RU2585234C2 - Method of producing compactin - Google Patents

Abstract

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 %.

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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 the Penicillium citrinum VKPM F-1099 strain. The claimed invention provides a highly profitable industrial production of compactin with a high degree of purification.

Compact 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, IIIb, 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 the 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, its contamination - difficult to separate impurities, such as a 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 to the method for producing compactin from a native solution is a method for producing compactin, in which the culture fluid of the producer strain is subjected to alkaline treatment after fermentation. After this, the target product is isolated under acidic conditions by extraction with a hydrophobic solvent, the extract is concentrated and then back-extracted 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 for producing compactin based on the producer strain Penicillium citrinum VKPM F-1099 - is to obtain a product with high yield and high purity.

To achieve the technical result and ensure high-performance production of compactin, a method for producing compactin is proposed, which involves cultivating the producer strain under aeration on a nutrient medium containing carbohydrates, nitrogen sources and mineral salts, isolating compactin by delactonization of compactin in an alkaline medium, extraction with an organic solvent, and purification of the target product by crystallization and recrystallization, and the Penicillium strain is used as a producer citrinum VKPM F-1099; the resulting culture fluid is treated with alkali at a pH of 12.0 and a temperature of 30-50 ° C for 2-3 hours, then the mycelium is separated, compactin is sorbed from the native solution on a column with a hydrophobic nonionic sorbent with elution with aqueous isopropanol, followed by removal of the solvent by distillation under vacuum; from the resulting solution, compactin is extracted with butyl acetate with acidification to a pH of 3.0-5.0; the resulting extract is purified with activated carbon; compactin is concentrated and lactonized with partial distillation of the solvent at a residual pressure of 10-14 kPa and heating at 75-95 ° C in the presence of mineral acid; compactin-lactone is crystallized from an aqueous solution of isopropanol at room temperature with periodic stirring for 1-2 hours, then at a temperature of 6-10 ° C for at least 4 hours.

According to the following, the optimal mode of concentration and lactonization provides a degree of lactonization of at least 99% in 3-6 hours with a minimum content of impurities, which leads to an increase in the yield of the product.

In addition, the strain used 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, a product is obtained with a basic substance content of not less than 98%, 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 not less than 65%.

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. The strain is deposited in the All-Russian collection of industrial microorganisms (VKPM) FSUE State Research Institute of Genetics under the number F-1099. (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 - a compactin producer 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, from 72-75, 2006).

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 dry cleaning process was developed, providing 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 the initial seed. 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 adjusting the speed of mixing the medium, aeration and overpressure in the range of 0.2-0.5 atm.

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 maintain the pH of the culture fluid at a constant level.

Thus obtained culture fluid with a compactin content of 10-12 mg / g is passed 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. Thus, the present invention claims the technology of isolation and chemical purification of compactin in the form of a lactone using a synthetic sorbent, and the production of highly purified compactin sodium salt from it.

After biosynthesis is complete, compactin in the culture fluid is in the form of acid and lactone. To carry out the process of sorption of the drug on a hydrophobic polymer sorbent such as Amberlite® XAD-16 or its analog Diaion НР-20, it is required to convert the intermediate into an aqueous solution in the form of sodium salt, which is achieved by hydrolysis of compactin lactone (delactonization) in an alkaline medium at pH 10-12 , and a temperature of 30-50 ° C for 2-3 hours.

Then, the delactonized culture fluid is vacuum filtered and compactin is sorbed from the resulting native solution on a hydrophobic polymer sorbent of the Amberlite® XAD-16 type or its analog Diaion HP-20, followed by elution with aqueous isopropanol.

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

The purification of the extract proposed in the described method before the stage of concentration and lactonization with activated carbon, as well as the use of mineral acid as a catalyst for the lactonization process, the optimal concentration and lactonization mode, provides a degree of lactonization of at least 99% in 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.

The advantages of the claimed method of lactonization are:

- almost complete transition of compactin to the lactone form;

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

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, the stage of recrystallization from an aqueous solution of isopropanol or the conversion of lactone to the compactin sodium salt with additional purification of the target product with activated carbon is additionally included. 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 a yield of 88% from the theory can be obtained from compactin-lactone. To do this, compactin-lactone is dissolved in a mixture of isopropyl alcohol and water, then delactonization is carried out with the addition of NaOH (pH 11-12) and a temperature of 40 ° C. Then the water-isopropanol mixture is neutralized to a pH of 9.0-9.5 and isopropyl alcohol is distilled off under reduced pressure and a temperature of 45 ° C on a rotary evaporator. The compactin is extracted with butyl acetate from the aqueous solution at an acidic pH, the aqueous phase is separated off, 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.

