Method for purifying fermentation broth

FIELD: biotechnology, pharmacy.

SUBSTANCE: statin compounds from fermentation solution are purified by extraction and crystallization. Fermentation broth is subjected for preliminary treatment that involves preliminary alkaline treatment followed by extraction of nonpolar impurities. After preliminary treatment the statin compound is extracted into hydrophobic solvent that is separated after extraction. Then solution of hydrophobic organic solvent is concentrated and washed out, if necessary, with a base-containing aqueous solution. Then extracted statin compound is purified by crystallization. Invention provides isolating statins from fermentation broth with high degree of effectiveness and under pharmaceutically acceptable level of purity.

EFFECT: improved purifying method.

28 cl, 8 ex

 

The SCOPE of the INVENTION

This invention relates to a method of purification of compounds from fermentation broth. In particular, this invention relates to the selection of statins from fermentation broth in crystalline form.

BACKGROUND of the INVENTION

Complications of cardiovascular diseases such as myocardial infarction, sudden attacks and peripheral vascular diseases account for half of the deaths in the United States. High levels of low-density lipoprotein (low density lipoprotein, LDL) in the blood stream leads to the formation of coronary lesions that obstruct the flow of blood and can rupture and cause thrombosis (Goodman. Gilman. The Pharmacological Basis of Therapeutics 879 (Joel G.Hardman et al., eds. 9th ed. 19%)). It was shown that lowering the levels of LDL in the plasma reduces the risk of clinical cases of patients with cardiovascular disease and in patients who do not have cardiovascular disease but who have hypercholesterolemia (Scandinavian Simvastatin Survival Study group, 1994; Lipid Research Clinics Program, 1984a, 1984b).

Drugs statins are currently the most therapeutically effective drugs available to reduce the level of LDL in the bloodstream of patients with an increased risk of cardiovascular disease. This class of drugs includes, inter alia, compactin, lovastatin, who simvastain, pravastatin and fluvastatin. The mechanism of action of statin drugs is explained in some detail. They violate the synthesis of cholesterol and other sterols in the liver, with the competitive inhibition of the enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme a reductase (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, "HMG-CoA reductase"). HMG-CoA reductase catalyzes the conversion of HMG-CoA in the mevalonata, which is the rate-determining step by step in the biosynthesis of cholesterol. Therefore, this inhibition leads to a reduction in the rate of formation of cholesterol in the liver.

Compactin is the common medical name of a chemical 1,2,3,7,8,8A-hexahydro-7-methyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-Piran-2-yl)-ethyl-1-naftalinovogo ether 2-methylbutanoic acid with the formula

It was shown that compactin, also called mevastatin is the first statin drug - inhibitor of HMG-CoA reductase. Compactin was purified from Penicillium citrinum and Penicillium adametzioides (see U.S. patent No. 3983140, 4049495 and 5691173 that described in this invention as a reference).

Lovastatin is the common medical name of a chemical 1,2,3,7,8,8A-hexahydro-3,7-dimethyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-Piran-2-yl)-ethyl]-1-naftalinovogo ether 2-methylbutanoic acid with the formula

Lovastatin, that is also called mevinolin, differs from compactin only in the presence of a methyl group and may be emitted from Aspergillus terreus (see U.S. patent No. 4294926, 4420491, 4319039 and 4294896 that described in this invention as a reference). Lovastatin was also highlighted some other microorganisms (see British patent No. 2046737, German patent No. 4402591, canadian patent No. 2129416 and Hungarian patent No. 208997).

Compactin and lovastatin and other statins exist in the form of hydroxy acids with an open ring and a lactone, as shown:

The balance between the lactone and oxicology makes cleaning difficult, because of the form of the free acid and lactone compounds statin have different polarity. The way to clean one form, obviously, is to remove the other form, which thereby reduces the overall yield. Therefore, great care is usually necessary to show, when clear connection statin to select them with great output.

U.S. patent No. 5202029 relates to a method of cleaning the lactone form of the statin compounds by HPLC (HPLC). The crude fermentation broth (e.g., lovastatin, simvastatin, pravastatin, fluvastatin and mevastatin) is dissolved in an organic solvent and elute through HPLC-column (HPLC-column). Statin elute from the column as dissolved in eluent prophetic is TBA. Eluent partially evaporated and subsequently add water to cause crystallization. The main disadvantage of the production on an industrial scale is a great value chromatographic columns.

U.S. patent No. 5616595 relates to a continuous method of selection of a wide range of water-insoluble compounds from fermentation broth by tangential filtration. The method can be applied to the lactone form of lovastatin. pravastatin and simvastatin. The method involves the periodic transmission of fermentation broth through a filter that retains insoluble compound. The compound is dissolved in a solvent and the solution is filtered. A solution of the desired compound are collected in the form of filtrate and the desired compound can then be subjected to additional purification. Because of the insolubility of the compounds in water method requires a solvent to dissolve and requires numerous filtration membrane. Re-filtering makes the method very expensive for large-scale production.

