Preparation method of mecobalamine crystal with high crystallization yield

Abstract

The invention provides a preparation method of a mecobalamin crystal with high crystallization yield. The preparation method comprises the following steps: carrying out resin adsorption and desorption on a product crude solution; carrying out anti-solvent crystallization on a concentrated eluent; and separating to obtain the mecobalamin crystal. According to the method, the mecobalamin crude mother liquor obtained through biological fermentation and a semi-synthesis method is used as a raw material, resin is utilized for adsorption and desorption to purify the mecobalamin crude mother liquor, the mother liquor before mecobalamin crystallization is obtained through reduced pressure evaporation and concentration, then the mother liquor is crystallized through an anti-solvent method, and mecobalamin crystals with high crystallization yield are obtained. Related impurities in the mecobalamin crude mother liquor are removed through adsorption and desorption, then organic matters in the impurities are further removed through vacuum concentration, the saturation degree of the mother liquor before crystallization is improved, finally, mecobalamin crystallization is further promoted through stirring in an anti-solvent, and a mecobalamin crystal product with high crystallization yield is obtained through drying. The method is simple to operate, low in cost, environment-friendly in solvent, short in production period, high in crystallization yield and good in repeatability.

Description

Technical Field

[0001] The present invention relates to the technical field of methylcobalamin production, and specifically, to a method for preparing methylcobalamin crystals with a high crystallization yield. Background Art

[0002] As the active form of coenzyme vitamin B 12 , methylcobalamin has important clinical applications in the treatment of peripheral neuropathy and megaloblastic anemia. However, the optimization of the methylcobalamin crystallization process remains a difficult point in the pharmaceutical field. Traditional crystallization methods (such as evaporation crystallization and cooling crystallization) often result in low purity and yield of methylcobalamin crystals, uneven particle size distribution, and irregular crystal morphology, which in turn affect the fluidity and tableting performance of subsequent preparations, increase the process complexity and cost, lead to quality fluctuations between product batches, and restrict large-scale production. Currently, the anti-solvent crystallization technology has attracted much attention because it can precisely control supersaturation and improve crystal morphology. However, when applied to methylcobalamin, the existing technology still has the following problems: (1) The matching between the rate of adding the anti-solvent and the crystallization kinetics is insufficient, which easily causes secondary nucleation or the formation of solvates; (2) A suitable anti-solvent has not been selected, resulting in a low crystal yield.

[0003] The post-treatment in the existing technology generally adopts methods such as anti-solvent acetone crystallization, or gel filtration adsorption, column chromatography separation and purification, etc. However, the crystallization yield of methylcobalamin prepared by domestic enterprises at present is relatively low, and there are also problems such as serious mixed crystals and irregular crystal morphology.

[0004] Therefore, a method for preparing methylcobalamin crystals with a high crystallization yield is provided, which focuses on improving the crystallization yield of methylcobalamin crystals, and forms a method for preparing methylcobalamin crystals with simple operation, low cost, short production cycle, high product crystallization yield, and good repeatability. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing methylcobalamin crystals with a high crystallization yield. According to the principle of anti-solvent crystallization, by utilizing the solubility mutation characteristic of methylcobalamin in acetonitrile, a binary gradient solvent system of acetonitrile / water is constructed to prepare methylcobalamin crystals with a high crystallization yield, and by designing a closed-loop solvent recovery system, the solvents in the system can be recycled.

[0006] The present invention provides a method for preparing methylcobalamin crystals with a high crystallization yield, comprising the following steps:

[0007] (1) Performing resin adsorption and desorption on the crude product liquid: After filtering the methylcobalamin crude mother liquor, pumping it into a resin adsorption column using a peristaltic pump for adsorption, then using an eluent to perform elution on the resin until there is no obvious red liquid, and then concentrating and membrane-filtering the eluent to obtain a concentrated eluent. During concentration, the eluent is recovered for standby;

[0008] (2) Anti-solvent crystallization of the concentrated eluate: The concentrated eluate obtained in step (1) is injected into the anti-solvent by a peristaltic pump for crystallization separation, while stirring. After the feeding is completed, continue stirring for a certain period of time until no more crystals of mecobalamin precipitate out to obtain a solid-liquid mixture. Stop stirring and let it stand for layering;

[0009] (3) Separate and obtain mecobalamin crystals: Withdraw the supernatant of the solid-liquid mixture obtained in step (2), perform suction filtration on the lower solid-liquid mixture, and dry the filter cake obtained by suction filtration to obtain mecobalamin crystals.

