CN115838363A - Method for purifying mycophenolic acid - Google Patents
Method for purifying mycophenolic acid Download PDFInfo
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- CN115838363A CN115838363A CN202310027535.7A CN202310027535A CN115838363A CN 115838363 A CN115838363 A CN 115838363A CN 202310027535 A CN202310027535 A CN 202310027535A CN 115838363 A CN115838363 A CN 115838363A
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- mycophenolic acid
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- 229960000951 mycophenolic acid Drugs 0.000 title claims abstract description 114
- HPNSFSBZBAHARI-RUDMXATFSA-N mycophenolic acid Chemical compound OC1=C(C\C=C(/C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-RUDMXATFSA-N 0.000 title claims abstract description 114
- HPNSFSBZBAHARI-UHFFFAOYSA-N micophenolic acid Natural products OC1=C(CC=C(C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-UHFFFAOYSA-N 0.000 title claims abstract description 112
- 238000000034 method Methods 0.000 title claims abstract description 42
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000013078 crystal Substances 0.000 claims abstract description 27
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims abstract description 26
- 238000000855 fermentation Methods 0.000 claims abstract description 26
- 230000004151 fermentation Effects 0.000 claims abstract description 26
- 238000001914 filtration Methods 0.000 claims abstract description 26
- 238000003756 stirring Methods 0.000 claims abstract description 19
- 238000001035 drying Methods 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims abstract description 13
- 235000019799 monosodium phosphate Nutrition 0.000 claims abstract description 13
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims abstract description 13
- 235000017557 sodium bicarbonate Nutrition 0.000 claims abstract description 13
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims abstract description 13
- 238000000605 extraction Methods 0.000 claims abstract description 12
- 238000005406 washing Methods 0.000 claims abstract description 11
- 238000001816 cooling Methods 0.000 claims abstract description 8
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 6
- 239000000706 filtrate Substances 0.000 claims description 30
- 239000000047 product Substances 0.000 claims description 24
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 12
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- 238000002425 crystallisation Methods 0.000 claims description 9
- 230000008025 crystallization Effects 0.000 claims description 9
- DKPFZGUDAPQIHT-UHFFFAOYSA-N butyl acetate Chemical compound CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 claims description 8
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 6
- 241000228145 Penicillium brevicompactum Species 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 229940098377 penicillium brevicompactum Drugs 0.000 claims description 4
- GJRQTCIYDGXPES-UHFFFAOYSA-N iso-butyl acetate Natural products CC(C)COC(C)=O GJRQTCIYDGXPES-UHFFFAOYSA-N 0.000 claims description 3
- FGKJLKRYENPLQH-UHFFFAOYSA-M isocaproate Chemical compound CC(C)CCC([O-])=O FGKJLKRYENPLQH-UHFFFAOYSA-M 0.000 claims description 3
- OQAGVSWESNCJJT-UHFFFAOYSA-N isovaleric acid methyl ester Natural products COC(=O)CC(C)C OQAGVSWESNCJJT-UHFFFAOYSA-N 0.000 claims description 3
- 239000012535 impurity Substances 0.000 abstract description 11
- 238000002360 preparation method Methods 0.000 abstract description 8
- 239000003814 drug Substances 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 38
- 238000000746 purification Methods 0.000 description 22
- 230000000052 comparative effect Effects 0.000 description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 229960004866 mycophenolate mofetil Drugs 0.000 description 3
- RTGDFNSFWBGLEC-SYZQJQIISA-N mycophenolate mofetil Chemical compound COC1=C(C)C=2COC(=O)C=2C(O)=C1C\C=C(/C)CCC(=O)OCCN1CCOCC1 RTGDFNSFWBGLEC-SYZQJQIISA-N 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical class O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical group CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- GRSZFWQUAKGDAV-KQYNXXCUSA-N IMP Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(NC=NC2=O)=C2N=C1 GRSZFWQUAKGDAV-KQYNXXCUSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- XZTYGFHCIAKPGJ-UHFFFAOYSA-N Meclofenoxate Chemical compound CN(C)CCOC(=O)COC1=CC=C(Cl)C=C1 XZTYGFHCIAKPGJ-UHFFFAOYSA-N 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 235000013902 inosinic acid Nutrition 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229960003442 meclofenoxate Drugs 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000036963 noncompetitive effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000013037 reversible inhibitor Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
Landscapes
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention belongs to the technical field of medicine preparation, and particularly relates to a method for purifying mycophenolic acid. The invention provides a method for purifying mycophenolic acid, which comprises the following steps: s1, filtering a fermentation culture solution containing mycophenolic acid, adding sodium dihydrogen phosphate, stirring, adjusting the pH value, adding an extracting agent for extraction, and removing a water layer to leave an extract for later use; s2, filtering the extract obtained in the step S1, washing the extract with a sodium bicarbonate solution, and removing a water layer; s3, concentrating the solution washed in the step S2 under reduced pressure, adding triethylamine, stirring, cooling, crystallizing and filtering; and S4, drying the mycophenolic acid crystal obtained in the step S3 under reduced pressure to obtain the mycophenolic acid crystal. The method for purifying the mycophenolic acid provided by the invention is simple, and can efficiently remove Z isomer which is a key impurity of the mycophenolic acid, so that the content of the Z isomer in the mycophenolic acid is below 0.10%.