The allocation method lovastatin in the form of a lactone described in U.S. patent No. 5712130. In the specified way lovastatin extracted from the fermentation broth by butyl acetate. The resulting solution was then centrifuged and the aqueous phase discarded. The organic phase is distilled in vacuum at 40°C, h what about the addition of concentrerebbe solution promotes the formation of the lactone with the removal of water. Crystals of lovastatin lactone formed upon cooling and is recrystallized to purity of 90% or higher. However, to use as a medicinal product crystallized lovastatin should additionally be purified, which will reduce the overall yield and will add additional costs for implementing the method.

The PURPOSE AND ESSENCE of the INVENTION

Known methods for isolating statin from fermentation broth not reach a pharmaceutically acceptable level of purity by the way, economical on an industrial scale, or require chromatographic separation to achieve high purity. The purpose of this invention is to develop a simple, rapid method with high yield allocation of statins from the fermentation broth with pharmaceutically acceptable level of purity (i.e., not less than 98,5%).

Another purpose of this invention is a method of separation connections statin from a fermentation broth, where the connection statin contains carboxylic acid. capable of forming a lactone and a condensed bicyclic ring. The method comprises the stages: extraction connection statin from the fermentation broth during the processing of the extraction solvent and extraction connection statin from the fermentation broth in the extraction solvent, where the extraction of the races is oritel is hydrophobic organic extraction solvent; the separation of hydrophobic organic extraction solvent from the fermentation broth; the concentration of a solution of hydrophobic organic extraction solvent containing the extracted connection statin; and purification of extracted compounds statin crystallization.

Another purpose of this invention is a method of separation connections statin from a fermentation broth, where the connection statin contains a carboxylic acid capable of forming a lactone and a condensed bicyclic ring, the method involves the following stages:

(a) pre-processing of the fermentation broth under alkaline conditions to remove non-polar impurities;

(b) extraction of the compounds of the statin from a fermentation broth in a hydrophobic organic extraction solvent;

(C) separating the hydrophobic organic extraction solvent from the fermentation broth;

(g) concentrating the solution of hydrophobic organic extraction solvent containing the extracted connection statin;

(d) washing the concentrated solution of hydrophobic organic extraction solvent an aqueous solution containing the base for cleaning lactone; and

(e) purification of extracted compounds statin crystallization.

DETAIL THE description of the INVENTION

This invention relates to a process for obtaining highly purified crystals of statin compounds from the fermentation broth, as illustrated when cleaning compact and lovastatin. But experts it is clear that the method described in this invention can be used for cleaning other compounds that receive microbiological or enzymatic methods, which contain a lactone, such as simvastatin, pravastatin and fluvastatin. Experts also clear that the optimal conditions cleaning compound statin can vary depending on the given allocated connection statin, fermentation broth of a microorganism producing the connection.

A preferred variant of the present invention involves the following stages:

(I) pre-processing of the fermentation broth under alkaline conditions to remove non-polar impurities;

(II) extraction of the statin drug in the acidic conditions in the form of a hydroxy acid or lactate from fermentation broth in a hydrophobic organic extraction solvent;

(III) separating the hydrophobic organic extraction solvent from the fermentation broth;

(IV) the concentration of hydrophobic organic extraction solvent containing the extracted connection statin, with the formation of the lactone; and

(V)cleaning of extracted compounds statin crystallization.

In another embodiment of the invention stage pre-treatment of fermentation broth under alkaline conditions is lowered.

In another embodiment, prior to crystallization of the concentrated hydrophobic organic solvent is washed with an aqueous solution containing the base, to increase the output. Preferred alkaline solutions containing ammonium hydroxide (NH4OH, the pH from 7.5 to 10) or 1-5% (wt./weight.) a solution of sodium bicarbonate (NaHCO3) or sodium carbonate (Na2CO3).

Pre-treatment of fermentation broth under alkaline conditions

Pre-treatment of fermentation broth includes processing of fermentation broth in alkaline conditions, followed by extraction in a hydrophobic organic extraction solvent or mixture of solvents. Alkaline conditions a pre-processing stage hydrolyzing statin lactone to the corresponding hydroxy (Aha) statin (Andrew Streitweiser, Jr. Clayton Heathcock, Introduction to Organic Chemistry. 858-60 MacMillan Publishing Co., 2d Ed. 1981). The hydroxy acids statin keep in aqueous fermentation broth, while fatty and oily substances, as well as other non-polar organic impurities from the fermentation broth are distributed in a hydrophobic organic extraction solvent or solvent mixture pre-treatment. Thus the om, pre-treatment of fermentation broth under alkaline conditions leads to a complete cleaning of the oxyacids of statin by separating it or impurities.

Remove non-polar impurities improves the overall yield of crystallization, because I believe that the presence of non-polar impurities in the fermentation broth reduces the overall yield by crystallization. Thus. pre-treatment of fermentation broth under alkaline conditions remove non-polar impurities from the fermentation broth, thereby significantly increases the total output during crystallization.

During the pre-treatment pH of fermentation broth or a mixture of fermentation broth and the hydrophobic organic extraction solvent is brought to an alkaline pH. Preferably, when the pH was adjusted by addition of an inorganic base or an organic amine. Preferred inorganic bases or organic amines include NaOH, KOH, LiOH, Ca(OH)2, NH4OH and triethylamine. The most preferred base is NaOH, pH 7.0 to 13.9. Preferably, when the pH to compactin and lovastatin regulate in the range from 8.5 to 10.0. Most preferably, when the pH to compactin and lovastatin is from 9.0 to 9.6,

Pre-incubation in alkaline conditions can be carried out at t is mperature from 15° With up to 100°C. a temperature of 15-20°With result in reduced overall yield of purified statin.

