Title of Invention	A PROCESS FOR THE ISOLATION AND PURIFICATION OF COMPACTIN FROM A LIQUID MEDIUM
Abstract	The present invention is related to a new method of producing compaction (ML-236B), obtained from a liquid medium. Compacting is used as a precursor of pravastatin sodium, which is potent cholesterol-lowering agent, used in treatment for hypercholesterolemia.

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Novel Process For The Purification of Compactin. The present invention relates to an improved method for the purification of Compactin. Compactin is an antihypercholesterolemic and antiartereosclerotic agent from the statin family. Compactin is also known to exhibit anti-fungal properties. The fermented broth or the extract obtained after fermentation is adjusted to a pH of 7 - 12 to dissolve the Compactin obtained by fermentation. The microbial biomass and other undissolved solids are removed by microfiltration. The filtrate so obtained may be subjected to ultrafiltration to remove the proteins. The pH of the filtrate is adjusted to 1.5 to 3.0; optionally adding a precipitating agent and the precipitate so obtained is filtered. The filtered precipitate is dissolved in an organic solvent or an aqueous buffer and concentrated. The compactin so obtained in solution is further processed to get pure compactin or used as a feed for the fermentation to get Pravastatin. Advantages: 1. The yield of Compactin by purifying by this method is higher than other reported methods. 2. This method for the production involves less number of steps and thus consumes less time. 3. This method for the production involves less quantity of organic solvents. 4. The product obtained by this method is economically attractive. THE PATENTS ACT, 1970 COMPLETE SPECIFICATION TITLE:"A process for the isolation and purification of Compactin from a liquid medium" APPLlCANT:Biocon India Limited An Indian Company,of 20th Km Hosur Road, Hebbagodi, Bangalore-561 229, Karnataka, India INVENTORS: 1. KULKARNI Madhav 2. MELARKODE Ramkrishnan 3. GOEL Anju 4. GURURAJA Ramavana 5. SURYANARAYAN Shrikumar The following Specification particularly describes the nature of this invention and the manner in which it is to be performed:- The present invention discloses a novel method for the isolation and purification of Compactin from a liquid medium. Background Lovastatin, Compactin, Pravastatin, Simvastatin, their derivatives and analogs are known as HMG-CoA reductase inhibitors and are used as antihypercholesterolemic agents. They are produced by fermentation as well as synthetically. Following synthesis or fermentation, these compounds are to be separated and purified using different methods. The processes for the isolation and purification of the antihypercholesterolemic agents are disclosed in the earlier patent applications through a family of patents by Sankyo and others (WO 92/16276, WO 99/42601) The reported methods for purification comprise different combinations of extraction, chromatography, distillation, lactonization and crystallization methods. The purity of the final product thus obtained is pharmaceutically acceptable but the yields of the desired product are then unacceptably low when scaled up to an industrial scale. The method for dissolution disclosed in patent application WO 97/20834 comprises treatment of fermentation broth with alkaline base to pH 11.5 and stirring for 3 hours. The WO 97/06128 discloses a method that dissolution may be done with alkalifying the fermentation broth to pH 10 to 13, Also temperature between 60 to 95° C is applied. Too long exposure to such rigorous conditions causes degradation ot (compactin. L he dissolution may oe carried out also at pH lower than 9 and especially lower than 6 but the use of large amount of organic solvents is necessary in this case. Detailed description of the invention The present invention is related to a new method of producing compactin (ML-236B), obtained from culture broth of Penicillium sp. Compactin is used as a precursor of Pravastatin sodium, which is potent cholesterol-lowering agent, used in treatment for hypercholesterolemia. The present invention discusses a process for isolation and purification of compactin from a liquid medium of the kind as herein described, comprising of: a) adjusting pH of the liquid medium to 7 to 12 to dissolve compactin, b) separating the biomass and the broth c) ultrafiltration of the broth for removing proteins d) acidifying the filtrate to pH of 1.5 to 3 to get a precipitate, e) separation of the precipitate, f) contacting the precipitate of step (e) with a water, organic solvent or aqueous buffer at a pH ranging between 7-10, g) concentration of the organic solvent of (f) h) filtration and drying to get pure Compactin The liquid medium is a fermentation broth water or an aqueous buffer. The biomass is separated in step (b) from the broth by filtration, decantation or centrifugation. The filtrate is optionally clarified by microfiltration after separation. The precipitate in step (e) is separated by filtration, optionally using filter aid. The filter aid is selected from diatomaceous earth material. The pH in step (d) is 2-3. The pH in step (f) is 8-9. The precipitate in step (e) is separated by microfiltration or ultrafiltration. The precipitate obtained is contacted with an organic solvent in step (f) selected from ethyl acetate, butyl acetate or toluene. The precipitate obtained in step (e) is contacted with water or an aqueous buffer. The organic solvent containing Compactin is concentrated in step (g) by distillation. The compactin so obtained is further processed to get pravastatin or its analogues. Since Compactin is found to be an intra- and extra cellular product, it is required to dissolve it from mycelium into the aqueous medium in which the cells are suspended. In the present invention it was found that treatment at pH 12 for 30 minutes is more than sufficient to reach the plateau in extraction at room temperature. Two subsequent extractions for time not more than 30 minutes dissolves all the Compactin from mycelium into the aqueous medium. After dissolution, the mycelium can be separated by simple filtration with and without the filter aid. The filtrate obtained by