A process for the preparation of a stable crystalline form of daptomycin

Abstract

The application belongs to the technical field of separation and crystallization, and relates to a preparation method of a stable crystal form of daptomycin. Daptomycin raw materials are dissolved in water and mixed to obtain a daptomycin aqueous solution, acetic acid buffer is added to the daptomycin aqueous solution and mixed to obtain a mixed solution, the mixed solution is vacuum concentrated to obtain a concentrated solution, the concentrated solution is heated and dissolved to obtain a saturated solution system, the saturated solution system is cooled, and after solid is precipitated, stirring is performed at the current crystallization temperature to crystallize; after the crystallization is completed, the system is continuously cooled to obtain a crystallization mother liquor containing daptomycin crystals; finally, the daptomycin crystals are washed at low temperature, daptomycin crystal crude products are collected by centrifugation, and the crude products are dried to obtain daptomycin crystals. The order of the daptomycin crystals prepared by the method is obviously better than that of the daptomycin crystals prepared by the crystallization method reported in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of separation and crystallization technology, and relates to a method for preparing a stable crystal form of daptomycin. Background Technology

[0002] Daptomycin (CAS: 103060-53-3, molecular formula: C) 72 H 101 N 17 O 26 (Molecular weight: 1620.67 g / mol) Daptomycin contains a cyclic decalipeptide formed by ten amino acids (3-alanine-S-lactone, 3-methyl-L-glutamyl, D-seryl, L-threonyl, L-aspartylglycyl, D-alanyl, L-aspartyl, L-ornithine, glycyl) linked together, and also contains a branched chain with a decyl group and three amino acids (L-aspartyl, L-asparagine, L-tryptophanyl), for a total of 13 amino acids.

[0003]

[0004] Daptomycin has a complex structure and is difficult to synthesize chemically. It is usually produced by fermentation of *Streptomyces roseosporus*. In 1961, Falcao and Dailia isolated and screened *Streptomyces roseosporus* from soil samples in Ararat, Turkey, to produce daptomycin through fermentation. The daptomycin molecule has a complex structure, containing both hydrophilic carboxyl and amino groups, and hydrophobic benzene rings and saturated alkane chains, giving it both hydrophilic and lipophilic properties. Daptomycin is a water-soluble antibiotic, and its aqueous solution generally has a pH of around 3.5. When the pH is maintained between 3.5 and 9.0, daptomycin can maintain a good state of incorporation. At room temperature, daptomycin aqueous solutions can remain relatively stable for more than a week. However, if the pH undergoes extreme changes (greater than 9.0 or less than 2.0), daptomycin will denature, and this denaturation is irreversible. Daptomycin has relatively poor solubility in acetone and chloroform compared to other organic solvents, and is almost insoluble in acetonitrile.

[0005] Obtaining a stable crystal form of daptomycin has significant patent value in drug development and formulation. As a glycopeptide antibiotic, the efficacy of daptomycin is affected by its crystal form stability, solubility, and bioavailability. Obtaining a stable crystal form can effectively improve its chemical stability, preventing degradation or quality loss during storage and transportation; simultaneously, it improves solubility and bioavailability, thereby enhancing the drug's therapeutic effect. Optimization of the stable crystal form also provides a foundation for dosage form innovation of daptomycin, especially in oral and injectable formulations, where crystal form control enables better dosage accuracy and sustained efficacy. Furthermore, the stable crystal form technology involved in this invention can not only be applied to daptomycin but also provides valuable reference for the crystal form improvement of other glycopeptide antibiotics, showing broad application prospects. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to address the problems of poor orderliness, poor stability, and easy moisture absorption of daptomycin crystals when placed at room temperature in the prior art. The present invention provides a method for preparing a stable crystal form of daptomycin.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] This invention discloses a method for preparing a stable crystal form of daptomycin, comprising the following steps:

[0009] (1) Dissolve daptomycin raw material in water and mix well to obtain daptomycin aqueous solution;

[0010] (2) Add acetate buffer to the daptomycin aqueous solution obtained in step (1), mix well, and obtain a mixed solution;

[0011] (3) Vacuum concentrate the mixed solution obtained in step (2) until solid precipitates out, then stop vacuum concentration to obtain concentrated liquid.

