RU2585233C2 - Industrial method of producing compactin - Google Patents

Abstract

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

Description

The invention relates to biotechnology, in particular to the production of highly purified compactin, an intermediate product for the production of one of the inhibitors of cholesterol biosynthesis in the body. Specifically, the invention relates to a method for producing compactin based on a compactin producer strain, Penicillium citrinum. The claimed invention provides a highly profitable industrial production of compactin with a high degree of purification.

Compactin is an inhibitor of the enzyme beta-hydroxymethyl-glutaryl-CoA reductase (HMG-CoA) and is able to inhibit the early stages of cholesterol biosynthesis. Therefore, compactin derivatives and related compounds (statins) are widely used as the active principle of pharmaceuticals to reduce high blood cholesterol.

It is known that compactin is the initial fermentation raw material in the technology for producing the pharmaceutical substance pravastatin, one of the most effective drugs used to treat hyperlipoproteinemia of IIa, IIb and III phenotypes.

Compactin undergoes conversion to pravastatin as a result of biological transformation, and it is very important not to add an additional amount of related impurities together with compactin, especially such difficult to separate as compactin dimer, which reduces the cost of cleaning the final product - the pharmaceutical substance of pravastatin.

Currently, the development of new technologically advanced and cost-effective methods for producing compactin, which would consistently provide a high yield of the product during its industrial production, is of particular importance.

Known strains producing compactin of the species Penicillium citrinum and Penicillium adametzioides (US 3983140, 4049495, 5691173).

However, the productivity of known strains is low, which does not ensure the profitability of the process and the high yield of the product at the final stage.

Known methods for producing compactin by cultivating a producer strain of compactin and multi-stage isolation of the target product, most often in the form of a lactone, with a final crystallization stage (RU 2114912, WO 98/50572). The main disadvantages of the described methods are:

- insufficient purity of the target product, contamination with difficult to separate impurities, such as dimer and acid forms of compactin;

- low economic indicators, insufficient yield of the target product (50-60%);

- the complexity and multi-stage technology, the use of expensive equipment.

The closest analogue of the industrial method for producing compactin is a method for producing compactin, in which the culture fluid of the producer strain after fermentation is subjected to alkaline treatment and purified using alkali. After that, the target product is isolated under acidic conditions by extraction with a hydrophobic solvent, after which crystallization is carried out, the extractant is concentrated and then extraction is carried out with a weak base, and the target product is isolated by crystallization (WO / 2001/062949).

The known method does not provide the target product of a sufficient degree of purity and with a high yield, the method is multi-stage, time-consuming, requires the use of large volumes of toxic organic solvents.

The technical result of the claimed invention - an industrial method of producing compactin based on a producer strain - Penicillium citrinum VKPM F-1099 - is to obtain a high-quality product with a total impurity content of not more than 1.0%, a single maximum impurity (dimer) of not more than 0.5 % and a product yield of at least 65%.

To achieve the specified technical result and ensure high-performance production of compactin, an industrial method for producing compactin based on the use of the producer strain Penicillium citrinum VKPM F-1099 is proposed, which involves culturing the specified producer strain under aeration conditions on a nutrient medium containing carbohydrates, nitrogen sources and mineral salts, isolation of compactin by extraction with an organic solvent, purification of the target product by crystallization and recrystallization, posing as the producer using the Penicillium citrinum VKPM F-1099 strain;

- the resulting culture fluid is acidified with mineral acid to a pH of 2.5-3.5 and incubated for 1-2 hours;

- compactin is extracted from mycelium with an organic solvent - butyl acetate in three stages;

- extracts are purified with activated carbon;

- the purified extract is concentrated by distillation of butyl acetate under reduced pressure, then mineral acid is added and heated to 85-90 ° C under reduced pressure until the compactin is completely lactonized;

- after crystallization, compactin-lactone is recrystallized from an aqueous solution of isopropanol at room temperature and periodically stirred for 1-2 hours, then at 6-10 ° C for at least 4 hours with the release of compactin.

