Method for purifying abamectin crystal

Abstract

The invention provides a purification method of an abamectin crystal, and relates to the technical field of biological pharmacy. The invention relates to a purification method of an abamectin crystal. The purification method comprises the following steps: S1, crystallizing an abamectin extract to obtain a crystal; s2, carrying out crystallization treatment on the crystal by using methanol with the pH value of 4.0-6.5 and the mass concentration of 80-95% to obtain a recrystallized crystal; and S3, carrying out crystallization treatment on the recrystallized crystal by using methanol with the mass concentration of 80-95% to obtain an abamectin crystal. According to the method, the content of impurities influencing clarity in the crystal can be reduced, and the clarity of the abamectin crystal is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to a method for purifying avermectin crystals. Background Art

[0002] Avermectin is a macrolide antibiotic produced by fermentation of Streptomyces avermitilis and widely used in agricultural pest control and veterinary medicine. Because it typically exists in nature as a mixture of various structural analogs (such as A1a, A2a, B1a, and B2a), and because impurities are easily introduced during the production process, the purification process has a crucial impact on the quality and effectiveness of the final product. Avermectin can be used as a precursor to synthesize ivermectin, a novel broad-spectrum, highly effective, and low-toxic antibiotic antiparasitic drug. The clarity of avermectin crystals directly affects the clarity of ivermectin.

[0003] In the prior art, methods for crystallizing and purifying avermectin using composite solvents are available. This method utilizes a composite solvent system composed of two or more organic solvents of different polarities as the crystallization solvent. During the crystallization process, the synergistic effect between the solvents is exploited to reduce the polarity of poorly water-soluble non-polar impurities, thereby increasing their solubility in the mother liquor. These impurities remain in the mother liquor during the crystallization process, effectively separating the avermectin crystals from the impurities and achieving the desired purification.

[0004] However, this composite solvent crystallization method still has many shortcomings: First, the changes in the proportions of the various components of the composite solvent are extremely sensitive to the crystallization process. Even slight fluctuations in the ratio may significantly affect the formation process of avermectin crystals, leading to problems such as uneven crystal form, wide crystal particle size distribution, and decreased yield, thereby affecting the stability of product quality. Second, the selective removal ability of the composite solvent system is limited for certain specific impurities (such as high molecular weight polymers, pigments, and some structural analogs with similar polarity), making it difficult to effectively improve the clarity and color of the crystals, and it is difficult to meet the purity and appearance requirements of high-end formulations. Third, the composite solvent system usually contains multiple organic solvents with similar boiling points, which easily form azeotropic mixtures, making solvent recovery difficult and energy consumption high, which not only increases production costs but also brings greater environmental pressure. Summary of the Invention

[0005] The object of the present invention is to provide a method for purifying avermectin crystals to solve the problem of poor clarity of avermectin crystals in the prior art, thereby improving the quality of avermectin crystals.

[0006] The present invention is achieved through the following technical solutions: A method for purifying avermectin crystals comprises the following steps: S1, crystallizing the avermectin extract to obtain crystals; S2, crystallizing the crystals using methanol having a pH value of 4.0-6.5 and a mass concentration of 80-95% to obtain recrystallized crystals; S3. Using methanol with a mass concentration of 80-95% to crystallize the recrystallized crystals to obtain avermectin crystals.

[0007] Furthermore, in steps S2 and S3, the amount of methanol added is 10-16 times the weight of avermectin.

[0008] Furthermore, the crystallization process of step S2 is as follows: mixing the crystals with methanol having a pH value of 4.0-6.5 and a mass concentration of 80-95%, heating to 65°C, stirring and dissolving at a speed of 40-60 r / min, then cooling to 25°C for crystallization, centrifuging, and obtaining recrystallized crystals.

[0009] Furthermore, the crystallization process of step S3 is as follows: mixing the recrystallized crystals with methanol having a mass concentration of 80-95%, heating to 65° C., stirring and dissolving at a speed of 40-60 r / min, then cooling to 25° C. for crystallization, and centrifuging to obtain avermectin crystals.