Obtaining compactin-lactone using a synthetic sorbent.

1. Delactonization of compactin in the culture fluid to obtain the sodium salt.

After completion of biosynthesis, compactin in the culture liquid is in the form of acid and lactone. To carry out the process of sorption of the drug on a hydrophobic polymer sorbent of the Amberlite® XAD-16 type or its analog Diaion НР-20, it is required to convert the intermediate into an aqueous solution in the form of sodium salt, which is achieved by hydrolysis of compactin lactone at pH 12 for 3 hours.

2. Filtration of the culture fluid under vacuum.

3. Sorption of compactin on a hydrophobic non-ionic adsorbent type Amberlite® XAD-16 with elution with its aqueous isopropanol.

4. The distillation of isopropyl alcohol from the eluate under vacuum at a temperature of 40 ° C.

5. Extraction of compactin acid with butyl acetate and purification of the extract with activated carbon.

6. Concentration on a rotary vacuum evaporator and lactonization with partial distillation of the solvent at a residual pressure of 10-14 kPa and heating T = 75-95 ° C, for 3 hours in the presence of an organic or mineral acid.

7. Washing the lactonized mass with an aqueous alkaline solution.

8. Crystallization from aqueous isopropanol.

In order to obtain a highly purified target product, a recrystallization step from aqueous isopropanol is additionally included. Get a product with a basic substance content of up to 98% (HPLC method), the amount of impurities is not more than 0.6%, a single maximum impurity (dimer) is not more than 0.4% and the product yield is not less than 65%.

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 main substance is not less than 98% (LENGTH); 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 biosynthesis of compactin.

The cultivation of seed material of the strain Penicillium citrinum VKPM F-1099 is carried out in a device 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 pH 6.0-6.2 The seed medium is sterilized in the apparatus at 120 ° C for 30 minutes.

Sowing of the sowing apparatus is carried out with spore seed grown on an agar medium, the 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 was 15.2%. Seed is transferred to fermentation in the amount of 8% of the loaded fermentation medium.

The culture of the strain Penicillium citrinum VKPM F-1099 is grown in a device 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 pH 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 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 was 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 was 70.2 g.

The culture fluid is alkalinized with a 40% NaOH solution to a pH of 11.8; incubated with constant stirring for 3 hours, the native solution is separated by filtration under vacuum and the spent mycelium is washed with water with a pH of 12.0.

550 ml of a native solution containing 4.57 g of compactin is passed through a column with 100 ml of Amberlite XAD - 16 sorbent at a rate of 1 ml / min. Compactin is eluted with an aqueous IPA solution. Obtain 293 ml of an eluate containing 4.48 g of compactin. IPA is distilled off from the eluate under vacuum, 150 ml of butyl acetate are poured into an aqueous solution of Na - compactin salts and acidified with a solution of H ₂ SO ₄ with constant stirring to pH 3.0-5.0. After 30 minutes, BAE is treated with 2.17 g of activated carbon at T = 40 ° C for 1 hour. The coal is washed with butyl acetate, the washing is attached to the main extract. The clarified extract is concentrated in vacuo at T = 60 ° C to a compactin content of 100-150 mg / ml, after which 1.2 ml of mineral acid are added and lactonization is carried out under vacuum for 4 hours. The lactonized mass is washed with an aqueous alkaline solution.

The obtained raw compactin-lactone is dissolved in 18 ml of aqueous IPA and crystallized. The crystals are separated by filtration under vacuum; 3.46 g of paste are obtained with a compactin content of 93.04%, a total of impurities of 1.07%, a single impurity content of 0.38%. After recrystallization, the compactin content is 98.2%, the amount of impurities is 0.26%, a single impurity is 0.15%. The total yield of 63%.

