MXPA00001032A - Purification of lipstatin - Google Patents

Abstract

The present invention relates to a method for the purification of lipstatin. More particularly, this invention relates to a method combining liquid-liquid extraction in form of a double-current extraction with a re-extraction in form of a counter-current extraction, as means for isolating lipstatin from crude lipstatin in exceptionally high yield and purity.

Description

PROCESS FOR THE PURIFICATION OF LIPSTATINE DESCRIPTION OF THE INVENTION The present invention relates particularly to a method for the purification of lipstatin. More particularly, this invention relates to a method that combines a liquid-liquid extraction in the form of a double stream extraction with a reextraction in the form of a countercurrent extraction, as a means to isolate lipstatin from crude lipstatin with a yield and purity Exceptionally high Lipstatin is of considerable importance as an intermediate key for the preparation of tetrahydrolipstatin (THL, Orlistat), which is useful in the prophylaxis and treatment of diseases associated with obesity. Lipstatin, a fermentative process for its production, a process for its isolation of microorganisms and a process for its hydrogenation to tetrahydrolipstatin are known and described for example in US Pat. No. 4,598,089. Lipstatin is described as follows: -, - .--, - »--r- < ? REF .: 32594 A process for the preparation of crude lipstatin has also been described in European Patent Application No. 96106598. This process comprises aerobically culturing a microorganism on the order of the actin icetos, for example, Streptomyces toxytri cinl, which produces lipstatin in an aqueous medium. which is substantially free of fats and oils, and which contains convenient carbon and nitrogen sources and inorganic salts, until the initial growth phase is substantially completed and sufficient cell mass has been produced. Then, the linoleic acid together with an antioxidant, and optionally together with the caprylic acid, and N-formyl-L-leucine or preferably L-leucine are added to the broth. After the fermentation is carried out, the fermentation broth is extracted. The crude lipstatin produced can be further enriched and purified, for example by chromatographic methods, described in U.S. Patent No. 4,598,089. Multi-step chromatographic protocols or liquid-liquid extractions for enriching lipstatin in combination with multi-step chromatography to obtain lipstatin characterize the prior art methods for the purification of crude lipstatin. These methods are normally used for the isolation of fermentation of metabolites on a laboratory scale. However, these methods are generally not appropriate for a large-scale economic process. Efforts to purify crude lipstatin by distillation failed. Lipstatin is stable for several hours at 60 ° C, but due to its low vapor pressure (7 x 10"mbar) vacuum distillation (<2 mbar) at this temperature is not feasible. At a higher temperature, lipstatin is degraded by the removal of carbon dioxide. Crude lipstatin can also be purified by the crystallization of lipstatin at temperatures below -20 ° C. However, an expensive technical equipment is required for low temperature crystallization and the yield of this crystallization depends mainly on the quality of crude lipstatin. Especially, with the low quality of crude lipstatin, lipstatin is obtained with a low yield by crystallization. The present invention provides a novelty, simple, and economical process for the purification of lipstatin from crude lipstatin with a high purity and yield, even with the low quality of the raw material The process of the present invention for the purification of lipstatin from crude lipstatin comprises a) the liquid-liquid extraction of lipstatin from a non-polar solvent selected from an aliphatic or aromatic hydrocarbon in a selected polar solvent of a carboxylic acid, an alcohol, a mono-ethylene glycol or O-mono-substituted polyethylene glycol, a diol or a dipolar aprotic solvent followed by b) diluting the phase of the polar solvent with water or changing the proportion of the phase and reextraction of lipstatin in a fresh non-polar solvent The following definitions are indicated to illustrate and define the Ignited and scope of the various terms used to describe the invention herein. The term "dual current extraction" means an extraction. countercurrent of two solvents. The extraction has been done as shown in the following figure: the light phase) + impurities extraction wash 1st heavy phase (extract; Two solvents whose mutual solubility is low are introduced to the top and bottom of the extraction unit. The mixture to be separated (feed) can be introduced anywhere, except in combination with the washing solvent, preferably in the middle of the extraction unit. In the previous figure the extraction has been carried out in such a way that the extraction solvent and the extract, respectively, contain the solvent with a higher density (heavy phase) and the washing and refining solvent, respectively, contain the solvent with Lower density (light phase) The term "countercurrent extraction" means that the substance to be extracted (feed) is added together with one of the solvents used. In the case of the separation of impurities from the compound to be purified is not as high as in the case of a double current extraction, when the washing section is omitted. The countercurrent extraction has been carried out as shown in the following figure: output of the light phase (extract) output of the heavy phase (extract) A solvent together with the feed and another solvent with low mutual solubility are introduced into the top and bottom of the extraction unit. In an outlet, the extraction solvent enriched with the indicated substance that starts from the column (extraction). In the other outlet the solvent, which was used to introduce the feed, was emptied into the indicated substance (raffinate) that starts from the column. In the previous figure the extraction is demonstrated in the way that the feed and the refining, respectively, contain the solvent with a higher density (heavy phase) and the solvent used for the extraction and the extract, respectively, contain the solvent with the lower density (light phase). The term "crude lipstatin" means the crude product containing lipstatin and impurities, being the result of the fermentation process after separation of the cell mass, extraction and concentration. The present invention relates to a method that combines two liquid-liquid extractions, as means to isolate lipstatin from crude lipstatin in a high purity and practically quantitative yield. In general, the process for the purification of lipstatin from crude lipstatin comprises a liquid-liquid extraction of lipstatin from a non-polar solvent selected from an aromatic or aliphatic hydrocarbon in a polar solvent selected from a carboxylic acid, an alcohol, a mono- oligoethylene glycol or substituted O-mono-oligoethylene glycol, a diol or a dipolar aprotic solvent followed by diluting the phase of the polar solvent with water or changing the proportion of the phase and the reextraction of lipstatin in a fresh non-polar solvent.