[0010] Preferably, the resin in step (1) is macroporous resin, the pore diameter of the macroporous resin is 100 - 500 nm, and the volume ratio of the loading amount of the macroporous resin to the volume of the crude mecobalamin mother liquor is 50:(20 - 80).

[0011] Preferably, the membrane filtration treatment in step (1) uses an organic membrane, and the pore diameter of the organic membrane is 0.22 μm.

[0012] Preferably, the eluate in step (1) is an acetonitrile aqueous solution, and the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is (70 - 90):(10 - 30).

[0013] Preferably, the concentration treatment in step (1) is carried out by rotary evaporation under reduced pressure in a rotary evaporator, the evaporation temperature is 50 - 70 °C, the rotation speed is 50 - 150 rpm, and the acetonitrile in the eluate is recovered for reuse during the concentration process.

[0014] Preferably, the injection rate of the concentrated eluate in step (2) is 20 - 80 ml / min,

[0015] Preferably, the anti-solvent in step (2) is acetonitrile, and the volume ratio of the anti-solvent to the concentrated eluate is (70 - 90):(10 - 30).

[0016] Preferably, the stirring treatment in step (2) adopts a mechanical stirring method, the stirring rate is 20 - 60 rpm, the time for continuous stirring treatment is 1 - 5 h, and the standing time after stopping stirring is 1 - 3 h.

[0017] Preferably, the drying treatment in step (3) is vacuum drying, the drying temperature is 50 - 80 °C, and the drying time is 5 - 8 h.

[0018] Preferably, the anti-solvent in step (2) is the acetonitrile recovered during concentration in step (1).

[0019] Working principle of the present invention: The present invention uses the crude mother liquor of mecobalamin obtained by biological fermentation and semi-synthesis as raw material, removes some impurities in the crude mother liquor of mecobalamin through adsorption and desorption, then uses the eluent to wash to reduce the inclusion and residue of impurities in the mother liquor, and further removes the organic matter in the impurities and increases the saturation of the mother liquor before crystallization during the vacuum concentration process. Stirring during the anti-solvent crystallization process is beneficial to obtaining the effective crystal form of the required mecobalamin solid preparation, improving the yield and purity of mecobalamin. Therefore, this application uses physical and chemical methods to change the solubility of mecobalamin in the solution and obtain mecobalamin crystals with high crystallization yield.

[0020] Beneficial effects of the present invention: Compared with the traditional process, this method has the advantages of simple operation, low cost, friendly solvent environment, short production cycle, high crystallization yield, and good repeatability.

[0021] (1) Readily available raw materials: Using the crude mother liquor of mecobalamin obtained by biological fermentation and semi-synthesis as raw material, the eluent and anti-solvent are acetonitrile. The raw material source is reliable, with a large output and easy availability.

[0022] (2) Cost savings: The crystallization process of mecobalamin is simple, the crystallization precipitation time is short, and the precipitation amount is sufficient, reducing the time cost during crystallization. Moreover, the raw material liquid after crystallization can be concentrated and returned to the front section of this crystallization process, reducing the cost of raw material waste during production.

[0023] (3) Green and environmental protection: Crystallizing mecobalamin by using the anti-solvent acetonitrile generates no waste acid or waste alkali, providing a new method for the environmental protection production of mecobalamin. Moreover, the anti-solvent used can be recycled and reused, greatly reducing the discharge of organic waste liquid, achieving green environmental protection and resource recycling.

[0024] (4) Excellent products: By using the anti-solvent to crystallize mecobalamin, the crystallization yield of the obtained mecobalamin crystals is as high as 90.5 - 93.4%. Specific embodiments

[0025] In order to make the technical solution of the present invention easier to understand, the technical solution of the present invention is clearly and completely described by using specific embodiments. Since mecobalamin is easily decomposed by light, all operations are carried out under dark red light.

[0026] Example 1

[0027] The preparation method of the mecobalamin crystals with high crystallization yield in this example includes the following steps:

[0028] 1. Take 5 ml of the crude mother liquor of mecobalamin produced by biological fermentation and semi-synthesis, filter to remove solid particles, and collect the filtrate for use.

[0029] 2. The filtrate obtained in step 1 is pumped into a resin adsorption column through a peristaltic pump. The filling size of the adsorption column is Φ10×200mm, the filler is a non-polar macroporous resin with a pore size of 100nm, and the resin dosage is 3ml.

[0030] 3. Use acetonitrile aqueous solution as eluent to elute the resin after adsorption in step 2. The volume ratio of acetonitrile / water in the eluent is 70:30, and elute until there is no obvious red liquid.