[0012] (4) The concentrated liquid obtained in step (3) is heated and dissolved, and the solid part in the concentrated liquid is completely dissolved to obtain a saturated solution system.

[0013] (5) Cool down the temperature of the saturated solution system obtained in step (4) while stirring. After cooling down to the point where solid precipitates, stir at the current crystallization temperature to crystallize.

[0014] (6) After the crystallization in step (5) is completed, the system temperature is further cooled to obtain a crystallization mother liquor containing daptomycin crystals;

[0015] (7) Wash the mother liquor containing daptomycin crystals obtained in step (6) at low temperature, centrifuge and dry to obtain daptomycin crystals.

[0016] In some embodiments, the daptomycin raw material is amorphous daptomycin.

[0017] In some embodiments, in step (1), the mass-to-volume ratio of the daptomycin raw material to water is 1g:10mL to 3g:10mL.

[0018] The concentration of daptomycin in the daptomycin aqueous solution is not less than 100 g / L, in order to reduce the evaporation volume of the solvent.

[0019] In some embodiments, preferably, in step (1), the mass-to-volume ratio of the daptomycin raw material to water is 1 g: 10 mL.

[0020] In step (1), during the process of dissolving daptomycin raw material in water and mixing, in order to accelerate the dissolution process of daptomycin raw material, it can be continuously stirred, and heated at 40℃~60℃ to assist in the dissolution of daptomycin raw material until the solution is clear and free of solids.

[0021] In some embodiments, the acetate buffer pair is a mixture of sodium acetate and acetic acid; in the acetate buffer pair, the mass ratio of sodium acetate to acetic acid is 1.0:(1.0-1.2); the mass of the acetate buffer pair accounts for 1% to 10% of the mass of the daptomycin raw material, preferably 1% to 8%, more preferably 4% to 8%.

[0022] In some embodiments, preferably, the mass ratio of sodium acetate to acetic acid in the acetate buffer pair is 1.0:1.0.

[0023] In some embodiments, in step (3), the vacuum concentration is carried out at 40°C to 70°C, preferably at 40°C to 60°C; the vacuum concentration is carried out until solid precipitates out, and the mass of the precipitated daptomycin accounts for 0.2% to 1.0% of the mass of the daptomycin raw material, preferably 0.2% to 0.8%, then the vacuum concentration is stopped and a concentrated liquid is obtained.

[0024] In some embodiments, more preferably, in step (3), the vacuum concentration is carried out at 50°C to 60°C.

[0025] In step (3), the vacuum degree of the vacuum concentration is -0.099 to -0.1 MPa.

[0026] In some embodiments, in step (3), more preferably, the vacuum concentration is stopped when solid precipitates out, and the mass of the precipitated daptomycin accounts for 0.2% to 0.5% of the mass of the daptomycin raw material, and the concentrated liquid is obtained.

[0027] In some embodiments, in step (4), the concentrated liquid is heated to dissolve, and the heating temperature is 80℃~90℃, preferably 85℃~90℃.

[0028] In step (4), the concentrated liquid is heated to dissolve it, and the heating time is sufficient to completely dissolve the solid portion of daptomycin in the liquid.

[0029] In some embodiments, in step (5), the cooling process involves first cooling the saturated solution system to 50°C–58°C, and then gradually cooling it down to 35°C–50°C, preferably 35°C–46°C; the stirring process involves a stirring rate of 100 rpm–800 rpm, preferably 200 rpm–600 rpm; the crystallization temperature is 35°C–50°C, preferably 35°C–46°C; and the crystallization process involves stirring for 1–4 hours, preferably 1–3 hours.

[0030] In step (5), the temperature of the saturated solution system is first cooled to 50℃~58℃. During this cooling process, there is no need to perform gradient cooling. The circulating water bath can be set to the required temperature directly, and crystals will not precipitate within the temperature range of 50℃~58℃.

[0031] In some embodiments, in step (6), the cooling process involves gradually reducing the system temperature to 1°C to 10°C.

[0032] In some embodiments, in step (5) or step (6), the gradient cooling rate is 0.5℃ / h to 5℃ / h, preferably 2℃ / h to 5℃ / h.

[0033] In some embodiments, in step (7), the low temperature is 0°C to 10°C; the solvent used in washing the daptomycin crystals is any one or a combination of acetonitrile, acetone and chloroform, preferably acetonitrile.