[0010] Furthermore, the crystallization process of step S1 is as follows: (1) adding anhydrous methanol with a mass of 4-5 times the weight of avermectin to the avermectin extract, heating to 65°C, stirring and dissolving at a speed of 40-60 r / min, then cooling to crystallize, and maintaining stirring for 30 minutes when the temperature is cooled to 25°C, centrifuging to obtain primary coarse crystals; (2) adding anhydrous methanol with a mass of 10-14 times the weight of avermectin to the primary coarse crystals, heating to 65°C, stirring and dissolving at a speed of 40-60 r / min, then cooling to 25°C for secondary crystallization, centrifuging to obtain secondary crystals.

[0011] Furthermore, the preparation method of the avermectin extract is as follows: (1) adding anhydrous methanol with a mass of 10-14 times the mass of avermectin to the avermectin mycelium for extraction, and combining the extracts after extraction three times; (2) evaporating the solvent from the extract at a speed of 40-60 r / min and a temperature of 60-65° C. to obtain a concentrated paste; (3) adding 70-80° C. hot water with a mass of 8-10 times the mass of avermectin to the concentrated paste for desugaring treatment, stirring and layering to obtain a concentrated paste; (4) adding anhydrous methanol with a mass of 4-5 times the mass of avermectin to the concentrated paste for dehydration under reduced pressure.

[0012] Furthermore, the avermectin mycelium is obtained by filtering the avermectin fermentation liquid through a plate and frame filter and then drying it with hot air at 110-140°C.

[0013] In his research on the application of composite solvents in the crystallization of avermectin, Zhang Hangzhou mentioned that the composite solvents of isopropyl alcohol and chloroform were used to purify the avermectin crystals. However, 1. The composite solvent is prone to residues during the crystallization and purification of avermectin; 2. The composite solvent itself will carry impurities, which may be encapsulated during crystallization; 3. The composite solvent may also form co-crystallized solvates, changing the crystal structure. CN106380500A A method for extracting avermectin B1a powder to improve its production capacity and intrinsic quality, wherein the crude product is purified by recrystallization using methanol at a mass concentration of 99%. However, 1. Since avermectin crystals and impurities may be adsorbed on each other due to charge effects, recrystallization cannot affect the charge distribution on the surface of the crystals and impurities, making it difficult to separate the impurities from the crystals, and the effect on removing such impurities is limited; 2. Methanol with a mass concentration of 99% evaporates quickly, and avermectin crystallizes quickly, which may form small, irregularly shaped or aggregated crystals. This form will increase scattering on the crystal surface and scattering on irregular internal interfaces, resulting in a decrease in clarity.

[0014] Based on this, the inventors first removed soluble impurities, solvent molecules, pigments or other tiny particles on the crystal surface through secondary crystallization to reduce interference with subsequent crystallization. Then, during the tertiary crystallization, methanol with a pH value of 4.0-6.5 and a mass concentration of 80-95% was used as the crystallization solvent. This crystallization solvent greatly increased the solubility of impurities that were insoluble in methanol and affected the clarity of the product, allowing them to dissolve in the mother liquor and separate from the avermectin crystals. Acidic conditions can change the charge distribution of avermectin crystals and impurities. Abamectin crystals and impurities may originally attract each other due to the effect of charge, and during the crystallization process, they are wrapped inside the crystals or adsorbed on the crystal surface. Under acidic conditions, their surface charge distribution changes, weakening the mutual attraction, thereby expelling impurities from the crystals. The presence of water slows down the evaporation rate of the solvent, resulting in a relatively slow crystallization process for avermectin. The slow growth rate allows molecules / ions more time to arrange themselves in an orderly manner, which is conducive to more regular crystal growth. Impurities are less likely to be trapped inside the crystals, and more of them remain in the mother liquor, thereby improving the separation of impurities from avermectin and increasing the purity of the avermectin crystals. The solubility of avermectin solution in methanol decreases as the methanol concentration decreases. As the methanol evaporates, the solubility of avermectin in the mother liquor continues to decrease, resulting in a higher avermectin yield.