The liquid-liquid extractions are carried out in the following solvent systems: The non-polar solvent is selected from an aliphatic or aromatic hydrocarbon. Preferred aliphatic hydrocarbons are aliphatic hydrocarbons of 5-8 carbon atoms, more preferably aliphatic hydrocarbons of 6-7 carbon atoms such as hexane or heptane. Aromatic benzene hydrocarbons, optionally substituted by 1 to 3 methyl groups, may be selected. Preferred aromatic hydrocarbons are benzene and toluene. The polar solvent may be selected from carboxylic acids, for example, alkylcarboxylic acid or acids of 1 to 3 carbon atoms, such as acetic acid or propionic acid, or alcohols of 1 to 3 carbon atoms, such as methanol, ethanol, propanol, or mono-oligoethylene glycols or O-mono-substituted oligethylene glycols, such as ethylene glycol monomethyl ether or diols of 2-3 carbon atoms, such as ethanediol or 1,3-propanediol, or furfuryl or tetrahydrofurfuryl alcohol. A dipolar aprotic solvent comprises solvents such as dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, acetonitrile, sulfolane, nitromethane, etc. Preferred polar solvents are water-soluble organic carboxylic acids, alcohols or mono-polyethylene glycols or O-monosubstituted polyethylene glycols for example acetic acid, methanol or ethylene glycol mono ethyl ether, especially acetic acid is preferred. The polar solvent can either be used directly or as a mixture with water. In the first stage, non-polar extraction step and in the second polar extraction stage impurities are removed from the crude lipstatin. The extraction of lipstatin in a polar solvent results in the removal of non-polar impurities and the extraction of lipstatin in a non-polar solvent results in the removal of polar impurities. In a preferred embodiment the same solvents are used in both extractions. The first extraction is followed by a second step comprising diluting the polar solvent with water or changing the proportion of the phase to change the lipstatin distribution. In a further preferred embodiment the first extraction was performed in such a way that the lipstatin is extracted in the polar solvent followed by the reextraction of lipstatin from the polar solvent with a non-polar solvent, preferably the same non-polar solvent as used in the first step of extraction. In a preferred embodiment the liquid-liquid extraction of the first stage is characterized in that the polar solvent contains about 1 to about 20% water, more preferably about 5% water. In a further preferred embodiment, the non-polar solvent is an aliphatic hydrocarbon. Especially heptane is preferred. In the most preferred embodiment of the present invention, the liquid-liquid extraction of the first stage has been carried out as a double current extraction and the lipstatin is extracted from the polar solvent. The feed is crude lipstatin, which can be diluted by one of the solvents used. Lipstatin is extracted from the polar phase (heavy); non-polar impurities such as fatty acids or glycerides remain in the non-polar (light) phase. Since the partition coefficients of lipstatin and impurities depend on the concentration, the proportion of the phase of the two solvents, the number of theoretical phases of the extraction system and the feed ratio for the solvents have to be adjusted in an appropriate manner. Reference is made to Lo, TC, Bairds, MHI, Hafez, M, Hanson, C (1983): Handbook of solvent extraction. Wiley, New York. The dependence of the partition coefficient on the concentration varies significantly with the type of polar solvent used in the extraction system. Aqueous acetic acid provides very good results in order to achieve high concentration and therefore high yield and minimal use of solvent. The extraction can be carried out either by using a continuous extraction apparatus such as a mixing decanter system or an extraction column such as a pulsed plate puller or a stirred column. In the preferred embodiment prior to reextraction (the second purification step) the polar solvent is diluted to about 70% to 90%, preferably to about 80% by adding water. In the most preferred embodiment of the present invention, the reextraction of the second purification step has been performed as a countercurrent extraction and the lipstatin is extracted in the non-polar solvent. In that case the extract from the first extraction can be used directly. In contrast to the extraction of double current, where the feeding of concentrated lipstatin is required, in the case of performing the second extraction as a countercurrent extraction in a continuous process, no concentration of lipstatin is necessary after the first extraction. Lipstatin is extracted in the non-polar phase (light) and the polar impurities remain in the polar phase (heavy). During the fermentation, several lipstatin analogue amino acids are formed. Lipstatin itself contains N-formyl leucine (S) as a side chain, while the derivatives contain other N-formyl amino acids. One of these derivatives is lipstatin of methionine analogues. Crude lipstatin