Description
Technical Field
The invention belongs to the technical field of medicine preparation, and particularly relates to a method for purifying mycophenolic acid.
Background
Mycophenolic acid (mycophenolic acid, MPA) is an antibiotic with antifungal, antitumor and immunosuppressive effects produced by penicillium brevicompactum, is a high-efficiency, selective, noncompetitive and reversible inhibitor of inosine monophosphate dehydrogenase (MPDH), and can inhibit the initial synthetic pathway of guanine nucleotide, exhaust guanine nucleotide and further block DNA synthesis. The Mycophenolate Mofetil (MMF) and the mycophenolate sodium (MPS) are frequently used clinically. The production method of the mycophenolic acid has two ways of chemical synthesis and biological synthesis, the chemical synthesis method is difficult to industrialize due to multiple steps and low yield, and the mycophenolic acid is obtained by extracting and purifying a fermentation liquid containing the mycophenolic acid after the penicillium microbial fermentation production method is adopted for international large-scale production.
In the fermentation, extraction and purification processes of mycophenolic acid, an impurity Z isomer is generated at the same time, is a key impurity in mycophenolic acid, is difficult to remove due to the high similarity of the structure of mycophenolic acid, and is finally carried into mycophenolate mofetil or mycophenolate sodium to influence the quality one of a finished product. The Z isomer (meclofenoxate sodium European pharmacopoeia 10.3 impurity B) has the following structure:
chinese patent CN109020933B discloses a method for purifying mycophenolic acid, which comprises the following steps: 1) Adding mycophenolic acid into a mixed solution of acetone and hydrochloric acid, heating, stirring and dissolving; 2) Adding active carbon, stirring and decoloring; 3) Filtering; 4) Dropwise adding a solvent B into the filtrate; 5) Heat preservation and crystallization; 6) Filtering, washing and drying to obtain the pure mycophenolic acid. Chinese patent application CN103880798A discloses a method for purifying mycophenolic acid. The method comprises the following steps: 1) Providing an ethanol solution of mycophenolic acid at a temperature of 50 ℃ to 70 ℃; 2) Cooling the ethanol solution obtained in the step 1) to 10-30 ℃ to separate out mycophenolic acid crystals, and separating to obtain mycophenolic acid crystals; 3) Dissolving the mycophenolic acid crystals obtained in the step 2) with ethanol, mixing the obtained solution with water at the temperature of 5-12 ℃, recrystallizing and separating mycophenolic acid, and separating to obtain mycophenolic acid recrystallized crystals.
In the disclosed extraction and purification technology of mycophenolic acid, although the mycophenolic acid has a certain purification effect, the purification technology has the defects of complexity, poor purification effect and the like, and only aims at the removal technology of the impurity A, and a method for efficiently removing the Z isomer as the key impurity of the mycophenolic acid is not disclosed.
Therefore, a method for purifying mycophenolic acid needs to be developed, the impurity Z isomer generated in the fermentation, extraction and purification processes of mycophenolic acid can be effectively removed, and the preparation method is simple and controllable and has a good purification effect.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a method for purifying mycophenolic acid. The purification method of mycophenolic acid provided by the invention is simple, and can efficiently remove the Z isomer which is a key impurity of mycophenolic acid through the synergistic effect of various steps of the purification method, so that the Z isomer content in mycophenolic acid is below 0.10%.