[0015] During the quaternary crystallization, methanol at a concentration of 80-95% by mass is used as the crystallization solvent. The presence of water greatly increases the solubility of methanol-insoluble impurities that affect product clarity, allowing them to dissolve in the mother liquor and separate from the avermectin crystals. This releases impurities trapped within the tertiary crystals into the solution, allowing the target compound to precipitate and form crystals. During this process, more impurities remain in the mother liquor rather than being encapsulated in the newly formed crystals, thereby improving crystal clarity. Similarly, the presence of water slows solvent evaporation, slowing the crystallization of avermectin. This allows the target compound to selectively and slowly precipitate, promoting more regular crystal growth and making it less likely that impurities will be encapsulated within the crystals, leaving more of them in the mother liquor. This improves the separation of impurities from avermectin and increases the purity of the avermectin crystals. The solubility of avermectin solution in methanol decreases with decreasing methanol concentration. As methanol evaporates, the solubility of avermectin in the mother liquor continues to decline, further increasing the yield of avermectin.

[0016] The reason for changing the solvent for the third and fourth crystallizations is to selectively remove impurities such as proteins that affect clarity. The initial crystals contain a large number and variety of impurities, and the removal efficiency is low. However, after the first and second crystallizations, most of the impurities have been removed, and there is less interference.

[0017] The technical solution of the present invention has at least the following advantages and beneficial effects: By replacing the solvent for tertiary and quaternary crystallization with methanol of varying pH values, the problem of poor clarity in avermectin crystals was effectively resolved. Methanol of varying pH values increases the solubility of various proteinaceous and other substances, reducing the impurities that affect clarity during the crystallization process, significantly improving the clarity of avermectin crystals. This is of great significance for improving the quality of ivermectin, reducing insoluble matter and enhancing its appearance, therapeutic efficacy, and safety. Furthermore, this process is simple to operate and readily applicable in actual production, with promising economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the clarity of avermectin crystals obtained in Example 1 of the present invention and Comparative Example 1. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with specific embodiments, but the examples do not limit the present invention in any form. Unless otherwise stated, the raw materials and reagents used in the examples of the present invention are conventionally purchased raw materials and reagents.

[0020] Example 1 A method for purifying avermectin crystals comprises the following steps: S1. Preparation of avermectin extract: (1) After the avermectin fermentation liquid is filtered through a plate and frame, it is hot-air dried at 110°C to obtain avermectin mycelium, and anhydrous methanol with a mass of 10 times the mass of avermectin is added to the avermectin mycelium for extraction. After extraction three times, the extracts are combined; (2) The extract is evaporated at a speed of 40 r / min and a temperature of 60°C to obtain a concentrated paste; (3) 70°C hot water with a mass of 8 times the mass of avermectin is added to the concentrated paste for desugaring treatment, and after stirring and layering, a concentrated paste is obtained; (4) Anhydrous methanol with a mass of 4 times the mass of avermectin is added to the concentrated paste for dehydration by reduced pressure evaporation to obtain an avermectin extract; S2, primary crystallization: add anhydrous methanol with a mass of 4 times the weight of avermectin to the avermectin extract, heat to 65 ° C, stir and dissolve at a speed of 40 r / min, then cool and crystallize, keep stirring for 30 minutes when the temperature drops to 25 ° C, centrifuge to obtain primary coarse crystals; S3, secondary crystallization: add anhydrous methanol with a mass 10 times the weight of avermectin to the primary coarse crystals, heat to 65 ° C, stir and dissolve at a speed of 40 r / min, then cool to 25 ° C for secondary crystallization, centrifuge to obtain secondary crystals; S4, tertiary crystallization: add methanol with a mass 10 times that of avermectin, a pH value of 5.5, and a mass concentration of 80% to the secondary crystals, heat to 65°C, stir and dissolve at a speed of 40 r / min, then cool to 25°C for crystallization, centrifuge to obtain tertiary crystals; S5. Quaternary crystallization: Add methanol with a mass 10 times that of avermectin and a mass concentration of 80% to the tertiary crystals, heat to 65°C, stir and dissolve at a speed of 40 r / min, then cool to 25°C for crystallization, centrifuge to obtain avermectin crystals.