contains up to 3% of this derivative in relation to lipstatin. For the purification of lipstatin it is of great importance to remove this impurity. On the other hand the subsequent hydrogenation could be inhibited by this sulfur-containing compound. According to the present invention the purification of crude lipstatin is further improved, if the lipstatin of methionine analogues in crude lipstatin is oxidized prior to extraction. It is not possible to remove the lipstatin from methionine analogs directly by crystallization or extractive purification of lipstatin, except after oxidation for the corresponding sulfoxide or sulphone. This oxidation is done by a conventional method, for example using peracetic acid in acetic acid. The lipstatin of methionine analogs can either be oxidized to the sulfoxide or to the sulfone: The oxidizing agent is used in an equimolar amount or up to 1.5 equivalent to the amount of lipstatin of methionine analogues, if the sulfoxide is required or double amounts if the sulfone is required. The larger excess should be avoided to prevent the oxidative degradation of lipstatin. Oxidation was carried out at 0 ° to 50 ° C, preferably at room temperature; the reaction is completed in a few minutes. The solvent used for oxidation is not critical, preferably one of the solvents used during the extractive purification process for oxidation is also used. The lipstatin of methionine analogs can be oxidized either in the crude lipstatin stage or after the first stage of extractive purification. Preferably the oxidation is carried out with crude lipstatin in high concentration heptane solution and the resulting mixture is used as feed for the first stage of extractive purification. Lipstatin of oxidized methionine analogues is a polar impurity and is removed during the second extraction, where it remains in the polar phase. It is sufficient that the second extraction stage is carried out as a countercurrent extraction using the extract from the first extraction stage directly after the addition of water, if the heptane / aqueous acetic acid system is used for extractive purification, due to the difference between the partition coefficients of lipstatin and the lipstatin of oxidized methionine analogs. Therefore in the most preferred embodiment, the lipstatin purification of crude lipstatin is achieved by the extraction of crude lipstatin double stream of approximately 95% acetic acid / heptane, followed by dilution of the lipstatin solution with water to approximately 80% acetic acid and the countercurrent extraction of the solution with heptane. The purified lipstatin of heptane can then be isolated by concentration or crystallization. This preferred process is illustrated in the following figure: heptane. non-polar impurities aqueous acetic acid crude lipstatin (after - first extraction of the oxidation of the derivative containing raetionine) purified lipstatin in heptane heptane lipstatin - polar impurities in aqueous acetic acid ag tua second hept non-extraction aqueous acetic acid + polar impurities As noted above, lipstatin prepared by any of the processes as described above, can be converted to tetrahydrolipstatin (Orlistat) by hydrogenation. The hydrogenation of lipstatin can be carried out according to methods which are known per se, for example as described in US Pat. No. 4,598,089, in the presence of an appropriate catalyst. Examples of catalysts that can be used are carbon / palladium, platinum oxide, palladium and the like. The solvent used for hydrogenation is not critical. The most preferred solvent is that used by the last extraction step. In that case the extract containing the lipstatin is used directly or after the concentration for hydrogenation. Other suitable solvents are, for example, lower alcohols such as methanol and ethanol, others such as tert-butyl methyl ether or tetrahydrofuran, acetic acid or halogenated solvents such as dichloromethane. The hydrogenation is preferably carried out at low hydrogen pressures and at room temperature. Accordingly, the present invention also comprises the above extraction processes followed by the hydrogenation of lipstatin for tetrahydrolipstatin. Preferably, the reaction is carried out with a hydrogenation catalyst containing a precious metal at a temperature of 25 ° C at low hydrogen pressure. Tetrahydrolipstatin can be purified and isolated by crystallization. Preferably a non-polar solvent such as hexane or heptane is used for hydrogenation and crystallization. In summary, the present invention relates to a process as defined above, comprising a) the oxidation of lipstatin of methionine analogs b) the extraction of double stream of crude lipstatin at approximately 95% acetic acid / heptane; c) diluting the lipstatin solution with water to approximately 80% acetic acid, and d) countercurrent extraction of the heptane solution. This process can be followed by the hydrogenation of lipstatin for tetrahydrolipstatin, optionally followed by the crystallization of tetrahydrolipstatin.