At 80-100°With the preferred conditions of the pre-incubation for 15 minutes at pH ranging from 12.0 to 13.9. When 55-65°With the preferred conditions of the pre-incubation for 2 hours at pH from 9.0 to 9.6. When 15-25°With the preferred conditions of the pre-incubation of 48 hours at a pH of from 9.0 to 9.6.

Hydrophobic organic extraction solvent pre-treatment include, but are not limited to, isobutyl acetate, n-butyl acetate, tert-butyl acetate, ethyl acetate, propyl, ethyl formate, butylmethylether, dichloromethane, chloroform, carbon tetrachloride, dichloroethane, toluene, acetonitrile, methylformate, methanol, ethanol, isopropanol, n-propanol, n-butanol, Isobutanol, tert-butanol, amyl alcohol, and benzyl alcohol.

Or hydrophobic organic extraction solvent pre-treatment may include any mixture of the aforementioned solvents. Organic solvents for the alkali extraction include, but are not limited to, isobutyl acetate, n-butyl acetate, tert-butyl acetate, ethyl acetate, propyl, ethyl formate, butylmethylether, dichloromethane, chloroform, carbon tetrachloride, dichloroethane, toloo the om and benzyl alcohol. Preferred hydrophobic organic extraction solvent pre-treatment is isobutyl acetate, ethyl acetate and toluene. The most preferred hydrophobic organic extraction solvent is isobutyl acetate. Alcohols are (justified), but not preferred because of the regeneration of the solvent. However, the alcohols are present in the case of acetate at alkaline pH. Spirits help the opening lactoovo rings in alkaline conditions. Pre-processing can also be carried out without extraction solvents.

In this preferred embodiment, after pre-treatment of hydrophobic organic extraction solvent is in contact with the fermentation broth under alkaline conditions until such time as fatty and oily substances, as well as other non-polar organic impurities will not be extracted from the fermentation broth. To evaluate the extraction of non-polar impurities from the fermentation broth can be used in thin layer chromatography or any other method, including subjective opinion. For optimal removal of impurities can be repeated extraction. However, a highly effective one-two extraction when using the isobutyl acetate for alkaline extraction. Preferably, when the alkaline ek the traction to hold the volume of the solvent, which is from 20% to 50% (vol./about.) the amount of fermentation.

the pH adjustment prior to the extraction of fermentation broth in acidic conditions

Preferably, when the pH of the purified fermentation broth was adjusted to a pH of from 1.0 to 6.4 a strong acid prior to the extraction of compounds of the statin in the hydrophobic organic extraction solvent. The preferred range of pH for lovastatin and compactin is from 2.0 to 4.5. The preferred range of pH for pravastatin is from 4.5 to 6.0. Preferred acids for pH regulation are sulfuric and phosphoric acid. Or extraction of the compounds of the statin can be when bringing the pH of the hydrophobic extraction solvent to the interval from 1.0 to 6.4.

Extraction connection statin drug in the acidic conditions in the form of a hydroxy acid or lactone

Hydrophobic organic extraction solvent is in contact with the purified fermentation broth in acidic conditions up until the hydroxy acid and the lactone will be almost completely extracted from the fermentation broth. To evaluate the extraction of the lactone in the hydrophobic organic extraction solvent can be used in thin layer chromatography or any other method, including subjective opinion. For optimal extraction can be repeated extraction. However, Vysokoe the active two-three extraction, when the hydrophobic organic extraction solvent is isobutyl acetate and the pH is in the optimum range, as described above. Preferably, when the extraction is carried out with a volume of organic extraction solvent, which is two times less than the volume of fermentation broth.

The most preferred hydrophobic organic extraction solvent for extraction compactin is isobutyl acetate. Other suitable hydrophobic organic extraction solvents include, but are not limited to, n-butyl acetate, tert-butyl acetate, ethyl acetate, propyl, ethyl formate, butylmethylether, dichloromethane, chloroform, carbon tetrachloride, dichloroethane and toluene. Preferred extraction solvents are isobutyl acetate, n-butyl acetate, ethyl acetate and butylmercaptan. When the acid extraction solvent mixture is not applied.

The separation of the phases of fermentation broth and the hydrophobic organic extraction solvent

The fermentation broth can be separated from the hydrophobic organic extraction solvent by known methods. Preferred methods of separation include countercurrent extraction. Apparatus for decanting are known for this equipment. P the following phase separation, the purity of the solvent can be improved by washing with water.

The concentration of hydrophobic organic extraction solvent

The volume of the hydrophobic organic extraction solvent after separation of the phases is reduced. Volume reduction can be achieved by evaporation under reduced pressure at a temperature of from 30 to 90°C. Preferably, when the evaporation is carried out at reduced pressure and a temperature of from 40 to 70°C. Or evaporation of the hydrophobic solvent is carried out at elevated temperature (below 90° (C) under reduced or atmospheric pressure.