[0021] Example 2 A method for purifying avermectin crystals comprises the following steps: S1. Preparation of avermectin extract: (1) After the avermectin fermentation liquid is filtered through a plate and frame, it is hot-air dried at 140°C to obtain avermectin mycelium, and anhydrous methanol with a mass of 14 times the mass of avermectin is added to the avermectin mycelium for extraction. After extraction three times, the extracts are combined; (2) The extract is evaporated at a speed of 60 r / min and a temperature of 65°C to obtain a concentrated paste; (3) 80°C hot water with a mass of 10 times the mass of avermectin is added to the concentrated paste for desugaring treatment, and after stirring and layering, a concentrated paste is obtained; (4) Anhydrous methanol with a mass of 5 times the mass of avermectin is added to the concentrated paste for dehydration by reduced pressure evaporation to obtain an avermectin extract; S2, primary crystallization: add anhydrous methanol with a mass of 5 times the weight of avermectin to the avermectin extract, heat to 65 ° C, stir and dissolve at a speed of 60 r / min, then cool and crystallize, keep stirring for 30 minutes when the temperature drops to 25 ° C, centrifuge to obtain primary coarse crystals; S3, secondary crystallization: add anhydrous methanol with a mass 14 times the weight of avermectin to the primary coarse crystals, heat to 65 ° C, stir and dissolve at a speed of 60 r / min, then cool to 25 ° C for secondary crystallization, centrifuge to obtain secondary crystals; S4, tertiary crystallization: add methanol with a mass 16 times that of avermectin, a pH value of 6, and a mass concentration of 95% to the secondary crystals, heat to 65°C, stir and dissolve at a speed of 60 r / min, then cool to 25°C for crystallization, centrifuge to obtain tertiary crystals; S5. Quaternary crystallization: Add methanol with a mass 16 times the weight of avermectin and a mass concentration of 95% to the tertiary crystals, heat to 65°C, stir and dissolve at a speed of 60r / min, then cool to 25°C for crystallization, centrifuge to obtain avermectin crystals.

[0022] Example 3 A method for purifying avermectin crystals comprises the following steps: S1. Preparation of avermectin extract: (1) After the avermectin fermentation liquid is filtered through a plate and frame, it is hot-air dried at 130°C to obtain avermectin mycelium, and anhydrous methanol with a mass of 11 times the mass of avermectin is added to the avermectin mycelium for extraction. After extraction three times, the extracts are combined; (2) The extract is evaporated at a speed of 50 r / min and a temperature of 63°C to obtain a concentrated paste; (3) 74°C hot water with a mass of 9 times the mass of avermectin is added to the concentrated paste for desugaring treatment, and after stirring and layering, a concentrated paste is obtained; (4) Anhydrous methanol with a mass of 4.5 times the mass of avermectin is added to the concentrated paste for dehydration by reduced pressure evaporation to obtain an avermectin extract; S2, primary crystallization: add anhydrous methanol with a mass of 4.5 times the weight of avermectin to the avermectin extract, heat to 65 ° C, stir and dissolve at a speed of 50 r / min, then cool and crystallize, keep stirring for 30 minutes when the temperature drops to 25 ° C, centrifuge to obtain primary coarse crystals; S3, secondary crystallization: add anhydrous methanol with a mass 13 times the weight of avermectin to the primary coarse crystals, heat to 65 ° C, stir and dissolve at a speed of 50 r / min, then cool to 25 ° C for secondary crystallization, centrifuge to obtain secondary crystals; S4, tertiary crystallization: add methanol with a mass 12 times that of avermectin, a pH value of 6.2, and a mass concentration of 85% to the secondary crystals, heat to 65°C, stir and dissolve at a speed of 50 r / min, then cool to 25°C for crystallization, centrifuge to obtain tertiary crystals; S5. Quaternary crystallization: Add methanol with a mass of 10-16 times the weight of avermectin and a mass concentration of 90% to the tertiary crystals, heat to 65°C, stir and dissolve at a speed of 50 r / min, then cool to 25°C for crystallization, centrifuge to obtain avermectin crystals.