The Penicillium citrinum VKPM F-1099 strain used to obtain compactin was obtained from the Penicillium citrinum SANK 18767 strain by the method of sequential induced mutagenesis. (Ukraintseva SN, Voinova T.M., Dzhavakhiya VG "Obtaining the highly productive mutants Penicillium citrinum producing compactin and optimization of fermentation process in shaken flasks" Biotechnology in Biology and Medicine. Editor AM Egorov and G. Zaikov, New York, 2006; Ukraintseva CH, Voinova TM, Javakhia VG “Increasing the activity of the fungus Penicillium citrinum - producer of compactin by the method of induced mutagenesis and optimizing the cultivation conditions of highly productive strains.” Materials of the 3rd international conference from the series “Science and Business”, June 19-21 2006, Pushchino, pp. 72-75, 2006)

The strain is deposited in the All-Russian collection of industrial microorganisms (VKPM) FSUE State Research Institute of Genetics under the number F-1099.

The technical result of the given set of essential features is to obtain a product with a basic substance content of at least 97%, a total of impurities of not more than 1.0%, a unit maximum admixture of a dimer of not more than 0.5%. The product yield is at least 65%.

In addition, the strain Penicillium citrinum VKPM F-1099 is highly productive in compactin and stable in its cultural, physiological and biochemical characteristics, which allows it to be effectively used in the process of compactin biosynthesis and to obtain a culture fluid with a high compactin content (10-12 mg / g) . The high productivity of this strain provides a high yield of the target product and the profitability of its production.

Thus, the advantage of the claimed method is the almost complete transition of compactin to the lactone form and the minimum content of impurities in the product in the form of a dimer, which is confirmed by the examples presented in the description.

The proposed compactin production technology has been tested on pilot fermentation units of 10 and 100 liters and a pilot chemical cleaning plant.

As a result of the study, the technology of the fermentation and chemical cleaning process was developed, which provides the set goals - obtaining a high-quality and cost-effective product.

The inventive method is as follows.

The cultivation of seed and hardware fermentation is carried out with dosed or constant introduction of an additional source of carbohydrate nutrition, regulation of dissolved oxygen, indirect regulation of the acidity of the culture fluid. This allows you to get a culture fluid with a compactin concentration of 10-12 mg / g and a low content of related impurities.

The cultivation of seed is carried out in an inoculator on a seed nutrient medium for 48-55 hours at 24 ° C, with constant stirring and aeration. A spore culture grown on an agar medium or barley is used as a seed source. The spore load during sowing of the sowing apparatus is 0.75 · 10 ⁵ -1.25 · 10 ⁵ spores / ml.

The biosynthesis process is carried out on a fermentation nutrient medium for 192-264 hours. Seed from the inoculator is transferred to fermentation in the amount of 8-10% of the loaded fermentation medium. The concentration of dissolved oxygen is maintained at a level of 25-40% by controlling the speed of mixing the medium, aeration and overpressure in the range of 0.2-0.5 ati.

In the fermentation process, the following is added to the culture fluid:

1) 50% sucrose solution in a volume equal to 11-12% of the volume of the initially loaded nutrient medium. Repeat with a decrease in the concentration of sucrose in the culture fluid;

or

2) 50% sucrose solution with a variable speed of 0.0025-0.0045 m ³ / m ³ · h (additive volume / volume of charged medium per hour) to maintain the sucrose concentration in the culture fluid at a constant level, which also allows you to maintain The pH of the culture fluid is constant.

Thus obtained culture fluid with a compactin content of 10-12 mg / g is transferred to the stage of isolation and chemical purification.

As a rule, the main form of using compactin is lactone, since it crystallizes easily, cleans well of impurities, and is stable during storage; however, the sodium form of compactin is also used. Therefore, obtaining the target product in the form of a lactone or sodium salt has its own characteristics.

Obtaining compactin-lactone by extraction

In the proposed method for isolating compactin-lactone, extraction of compactin is carried out with an organic solvent not from a fermentation broth, which requires equipment such as decanters, but from mycelium, on which compactin present in the culture fluid is deposited in lactone and acid form when the culture fluid is acidified with mineral acids to pH 2.5- 3.5 and exposure for 1-2 hours.

To achieve almost 100% yield per stage, in this invention, extraction with an organic solvent is carried out in 3 stages, subject to the optimal conditions at each stage.

Since compactin is obtained in the form of a lactone, the main stage of the process is the stage of lactonization.