filtration is optionally subjected to microfiltration to remove the cell mass and clarify the broth. The filtrate obtained by simple filtration as well as the micro-filtrate is subjected to ultrafiltration. This removed high molecular weight compounds in the filtrate, which in turn increased the purity of the product. The pH of the ultrafiltration permeate is adjusted to a value in a range of 1.5 to 4, preferably between 2 to 3 and kept under stirring for 0.5 to 4 hours. Precipitate is separated by simple filtration, microfiltration or ultrafiltration. To recover maximum amount of the product, this step was repeated once more. The precipitate thus obtained was dissolved into aqueous medium at alkaline pH, between 7 to 10, preferably between 8 to 9. This Compactm solution was used as precursor feed for Pravastatm analog fermentation. The precipitate obtained by filtration was also dissolved into an organic solvent, ethyl acetate and concentrated to get lactone analog of Compactin, which is further processed to get Compactin crystals. In this particular process, the ethyl acetate quantity required is very less as compared to the conventional process where the large quantity of ethyl acetate used to extract the Compactin from broth by adjusting the pH to acidic side, preferably below 6 and then is concentrated by distillation and lactonized. The following examples illustrate the process of instance invention and are not to be considered as limiting the invention set forth in the claims appended hereto. Example 1 The broth (500 ml) with a Compactin concentration of 1.9 mg/ml obtained by fermentation with Penicillium sp.(Q\CC 6827) was adjusted to pH 12 and stirred for 30 minutes. The broth was filtered. The cells obtained were suspended in water, pH of which was adjusted to 12. The suspension was stirred for 30 minutes and filtered. This procedure was repeated for maximal extraction. The filtrates thus obtained were pooled together and microfiltration was done using 0.1 micron membrane. The microfiltration permeate was then subjected to ultrafiltration. pH of the ultrafiltration permeate was adjusted to 2.2 and stirred for 1 hour. The precipitate was recovered over filter aid, celite. The filtrate pH was adjusted to 1.8, stirred for 1 hour and then filtered over celite. The Compactin thus obtained was of acceptable purity and the recovery was over 90%. Example 2 The broth (500 ml) with a Compactin concentration of 2.5 mg/ml obtained by fermentation with Penicillium sp. (BICC 7226) was adjusted to pH 12 and stirred for 30 minutes. The broth was filtered. The cells obtained were suspended in water, pH of which was adjusted to 12. The suspension was stirred for 30 minutes and filtered. This procedure was repeated once more. The filtrates thus obtained were pooled and filtrate was subjected to ultrafiltration using UF membrane. pH of the ultrafiltration permeate was adjusted to 2.0 and stirred for 1 hour. The precipitate was recovered over filter aid, celite. pH of the filtrate was adjusted to 1.8, stirred for 1 hour and then filtered over celite. The precipitates thus obtained were combined and dissolved in ethyl acetate. The ethyl acetate was then concentrated and refluxed to get Compactin lactone. When the lactonization was complete, the product was recovered by crystallization. The Compactin thus obtained was of acceptable purity and the recovery was over 86%. Example 3 The broth (500 ml) with a Compactin concentration of 2.0 mg/ml obtained by fermentation with Penicillium sp. was adjusted to pH=ll and stirred for 30 minutes. The broth was filtered using celite as filter aid. The cells obtained were suspended in water, pH of which was adjusted to 11. The suspension was stirred for 30 minutes and filtered. This procedure was repeated once more. The filtrates thus obtained were pooled and filtrate was subjected to microfiltration using 0.1 micron membrane and then ultrafiltered using UF membrane. The pH of the ultrafiltration permeate was adjusted to 2.0 and stirred for 1 hour. The precipitate was recovered by ultrafiltration and was diafiltered with pH 2.0 water. The Compactin thus' obtained was of acceptable purity and the recovery was over 84%. We Claim: 1. A process for the isolation and purification of Compactin from a liquid medium of the kind as herein described, comprising of a) adjusting pH of the liquid medium to 7 to 12 to dissolve compactin, b) separating the biomass and the broth c) ultrafiltration of the broth for removing proteins d) acidifying the filtrate to pH of 1.5 to 3 to get a precipitate, e) separation of the precipitate, f) contacting the precipitate of step (e) with a water, organic solvent or aqueous buffer at a pH ranging between 7-10, g) concentration of the organic solvent of (f) h) filtration and drying to get pure Compactin 2. The process according to claim 1 wherein the liquid medium is a fermentation broth. 3. The process according to claim 1 wherein the liquid medium is water or an aqueous buffer. 4. The process according to claim 1 wherein the biomass is separated in step (b) from the broth by filtration, decantation or centrifugation. 5. The process according to claim 1, wherein the filtrate is optionally clarified by microfiltration after separation. 6. The process according to claim 1, wherein the precipitate in step (e) is separated by filtration, optionally using filter aid. 7. The process according to claim 6 wherein the filter aid is selected firom diatomaceous earth material. 8. The process as claimed in claim 1 wherein the pH in step (d) is 2-3. 9. The process as claimed in claim 1 wherein the pH in step (f) is 8-9. 10. The process as claimed in claim 1 wherein the pH in step (e) is separated by microfiltration or ultrafiltration. 11. The process as claimed in claim 1, wherein the precipitate obtained is contacted with an organic solvent in step (f) selected from ethyl acetate, butyl acetate or toluene. 12. The process according to claim 1, wherein the organic solvent containing Compactin is concentrated in step (g) by distillation. 13. A process for isolation and purification of Compactin substantially as herein described with reference to the foregoing examples.

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