifluoroacetic acid, chlorotbrm-methanol gradient with 1% trifluoroacetic acid for silica resin, Acetic acid-water mixture with 1-2% trifluoroacetic acid, water-methanol gradient with 1-2% trifluoroaceuc acid for XAD or HP20 resin.
The following Examples illustrate the present invention without, however, limiting the same
thereto.

EXAMPLE 1. Production of lovastatin at 20L scale
Step 1 Slant culture preparation
A pure culture of lovastatin producing strain was streaked onto slants of Yeastextract
maltextract agar and grown at 28°C for 11 days to get a sporulated slants.
Step 2. Spore formation on rice
"Parimal sela" was washed with water in a stainless steel container until water was clear. The water was drained and 25g of rice was taken in 250 ml Erlenmeyer flask sterilised by autoclaving at 121°C for 25 min at 15 Ib. pressure. After cooling the sterilised rice flask was inoculated with 3 x 107 spores obtained by suspending well sporulated slant from stepl in 5 ml physiological saline (0.85% NaCl) suspension.The flask was shaken and allowed to incubate at 28C for 12 days. Spores were recovered by adding 200 ml of saline and 0.05% Tween 80 mixture in the flask, shaking it well and the filtering it through sterile stainless steel mesh.
Step .v Fermenter Seed Preparation
The seed culture was prepared in 20L Fermenter with working volume of 12L in Fermenter seed medium. The vessel was inoculated with 9.5 x 10g spores obtained from rice. The Fermenter was maintained at 28°C at aeration rate of 7 1pm, for 36 to 48h.
Step 4 Production at Fermenter level
The production medium is prepared in 20L Fermenter with working volume of 15L. The production batch was inoculated using 450 ml transfer volume of seed. The Fermenter was maintained at 28C and .0.5 wm aeration. The agitation and aeration was increased to maintain 50% dissolved oxygen. 2L of 50% Dextrose solution was prepared and sterilised in a glass dosing vessel. It was attached to the Fermenter. The dextrose feeding was started when residual sugar level false to 25g/l and pH of fermenter starts rising. The pH is maintained between 5.8 to 64 by addition of sugar when pH starts rising. The dosing is stopped intermittently if pH starts dropping. The batch is harvested after 200 to 240h when the activity use of lovastatin stops. The harvest activity of 7 g/1 could be obtained.
Step 5: Extraction of mevinolinic acid or its salt
The 10L of harvested broth is mixed with 5L of ethylacetate and refluxed for 30 minutes. The refluxed material was passed through westafalia extractor and separator. The ethylacetate layer was separated and back extracted using 0.2 M alkali solution. The alkali solution is acidified to pH 5 and is back extracted in ethylacetate-hexane mixture 1:1 ratio. The organic layer is used to get lactone.
Step 6: Lactonisation of mevinolinic acid or its salt
The organic layer obtained is concentrated under vaccum. It is dissolved in minimum volume of chloroform. I50g of Silica gel (100-200 mesh) is used in chloroform to get a column. The compound is loaded on the column and elution is done using gradient of
chlorotbrm-ethylacetate both prepared in 1% triflouroacetic acid. The elute was collected in 100 mi fractions and each fraction was checked for presence of lovastatin. The fractions showing lovastatin are free from its acid or salt. The compound comes between 10-30% of ethylacetate-chloroform- 1% triflouroacetic acid mixture. The fractions containing the mevinolinic acid lactone are pooled and concentrated under vaccum. The concentrate is crvstalised using alcohol. The lovastatin crystals are obtained. The conversion obtained is more than 99.8% and HPLC purity obtained is 99.5%
EXAMPLE 2. Lovastatin production at shake flask
Step 1 Shake Flask Inoculum
The seed flasks was prepared in 250 ml Erlenmeyer flasks containing 35 ml seed medium and autoclave sterilised at 121°C for 20 min at 15 lb.. The contents of well sporulated agar slant*from example 1, stepl) was scraped with 5 ml saline (.85% NaCl). One ml of this spore suspension was used to inoculate the seed flask. The seed flask was incubated at 28"C for 42 to 4d h on a rotary shaker at 240 RPM..