The technical scheme of the invention is as follows:
a method for purifying mycophenolic acid comprises the following steps:
s1, filtering a fermentation culture solution containing mycophenolic acid, measuring the product amount in the filtrate, adding sodium dihydrogen phosphate into the filtrate, stirring, adjusting the pH, then adding an extracting agent for extraction, and separating and removing a water layer to leave an extract for later use; the product amount is the mass of the mycophenolic acid in the filtrate;
s2, filtering the extract obtained in the step S1 by using kieselguhr, washing by using a sodium bicarbonate solution, and removing a water layer;
s3, concentrating the solution washed in the step S2 under reduced pressure to obtain a concentrated solution, adding triethylamine, stirring, cooling for crystallization, and filtering to obtain mycophenolic acid crystals;
and S4, drying the mycophenolic acid crystal obtained in the step S3 under reduced pressure to obtain the purified mycophenolic acid crystal.
Further, the fermentation broth containing mycophenolic acid in the step S1 is a fermentation broth containing mycophenolic acid obtained by fermenting conventional Penicillium Brevicompactum. The specific preparation method is disclosed in example 2 in the specification of a fermentation process of mycophenolic acid in Chinese patent CN 109929890B.
Furthermore, the filtering mode of the fermentation culture solution containing mycophenolic acid in the step S1 is plate-and-frame filtering.
Further, the adding amount of the sodium dihydrogen phosphate in the step S1 is 0.8-1.5% of the amount of the product in the filtrate.
Further, the amount of sodium dihydrogen phosphate added in step S1 is 1% of the amount of the product in the filtrate.
Further, the stirring time after the sodium dihydrogen phosphate is added in the step S1 is 1 to 2 hours; after stirring, the pH value is adjusted to 4.0-5.0 by adopting phosphoric acid solution with the mass concentration of 85%.
Further, the extractant in step S1 is one of ethyl acetate, toluene, n-butyl acetate, and isobutyl acetate.
Furthermore, the addition amount of the extracting agent in the step S1 is 1.5 to 2.5 times of the volume of the filtrate, and the extraction time is 2 to 3 hours.
Furthermore, the mass concentration of the sodium bicarbonate solution in the step S2 is 0.3-1.0%, the addition amount is 25-35% of the volume of the extraction liquid obtained in the step S1, and the washing time by the sodium bicarbonate solution is 0.5-2 h.
Furthermore, the sodium bicarbonate solution in step S2 has a mass concentration of 0.5% and is added in an amount of 30% of the volume of the extract obtained in step S1.
Further, in the step S3, the solution washed in the step S2 is concentrated under reduced pressure to 25 times of the product amount; the product amount is by mass and the concentrate in step S2 is by volume.
Further, the addition amount of triethylamine in the step S3 is 6-10% of the volume of the concentrated solution, and the stirring time is 30-60 min.
Further, the temperature in the step S3 is 40-55 ℃ when the concentration is carried out under reduced pressure, and the temperature in the step S3 when the crystallization is carried out under reduced temperature is 5-15 ℃.
Further, the temperature of the reduced pressure drying in the step S4 is 40-55 ℃, and the mycophenolic acid crystal is dried under reduced pressure until the Loss On Drying (LOD) is less than or equal to 1%.
The invention provides a method for purifying mycophenolic acid, which has simple integral purification process and excellent removal effect on Z isomer impurities, and ensures that the content of the Z isomer in the mycophenolic acid reaches below 0.10 percent. In the purification process, firstly, the bacterial residues and the fermentation residues in the mycophenolic acid fermentation culture solution are better removed by adopting a plate-and-frame filtration mode and the like, so that the filtrate containing the product is collected, and then, Z isomer impurities in the mycophenolic acid can be effectively removed by adding sodium dihydrogen phosphate before extraction, washing the extract liquor by using a sodium bicarbonate solution after extraction and adding a triethylamine solution with a specific addition amount before crystallization.
Compared with the prior art, the method for purifying mycophenolic acid provided by the invention has the following advantages:
(1) The method for purifying the mycophenolic acid provided by the invention is simple and easy to operate, simple and easy to control, low in production cost, safe and environment-friendly, and beneficial to industrial popularization and application, and the extracting agent can be recycled.