EXAMPLE 2

The cultivation of seed material of the strain Penicillium citrinum VKPM F-1099 is carried out in a device with a capacity of 10 l on a seed nutrient medium, for 36 hours at 24 ° C, stirring and aeration. The apparatus is seeded with spore seed grown on barley, the spore load was 1.5 × 10 ⁵ spores / ml. At the time of transfer of the seed to the fermentation apparatus, the producer biomass was 17.9%. Seed is transferred to fermentation in the amount of 8% of the loaded fermentation medium.

The culture of the strain Penicillium citrinum VKPM F-1099 is grown in a device 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 a level of 25-40%. At 73, 103 and 132 hours of growth, a 50% sucrose solution is added in a volume of 10% to the initial volume of the medium (650 ml each with an initial volume of 6.5 l). The mass fraction of compactin in the culture fluid at the time of discharge was 9.77 mg / g with a trace amount of carbohydrates, a volume biomass of 52.0% and a pH of 5.94. 6.83 kg of culture fluid was drained from the apparatus; the actual removal of compactin in the culture fluid was 66.7 g.

500 ml of culture fluid with a compactin content of 4.85 g is alkalinized with a 40% NaOH solution to a pH of 12.0; incubated with constant stirring for 3 hours, the spent mycelium is separated by filtration under vacuum and washed with alkaline water to a pH of 12.0. Get 518 ml of native solution, which is passed through a column with 100 ml of sorbent Amberlite XAD - 16. The elution of compactin is carried out with an aqueous solution of isopropyl alcohol (IPA). 300 ml of an eluate containing 4.38 g of compactin are obtained. IPA is distilled off from the eluate under vacuum, 180 ml of butyl acetate are poured into an aqueous solution of Na - compactin salts in an amount of 60 ml and, with constant stirring, a 20% solution of H2SO4 is poured dropwise to a pH of 3.0-5, 0. After 30 minutes, butyl acetate the extract (BAE) is treated with 2.4 g of activated carbon at T = 40 ° C for 1 hour. The coal is washed with 5 ml of butyl acetate, the washing is attached to the main extract. The clarified extract is concentrated in vacuo at T = 60 ° C to a compactin content of 100-150 mg / ml, after which 3-5 ml of mineral acid are added and lactonization is carried out under vacuum for 4 hours.

The obtained raw compactin - lactone is dissolved in 20.7 ml of 90% IPA, followed by crystallization. The crystals are separated by filtration under vacuum; get 3.63 g of paste with a compactin content of at least 90%. The paste is dissolved in 18.4 ml of IPA, treated with 4 g of activated carbon for 1 hour, the coal is washed and squeezed thoroughly; compactin - lactone is crystallized from the resulting solution.

Get 3.21 g of compactin - lactone with a basic substance content of at least 98%; the amount of impurities is not more than 1%; single impurity - not more than 0.5%.

The yield from the content in the culture fluid is 66.2%.

EXAMPLE 3

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 reaction mixture is neutralized with an acid pH to 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 was extracted from the aqueous solution with butyl acetate at pH = 3.0, the aqueous phase was separated, and the butyl acetate extract was purified with activated carbon, filtered and washed. To obtain the sodium form of compactin, NaOH is added to the extract with stirring to pH = 9.0-9.6, incubated for 30 minutes, the organic phase is removed. In order to concentrate the aqueous 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. Get 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%.

Claims (1)

A method of producing compactin, comprising cultivating a producer strain under aeration conditions on a nutrient medium containing carbohydrates, nitrogen sources and mineral salts, isolating compactin by delactonization of compactin in an alkaline medium, extraction with an organic solvent and 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 treated with alkali at pH 12.0 and a temperature of 30-50 ° C for 2-3 hours, then the mycelium is separated,
- conduct sorption of compactin from the native solution on a column with a hydrophobic nonionic sorbent with elution with aqueous isopropanol, followed by removal of the solvent by distillation under vacuum;
- from the resulting solution, compactin is extracted with butyl acetate with acidification to a pH of 3.0-5.0;
- the resulting extract is purified with activated carbon;
- compactin is concentrated and lactonized with partial distillation of the solvent at a residual pressure of 10-14 kPa and heating of 75-95 ° C in the presence of mineral acid;
- compactin-lactone is crystallized from an aqueous solution of isopropanol at room temperature with periodic stirring for 1-2 hours, then at a temperature of 6-10 ° C for at least 4 hours.