[0034] In some embodiments, in step (7), the centrifugation is carried out at a centrifugation speed of 3000 rpm to 10000 rpm for 10 min to 30 min; the drying is carried out by forced air drying or vacuum drying at 40℃ to 80℃ for 2 h to 8 h.

[0035] In some embodiments, preferably, in step (7), the centrifugation is performed at a centrifugation speed of 3000 rpm to 7000 rpm for 10 min to 30 min; the drying is performed by forced air drying or vacuum drying at 40℃ to 65℃ for 2 h to 8 h.

[0036] The daptomycin crystals prepared by the above method have a purity ≥99.0% and a yield ≥60.0%.

[0037] Beneficial effects:

[0038] Compared with the prior art, the present invention has the following advantages:

[0039] (1) The daptomycin of the present invention is obtained by using an aqueous system to obtain crystals, thus avoiding the use of a large amount of organic solvents.

[0040] (2) The daptomycin crystals prepared by this invention have higher storage stability, are less prone to moisture absorption, and have higher stability.

[0041] (3) The daptomycin crystals prepared by the method of the present invention have significantly better order than those prepared by crystallization methods reported in the prior art.

[0042] (4) The process for preparing stable daptomycin crystal form of the present invention is simple, the separation process is simple to operate, the purity of the crystal product is not less than 99.0%, and the yield is greater than 60.0%. The crystal obtained by the present invention is more stable than the crystal prepared by the prior art. The method for preparing stable daptomycin crystal form of the present invention has important technological innovation and market application potential. Attached Figure Description

[0043] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0044] Figure 1 The XRD pattern of daptomycin crystals prepared in an embodiment of the present invention.

[0045] Figure 2 The XRD pattern of daptomycin crystals prepared by Comparative Example 1 using ethanol as the antisolvent is shown.

[0046] Figure 3 The XRD pattern of daptomycin crystals prepared using isopropanol as the antisolvent is shown in Comparative Example 2. Detailed Implementation

[0047] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0048] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0049] The daptomycin raw material used in the embodiments of this invention is an amorphous powder from Hubei Huizepu Pharmaceutical Technology Co., Ltd., batch number HZP202209, with a purity of over 98%.

[0050] In this powder example, daptomycin with a purity of over 99% was used as a standard. A standard curve of daptomycin concentration and absorbance with absorbance between 0.2 and 0.8 at 268 nm was plotted and used to calibrate the purity of daptomycin.

[0051] Example 1:

[0052] (1) Dissolve 10g of amorphous daptomycin raw material in 100mL of aqueous solution and stir continuously. To accelerate the dissolution process, heat to 40℃ until the solution is clear and free of solids to obtain daptomycin aqueous solution.

[0053] (2) Add 0.1 g of acetate buffer to the daptomycin aqueous solution obtained in step (1) to completely dissolve sodium acetate in the daptomycin aqueous solution to obtain a mixed solution; wherein, the acetate buffer is a mixture of acetic acid and sodium acetate, and the mass ratio of acetic acid to sodium acetate in the mixture is 1:1.

[0054] (3) The mixed solution obtained in step (2) is vacuum concentrated. The vacuum degree is set to -0.095Mpa to -0.1Mpa, and the vacuum evaporation temperature is set to 40℃. Vacuum concentration is stopped when solid daptomycin is precipitated in the concentrated solution and the mass of the precipitated daptomycin accounts for about 0.2% of the initial amount of daptomycin.

[0055] (4) The concentrated liquid obtained in step (3) is heated at 80°C for 0.5 hours to completely dissolve the solid portion of daptomycin in the liquid and obtain a saturated solution system. Then the temperature of the system is rapidly reduced to 55°C and then reduced to 35°C at a rate of 5°C / h. During the cooling process, the system is stirred at a stirring rate of 200 rpm to 600 rpm until the solid precipitates. After the solid precipitates, the system is stirred and maintained at the current crystallization temperature of 35°C for 1 hour to crystallize. After the crystallization is completed, the system temperature is reduced to 1°C at a gradient of 5°C / h to obtain a crystallization mother liquor containing daptomycin crystals.

[0056] (5) In order to improve the purity of daptomycin crystals and reduce the influence of impurities in the crystallization mother liquor obtained in step (4) on the crystals, the daptomycin crystals were washed with pure acetonitrile at a low temperature of 0°C, centrifuged at 3000 rpm for 10 min, and the solid was collected.