[0023] Example 4 In this embodiment, the crystallization solvent for the three crystallizations is methanol with a pH value of 4.0 and a mass concentration of 88%, and the rest is the same as in Example 1.

[0024] Comparative Example 1 In this comparative example, the crystallization solvent for the tertiary crystallization and the quaternary crystallization is methanol with a mass concentration of 99.97%-99.99%, and the rest is the same as in Example 1.

[0025] Comparative Example 2 In this comparative example, the crystallization solvent for the three crystallizations was methanol with a mass concentration of 80%, and the rest was the same as in Example 1.

[0026] Comparative Example 3 In this comparative example, the crystallization solvent for the four crystallizations was methanol with a pH value of 4.5 and a mass concentration of 82%, and the rest was the same as in Example 1.

[0027] The crystal content, clarity and crude protein content of the avermectin prepared in the examples and comparative examples were tested.

[0028] Abamectin content detection, refer to "GB 29695-2013 Determination of Abamectin and Ivermectin Residues in Aquatic Products - High Performance Liquid Chromatography Method", the details are as follows: 1. Reagents and solutions: Water, freshly double-distilled water; methanol, chromatographic grade; acetonitrile, chromatographic grade; avermectin standard sample, with a known avermectin B1 (B1a+B1b) mass fraction ≥95.0%.

[0029] 2. Instruments High-performance liquid chromatograph with variable-wavelength ultraviolet detector; chromatographic data processing system; chromatographic column, 250 mm × 4.6 mm (id), stainless steel column filled with C18, 5 μm filler (or chromatographic column with equivalent effect); filter, membrane pore size approximately 0.45 μm; quantitative injection tube, 5 μl; ultrasonic cleaner.

[0030] 3. HPLC operating conditions Column temperature, room temperature (temperature variation should be no more than 2°C); wavelength, 245 nm; mobile phase, methanol:acetonitrile:water = 45:40:15 (V:V:V); flow rate, 1.0 mL / min; injection volume, 5 μL; retention time, B1a approximately 19.5 min, B1b 15.2 min; 4. System adaptability (after injecting the standard solution, record the chromatographic peak response value according to the operating steps) Resolution: The resolution (R) between B1a and B1b is not less than 3.0; Column efficiency: The theoretical plate number of the chromatographic peak of component B1a is not less than 2000; Tailing factor: The tailing factor of the chromatographic peak of component B1a is not greater than 2.5; Relative deviation: The relative deviation of the B1a peak area of two consecutive injections of standard solution should not exceed 1.0%. 5. Measurement steps 5.1 Preparation of standard solution: Weigh 0.05 g (accurate to 0.0002 g) of avermectin standard sample into a 50 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well.

[0031] 5.2 Preparation of sample solution: Weigh approximately 0.05 g (accurate to 0.0002 g) of sample containing avermectin into a 50 ml volumetric flask. Dissolve with methanol, dilute to volume, and shake well. Prepare two solutions in this manner.

[0032] 5.3 Determination: Under the above operating conditions, after the instrument stabilizes, continuously inject several injections of standard solution until the relative change in the peak area of avermectin B1 (B1a+B1b) between two adjacent injections is less than 1.5%. Then, perform liquid chromatography analysis in the order of standard solution, test solution, and standard solution. If the standard solution is more than 24 hours old, re-inject the standard solution.

[0033] 6. Calculation The mass fraction X1 (%) of avermectin B1a or B1 (B1a+B1b) in the sample is calculated according to formula (1): (1) Where: X1—mass fraction of avermectin B1a or B1 (B1a+B1b) in the sample, %; A1—average peak area of avermectin B1 (B1a+B1b) in the standard solution; A2—the average value of the peak area of avermectin B1a or B1 (B1a+B1b) in the sample solution; m1—mass of standard sample, g; m2—mass of the sample, g; p—mass fraction of avermectin B1 (B1a+B1b) in the standard sample, %.