The present invention also comprises a process for the preparation of tetrahydrolipstatin comprising a) the oxidation of lipstatin of methionine analogues b) the extraction of double stream of crude lipstatin at about 95% acetic acid / heptans; c) diluting the lipstatin solution with approximately 80% acetic acid water, and d) countercurrent extraction of the heptane solution, followed by e) the hydrogenation of lipstatin for tetrahydrolipstatin. In addition, the invention relates to the use of the above methods for the preparation of lipstatin and tetrahydrolipstatin. The following examples will illustrate the preferred embodiments of the present invention without wishing to limit the scope of the invention.

EXAMPLES EXAMPLE 1 Raw lipstatin from the fermentation was concentrated after separation of the biomass and extraction. The material used contained 58% (weight / weight) lipstatin (HPLC analysis) and lipstatin 0.9% (weight / weight) methionine analogues (H-NMR analysis). to. Lipid oxidation of methionine analytes: 55.7 g of crude lipstatin were diluted with 77 ml of heptane. 212 μl of a 37% peracetic acid solution in acetic acid was added and the mixture was stirred at room temperature for 30 minutes. b. double-run extraction with heptane / 95% acetic acid: for the simulation of an extractor with 7 separation stages, 7 separation funnels were used. The mixture resulting from the oxidation process described in a. was divided into 6 portions and each portion was fed into the fourth separation funnel (sf-4). Starting with the feeding of the sf-4, 50 ml of heptane and 100 ml of 95% aqueous acetic acid were added, which is equivalent to a ratio of the heptane / aqueous acetic acid phase of 1/2. The mixture was stirred and the phases were separated. The upper phase was transferred in the next phase and the lower phase in the previous separation funnel. Fresh initial solvents were used, which were replaced step by step with the phases of the previous and subsequent separating funnel. When all separation funnels were filled in this manner, heptane was added in the 1st stage and the aqueous acetic acid was separated only in the seventh separatory funnel. The extract containing lipstatin was combined. Lipstatin was monitored by thin layer chromatography to maintain its low content at 1 g per liter and to control the proportion and concentration of the precalculated phase. When all the crude lipstain was fed, both discharged solvents are added until all the lipstatin is washed out of the system. The following figure illustrates this extraction: Hepta crude lipstatin heptane + non-polar derivatives (refinements) sf-i sf-sf- Sf-I 3f-5 sf-6 lipstatin in acetic acid t 95? of aqueous acetic acid (exrtact) 95% Aqueous Acetic Acid The extract was concentrated to a volume of approximately 600 ml by distilling off the solvent under reduced pressure. The water content, at 3.5% was determined by the Karl Fischer method. c. Countercurrent extraction: for the simulation of an extractor with 7 separation stages were used against 7 separation funnels. The resulting extract of b (600 ml) was divided into 6 portions (each of 100 ml) and each portion was added together with 30 ml of water in the 1st separatory funnel. The extraction was performed as the simulation of a continuous process similar to section b) by transferring the upper phase to the next and the lower phase to the previous separation funnel. The lipstatin was monitored in the raffinate by thin layer chromatography to maintain its low content of 1 g per liter and to control the proportion and concentration of the precalculated phase. When all the solution extracted from the first extraction step was fed, both solvents were added until all the lipstatin was washed. The following figure illustrates this extraction: heptane purified lipstatin heptane in heptane (extract) aqueous acetic acid lipstatin in acetic acid + aqueous polar impurities (first extract) + water (raffinates) 95% aqueous acetic acid The purified lipstatin was combined and concentrated in the heptane (extract) under reduced pressure to a volume of 250 ml. This solution contained 35.3 g of purified lipstatin with a purity of 90% (w / w) (HPLC). This is equivalent to 98 ° of the calculated yield of the lipstatin content in the purified product, relative to the lipstatin in the crude product. d. Hi drogenation: 250 ml of the purified lipstatin solution was hydrogenated in a stainless steel autoclave at 25 ° C at a pressure of 5 bar for a total of 6 hours with 3.5 g of palladium or 5% charcoal. The catalyst was filtered and the filtrate was evaporated. The residue was dissolved in 700 ml of heptane and the tetrahydrolipstatin was crystallized at 0 ° C. The product was filtered, washed with cold heptane and dried under vacuum at 25 ° C. 28 g of tetrahydrolipstatin of 96% (w / w) purity (HPLC) were obtained. This is equivalent to 85% yield, relative to lipstatin in the purified product. The purity of the tetrahydrolipstatin after hexane recrystallization was 97% (weight / weight by HPLC).

