Production method of high-purity cyclosporine A

Abstract

The invention relates to a production method of high-purity cyclosporin A. The cyclosporin A is obtained by carrying out flash drying, organic solvent extraction, aqueous alkali treatment, silica gel column chromatography and spray drying on wet cyclosporin A thalli. The cyclosporine A production process is optimized, only one organic solvent is used in the process, solvent residues are remarkably reduced, meanwhile, the product purity is larger than 98.5%, the yield is 85.0% or above, and the cyclosporine A is more suitable for industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a method for producing high-purity cyclosporin A. Background Art

[0002] Cyclosporin A belongs to immunosuppressive drugs and is a cyclic peptide composed of 11 amino acids isolated from the culture broth of filamentous fungi. This drug was discovered in 1969, and in 1976, Sandoz Company in Switzerland first reported that cyclosporin A was produced by Trichoderma polysporum (later renamed Beauveria nivea and Cylindrocarpon lucidum) of the genus Dematiaceous fungi.

[0003] Chinese Name: Cyclosporin A, CsA

[0004] English Name: Ciclosporin A

[0005] CAS Registry Number: 59865-13-3

[0006] The chemical structural formula of cyclosporin A is as follows:

[0007]

[0008] At present, cyclosporin A is mainly obtained by microbial fermentation. In the fermented broth after fermentation, in addition to containing cyclosporin A, it also contains several other cyclosporins such as B, C, D, G, H, as well as other impurities such as culture medium components and various bacterial metabolites. The main process route for purifying cyclosporin A in the prior art is to first extract with organic solvents, and then separate and purify by different types of column chromatography or in combination with crystallization. For example, CN102086226A discloses a separation method combining a macroporous adsorption resin column and a silica gel column chromatography. Cyclosporin A is directly extracted from the fermented broth by adding an organic solvent, and then subjected to two chromatographic purifications with a macroporous adsorption resin and a silica gel column in sequence, and finally obtained a finished product with a purity greater than 98.5% through crystallization. This method requires two chromatographies using macroporous adsorption resin and silica gel, with a long process cycle, involving many types of organic solvents, being environmentally unfriendly and having high production costs, and is not suitable for industrial production.

[0009] Therefore, it is urgent to develop a method for producing high-purity cyclosporin A, which is of extremely important significance for its production and sales. Summary of the Invention

[0010] The purpose of the present invention is to provide a method for producing high-purity cyclosporin A. By means of flash drying and organic solvent extraction methods, combined with silica gel column chromatography and spray drying methods, the production process of cyclosporin A is simplified, the solvent residue is significantly reduced, the product quality is effectively improved, and it is more suitable for industrial production.

[0011] The technical solution adopted to achieve the above-mentioned invention purpose is as follows:

[0012] A purification method of cyclosporin A, comprising the following steps:

[0013] 1) Flash-drying the wet mycelium obtained by filtering the cyclosporin A fermentation broth to obtain dry mycelium;

[0014] 2) Extracting the dry mycelium obtained in step 1) with a first solvent, separating the solid and liquid to obtain an extract, washing successively with an alkali solution and water, and separating the phases to obtain an extraction solution;

[0015] 3) Concentrating the extraction solution obtained in step 2), passing it through a silica gel column, eluting with a second solvent, and collecting the cyclosporin A eluate;

[0016] 4) Concentrating the cyclosporin A eluate obtained in step 3) and then spray-drying to obtain the finished product of cyclosporin A;

[0017] Among them, the first solvent in step 1) is the same as the second solvent in step 3).

[0018] Among them, the flash-drying temperature in step 1) is 110°C to 130°C.

[0019] Among them, the first solvent in step 2) is ethyl acetate or acetone, and the dosage of the first solvent is 2-3 times the weight of the dry mycelium.

[0020] Among them, the alkali solution in step 2) is a 2-4% sodium hydroxide solution or potassium hydroxide solution, and the dosage of the alkali solution is 2.0-2.5 times the volume of the extraction solution.

[0021] Among them, the silica gel in step 3) is 400-500 mesh spherical silica gel.

[0022] Among them, the diameter-height ratio of the silica gel column in step 3) is 1:25-30.

[0023] Among them, the sample loading amount of the silica gel in step 3) is 10%-20% of the column volume.

[0024] Among them, the second solvent in step 3) is ethyl acetate or acetone.

[0025] Among them, the concentration of the eluate in step 4) is vacuum decompression concentration, and the concentration temperature is 60°C to 70°C.

[0026] Among them, the inlet air temperature of the spray drying in step 4) is 100-120°C

[0027] The technical solution of the present invention has at least the following beneficial technical effects:

[0028] 1. After filtering the fermentation broth, the present invention uses high-temperature flash evaporation technology to rapidly dry the bacterial cells to obtain dry bacterial cells. During this process, a large amount of proteins or other metabolites in the fermentation broth are denatured and inactivated, which is extremely beneficial for the removal of the above-mentioned proteins during subsequent ethyl acetate extraction. After the dry bacterial cells are extracted with ethyl acetate, they are washed successively with an alkaline solution and water to further remove some large-polarity impurities.