Purification of extracted compounds statin crystallization

Hydrophobic organic extraction solvent evaporated until such time as the concentration of the statin during crystallization reaches 50-250 g/l Lovastatin optimally crystallized at a concentration of 80-100 g/l, compactin optimally crystallized at a concentration of 130-170 g/l, and pravastatin optimally crystallized at a concentration of 80-120 g/l

The crystallization can be performed at room temperature over night. The preferred temperature interval is (-10)-5°C. the Crystallization is most preferably carried out at -10°C for 20 hours.

Crystallization can be carried out in any of the following solvents or combinations of the following solvents: ethanol, isopropanol, n-Pro is anal, Isobutanol, n-butanol, tertbutanol, ethyl acetate, acetone, methanol, acetonitrile, ethyl formate, isobutyl acetate, tributyltin, n-butyl acetate, toluene, propyl and butylmercaptan. Preferred solvents include toluene, isopropanol, isobutyl acetate, or a mixture of ethanol-water. The most preferred solvents are isobutylether and the mixture ethanol-water.

The crystals obtained using the preferred option, contain less than 3.6% (wt./weight.) impurities and receive with the release of above 75% (wt./weight.). For cleaning statin obtained by recrystallization from a mixture of water:ethanol, the preferred ratio of water to ethanol is from 0.8 to 2.0. The most preferred ratio of water to ethanol is from 0.9 to 1.2. The crystals obtained using the preferred option, have a purity of at least 98.5% of (wt./weight.).

The invention will be further explained by the following examples. Unless otherwise noted, all outputs are given in % (wt./weight.) and they represent communities of the output or outputs of all stages. However, this invention these examples are not limited. Professionals understand how to modify the examples ways to get the desired results.

EXAMPLES

EXAMPLE 1: Allocation of Compactin

Fermentation broth (70 m3containing 350 kg is impacting, received in a known manner (See, for example, U.S. patent No. 3983140, 4049495 and 5691173). To the aqueous fermentation broth (70 m3) was continuously added isobutyl acetate (35 m3) and water (35 m3). the pH was brought to 9.0 to 9.6 by addition of concentrated NaOH. The mixture was then heated to 60°C and kept at this temperature for 2 hours. The resulting organic and aqueous phases are then separated, using countercurrent extraction. While separation can help to break down the emulsion, was added sodium lauryl sulfate.

Purified fermentation broth was acidified to pH 2.0 to 4.5 with sulfuric acid. Then continuously added isobutyl acetate (35 m3and after mixing in situ isobutylacetate phase containing compactin, continuously separated.

Isobutylacetate phase was concentrated under vacuum to a volume of about 2150 L. the Concentrated solution was left to stand at 0-5°during the night, while compactin was led with 78% yield (weight/weight.) and 93% purity (wt./weight.). The crude compactin then recrystallized from a mixture of ethanol:water, equal to 1.2:0,9, with 75% (wt./weight.) output when the purity of 99,0% (wt./weight.).

EXAMPLE 2: Allocation of Compactin without preliminary purification of fermentation broth under alkaline conditions

Fermentation broth containing compactin, received as, the example1. Fermentation broth (50 l) was acidified to pH 2.0 to 4.5 with sulfuric acid. Then were added isobutyl acetate (25 l) and water (25 l) and after stirring for 0.5 hours was separated isobutylacetate phase containing compactin. The extraction was repeated with pure isobutyl acetate (25 l). United isobutylacetate phase was concentrated under vacuum to a volume of about 1.5 liters of a Concentrated solution was left to stand at 0-5°during the night, while compactin was led with 33% (wt./weight.) output when the purity of 94.5% (wt./weight.). While example 1 is an example of the application of alkaline extraction, example 2 differs in that it illustrates the use of technology without alkaline extraction.

EXAMPLE 3: Selection Compactin: pre-treatment of fermentation broth under alkaline conditions concentrated isobutyl acetate and washing NaHCO3

Fermentation broth was obtained as in example 1. To the aqueous fermentation broth (50 l) were added isobutyl acetate (25 l) and water (25 l). the pH was brought to 9.0 to 9.6 by addition of concentrated NaOH. The mixture was then heated to 60°C and kept at this temperature for 2 hours. Then separate the phases. While separation can help to break down the emulsion was added dodecyltrimethylammonium chloride. Purified fermentation broth was acidified to pH 2.0 to 4.5 the use of sulfuric acid. Then were added isobutyl acetate (25 l) and after stirring for 0.5 hours was separated isobutylacetate phase containing compactin. Acidic extraction was repeated with pure isobutyl acetate (25 l).

United isobutylacetate phase was concentrated under vacuum to a volume of about 1.5 liters of a Concentrated solution was diluted to a volume of about 8 liters and washed with a saturated solution of sodium bicarbonate. The washed solution was again concentrated to 1.5 l and left to stand at 0-5°during the night, while compaction has led to a 76.5% yield (weight/weight.) and 98.9% purity (wt./weight.).

In the absence of preliminary alkaline extraction compactin received only 39% (wt./weight.) output when the purity of 99.1% (wt./weight.).