The proposed purification of the extract before the stage of concentration and lactonization with activated carbon, as well as the use of mineral acid as a catalyst for the process of lactonization, the optimal mode of concentration and lactonization provide a degree of lactonization of at least 99% for 3-6 hours with a minimum content of impurities. Additionally, lactone crystals are washed to completely get rid of impurities with an aqueous alkaline solution.

An advantage of the claimed method of lactonization is:

- almost complete transition of compactin to the lactone form;

- the minimum content of impurities in the product in the form of a dimer,

Unlike the closest analogue, the crystallization of the target product is carried out from an aqueous solution of isopropanol at room temperature (25 ° C) with periodic stirring for 1-2 hours, then at T = 6-10 ° C for at least 4 hours. A product is obtained with a purity of not less than 90%, a yield of 75%, a total impurity content of not more than 1.5%, a single maximum impurity (dimer) of not more than 0.5%.

In order to obtain a highly purified target product, an additional step is the recrystallization stage in an organic solvent, or the conversion of lactone to compactin sodium salt with additional purification of the target product with activated carbon. A product is obtained with a basic substance content of at least 95%, a total of impurities of not more than 1.0%, a single maximum impurity (dimer) of not more than 0.5%, and a product yield of not less than 65%.

Obtaining sodium salt of compactin

Compactin sodium salt with a basic substance content of at least 98% (HPLC), a total of impurities of not more than 1.0%, a single maximum impurity (dimer) of not more than 0.5%, and 88% yield from theory are obtained from compactin-lactone. Compactin-lactone is dissolved in a mixture of isopropyl alcohol and water, then delactonization is carried out by adding NaOH (pH 11-12) and at a temperature of 40 ° C. Then the water-isopropanol mixture is neutralized to pH 9.0-9.5 and isopropyl alcohol is distilled off on a rotary evaporator under reduced pressure and a temperature of 45 ° C. The compactin is extracted with butyl acetate from the aqueous solution at an acidic pH, the aqueous phase is separated, the extract is purified with activated carbon, filtered and washed. To obtain the sodium form of compactin, NaOH is added to the extract to a pH of 9.0–9.5, and the organic phase is removed. In order to concentrate the aqueous solution and crystallize, water is distilled off in the form of an azeotrope with isopropyl alcohol under reduced pressure and a temperature of 45 ° C. Precipitated crystals of compactin sodium salt are filtered, washed and dried.

The advantages of this technology are:

- economical method;

- lack of by-products;

- yield of the final product - 88% of theory;

- high quality of the obtained sodium salt of compactin - the content of the basic substance is not less than 98% (HPLC); the amount of impurities is not more than 1.0%, a single maximum impurity dimer is not more than 0.5%;

- use of equipment typical for chemical production.

EXAMPLE 1

The cultivation of seed of the strain Penicillium citrinum F-1099 VKPM is carried out in an apparatus with a capacity of 10 l on a seed culture medium of the following composition (per 1 l of medium):

- soy flour - 20 g;

- peptone (tryptone) - 10 g;

- sucrose - 100 g;

- sodium nitrate - 2 g;

- magnesium sulfate seven-water - 1 g;

- distilled water - up to 1 l;

- alkali (KOH or NaOH) or acid (HCl) to a pH of 6.0-6.2.

The seed medium is sterilized in an apparatus at 120 ° C. for 30 minutes.

Sowing of the sowing apparatus is carried out with spore seed grown on an agar medium, spore load was 1.0 · 10 ⁵ spores / ml. The cultivation of seed in the sowing apparatus is carried out for 44 hours at 24 ° C, stirring and aeration. At the time of transfer of the seed to the fermentation apparatus, the producer biomass is 15.2%. Seed is transferred to fermentation in the amount of 8% of the loaded fermentation medium.

The culture of the strain Penicillium citrinum F-1099 VKPM is grown in an apparatus with a capacity of 10 l for 211 hours on a fermentation nutrient medium of the following composition (per 1 l of medium):

- soy flour - 30 g;

- peptone (tryptone) - 10 g;

- yeast extract - 3 g;

- sucrose - 100 g;

- sodium nitrate - 5 g;

- magnesium sulfate seven-water - 1 g;

- distilled water - up to 1 l;

- alkali (KOH or NaOH) or acid (HCl) to a pH of 5.8-6.0.

The fermentation medium is sterilized in an apparatus at 120 ° C. for 30 minutes.