[0029] 2. Compared with the prior art, while simplifying the process, the present invention only uses one ethyl acetate solvent during the production process, significantly reducing the solvent residue.

[0030] 3. The purity of the finished product obtained by this process is much higher than the pharmacopoeia standard, which is beneficial to the improvement of the drug quality. Finally, this process route is short, with high production efficiency, easy for industrial production, the product purity is greater than 98.5%, and the yield is above 85.0%. Detailed implementation manners

[0031] The following will clearly and completely describe the implementation schemes of the present invention in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. The description of at least one exemplary embodiment below is actually only illustrative and in no way constitutes a limitation to the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0032] The HPLC detection method for the following examples and comparative examples: Chromatographic conditions: Octadecylsilane chemically bonded silica gel is used as the filler (a stainless steel tube with a diameter of φ0.25mm×1000mm is connected in front of the column); acetonitrile-water-tert-butyl methyl ether-phosphoric acid (430∶520∶50∶1) is used as the mobile phase, the stainless steel tube and the column temperature are 70°C; the detection wavelength is 210nm; the injection volume is 20μl. Calculated by the peak area normalization method.

[0033] Example 1

[0034] After filtering 25L of fermentation broth (the concentration of cyclosporin A is 11.2g / L), 10.25kg of wet bacterial cells are obtained, flash-dried at 120°C to obtain 5.1kg of dry bacterial cells, and 10.2kg of ethyl acetate is taken for stirring extraction for 2 hours. The bacterial cells are filtered off to obtain 11.2L of extraction solution, and the obtained extraction solution is washed successively with 22.5L of 3% sodium hydroxide aqueous solution and 22.5L of aqueous solution, and 10.3L of extraction solution is obtained after layering.

[0035] The obtained extraction solution is vacuum concentrated at a vacuum degree of -0.08Mpa and a temperature of 45°C. After concentration, the content of cyclosporin A is 33.4g / L, and it is ready for column loading.

[0036] The 400 - 500 mesh spherical silica gel was packed into the column by wet method (diameter - height ratio = 1:25, φ50 * 1250 mm). After the column packing was completed, the concentrated solution was loaded onto the column with a sample loading amount of about 11%, and then eluted with ethyl acetate. The eluate with a purity higher than 98.5% was collected.

[0037] After the eluate was concentrated under vacuum at 45°C, a concentrated solution was obtained. Then, it was spray - dried in a spray dryer at an inlet air temperature of 110°C to obtain 242.2 g of white powder as the finished product. The total yield was calculated to be 85.55%. The purity was detected by HPLC to be 98.9%. When detecting the solvent residue in the product, only the ethyl acetate residue was detected to be 0.03%.

[0038] Example 2

[0039] After filtering 30 L of fermentation broth (cyclosporin A concentration was 11.7 g / L), 12.9 kg of wet mycelium was obtained. It was dried by flash evaporation at 120°C to obtain 6.5 kg of dry mycelium. 19.5 kg of ethyl acetate was taken and stirred for extraction for 2 hours. After filtering off the mycelium, 21.2 L of the extraction solution was obtained. The obtained extraction solution was washed successively with 53 L of 3% sodium hydroxide aqueous solution and 53 L of aqueous solution, and 18.5 L of the extraction solution was obtained after stratification.

[0040] The obtained extraction solution was concentrated under vacuum at a vacuum degree of - 0.08 Mpa and a temperature of 45°C. After concentration, the content of cyclosporin A was 35.8 g / L, waiting to be loaded onto the column.

[0041] The 400 - 500 mesh spherical silica gel was packed into the column by wet method (diameter - height ratio = 1:30, φ50 * 1500 mm). After the column packing was completed, the concentrated solution was loaded onto the column with a sample loading amount of about 12%, and then eluted with ethyl acetate. The eluate with a purity higher than 98.5% was collected.

[0042] After the eluate was concentrated under vacuum at 45°C, a concentrated solution was obtained. Then, it was spray - dried in a spray dryer at an inlet air temperature of 110°C to obtain 298.5 g of white powder as the finished product. The total yield was calculated to be 85.12%. The purity was detected by HPLC to be 99.1%. When detecting the solvent residue in the product, only the ethyl acetate residue was detected to be 0.03%.