EXAMPLE 2: A mixing decanter system was used 7 stages for continuous extraction with the same solvents as used in example 1. The purity of crude concentrated lipstatin used was 65% (w / w), containing lipstatin 0.14% (w / w) of methionine analogs . to. Double-stream extraction with heptane / 90% aqueous acetic acid: 100 kg of crude lipstatin were diluted with 100 kg of heptane and this solution was used as a feed. The mixing decanter was filled with heptane and aqueous acetic acid at 95% in a proportion of the phase of 1/2 (volume / volume). For the extractive purification, a feed of 37.5 liters of heptane per hour, 75 liters per hour of 95% aqueous acetic acid and 15 liters per hour of crude lipstatin solution was added. In the first mixer heptane was introduced, a solution of lipstatin in the fourth mixer and aqueous acetic acid in the seventh mixer. The extract containing the lipstatin and the raffinate containing the non-polar impurities have been collected. The content of lipstatin in the raffinate was monitored by thin layer chromatography to maintain the low level of 1 g per liter. Feeding all the crude lipstatin, the feeding of both solvents was continued until all the lipstatin was washed. A sample of the extract (lower phase) was concentrated and gave lipstatin 71% (w / w) of purity. Totally, 1500 kg of the extract were obtained, the water content was 4.6% (Karl Fischer method). b. Lipid oxidation of methionine analytes: To the solution extracted from the previous extraction was added 60 ml of a 37% solution of peracetic acid in acetic acid and the mixture was stirred for 30 minutes at room temperature. c. Extraction with choke: 280 liters of water were added to adjust a water content of 20%. Due to the separation of an oily phase from lipstatin after adding water this solution was extracted with 600 liter of heptane. This extract was separated and later added to the heptane phase of continuous extraction. For the subsequent continuous extraction of the remaining solution of lipstatin in aqueous acetic acid, the same mixing decanter was used as in the first extraction. The continuous extraction was carried out by feeding 50 liter per hour of heptane in the first mixer and 50 liter per hour of the solution of lipstatin in aqueous acetic acid in the seventh mixer. The extraction of heptane was added to the extract obtained above and this solution was concentrated to a volume of approximately 1000 liter by distilling the solvent under reduced pressure. A sample was concentrated for lipstatin which provides dryness with 86% (w / w) purity. d. Hi drogenation: The solution obtained was hydrogenated in 3 batches of the same size. Hydrogenation was performed in a linear agitation hydrogenation vessel. For a batch of 2 kg, 5% palladium was used in charcoal. Hydrogenation was completed after 1 hour; the hydrogen pressure reached 5 bar. The catalyst was filtered and the filtrate was concentrated by distilling off the solvent. A dry sample of the product contained 90% (w / w) of tetrahydrolipstatin (the quality is higher than that of the purified lipstatin, due to the "convertible" derivatives that contain lipstatin, which are isomers with different position or a single bond double C = C, which are also transformed to tetrahydrolipstatin after hydrogenation). 600 kg of heptane and crystallized tetrahydrolipstatin were added at 0 ° C. The product was filtered, washed with cold heptane and dried under reduced pressure. One batch gave 17 kg of tetrahydrolipstatin of 97% (w / w) purity. This is equivalent to 76% yield of tetrahydrolipstatin, in relation to lipstatin in crude lipstatin.