During the fermentation process, the air supply is controlled, the mixing speed of the medium, the overpressure, the concentration of dissolved oxygen are maintained at 30-40%. At 73, 101, and 129 hours of growth, a 50% sucrose solution is administered in a volume of 9.2% of the initial volume of the medium (600 ml each with an initial volume of 6.5 l). The mass fraction of compactin in the culture fluid at the time of discharge is 10.61 mg / g with a mass fraction of carbohydrates of 0.28%, a volume biomass of 42.5% and a pH of 5.75. 6.2 kg of culture fluid were drained from the apparatus; the actual removal of compactin in the culture fluid is 70.2 g.

961.5 cm ³ (1000 g) of the culture fluid obtained by culturing Penicillium citrinum strain F-1099 VKPM containing 11 g of compactin, acidified with mineral acid to pH 2.5-3.5, incubated for 30 minutes. The biomass is filtered, washed with acidified water, and squeezed thoroughly. Extraction of compactin from mycelium by butyl acetate is carried out in three stages. The extracts are combined, purified with activated carbon (9.4 g). The purified extract is concentrated by distillation of butyl acetate under reduced pressure, 3 cm ^{3 of} mineral acid are added to the concentrate, and heating is continued under reduced pressure at a temperature of 85-90 ° C for 3 hours until the compactin is completely lactonized. To remove impurities, the reaction mass is washed with an alkaline solution and water. The washed crystals of compactin lactone are dissolved in isopropyl alcohol, crystallized and dried. 8.3 g of compactin are obtained with a content of at least 90%. Next, recrystallization from isopropyl alcohol is carried out. The yield of compactin is 65.6%, the content of the basic substance is 99% (HPLC).

EXAMPLE 2

10 g of compactin-lactone with a basic substance content of 93.6% are dissolved in a mixture of isopropyl alcohol and water and delactonization is carried out with stirring, at a temperature of 40 ° C and pH 11-12 (addition of NaOH). Then the acid is adjusted to pH 9.0-9.5 and isopropyl alcohol is distilled off from the mixture on a rotary evaporator under reduced pressure and a temperature of 45 ° C. Compactin was extracted from the aqueous solution with butyl acetate at pH 3.0, and the aqueous phase was separated. The butyl acetate extract is purified with activated carbon, filtered and washed. To obtain the sodium form of compactin, NaOH is added to the extract with stirring to a pH of 9.0–9.5, it is held for 30 minutes, and the organic phase is removed. In order to concentrate the solution and crystallize the compactin sodium salt on a rotary evaporator, water is distilled off in the form of an azeotrope with isopropyl alcohol under reduced pressure and a temperature of 45 ° C. Precipitated crystals of compactin sodium salt are filtered, washed and dried. Obtain 10.2 g of compactin in the form of sodium salt with a basic substance content of 97.5% (HPLC), the amount of impurities of 0.19%. The output from the theory is 87.7%.

EXAMPLE 3

The cultivation of seed material of the strain Penicillium citrinum F-1099 VKPM is carried out in a device with a capacity of 10 l on a seed culture medium for 43 hours at 24 ° C, stirring and aeration. The apparatus is seeded with spore seed grown on an agarized nutrient medium, the spore load is 0.8 · 10 ⁵ spores / ml. At the time of transfer of the seed to the fermentation apparatus, the producer biomass is 18.1%. Seed is transferred to fermentation in the amount of 8% of the loaded fermentation medium.

The culture of the strain Penicillium citrinum F-1099 VKPM is grown in an apparatus with a capacity of 10 l for 192 hours on a fermentation nutrient medium. Regulate the air supply, the speed of mixing the medium, overpressure, the concentration of dissolved oxygen is maintained at 25-40%. From 73 hours of growth until the end of fermentation, a 50% sucrose solution is continuously introduced at a specific rate of 0.0025-0.0035 m ³ / m ³ · h (additive volume / volume of charged medium per hour). The mass fraction of compactin in the culture fluid at the time of discharge is 9.74 mg / g with a trace amount of carbohydrates, volume biomass of 43.1% and a pH of 5.92. 6.3 kg of culture fluid were drained from the apparatus; the actual removal of compactin in the culture fluid is 61.4 g.