[0043] Example 3

[0044] After filtering 27 L of fermentation broth (cyclosporin A concentration was 12.7 g / L), 12.3 kg of wet mycelium was obtained. It was dried by flash evaporation at 120°C to obtain 6.2 kg of dry mycelium. 15.5 kg of ethyl acetate was taken and stirred for extraction for 2.5 hours. After filtering off the mycelium, 16.8 L of the extraction solution was obtained. The obtained extraction solution was washed successively with 33.6 L of 3% sodium hydroxide aqueous solution and 33.6 L of aqueous solution, and 15.1 L of the extraction solution was obtained after removing the bottom water layer after stratification.

[0045] The obtained extract was vacuum concentrated at a vacuum degree of -0.08 Mpa and a temperature of 45°C. After concentration, the content of cyclosporin A was 35.8 g / L and was ready for column loading.

[0046] The spherical silica gel of 400 - 500 mesh was wet-packed into a column (diameter-height ratio = 1:28, φ50*1400 mm). After column packing, the concentrated solution was loaded onto the column at about 15% of the sample loading amount, and then eluted with ethyl acetate. The eluate with a purity higher than 98.5% was collected.

[0047] After the eluate was vacuum concentrated at 45°C, a concentrated solution was obtained. Then, it was spray-dried at an inlet air temperature of 110°C in a spray dryer to obtain 305.5 g of white powder as the finished product. The total yield was calculated to be 88.38%. The purity was detected by HPLC to be 99.2%. The solvent residue of the product was detected, and only 0.02% of ethyl acetate residue was detected.

[0048] Comparative Example 1

[0049] After filtering 25 L of fermentation broth (cyclosporin A concentration of 12.3 g / L), 12.1 kg of wet mycelium was obtained. Then, 12.0 kg of ethyl acetate was taken and stirred for extraction for 2.5 hours. The mycelium was filtered off to obtain 18.5 L of extract. After layering and removing the aqueous layer, 10.5 L of extract was obtained.

[0050] The obtained extract was vacuum concentrated at a vacuum degree of -0.08 Mpa and a temperature of 45°C. After concentration, the content of cyclosporin A was 30.1 g / L and was ready for column loading.

[0051] The spherical silica gel of 400 - 500 mesh was wet-packed into a column (diameter-height ratio = 1:30, φ30*900 mm). After column packing, the concentrated solution was loaded onto the column at about 10% of the sample loading amount, and then eluted with ethyl acetate. The eluate with a purity higher than 98.5% was collected.

[0052] After the eluate was vacuum concentrated at 45°C, a concentrated solution was obtained. Then, it was spray-dried at an inlet air temperature of 110°C in a spray dryer to obtain 44.2 g of white powder as the finished product. The total yield was calculated to be 14.19%. The purity was detected by HPLC to be 98.7%. The solvent residue of the product was detected, and only 0.05% of ethyl acetate residue was detected.

[0053] Comparative Example 2

[0054] After filtering 25 L of fermentation broth (cyclosporin A concentration of 11.4 g / L), 12.6 kg of wet mycelium was obtained. It was dried by flash evaporation at 120°C to obtain 6.6 kg of dry mycelium. 12.5 kg of ethyl acetate was taken and stirred for extraction for 2.5 hours. The mycelium was filtered off to obtain 13.6 L of extract. The obtained extract was washed successively with 30 L of 3% sodium hydroxide aqueous solution and 30 L of aqueous solution. After layering, 12.2 L of extract was obtained.

[0055] After vacuum concentration of the obtained extract at a vacuum degree of -0.08 Mpa and a temperature of 45°C, the content of cyclosporin A after concentration was 35.8 g / L, waiting to be loaded onto the column.

[0056] The 400 - 500 mesh spherical silica gel was wet-packed into a column (diameter-height ratio = 1:28, φ50 * 1400 mm). After the column packing was completed, the concentrated solution was loaded onto the column with a sample loading amount of about 14%, and then eluted with ethyl acetate, and the eluate with a purity higher than 98.5% was collected.

[0057] After vacuum concentration of the eluate at 45°C, a concentrated solution was obtained, and then spray drying was carried out at an inlet air temperature of 110°C in a spray dryer to obtain 215.5 g of white powder as the finished product. The total yield was calculated to be 74.55%, and the purity was 98.60% by HPLC detection. The solvent residue of the detected product was only detected with ethyl acetate residue of 0.06%.

[0058] Comparative Example 3

[0059] After filtering 25 L of fermentation broth (cyclosporin A concentration of 12.4 g / L), 11.3 kg of wet bacterial cells were obtained, flash dried at 120°C to obtain 5.8 kg of dry bacterial cells, and 14.5 kg of ethyl acetate was taken and stirred for extraction for 2.5 hours. The bacterial cells were filtered off to obtain 15.7 L of extract, and the obtained extract was washed successively with 39 L of 3% sodium hydroxide aqueous solution and 39 L of aqueous solution, and after layering, the lower water layer was removed to obtain 14.6 L of extract.