[0031] 4. The eluate in step 3 was concentrated by vacuum distillation in a rotary evaporator to obtain a concentrated eluate at a concentration temperature of 50° C. and a rotation speed of 50 rpm. The acetonitrile in the recovered components during the concentration process was set aside.

[0032] 5. Filter the concentrated eluate in step 4 using an organic filter membrane to remove solid particles, and collect the concentrated filtrate for later use.

[0033] 6. The concentrated eluate filtered in step 5 was injected into the recovered acetonitrile in step 4 at a rate of 20 ml / min via a peristaltic pump, wherein the volume ratio of acetonitrile / concentrated eluate was 70:30, and mechanical stirring was performed at a stirring rate of 20 rpm. After the addition was completed, stirring was continued for 5 hours, stirring was stopped and allowed to stand for 1 hour, and crystallization was completed.

[0034] 7. The upper clear liquid was extracted, the lower solid-liquid mixture was filtered, the filter cake was rinsed with a small amount of acetonitrile, and then vacuum dried at 50° C. for 8 hours to obtain methylcobalamin crystals.

[0035] The calculated crystallization yield was 90.7%, and the liquid phase purity was 99.4%.

[0036] Example 2

[0037] The method for preparing methylcobalamin crystals with high crystallization yield of the present embodiment comprises the following steps:

[0038] 1. Take 50 ml of crude methylcobalamin mother liquor produced by biological fermentation and semi-synthesis, filter to remove solid particles, and collect the filtrate for standby use.

[0039] 2. The filtrate obtained in step 1 is pumped into a resin adsorption column through a peristaltic pump. The filling size of the adsorption column is Φ20×300mm, the filler is a non-polar macroporous resin with a pore size of 200nm, and the resin dosage is 25ml.

[0040] 3. Use acetonitrile aqueous solution as eluent to elute the resin after adsorption in step 2. The volume ratio of acetonitrile / water in the eluent is 75:25, and elute until there is no obvious red liquid.

[0041] 4. The eluate in Step 3 is concentrated by reduced pressure distillation in a rotary evaporator to obtain a concentrated eluate. The concentration temperature is 55 °C, the rotation speed is 80 rpm, and the acetonitrile in the recovered component is reserved during the concentration process.

[0042] 5. The concentrated eluate in Step 4 is filtered using an organic filter membrane to remove the solid particles therein, and the concentrated filtrate is collected for use.

[0043] 6. The concentrated eluate in Step 5 is injected into the recovered acetonitrile in Step 4 at a rate of 35 ml / min by a peristaltic pump, where the volume ratio of acetonitrile / concentrated eluate is 75:25. Meanwhile, stirring is carried out at a stirring rate of 30 rpm. After the feeding is completed, stirring is continued for 4 hours, then the stirring is stopped and left to stand for 1.5 h, and the crystallization is completed.

[0044] 7. The supernatant is drawn out, and the lower-layer solid-liquid mixture is subjected to suction filtration. The filter cake is rinsed with a small amount of acetonitrile, and then vacuum dried at 60 °C for 7 hours to obtain mecobalamin crystals.

[0045] The crystallization yield is calculated to be 90.7%, and the purity detected by liquid phase is 99.4%.

[0046] Example 3

[0047] The preparation method of mecobalamin crystals with a high crystallization yield in this example includes the following steps:

[0048] 1. Take 100 ml of the crude mother liquor of mecobalamin produced by biotransformation and semi-synthesis method, filter to remove the solid particles, and collect the filtrate for use.

[0049] 2. The filtrate obtained in Step 1 is pumped into a resin adsorption column by a peristaltic pump. The filling size of the adsorption column is Φ20×600 mm, the packing is a non-polar macroporous resin with a pore diameter of 300 nm, and the resin dosage is 50 ml.

[0050] 3. Use an acetonitrile aqueous solution as the eluate to elute the adsorbed resin in Step 2. The volume ratio of acetonitrile / water in the eluate is 80:20, and elute until there is no obvious red liquid.

[0051] 4. The eluate in Step 3 is concentrated by reduced pressure distillation in a rotary evaporator to obtain a concentrated eluate. The concentration temperature is 60 °C, the rotation speed is 100 rpm, and the acetonitrile in the recovered component is reserved during the concentration process.

[0052] 5. The concentrated eluate in Step 4 is filtered using an organic filter membrane to remove the solid particles therein, and the concentrated filtrate is collected for use.