EXAMPLE 4: Selection Compactin: the influence of low temperatures during the pre-treatment of fermentation broth under alkaline conditions

Fermentation broth was obtained as in example 1. To the aqueous fermentation broth (50 l) were added isobutyl acetate (25 l) and water (25 l). the pH was brought to 9.0 to 9.6 by addition of concentrated NaOH. The mixture was stirred at a temperature of 15-20°C for 2 hours. Then separate the phases. While separation can help to break down the emulsion was added dodecyltrimethylammonium chloride. Purified fermentation broth was acidified to pH 2.0 to 4.5 with sulfuric acid is. Then were added isobutyl acetate (25 l) and after stirring for 0.5 hours was separated isobutylacetate phase containing compactin. The extraction was repeated with pure isobutyl acetate (25 l). United isobutylacetate phase was concentrated under vacuum to a volume of about 1.5 liters of a Concentrated solution was left to stand at 0-5°during the night, while compactin crystallized. The crude compactin then recrystallized from a mixture of ethanol:water =1,2:0,9. The total yield was 67% (wt./weight.) when the purity of 99.1% (wt./weight.).

EXAMPLE 5: Selection Compactin: effect of acidic pH 1.0 to 2.0 by acid extraction and low temperature during crystallization

Fermentation broth was obtained as in example 1. To water (fermentation broth (50 l) were added isobutyl acetate (25 l) and water (25 l). the pH was brought to 9.0 to 9.6 by addition of concentrated NaOH. The mixture was stirred at a temperature of 60°C for 2 hours. Then separate the phases. While separation can help to break down the emulsion was added dodecyltrimethylammonium chloride. Fermentation broth was acidified to pH 1.0 to 2.0 with sulfuric acid. Then were added isobutyl acetate (25 l) and after stirring for 0.5 hours was separated isobutylacetate phase containing compactin. The extraction was repeated with pure isobutyl acetate (25 l).

United from ethylacetate phase was concentrated under vacuum until while the concentration of the compact amounted to about 150 g/l Concentrated solution was kept at a temperature of about - 10°C for 20 hours. Crystals (was filtered and washed with isobutyl acetate. The crude compactin then recrystallize from a mixture of ethanol:water =1,2:0,9. The compacts were received from 67% (wt./weight.) output when the purity of 98.8% (wt./weight.). Acidic extraction (at pH 1.0 to 2.0) reduced output.

If the method described in this example, pre-processing of the fermentation broth in alkaline conditions was carried out at 15°total output compactin decreased to 60% (wt./weight.) when the purity of 98.7% (wt./weight.).

EXAMPLE 6: Isolation of Lovastatin

Fermentation broth containing lovastatin, obtained in a known manner. (See, for example, U.S. patent No. 5403728, 4420491, 4342767, 4319039 and 4294846). To the aqueous fermentation broth (50 l) were added isobutyl acetate (25 l) and water (25 l). the pH was brought to 9.0 to 9.6 by addition of concentrated NaOH. The mixture was then heated to 60±5°C and kept at this temperature for 2 hours. Then the phases were separated. While separation can help to break down the emulsion was added dodecyltrimethylammonium chloride.

Purified fermentation broth was acidified to pH 2.0 to 4.5 with sulfuric acid. Then were added isobutyl acetate (25 l) and after stirring for 2 hours separated isobut acetato phase, contains lovastatin. The extraction was repeated with pure isobutyl acetate (25 l).

United isobutylacetate phase was concentrated under vacuum to a volume of about 2.7 liters of a Concentrated solution was left to stand at a temperature of - 10°C for 36 hours, while lovastatin crystallized from 76% yield (weight/weight.) and 96,4% purity (wt./weight.). The crude lovastatin then recrystallized from a mixture of ethanol:water =1,2:0,9 from 71.4% (wt./weight.) output when the purity of 98.7% (wt./weight.).

EXAMPLE 7: Isolation of Lovastatin without pre-treatment of fermentation broth under alkaline conditions

Fermentation broth containing lovastatin, was obtained as in example 6. Fermentation broth (50 l) was acidified to pH 2.0 to 4.5 with sulfuric acid. Then were added isobutyl acetate (25 l) and water (25 l) and after stirring for 2 hours separated isobutylacetate phase containing lovastatin. The extraction was repeated with pure isobutyl acetate (25 l). United isobutylacetate phase was concentrated under vacuum to a volume of about 2.7 liters of a Concentrated solution was left to stand at 10°C for 36 hours, while lovastatin crystallized from 26% yield (weight/weight.) when the purity 88,0% (wt./weight.).

EXAMPLE 8: Allocation of Lovastatin: the use of washing NaHCO3

Fermentation broth was obtained as in example 6. To the one fermentation broth (50 l) were added isobutyl acetate (25 l) and water (25 l). the pH was brought to 9.0 to 9.6 by addition of concentrated NaOH. The mixture was then heated to 60°C and kept at this temperature for 2 hours. Then separate the phases. While separation can help to break down the emulsion was added dodecyltrimethylammonium chloride. Purified fermentation broth was acidified to pH 2.0 to 4.5 with sulfuric acid. Then were added isobutyl acetate (25 l) and after stirring for 2 hours separated isobutylacetate phase containing lovastatin. The extraction was repeated with pure isobutyl acetate (25 l).