[0060] After vacuum concentration of the obtained extract at a vacuum degree of -0.08 Mpa and a temperature of 45°C, the content of cyclosporin A after concentration was 35.8 g / L, waiting to be loaded onto the column.

[0061] The 400 - 500 mesh spherical silica gel was wet-packed into a column (diameter-height ratio = 1:28, φ50 * 1400 mm). After the column packing was completed, the concentrated solution was loaded onto the column with a sample loading amount of about 15%, and then eluted with ethyl acetate, and the eluate with a purity higher than 98.5% was collected.

[0062] After vacuum concentration of the eluate to a paste at 45°C, 750 mL of acetone was added, and it was left to crystallize at -5°C for 24 h, and then filtered to obtain the wet product. The wet product was vacuum dried at 55°C for 48 h to obtain 231.2 g of white powder as the finished product. The total yield was calculated to be 73.76%, and the purity was 98.90% by HPLC detection. The solvent residue of the detected product was 0.7% for acetone and 0.3% for ethyl acetate.

[0063] Comparative Example 4

[0064] Take 25 L of cyclosporin A fermentation broth (the concentration of cyclosporin A is 12.6 g / L), add 5 mol / l NaOH to adjust the pH to neutral, add 1 volume of acetone, stir and extract at 30 °C for 2.5 hours, and collect the filtrate for column loading.

[0065] Pack a column with D101 macroporous adsorption resin (diameter-height ratio = 1:7, φ10*700 mm), wash and treat the resin with pure water, 3% sodium hydroxide solution and 3% hydrochloric acid solution respectively. After the treatment, load about 10 column volumes of the extract onto the column; wash the column with about 2 column volumes of 3% hydrochloric acid and wash with pure water to pH 6.5 - 7.5; elute with acetone, and collect about 1.5 column volumes of the eluate; add 0.5 volume of petroleum ether to the eluate for extraction and layering, remove the lower aqueous phase, and concentrate the upper liquid phase for loading onto a silica gel column.

[0066] After appropriately treating 400 - 500 mesh silica gel, pack the column by wet method (diameter-height ratio = 1:28, φ50*1400 mm), balance the silica gel column with 2 column volumes of acetone∶petroleum ether (volume ratio) = 30∶70. After the balance, load about 20% column volume of the concentrated solution onto the column, elute with the above acetone / petroleum ether mixed reagent, and collect the eluate with a purity higher than 97%. The eluate is concentrated under vacuum at 60 °C, crystallized at -5 °C for 24 hours and then filtered, and rinsed with cold acetone, and then dried under vacuum at 55 °C for 48 h to obtain the white powder product

[0067] 205.6, calculate the total yield to be 64.36%, and the purity is 98.6% by HPLC detection. Detect the solvent residues in the product, among which acetone is

[0068] 0.8%, and petroleum ether is 0.4%.

Claims

1. A purification method of cyclosporin A, comprising the following steps: 1) Flash drying the wet mycelium obtained by filtering the cyclosporin A fermentation broth to obtain dry mycelium; 2) Extracting the dry mycelium obtained in step 1) with a first solvent, separating the solid and liquid to obtain an extraction solution, washing successively with an alkali solution and water, and separating phases to obtain an extract; 3) Concentrating the extract obtained in step 2), passing it through a silica gel column, eluting with a second solvent, and collecting the cyclosporin A eluate; 4) Concentrating the cyclosporin A eluate obtained in step 3) and spray drying to obtain the finished product of cyclosporin A; wherein the first solvent in step 1) is the same as the second solvent in step 3).

2. The purification method according to claim 1, characterized in that the flash drying temperature in step 1) is 110°C to 130°C.

3. The purification method according to claim 1, characterized in that the first solvent in step 2) is ethyl acetate or acetone, and the dosage of the first solvent is 2 - 3 times the weight of the dry mycelium.

4. The purification method according to claim 1, characterized in that the alkali solution in step 2) is a 2 - 4% sodium hydroxide solution or potassium hydroxide solution, and the dosage of the alkali solution is 2.0 - 2.5 times the volume of the extract.

5. The purification method according to claim 1, characterized in that the silica gel in step 3) is 400 - 500 mesh spherical silica gel.

6. The purification method according to claim 1, characterized in that the ratio of the column diameter to the height of the silica gel column in step 3) is 1:25 - 30.

7. The purification method according to claim 1, characterized in that the sample loading amount of the silica gel in step 3) is 10% - 20% of the column volume.

8. The purification method according to claim 1, characterized in that the second solvent in step 3) is ethyl acetate or acetone.

9. The purification method according to claim 1, characterized in that the concentration of the eluate in step 4) is vacuum decompression concentration, and the concentration temperature is 60°C to 70°C.

10. The purification method according to claim 1, characterized in that the inlet air temperature of the spray drying in step 4) is 100 - 120°C.

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