1021 g of the culture fluid containing 9.9 g of compactin is acidified with sulfuric acid to a pH of 2.75, incubated for 1 hour. The biomass is filtered, washed with acidified water, and squeezed thoroughly. Extraction of compactin from mycelium by butyl acetate is carried out in three stages. The extracts are combined, purified with activated carbon (9.7 g). The purified extract was concentrated by distillation of butyl acetate under reduced pressure with 3.4 ml of sulfuric acid and a temperature of 85 ° C until complete lactonization of compactin. To remove impurities, the reaction mass is washed twice with a solution of soda and water. The washed crystals of compactin lactone are dissolved in isopropyl alcohol, crystallized and dried. 8.2 g of compactin are obtained with a content of 94.7%. Next, recrystallization from isopropyl alcohol is carried out. Get 7.12 g with a basic substance content of 98.3% (HPLC), the amount of impurities of 0.6%. The yield of compactin is 71.9%.

EXAMPLE 4

The cultivation of seed material of the strain Penicillium citrinum F-1099 VKPM is carried out in an apparatus with a capacity of 10 l on a seed culture medium for 42 hours at 24 ° C, stirring and aeration. The apparatus is seeded with spore seed grown on barley, the spore load is 1.0 · 10 ⁵ spores / ml. At the time of transfer of the seed to the fermentation apparatus, the producer biomass is 15.6%. Seed is transferred to fermentation in the amount of 8% of the loaded fermentation medium.

The culture of the strain Penicillium citrinum F-1099 VKPM is grown in a device with a capacity of 100 l for 193 hours on a nutrient medium KZ. Regulate the air supply, the mixing speed of the medium, overpressure, the concentration of dissolved oxygen is maintained at 35-40%. From 74 hours of growth until the end of the fermentation, a 50% sucrose solution is continuously introduced at a rate of 0.0025-0.0040 m ³ / m ³ · h (additive volume / volume of charged medium per hour). The introduction of additives to keep the pH of the culture fluid at a level of 6.0-6.4. The mass fraction of compactin in the culture fluid at the time of discharge is 9.4 mg / g with a mass fraction of carbohydrates of 0.4%, volumetric biomass of 47.8% and a pH of 5.93. 68 liters of culture fluid were drained from the apparatus; the actual removal of compactin in the culture fluid was 639 g.

The culture fluid is acidified with sulfuric acid to a pH of 2.67, incubated for 1 hour. The biomass is filtered, washed with acidified water, squeezed thoroughly. Compactin is extracted from mycelium 15.3 kg butyl acetate 86 l in three stages. The extracts are combined, cleaned with activated carbon. The purified extract is concentrated by distillation of butyl acetate on a Laborota-20 apparatus under reduced pressure, 194.3 cm ^{3 of} concentrated mineral acid is added to the concentrate and heating is continued under reduced pressure and a temperature of 85 ° C for 5 hours until the compactin is completely lactonized. To remove impurities, the lactonized mass is washed twice with an alkaline aqueous solution. The washed crystals of compactin lactone are dissolved in isopropyl alcohol, crystallized at a temperature of 6-8 ° C and dried. Get 484.7 g of compactin with a content of 92.1%. Next, recrystallization from an organic solvent is carried out. 421.7 g of lactone compactin is obtained with a basic substance content of 97.8% (HPLC), a total of 0.9% impurities, a single 0.3%. The yield of compactin from the culture fluid is 66%.

Claims (1)

An industrial method of producing compactin, which involves cultivating a producer strain under aeration on a nutrient medium containing carbohydrates, nitrogen sources and mineral salts, isolating compactin by extraction with an organic solvent, purification of the target product by crystallization and recrystallization, characterized in that
- as a producer using a strain of Penicillium citrinum VKPM F-1099;
- the resulting culture fluid is acidified with mineral acid to a pH of 2.5-3.5 and incubated for 1-2 hours;
- compactin is extracted from mycelium with an organic solvent - butyl acetate in three stages;
- extracts are purified with activated carbon;
- the purified extract is concentrated by distillation of butyl acetate under reduced pressure, then mineral acid is added and heated to 85-90 ° C under reduced pressure until the compactin is completely lactonized;
- after crystallization, compactin-lactone is recrystallized from an aqueous solution of isopropanol at room temperature and periodically stirred for 1-2 hours, then at 6-10 ° C for at least 4 hours with the release of compactin.