Isobutylacetate phase was concentrated under vacuum to a volume of about 2.7 liters of a Concentrated solution was diluted to a volume of about 11 l and washed with a saturated solution of sodium bicarbonate. The washed solution was again concentrated to 2.7 l and it was left to stand at 10°C for 36 hours, while lovastatin received from 73.4% (wt./weight.) output when the purity of 98.8% (wt./weight.).

1. The allocation method connection statin from a fermentation broth, where the connection statin contains a carboxylic acid capable of forming a lactone and a condensed bicyclic ring, comprising the following stages:

a) pre-processing of the fermentation broth to remove non-polar impurities, and this pre-processing includes processing fermentaciones the broth in alkaline conditions, followed by extraction of non-polar impurities in the hydrophobic organic extraction solvent pre-treatment,

b) extraction of the compounds of the statin from the fermentation broth during the processing of fermentation broth extraction solvent and subsequent extraction of the compounds from the fermentation broth in the extraction solvent, which is the hydrophobic organic extraction solvent

C) separating the hydrophobic organic extraction solvent from the fermentation broth,

g) concentrating the solution of hydrophobic organic extraction solvent containing the extracted connection statin, and

d) purification of extracted compounds statin crystallization.

2. The method according to claim 1, in which the connection statin drug is an inhibitor of HMG-CoA reductase.

3. The method according to claim 1, in which the connection statin selected from the group consisting of lovastatin, compactin and pravastatin.

4. The method according to claim 1, wherein during the pre-processing of the fermentation broth is maintained at

(a) a temperature of from 15 to 100°C

(b) a pH of from 7.0 to 13.9 and

(C) during the period of time from 0 to 48 hours

5. The method according to claim 1, wherein during the pre-processing of the fermentation broth is maintained at

(a) a temperature of from 55 to 65°C

(b) a pH of from 9.0 to 9.6 and

(C) during the period of time of about 2 hours

6. The method according to claim 1, in which stage of pre-processing includes the regulation of the pH of fermentation broth using inorganic bases selected from the group consisting of ammonium hydroxide, NaOH, KOH, LiOH and CA(Oh)2.

7. The method according to claim 1, in which stage extraction includes the processing of fermentation broth hydrophobic organic extraction solvent.

8. The method according to claim 1, wherein the hydrophobic organic extraction solvent for pre-treatment selected from the group consisting of isobutylacetate, n-butyl acetate, tert-butyl acetate, ethyl acetate, propyl, ethylformate, butylmethylamine, dichloromethane, chloroform, carbon tetrachloride, dichloroethane, toluene, acetonitrile, methylformate, methanol, ethanol, isopropanol, n-propanol, n-butanol, Isobutanol, tert-butanol, amyl alcohol and benzyl alcohol, and mixtures thereof.

9. The method according to claim 7, in which the hydrophobic organic extraction solvent is isobutyl acetate, n-butyl acetate, tert-butyl acetate, ethyl acetate, propyl, ethyl formate, butylmethylether, dichloromethane, chloroform, carbon tetrachloride, dichloroethane or toluene.

10. The method according to claim 1, in which stage extraction connection statin from fermentation broth additionally includes the bringing the pH of fermentation broth to 1.0-6,4.

11. The method according to claim 10, in which the pH of fermentation broth was adjusted to 2.0 to 4.5.

12. The method according to claim 10, in which the pH of fermentation broth lead acid selected from the group consisting of sulfuric and phosphoric acids.

13. The method according to claim 1, in which stage extraction connection statin from the fermentation broth further includes bringing the pH of the hydrophobic organic extraction solvent phase (b) to a pH of from 1.0 to 6.4.

14. The method according to claim 1, wherein the hydrophobic organic extraction solvent phase (b) is selected from the group consisting of isobutylacetate, n-butyl acetate, tert-butyl acetate, ethyl acetate, propyl, ethylformate, butylmethylamine, dichloromethane, chloroform, carbon tetrachloride, dichloroethane and toluene.

15. The method according to claim 1, in which, after stage (b) a hydrophobic organic extraction solvent is treated with an aqueous solution containing a base.

16. The method according to item 15, in which the base is selected from the group consisting of ammonium hydroxide, NaHCO3and Na2CO3.

17. The method according to claim 1, wherein the hydrophobic organic extraction solvent is isobutyl acetate and the connection statin is compaction.

18. The method according to claim 1, in which stage of concentrating the solution of hydrophobic organic extraction solvent, the content is the future of extracted connection statin, carried out under reduced pressure.

19. The method according to claim 1, in which stage of concentrating the solution of hydrophobic organic extraction solvent containing the extracted connection statin, is performed during the process of evaporation at a temperature below 90°C.

20. The method according to claim 1, in which stage crystallization is carried out at a temperature from 10 to 20°C.

21. The method according to claim 20, in which stage crystallization is carried out at a temperature of from 0 to 5°C.

22. The method according to claim 20, in which stage crystallization is carried out at a temperature of -10 to 0°C.

23. The method according to claim 1, in which stage of purification of extracted compounds statin crystallization is carried out with a solvent selected from the group consisting of ethanol, isopropanol, n-propanol, Isobutanol, n-butanol, tertbutanol, ethyl acetate, acetone, methanol, acetonitrile, ethylformate, isobutylacetate, tert-butyl acetate, n-butyl acetate, toluene, propyl and butylmethylamine and mixtures thereof.