[0053] 6. Inject the concentrated eluate in Step 5 into the recycled acetonitrile in Step 4 at a rate of 50 ml / min by a peristaltic pump, where the volume ratio of acetonitrile to concentrated eluate is 80:20. Meanwhile, conduct mechanical stirring at a stirring rate of 40 rpm. After the feeding is completed, continue stirring for 3 hours, stop stirring and let it stand for 2 h to complete crystal precipitation.

[0054] 7. Draw out the upper clear liquid, subject the lower solid-liquid mixture to suction filtration, wash the filter cake with a small amount of acetonitrile, and then dry it in vacuo at 65 °C for 6.5 hours to obtain crystalline mecobalamin.

[0055] Calculate the crystallization yield to be 91.4%, and the purity detected by liquid phase is 99.6%.

[0056] Example 4

[0057] The preparation method of crystalline mecobalamin with high crystallization yield in this example includes the following steps:

[0058] 1. Take 500 ml of the crude mother liquor of mecobalamin produced by biological fermentation and semi-synthesis method, filter to remove solid particles, and collect the filtrate for use.

[0059] 2. Pump the filtrate obtained in Step 1 into a resin adsorption column by a peristaltic pump. The filling size of the adsorption column is Φ40×700 mm, the packing is a non-polar macroporous resin with a pore diameter of 400 nm, and the resin dosage is 250 ml.

[0060] 3. Use an acetonitrile aqueous solution as the eluent to elute the adsorbed resin in Step 2. The volume ratio of acetonitrile to water in the eluent is 85:15, and elute until there is no obvious red liquid.

[0061] 4. Conduct vacuum distillation and concentration of the eluate in Step 3 in a rotary evaporator to obtain a concentrated eluate. The concentration temperature is 65 °C, the rotation speed is 120 rpm, and the acetonitrile in the recovered components during the concentration process is reserved for use.

[0062] 5. Filter the concentrated eluate in Step 4 using an organic filter membrane to remove the solid particles therein, and collect the concentrated filtrate for use.

[0063] 6. Inject the concentrated eluate in Step 5 into the recycled acetonitrile in Step 4 at a rate of 64 ml / min by a peristaltic pump, where the volume ratio of acetonitrile to concentrated eluate is 85:15. Meanwhile, conduct mechanical stirring at a stirring rate of 50 rpm. After the feeding is completed, continue stirring for 2 hours, stop stirring and let it stand for 2.5 h to complete crystal precipitation.

[0064] 7. Draw out the upper clear liquid, subject the lower solid-liquid mixture to suction filtration, wash the filter cake with a small amount of acetonitrile, and then dry it in vacuo at 70 °C for 6 hours to obtain crystalline mecobalamin.

[0065] The calculated crystallization yield was 90.7%, and the purity detected by liquid phase was 99.4%.

[0066] Example 5

[0067] The preparation method of the high-crystallization-yield methylcobalamin crystal in this example includes the following steps:

[0068] 1. Take 2500 ml of the crude mother liquor of methylcobalamin produced by biological fermentation and semi-synthesis method, filter to remove solid particles, and collect the filtrate for use.

[0069] 2. Pump the filtrate obtained in step 1 into the resin adsorption column through a peristaltic pump. The filling size of the adsorption column is Φ60×700 mm, the filler is a non-polar macroporous resin with a pore size of 500 nm, and the resin dosage is 1000 ml.

[0070] 3. Use acetonitrile aqueous solution as the eluent to elute the adsorbed resin in step 2. The volume ratio of acetonitrile / water in the eluent is 90:10, and elute until there is no obvious red liquid.

[0071] 4. Concentrate the eluent in step 3 by vacuum distillation on a rotary evaporator to obtain a concentrated eluent. The concentration temperature is 70°C, the rotation speed is 150 rpm, and the acetonitrile in the recovered component is recycled during the concentration process for use.

[0072] 5. Filter the concentrated eluent in step 4 using an organic filter membrane to remove the solid particles therein, and collect the concentrated filtrate for use.

[0073] 6. Inject the concentrated eluent in step 5 into the recycled acetonitrile in step 4 at a rate of 80 ml / min through a peristaltic pump, where the volume ratio of acetonitrile / concentrated eluent is 90:10. At the same time, perform mechanical stirring at a stirring rate of 60 rpm. After the feeding is completed, continue stirring for 1 hour, stop stirring and let it stand for 3 h to complete crystal precipitation.

[0074] 7. Draw out the upper clear liquid, filter the lower solid-liquid mixture by suction filtration, wash the filter cake with a small amount of acetonitrile, and then dry it in vacuum at 80°C for 5 hours to obtain methylcobalamin crystals.