EXAMPLE 3 Raw lipstatin from the fermentation was concentrated after separation and extraction of the biomass. The material contained 62% (weight / weight) of lipstatin and 0.1% (w / w) of lipstatin of the methionine analogues. to. Oxi ation of the lips of the meti onine analogous: 53 g of crude lipstatin were diluted with 75 ml of heptane. 24 μl of a 37% solution of peracetic acid in acetic acid was added and the mixture was stirred at room temperature for 30 minutes. b. Double extraction was carried out with 10% heptane / aqueous methanol: the same system of the 7 separation funnels was used to simulate a continuous extraction as described in example 1. The mixture resulting from the oxidation process described in a) was divided into 6 portions and each portion was fed into the fourth separation funnel. 100 ml of heptane was added in the first funnel and 10 ml of 10% aqueous methanol in the seventh separatory funnel, which is equivalent to a proportion of the heptane / aqueous methanol phase of 10/1. The extract was combined (upper phase, lipstatin in heptane). When all lipstatin was fed, the fresh solvents were added further until all the lipstatin was washed. The extract was concentrated to a volume of 110 ml by distilling off the solvent under reduced pressure. c. Additional double-wise extraction with heptane / aqueous methanol: The solution obtained from the previous extraction is divided into 6 portions. As was previously used with the same system of 7 separation funnels, the same type of extraction was carried out, but with a different phase proportion. Each feeding portion was added in the fourth separation funnel. 100 ml of heptane in the first funnel and 100 ml of 10% aqueous methanol in the seventh separatory funnel were added, which are equivalent to a proportion of the methanol / aqueous heptane phase of 1/1. The extract was combined (lower phase, lipstatin in aqueous methanol). When all the lipstatin was fed, the fresh solvents were added further until all the lipstatin was washed. The extract was concentrated to a volume of 250 ml by distilling off the solvent under reduced pressure. d. Lot of lipstick extraction in heptane: Water was added to the extraction solution obtained from c) to adjust a 50% water content. This mixture was extracted twice with 600 ml of heptane each. The extract was concentrated by distilling off the solvent. 34 g of lipstatin 86% were obtained (weight / weight) of purity. This is equivalent to 89% of * calculated yield on the lipstatin content in the purified product, relative to the lipstatin in the crude product. and. Hydrogenation: Hydrogenation and crystallization were performed similar to Example 1. Tetrahydrolipstatin was obtained with 97% (w / w) purity in 90% yield, relative to the lipstatin in the purified product.