24. The allocation method connection statin from a fermentation broth, where the connection statin contains a carboxylic acid capable of forming a lactone and a condensed bicyclic ring, comprising the following stages:

a) pre-processing of the fermentation broth to remove non-polar impurities, and this prior is the processing includes processing of fermentation broth in alkaline conditions, followed by extraction of non-polar impurities in the hydrophobic organic extraction solvent pre-treatment.

b) extraction of the compounds of the statin from the fermentation broth during the processing of fermentation broth extraction solvent and subsequent extraction of the compounds from the fermentation broth in the extraction solvent, which is the hydrophobic organic extraction solvent

C) separating the hydrophobic solvent from the fermentation broth,

g) concentrating the solution of hydrophobic solvent containing the extracted connection statin,

d) washing the concentrated solution of hydrophobic solvent with an aqueous solution containing the base, and

e) purification of extracted compounds statin crystallization.

25. The method according to paragraph 24, in which the aqueous alkaline solution contains ammonium hydroxide, NaHCO3and Na2CO3.

26. The method according to paragraph 24, in which the connection statin drug is an inhibitor of HMG-CoA reductase.

27. The method according to claim 1, in which the purity of the selected statin exceeds 99%.

28. The method according to paragraph 24, in which the purity of the selected statin exceeds 99%.



 

Same patents:

FIELD: biotechnology, organic chemistry, microbiology, pharmacy.

SUBSTANCE: invention describes a new highly productive strain of fungus Aspergillus terreus № 44-62 that is deposited in the Collection of the company Metkinen Oy, Littoinen, Finland, producing lovastatin. Also, invention relates to a method for isolation of lovastatin and a method for lactonization of statins, such as lovastatin and simvastatin. Method for isolation of lovastatin involves its extraction from raw obtained in culturing the above said fungus-producer, concentrating extract, lactonization of lovastatin in the absence of solvent, clearing and crystallization of the end product. The lactonization process of statins carrying out in the absence of solvent provides preparing their lactones in crystalline form directly and practically without impurities of dimmers and acid form. Invention provides highly profitable manufacturing lovastatin and allows preparing the end product corresponding to Pharmacopoeia purity, higher yield (above 70%) and low cost.

EFFECT: improved preparing method and enhanced quality of product.

48 cl, 2 dwg, 10 ex

The invention relates to a method of transforming isoflavonoid conjugates in isoflavonones the aglycones by processing a mixture containing isoflavone conjugates and water at a pH of about 6-13,5, a temperature of about 2-121oWith over a period of time sufficient to convert most of the conjugates in isoflavone conjugates, then contact with the enzyme capable of decomposing due isoflavonoid glycoside with isoflavonoid the glikona at pH of about 3 to 9 and a temperature of about 5-75oWith over a period of time sufficient to convert these isoflavonoid of glikona in isoflavonones the aglycones, as well as way of turning isoflavonoid conjugates and isoflavonoid of glikona in isoflavonones the aglycones after removal isoflavonoid conjugates and isoflavonoid of glikona from material of vegetable protein

FIELD: biotechnology, organic chemistry, microbiology, pharmacy.

SUBSTANCE: invention describes a new highly productive strain of fungus Aspergillus terreus № 44-62 that is deposited in the Collection of the company Metkinen Oy, Littoinen, Finland, producing lovastatin. Also, invention relates to a method for isolation of lovastatin and a method for lactonization of statins, such as lovastatin and simvastatin. Method for isolation of lovastatin involves its extraction from raw obtained in culturing the above said fungus-producer, concentrating extract, lactonization of lovastatin in the absence of solvent, clearing and crystallization of the end product. The lactonization process of statins carrying out in the absence of solvent provides preparing their lactones in crystalline form directly and practically without impurities of dimmers and acid form. Invention provides highly profitable manufacturing lovastatin and allows preparing the end product corresponding to Pharmacopoeia purity, higher yield (above 70%) and low cost.

EFFECT: improved preparing method and enhanced quality of product.

48 cl, 2 dwg, 10 ex

FIELD: biotechnology, pharmacy.

SUBSTANCE: statin compounds from fermentation solution are purified by extraction and crystallization. Fermentation broth is subjected for preliminary treatment that involves preliminary alkaline treatment followed by extraction of nonpolar impurities. After preliminary treatment the statin compound is extracted into hydrophobic solvent that is separated after extraction. Then solution of hydrophobic organic solvent is concentrated and washed out, if necessary, with a base-containing aqueous solution. Then extracted statin compound is purified by crystallization. Invention provides isolating statins from fermentation broth with high degree of effectiveness and under pharmaceutically acceptable level of purity.

EFFECT: improved purifying method.

28 cl, 8 ex

FIELD: distillation industry, in particular, process for preparing of raw grain material for alcoholic fermentation.

SUBSTANCE: method involves grinding grain; mixing ground grain with water and performing thermal processing of batch; introducing phytase into batch at mixing stage, said mixing process being carried out in two stages, first stage including heating batch to temperature of 45-55 C during 15-20 min, and second stage including heating batch in flow and holding at temperature of 60-70 C during 1.5-2 hours.