(2) The invention can remove the key impurity Z isomer of the mycophenolic acid with high efficiency by the synergistic effect of a plurality of steps of the purification method, so that the Z isomer content in the mycophenolic acid reaches below 0.10 percent.
Detailed Description
The present invention is further illustrated by the following description of specific embodiments, which are not intended to limit the invention, and various modifications and improvements can be made by those skilled in the art based on the basic idea of the invention, but the invention is within the protection scope of the invention.
In the following examples and comparative examples, the reagents not specifically described are conventional reagents and are available from conventional reagent production and distribution companies.
Example 1A method for purifying mycophenolic acid
The purification method of mycophenolic acid comprises the following steps:
s1, filtering a fermentation culture solution containing mycophenolic acid by using a plate-and-frame filter, collecting a filtrate, measuring the product amount in the filtrate, adding sodium dihydrogen phosphate with the product amount of 0.8 percent, stirring for 1 hour, adjusting the pH of the filtrate to 4.0-5.0 by using phosphoric acid with the mass concentration of 85 percent, extracting for 2 hours by using ethyl acetate with the volume of 1.5 times of the filtrate, separating a water layer, and leaving an extract for later use;
s2, filtering the extract obtained in the step S1 by using diatomite, adding a sodium bicarbonate solution with the volume of 30% of the extract and the mass concentration of 0.4%, washing for 1 hour at room temperature, and removing a water layer;
s3, concentrating the solution washed in the step S2 at 40 ℃ under reduced pressure to 25 times of the product amount (the product amount is calculated by mass, and the concentrated solution in the step S2 is calculated by volume), adding triethylamine with the volume of 6% of that of the concentrated solution, stirring for 30min, cooling to 5 ℃ for crystallization, and filtering to obtain mycophenolic acid crystals;
and S4, drying the mycophenolic acid crystals obtained in the step S3 at 40 ℃ under reduced pressure until the Loss On Drying (LOD) is less than or equal to 1%.
The fermentation culture solution containing mycophenolic acid in the step S1 is fermentation broth containing mycophenolic acid obtained by fermenting conventional Penicillium Brevicompactum. The specific preparation method is disclosed in example 2 in the specification of a fermentation process of mycophenolic acid in Chinese patent CN 109929890B.
Example 2 purification method of mycophenolic acid
The purification method of mycophenolic acid comprises the following steps:
s1, filtering a fermentation culture solution containing mycophenolic acid by using a plate-and-frame filter, collecting a filtrate, measuring the product amount in the filtrate, adding sodium dihydrogen phosphate with the product amount of 1%, stirring for 1.5h, adjusting the pH of the filtrate to 4.0-5.0 by using a phosphoric acid solution with the mass concentration of 85%, extracting for 3 hours by using n-butyl acetate with the volume 2 times that of the filtrate, separating a water layer, and leaving an extract for later use;
s2, filtering the extract obtained in the step S1 by using diatomite, adding a sodium bicarbonate solution with the volume of 30% of the extract and the mass concentration of 0.5%, washing for 1h at room temperature, and discarding a water layer;
s3, concentrating the solution washed in the step S2 at 45 ℃ under reduced pressure to 25 times of the product amount (the product amount is calculated by mass, and the concentrated solution in the step S2 is calculated by volume), adding triethylamine with the volume of 8% of that of the concentrated solution, stirring for 40min, cooling to 10 ℃ for crystallization, and filtering to obtain mycophenolic acid crystals;
and S4, drying the mycophenolic acid crystals obtained in the step S3 at 45 ℃ under reduced pressure until the Loss On Drying (LOD) is less than or equal to 1%.
The preparation method of the fermentation broth containing mycophenolic acid in the step S1 is the same as that of the example 1.