[0034] The clarity test method is as follows: Weigh 0.8g of avermectin into a 100ml beaker, add benzyl alcohol to 5.0g, heat (60°C), and stir until the avermectin is completely dissolved (approximately 10 minutes). Then, add propylene glycol to 50g and continue stirring for 5 minutes. Pour the solution into a colorless glass bottle, allow it to cool to room temperature, seal it, and store it in a -20°C refrigerator. After 24 hours, remove it from the bottle and allow it to stand at room temperature for observation.

[0035] Phenomenon observation: Observe under the light inspection table. If the solution is clear, without suspended matter, without oily matter floating on the surface of the solution, and without precipitation adhering to the bottom, it is qualified.

[0036] The crude protein detection method is: refer to "GB / T5511-2008 Cereals and Legumes Nitrogen Content Determination and Crude Protein Content Calculation Kjeldahl Method".

[0037] The test results are as follows: Table 1

[0038] According to Table 1 and Figure 1 It can be seen that the clarity of the avermectin crystals prepared in Comparative Example 1 is unqualified and the crude protein content is high. The results of Example 1 and Comparative Examples 2-3 show that by replacing the crystallization solvent in the tertiary crystallization or the quaternary crystallization with a single pH value of 4.0-6.5, 80-95% methanol or 80-95% methanol, although the clarity can be improved to a certain extent, the presence of suspended matter still cannot completely solve the clarity problem. Instead, it is necessary to use pH values of 4.0-6.5, 80-95% methanol and 80-95% methanol to replace the crystallization solvent in the tertiary crystallization or the quaternary crystallization to effectively remove protein impurities and solve the clarity problem.

[0039] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for purifying avermectin crystals, characterized in that: The following steps are involved: S1, crystallizing the avermectin extract to obtain crystals; S2, crystallizing the crystals using methanol having a pH value of 4.0-6.5 and a mass concentration of 80-95% to obtain recrystallized crystals; S3. Using methanol with a mass concentration of 80-95% to crystallize the recrystallized crystals to obtain avermectin crystals.

2. The method for purifying Avermectin crystals according to claim 1, wherein: In steps S2 and S3, the amount of methanol added is 10-16 times the weight of avermectin.

3. The method for purifying Avermectin crystals according to claim 1, wherein: The crystallization process of step S2 is as follows: mixing the crystals with methanol having a pH value of 4.0-6.5 and a mass concentration of 80-95%, heating to 65°C, stirring and dissolving at a speed of 40-60 r / min, then cooling to 25°C for crystallization, centrifuging, and obtaining recrystallized crystals.

4. The method for purifying Avermectin crystals according to claim 1, wherein: The crystallization process of step S3 is as follows: mixing the recrystallized crystals with methanol having a mass concentration of 80-95%, heating to 65°C, stirring and dissolving at a speed of 40-60 r / min, then cooling to 25°C for crystallization, and centrifuging to obtain avermectin crystals.

5. The method for purifying Avermectin crystals according to claim 1, wherein: The crystallization process of step S1 is as follows: (1) adding anhydrous methanol to the avermectin extract, heating to 65°C, stirring and dissolving at a speed of 40-60 r / min, then cooling and crystallizing, and maintaining stirring for 30 minutes when cooling to 25°C, centrifuging to obtain primary coarse crystals; (2) adding anhydrous methanol to the primary coarse crystals, heating to 65°C, stirring and dissolving at a speed of 40-60 r / min, then cooling to 25°C for secondary crystallization, centrifuging to obtain secondary crystals.

6. The method for purifying Avermectin crystals according to claim 1, wherein: The preparation method of the avermectin extract is as follows: (1) adding anhydrous methanol to the avermectin mycelium for extraction, and combining the extracts after extraction three times; (2) evaporating the solvent of the extract to obtain a concentrated paste; (3) adding 70-80°C hot water to the concentrated paste for desugaring treatment, stirring and layering to obtain a concentrated paste; (4) adding anhydrous methanol with a mass of 4-5 times the weight of avermectin to the concentrated paste and performing dehydration by reduced pressure evaporation to obtain the avermectin extract.

7. The method for purifying Avermectin crystals according to claim 6, wherein: The avermectin mycelium is obtained by filtering the avermectin fermentation liquid through a plate and frame filter and then drying it with hot air at 110-140°C.

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