EFFECT: intensified process for preparing of grain for alcoholic fermentation, increased yield of ready product and provision for adjustment of dough batch acidity within wide range of values.

1 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: ferment lovastatin esterase is proposed, which is immobilised on a water-insoluble hard carrier activated with a bifunctional agent. At the same time the hard carrier represents a modified di-(C1-6alkyl)amino-C1-6alkylcellulose, in another version the hard carrier represents a silica gel modified with amino-C1-6alkyl-tri(C1-6alkoxy)silane, and the bifunctional agent that activates the hard carrier represents O-sulfonate of cyanuric acid or acid halide of cyanuric acid. In the third version the hard carrier represents agarose, and the bifunctional agent is a compound that corresponds to the formula , as defined in the formula. Methods (versions) are proposed to immobilise the ferment lovastatin esterase on specified water-insoluble hard carriers. According to the methods, in process of mechanical mixing the bifunctional activating agent is brought in contact with the hard carrier in the dissolvent. The activated hard carrier is separated by filtration, then dried and suspended in a water mixture, containing the ferment lovastatin esterase, with performance of ferment immobilisation. The suspended substance is separated by filtration, washed with the buffer solution and dried. Also the method is proposed to treat simvastatin, including treatment of the simvastatin salt solution, containing the remaining amount of the lovastatin salt, immobilised witht the ferment lovastatin esterase, and a biocatalysed flow reactor is proposed with a layer for realisation of this method. The reactor comprises a reactor body (1) with the inner space (2), connected with a liquid inlet (3) and connected with a liquid outlet (4), in the inner space there is a perforated plate supporting the layer (5), containing the ferment lovastatin esterase, immobilised on the water-insoluble hard carrier.

EFFECT: immobilised lovastatin esterase according to the invention demonstrates at least 5 times higher hydrolytic activity in respect to lovastatin and lovastatin salts in presence of simvastatin and simvastatin salts than in respect to simvastatin and simvastatin salts.

18 cl, 2 dwg, 8 tbl, 20 ex

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology and agriculture. Disclosed are a pesticidal composition comprising an isolated strain of Burkholderia sp. NRRL No. B-50319, having pesticidal activity, as well as isolated pesticidal compounds such as templazole B, templamide A and B, derived from Burkholderia sp. NRRL No. B-50319. Said composition is used in a method of controlling plant pests, a method to combat emergence and/or growth of monocotyledonous or dicotyledonous sedge weeds as well as for seed coating. Also provided are methods for producing and isolating a compound selected from templazole A and B, templamide A and B, FR901465 and FR90128, by culturing Burkholderia sp. NRRL No. B-50319 and isolating said compound from culture supernatant.

EFFECT: said compounds are used to prepare compositions for controlling plant pests and/or control appearance and/or growth of monocotyledonous or dicotyledonous sedge weeds.

12 cl, 10 dwg, 12 tbl, 13 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. Disclosed is an industrial method of producing compactin. Method comprises culturing Penicillium citrinum VKPM F-1099 producer strain under conditions of aeration in a nutrient medium containing carbohydrate and nitrogen sources and mineral salts. Obtained culture fluid is acidified with mineral acid to pH 2.5-3.5 and held for 1-2 hours. Compactin is extracted from mycelium with butyl acetate in three steps. Extracts are purified with activated carbon. Purified extract is concentrated by distillation of butyl acetate at low pressure. Method then includes adding mineral acid and heating to 85-90 °C at low pressure to complete lactonisation of compactin. Compactin-Lactone crystallisation is carried out. Method then includes recrystallisation of compactin-lactone from aqueous solution of isopropanol at room temperature and periodic stirring for 1-2 hours, then at 6-10 °C for at least 4 hours with extraction of compactin. Product is obtained with compactin content of not less than 97 %, sum of impurities of not more than 1.0 %, single maximum dimer impurity of not more than 0.5 %.

EFFECT: output of product is not less than 65 %.

1 cl, 4 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. Disclosed is a method of producing compactin, involving culturing Penicillium citrinum VKPM F-1099 strain on a culture medium. Obtained culture liquid alkalised to pH 12.0 at 30-50 °C for 2-3 hours. Mycelium is then separated. Method then includes performing sorption of compactin from native solution on a column with hydrophobic nonionic sorbent with elution thereof with aqueous solution of isopropanol. Compactin is extracted from obtained solution with butyl acetate with acidification to pH 3.0-5.0. Extract is purified with activated carbon. Compactin is concentrated and lactonised with partial evaporation of solvent at residual pressure of 10-14 kPa and heating to 75-95 °C in presence of a mineral acid. Method includes crystallising compactin-lactone from aqueous solution of isopropanol at room temperature and periodic stirring for 1-2 hours, then at a temperature of 6-10 °C for at least 4 hours. Product is then recrystallised.

EFFECT: invention provides degree of lactonisation of compactin of not less than 99 % and obtaining a product with compactin content of not less than 98 %, sum of impurities of not more than 1 %, single maximum dimer impurity of not more than 5/10 % and output of product of not less than 65 %.

1 cl, 3 ex

Up!