Example 3A method for purifying mycophenolic acid
The purification method of mycophenolic acid comprises the following steps:
s1, filtering a fermentation culture solution containing mycophenolic acid by using a plate-and-frame filter, collecting a filtrate, measuring the product amount in the filtrate, adding sodium dihydrogen phosphate accounting for 1.5% of the product amount into the filtrate, stirring the mixture for 2 hours, adjusting the pH of the filtrate to 4.0-5.0 by using a phosphoric acid solution with the mass concentration of 85%, extracting the filtrate for 2 hours by using isobutyl acetate with the volume 2.5 times of that of the filtrate, and separating and discarding a water layer to leave an extract for later use;
s2, filtering the extract obtained in the step S1 by using diatomite, adding a sodium bicarbonate solution with the volume of 30% of the extract and the mass concentration of 0.8%, washing for 2 hours at room temperature, and removing a water layer;
and S3, concentrating the solution washed in the step S2 at 50 ℃ under reduced pressure to 25 times of the product amount (the product amount is calculated by mass, and the concentrated solution in the step S2 is calculated by volume), then stirring triethylamine with 10% of the volume of the concentrated solution for 60min, cooling to 15 ℃, crystallizing, and filtering to obtain the mycophenolic acid crystals.
And S4, drying the mycophenolic acid crystals obtained in the step S3 at 55 ℃ under reduced pressure until the Loss On Drying (LOD) is less than or equal to 1%.
The preparation method of the fermentation broth containing mycophenolic acid in the step S1 is the same as that of the example 1. Comparative example 1A conventional purification method for mycophenolic acid
The purification method of mycophenolic acid comprises the following steps:
s1, filtering a fermentation culture solution containing mycophenolic acid by using a plate-and-frame filter, collecting a filtrate, measuring the product amount in the filtrate, adjusting the pH of the filtrate to 4.0-5.0 by using a phosphoric acid solution with the mass concentration of 85%, extracting for 3 hours by using n-butyl acetate with the volume 2 times that of the filtrate, and removing a water layer to leave an extract for later use;
s2, filtering the extract liquid obtained in the step S1 by using diatomite;
s3, concentrating the filtrate extracted in the step S2 at 45 ℃ under reduced pressure to 25 times (V/W) of the volume of the product, cooling to 10 ℃ for crystallization, and filtering to obtain mycophenolic acid crystals;
and S4, drying the mycophenolic acid crystals obtained in the step S3 at 45 ℃ under reduced pressure until the Loss On Drying (LOD) is less than or equal to 1%.
The preparation method of the fermentation broth containing mycophenolic acid in the step S1 is the same as that of the example 1. Comparative example 2 method for purifying mycophenolic acid
In comparison with example 2, comparative example 2 is different in that triethylamine in the step S3 is replaced with n-hexane, and other parameters and operations are the same as those of example 2.
Comparative example 3 purification method of mycophenolic acid
In comparison with example 2, comparative example 2 is different in that no sodium dihydrogen phosphate is added in step S1, and other parameters and operations are the same as those of example 2.
Comparative example 4 method for purifying mycophenolic acid
In comparison with example 2, comparative example 2 is different in that no sodium bicarbonate solution is used for washing in step S2, and other parameters and operations are the same as example 2.
Test example I Performance testing of mycophenolic acid
1. Test materials: the mycophenolic acid crystals prepared in the examples 1-3 and the comparative examples 1-4.
2. The test method comprises the following steps: the content of Z isomer in the mycophenolic acid crystals prepared in the examples 1 to 3 and the comparative examples 1 to 4 is detected by using a related substance detection method of European pharmacopoeia 10.3 2813 (the content of Z isomer as an impurity in mycophenolic acid is not more than 0.10%).
3. Test results
The test results are shown in table 1.
TABLE 1Z isomer content of mycophenolic acid
| Group of | Z isomer containingAmount (%) |
| Example 1 | 0.04 |
| Example 2 | 0.02 |
| Example 3 | 0.05 |
| Comparative example 1 | 0.43 |
| Comparative example 2 | 0.18 |
| Comparative example 3 | 0.15 |
| Comparative example 4 | 0.14 |
As is clear from Table 1, the Z isomer content in the purified mycophenolic acid crystals obtained in examples 1 to 3 was 0.10% or less, and of these, example 2 was the most effective, only 0.02%, and is the most preferred example of the present invention. The Z isomer content in the mycophenolic acid crystal purified by the conventional mycophenolic acid purification method adopted in the comparative example 1 is 0.43 percent and far exceeds the quality standard of pharmacopoeia. In comparative examples 2 to 4, the Z isomer content in the purified mycophenolic acid crystals was 0.10% or more, respectively, after the purification conditions in the purification process were changed.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Those skilled in the art can modify or change the above-described embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. A method for purifying mycophenolic acid is characterized by comprising the following steps:
s1, filtering a fermentation culture solution containing mycophenolic acid, adding sodium dihydrogen phosphate into the filtrate, stirring, adjusting the pH value, adding an extracting agent for extraction, and separating and removing a water layer to leave an extract for later use;
s2, filtering the extract obtained in the step S1, washing the extract with a sodium bicarbonate solution, and removing a water layer;
s3, concentrating the solution washed in the step S2 under reduced pressure to obtain a concentrated solution, adding triethylamine, stirring, cooling for crystallization, and filtering to obtain mycophenolic acid crystals;
and S4, drying the mycophenolic acid crystal obtained in the step S3 under reduced pressure to obtain the purified mycophenolic acid crystal.