[0075] The calculated crystallization yield was 93.1%, and the purity detected by liquid phase was 99.5%.

[0076] The chromatographic conditions detected in the above examples: Use octadecylsilane-bonded silica gel as the filler (Luna C18 chromatographic column, 4.6 mm×250 mm, 5 μm or a chromatographic column with equivalent efficiency); use 0.03 mol / L potassium dihydrogen phosphate solution (adjust the pH value to 4.5 with 0.2 mol / L sodium hydroxide solution or phosphoric acid)-acetonitrile (84:16) as the mobile phase; the detection wavelength is 342 nm; the injection volume is 20 μl.

[0077] It should be noted that the embodiments described herein are only partial embodiments of the present invention, rather than all implementation manners of the present invention. The embodiments are only exemplary, and their function is only to provide a more intuitive and clear way to understand the content of the present invention, rather than a limitation on the technical solutions described in the present invention. Without departing from the concept of the present invention, all other implementation manners that can be thought of by those of ordinary skill in the art without creative efforts, as well as other simple substitutions and various changes to the technical solutions of the present invention, all fall within the protection scope of the present invention.

Claims

1. A method for preparing mecobalamin crystals with a high crystallization yield, characterized in that, It includes the following steps: (1) Resin adsorption and desorption of the crude product solution: After filtering the crude mother liquor of mecobalamin, it is pumped into the resin adsorption column using a peristaltic pump for adsorption, and then the resin is eluted with the eluent until no obvious red liquid remains. The eluent is then concentrated and membrane-filtered to obtain a concentrated eluate. During concentration, the eluent is recovered for reuse; (2) Anti-solvent crystallization of the concentrated eluate: The concentrated eluate obtained in step (1) is injected into the anti-solvent using a peristaltic pump for crystallization separation, and stirring is carried out simultaneously. After the feeding is completed, stirring is continued for a certain period of time until no more mecobalamin crystals precipitate to obtain a solid-liquid mixture. Stirring is stopped and left to stand for layer separation; (3) Separating and obtaining mecobalamin crystals: The supernatant of the solid-liquid mixture obtained in step (2) is drawn out, and the lower solid-liquid mixture is subjected to suction filtration. The filter cake obtained by suction filtration is dried to obtain mecobalamin crystals.

2. The preparation method of the mecobalamin crystal with high crystallization yield according to claim 1, characterized in that, In step (1), the resin is macroporous resin with a pore size of 100 - 500 nm, and the volume ratio of the macroporous resin loading to the volume of the crude mother liquor of mecobalamin is 50:(20 - 80).

3. The preparation method of the mecobalamin crystal with high crystallization yield according to claim 1, characterized in that, In step (1), the membrane filtration treatment uses an organic membrane with a pore size of 0.22 μm.

4. The preparation method of the mecobalamin crystal with high crystallization yield as described in claim 1, wherein, In step (1), the eluent is an acetonitrile aqueous solution, and the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is (70 - 90):(10 - 30).

5. The preparation method of the mecobalamin crystal with high crystallization yield according to claim 4, wherein, In step (1), the concentration treatment is carried out by rotary evaporation under reduced pressure in a rotary evaporator. The evaporation temperature is 50 - 70°C, and the rotary speed is 50 - 150 rpm. During concentration, the acetonitrile in the eluent is recovered for use.

6. The preparation method of the mecobalamin crystal with high crystallization yield as described in claim 1, wherein, In step (2), the injection rate of the concentrated eluate is 20 - 80 ml / min.

7. The preparation method of the mecobalamin crystal with high crystallization yield as described in claim 4, characterized in that, In step (2), the anti-solvent is acetonitrile, and the volume ratio of the anti-solvent to the concentrated eluate is (70 - 90):(10 - 30).

8. The preparation method of the mecobalamin crystal with high crystallization yield as described in claim 1, characterized in that, In step (2), the stirring treatment adopts a mechanical stirring method, the stirring rate is 20 - 60 rpm, the time for continuous stirring treatment is 1 - 5 h, and the standing time after stopping stirring is 1 - 3 h.

9. The preparation method of the mecobalamin crystal with high crystallization yield according to claim 1, characterized in that, In step (3), the drying treatment is vacuum drying at a drying temperature of 50 - 80°C for a drying time of 5 - 8 h.

10. The preparation method of the mecobalamin crystal with high crystallization yield according to claim 7, characterized in that, The anti-solvent in step (2) is the acetonitrile recovered during concentration in step (1).