Step 3. Shake Flask Production
The production flasks were prepared in 250 ml Erlenmeyer flasks containing 35 ml seed medium and autoclave sterilised at 121°C for 20 min at 15 Ib. 1.0 ml of this growth was inoculated into 250 ml Erlenmeyer flasks containing 35 ml production medium. The flasks were incubated at 28"C on a rotary shaker at 240 RPM. 0.6 ml Dextrose shots were given at 72 and 96 h of a 50%(w/v) solution. Incubation of flask is continued for 8 to 11 days. The activity was measured from 8 to 11 days. Activity ranging from 6.5 g/L to 7.5 g/L is obtained. The flasks were pooled and processed as per example 1, steps 5-6 to get lovastatin. The Lovastatin obtained has 99.5% HPLC purity.
EXAMPLE 3: Lactonisation using mevinolinic acid salt crude
Step 1. Preparation of salt of mevinolinic acid
The fermentation was performed as per example 1, step 1-4 to get whole broth containing
mevinolinic acid. The extraction is performed as per example 1, step 5. The organic layer
obtained is passed through sodium sulphate salt and acetone is added to get its 20%
concentration.
Ammonia gas is passed through the solution. The precipitate obtained is washed using acetone
to get ammonium salt of mevinolinic acid.
Step 2. Lactonisation
A coloumn was prepared using 100ml XAD1180 in water. The mevinolinic acid salt containing 5g of mevinolinic acid obtained from step 1 was dissolved in 50ml water. The salt solution was passed through the coloumn. Water wash was given to the coloumn and elution was done using methanol and 1.5% Triflouroacetic acid mixture. Elutes were checked for lactone by HPLC analysis and concentrated to obtain lovastatin. The concentrate is washed and crystalised using in alcohol to get lovastatin.
EXAMPLE 4: Lactonisation using mevinolinic acid or its Ammonium salt by silica gel method
Ammonium salt (5g) of Mevinolinic acid prepared according to any of the preceeding examples is dissolved in 250 ml water and extracted in 100ml chloroform twice. The chloroform layer is concentrated under vaccum. The concentrate is loaded on silica gel coloumn and processed as per example 4 to obtain lactone of mevinolinic acid in elute. The elute is concentrated and crystalised in isopropanol to obtain lovastatin.

We Claim :
1. An improved process for the preparation of Lovastatin having the Formula shown in Figure I of the accompanying drawings which process comprises fermentation of microfungus of genus Aspergillus in conventional culture media, adding assimilable carbon source, as herein described, continuously or in calculated batches during fermentation to maintain the pH of the fermentation broth between 5.5 to 7.5 and to maintain the residual sugar level in the fermentation broth between 0.1 to 2.8%, the fermentation broth is acidified, mixed with extraction solvent, such as herein described and refluxed at 60°C to obtain mevinolinic acid having the Formula shown in Figure II in the accompanying drawings or its salt, the mevinolinic acid or its salt thus obtained is then subjected to the lactonisation reaction wherein during the lactonisation reaction, the mevinolinic acid or its salt is bound to a solid support such as resin, as herein described and eluting by elutes containing catalyst, as herein described, to convert the mevinolinic acid or its salt to Lovastatin.
2. A process as claimed in claim 1 wherein the assimilable carbon source is mono-, di- or polysachharides or polyols.
3. A process as claimed in claim 2 wherein the assimilable carbon source is dextrose or
sucrose.
4. A process as claimed in claim 1 to 3 wherein the assimilable carbon source is added continuously or calculated batchwise during the fermentation process to maintain the pH of the fermentation broth between 5.8 to 6.2.
5.A process as claimed in claims 1 to 4 wherein the assimilable carbon source is added continuously or calculated batchwise during the fermentation process to maintain the residual sugar level between 1.5 to 1.8%.
6. A process as claimed in claims 1 to 5 wherein an acid catalyst is used while eluting to
convert the mevinolinic acid or its salt to Lovastatin.
7. The improved process for the preparation of Lovastatin substantially as herein described
and illustrated in the Examples stated herein.