2. The method for purifying mycophenolic acid as claimed in claim 1, wherein the amount of sodium dihydrogen phosphate added in step S1 is 0.8-1.5% of the amount of the product in the filtrate.
3. The method of claim 1, wherein the extractant in step S1 is one of ethyl acetate, toluene, n-butyl acetate, and isobutyl acetate; the addition amount of the extracting agent is 1.5 to 2.5 times of the volume of the filtrate, and the extraction time is 2 to 3 hours.
4. The method for purifying mycophenolic acid as claimed in claim 1, wherein the mass concentration of the sodium bicarbonate solution in the step S2 is 0.3-1.0%, and the addition amount is 25-35% of the volume of the extraction liquid obtained in the step S1; the washing time by sodium bicarbonate solution is 0.5-2 h.
5. The method for purifying mycophenolic acid as claimed in claim 1, wherein in step S3, the solution washed in step S2 is concentrated under reduced pressure to 25 times the product amount by mass and the concentrated solution in step S2 is measured by volume.
6. The method for purifying mycophenolic acid as claimed in claim 1, wherein the amount of triethylamine added in step S3 is 6-10% of the volume of the concentrated solution.
7. The method for purifying mycophenolic acid as claimed in claim 1, wherein the stirring time after the sodium dihydrogen phosphate solution is added in the step S1 is 1-2 h; after stirring, the pH value is adjusted to 4.0-5.0 by adopting phosphoric acid solution with the mass concentration of 85%.
8. The method for purifying mycophenolic acid as claimed in claim 1, wherein the temperature for concentration under reduced pressure in step S3 is 40-55 ℃, and the temperature for temperature reduction and crystallization is 5-15 ℃.
9. The method for purifying mycophenolic acid as claimed in claim 1, wherein the temperature for drying under reduced pressure in step S4 is 40-55 ℃, and the mycophenolic acid crystals are dried under reduced pressure until the loss on drying is less than or equal to 1%.
10. The method for purifying mycophenolic acid as claimed in claim 1, wherein the fermentation broth containing mycophenolic acid in step S1 is a fermentation broth containing mycophenolic acid obtained by conventional Penicillium Brevicompactum fermentation.
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| GB1158387A (en) * | 1967-06-13 | 1969-07-16 | Ici Ltd | Procedure for Isolation of Mycophenolic Acid |
| WO2008003637A2 (en) * | 2006-07-05 | 2008-01-10 | Dsm Ip Assets B.V. | Isolation and use of amine salts of mycophenolic acid |
| WO2009040828A1 (en) * | 2007-09-25 | 2009-04-02 | Biocon Limited | A process for purification of mycophenolic acid |
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| CN109020933A (en) * | 2017-06-09 | 2018-12-18 | 鲁南制药集团股份有限公司 | A kind of purification process of Mycophenolic Acid |
| CN110922371A (en) * | 2019-12-27 | 2020-03-27 | 广东蓝宝制药有限公司 | Preparation method of M2 crystal form meclofenol sodium |
| CN112645912A (en) * | 2020-12-27 | 2021-04-13 | 广东蓝宝制药有限公司 | Preparation method of high-purity M2 crystal form meclofenol sodium |
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| GB1158387A (en) * | 1967-06-13 | 1969-07-16 | Ici Ltd | Procedure for Isolation of Mycophenolic Acid |
| WO2008003637A2 (en) * | 2006-07-05 | 2008-01-10 | Dsm Ip Assets B.V. | Isolation and use of amine salts of mycophenolic acid |
| WO2009040828A1 (en) * | 2007-09-25 | 2009-04-02 | Biocon Limited | A process for purification of mycophenolic acid |
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