EXAMPLE 4 Using the same group of a seventh extraction step as described in Example 3, the crude lipstatin was purified from the same batch as in Example 3. Alternatively, 10% aqueous ethylene glycol monomethylether and heptane were used as solvents. The concentrations and proportions of the phase remained unchanged compared to Example 3. A sample of lipstatin after purification had an 80% (w / w) assay. Hydrogenation and crystallization of tetrahydrolipstatin were performed as described in the previous examples. The tetrahydrolipstatin was obtained with a purity of 96% (w / w).

EXAMPLE 5 Crude lipstatin was purified with a 55% (weight / weight) of lipstatin and 0.6% (w / w) of lipstatin methionine analogues on a Kuehni shaking column. to. Extraction of double stream with heptane / 95% aqueous acetic acid: For extraction, a Kuehni E60 column with the following dimensions was used: column I.D .: 6 cm, height of the column: 180 cm, number of practical phases: 50, area in free cross section of stator plates: 10%. The column was stirred at 130 rpm. The 95% fresh aqueous acetic acid was fed as the dispersed phase with 4.0 1 / h at the top entrance of the column. Fresh heptane was fed as a continuous phase with 1.75 1 / h at the lower entrance of the column. A crude 40% (w / w) lipstatin solution was fed to heptane at 0.775 kg / h in the twenty-seventh practical phase (counting from the bottom) in the column. In equilibrium the yield of lipstatin in the extract phase at the lower exit of the column was >; 99% and the purity of lipstatin was 70% (weight / weight). The lipstatin content of methionine analogs was 0.6% (w / w). b. oxidation with peracetic acid: The lipstatin of methionine analogues in 95% aqueous acetic acid extract was oxidized by the addition of an equimolar amount of 37% peracetic acid. c. The countercurrent extraction with heptane / 75% aqueous acetic acid: For extraction, a Kuehni column with the same dimensions was used as described under section a). The column was stirred at 205 rpm. The Lipstatin extract in 95% aqueous acetic acid (combination of several extraction batches, with a purity of 68% (w / w), of lipstatin, prepared according to section a)) was fed as a dispersed phase with 8.6 1 / h in the top entry column. The fresh deionized water was fed with 2.1 1 / h in the forty-seventh practical phase (counted from the bottom) of the column. Fresh heptane was fed as a continuous phase with 6.0 1 / h in the lower inlet column. In equilibrium, the lipstatin yield in the extract phase in the upper exit column was 97% and the lipstatin purity was 88% (w / w). This is equivalent to 96-97% of the calculated yield of the lipstatin content in the purified product, relative to the lipstatin in the crude product.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects- or products to which it refers. Having described the invention as above, the content of the following is claimed as a priority:

Claims (21)

1. A process for the purification of lipstatin from crude lipstatin, characterized in that it comprises a) the liquid-liquid extraction of lipstatin from a non-polar solvent selected from an aromatic or aliphatic hydrocarbon in a polar solvent selected from a carboxylic acid, an alcohol, a mono-ethylene glycol or O-mono-substituted polyethylene glycol, a diol or a dipolar aprotic solvent followed by b) diluting the polar solvent phase with water or changing the proportion of the phase and the reextraction of lipstatin in a fresh non-polar solvent.

2. The process according to claim 1, characterized in that the polar solvent of step a) contains about 1 to about 20% water.

3. The process according to claim 2, characterized in that the polar solvent of step a) contains about 5% water.

4. The process according to any of claims 1 to 3, characterized in that the non-polar solvent is an aliphatic hydrocarbon.

5. The process according to claim 4, characterized in that the non-polar solvent is heptane.

6. The process according to any of claims 1 to 5, characterized in that the polar solvent is an organic carboxylic acid soluble in water, an alcohol or a mono-ethylene glycol or O-monosubstituted oligoethylene glycol.

7. The process according to claim 6, characterized in that the polar solvent is acetic acid, methanol or ethylene glycol monomethyl ether.

8. The process according to any of claims 1-7, characterized in that the liquid-liquid extraction of step a) as defined in claim 1 is a double current extraction and the lipstatin is extracted in the polar solvent.

9. The process according to any of claims 1-8, characterized in that for the reextraction the polar solvent is diluted approximately 90 to approximately 70% by adding water.

10. The process according to claim 9, characterized in that the polar solvent is diluted to approximately 80% by adding water.

11. The process according to any of claims 1-10, characterized in that the reextraction as defined in step b) of claim 1 is a countercurrent extraction and the lipstatin is extracted in the non-polar solvent.

12. The process according to any of claims 1-11, characterized in that the analogous methionine derivative of crude lipstatin is oxidized prior to extraction.

13. The process according to any of claims 1-12, characterized in that it is followed by the hydrogenation of lipstatin for tetrahydrolipstatin.

14. The process according to claim 13, characterized in that the reaction of the hydrogenation has been carried out with a hydrogenation catalyst containing a precious metal at a temperature of 25 ° C at a low hydrogen pressure.

15. A process according to any of claims 1-12, characterized in that it comprises a) the oxidation of lipstatin of methionine analogs; b) the extraction of double stream of crude lipstatin in approximately 95% acetic / heptane; c) dilute the lipstatin solution with water to approximately 80% acetic acid; and d) the countercurrent extraction of the solution with heptane.

16. The process according to claim 15, characterized in that it is followed by the hydrogenation of lipstatin for tetrahydrolipstatin.

17. The process, according to claim 13, characterized in that it is followed by the crystallization of tetrahydrolipstatin.

18. A process for the purification of lipstatin from crude lipstatin characterized in that it comprises a) the oxidation of lipstatin of methionine analogues b) the extraction of double stream of crude lipstatin at approximately 95% acetic / heptane; c) dilute the lipstatin solution with water to approximately 80% acetic acid; and d) the countercurrent extraction of the solution with heptane.

19. A process for the preparation of tetrahydrolipstatin characterized in that it comprises a) the oxidation of lipstatin of methionine analogs: b) the extraction of double stream of crude lipstatin in about 95% acetic acid / heptane; c) dilute the lipstatin solution with water to approximately 80% acetic acid; and d) the countercurrent extraction of the solution with heptane followed by e) the hydrogenation of lipstatin for tetrahydrolipstatin. f) the crystallization of tetrahydrolipstatin

20. The use of a process according to any of claims 1-19, characterized by the preparation of lipstatin or tetrahydrolipstatin.

21. The invention characterized as described hereinabove.