[0057] (6) The solid obtained in step (5) is dried by blowing air at a temperature of 40°C for 8 hours to obtain daptomycin crystals with a purity of 99.5% and a yield of 62%.

[0058] The XRD pattern of the daptomycin crystals prepared in this invention is as follows: Figure 1 As shown.

[0059] Example 2:

[0060] (1) Dissolve 10g of amorphous daptomycin raw material in 100mL of aqueous solution and stir continuously. To accelerate the dissolution process, heat to 60℃ and continue until the solution is clear and free of solids to obtain daptomycin aqueous solution.

[0061] (2) Add 1.0 g of acetate buffer to the daptomycin aqueous solution obtained in step (1) to completely dissolve sodium acetate in the daptomycin aqueous solution to obtain a mixed solution; wherein, the acetate buffer is a mixture of acetic acid and sodium acetate, and the mass ratio of acetic acid to sodium acetate in the mixture is 1:1.

[0062] (3) The mixed solution obtained in step (2) is vacuum concentrated. The vacuum degree is set to -0.099Mpa to -0.1Mpa, and the vacuum evaporation temperature is set to 70℃. Vacuum concentration is stopped when solid daptomycin is precipitated in the concentrated liquid and the mass of the precipitated daptomycin accounts for about 1% of the initial amount of daptomycin.

[0063] (4) The concentrated solution obtained in step (3) is heated at 90°C for 0.5 hours to completely dissolve the solid portion of daptomycin in the solution and obtain a saturated solution system. Then the temperature of the system is rapidly reduced to 55°C and then reduced to 50°C at a rate of 0.5°C / h. During the cooling process, the system is stirred at a stirring rate of 200 rpm to 600 rpm until the solid precipitates. After the solid precipitates, the system is stirred and maintained at the current crystallization temperature of 50°C for 4 hours to crystallize. After the crystallization is completed, the system temperature is reduced to 10°C at a gradient of 0.5°C / h to obtain a crystallization mother liquor containing daptomycin crystals.

[0064] (5) In order to improve the purity of daptomycin crystals and reduce the influence of impurities in the mother liquor obtained in step (4) on the crystals, the daptomycin crystals were washed with acetone at a low temperature of 8°C and centrifuged at 10,000 rpm for 30 min to collect the solid.

[0065] (6) The solid obtained in step (5) is vacuum dried at a temperature of 80°C for 2 hours to obtain daptomycin crystals with a purity of 99.0% and a yield of 70.2%.

[0066] The XRD pattern of the daptomycin crystals prepared in this invention is the same as that of... Figure 1 .

[0067] Example 3:

[0068] (1) Dissolve 10g of amorphous daptomycin raw material in 100mL of aqueous solution, stirring constantly. To accelerate the dissolution process, heat to 50℃ and continue until the solution is clear and free of solids to obtain daptomycin aqueous solution.

[0069] (2) Add 0.5g of acetate buffer to the daptomycin aqueous solution obtained in step (1) to completely dissolve sodium acetate in the daptomycin aqueous solution to obtain a mixed solution; wherein, the acetate buffer is a mixture of acetic acid and sodium acetate, and the mass ratio of acetic acid to sodium acetate in the mixture is 1:1.

[0070] (3) The mixed solution obtained in step (2) is concentrated under vacuum. The vacuum degree is set to -0.098Mpa to -0.1Mpa, and the vacuum evaporation temperature is set to 50℃. Vacuum concentration is stopped when solid daptomycin is precipitated in the concentrated liquid and the mass of the precipitated daptomycin accounts for about 0.5% of the initial amount of daptomycin.

[0071] (4) The concentrated liquid obtained in step (3) is heated at 90°C for 0.5 hours to completely dissolve the solid portion of daptomycin in the liquid and obtain a saturated solution system. Then the temperature of the system is rapidly reduced to 55°C and then reduced to 50°C at a rate of 2°C / h. During the cooling process, the system is stirred at a stirring rate of 200 rpm to 600 rpm until the solid precipitates. After the solid precipitates, the system is stirred and maintained at the current crystallization temperature of 50°C for 3 hours to crystallize. After the crystallization is completed, the system temperature is reduced to 4°C at a gradient of 2°C / h to obtain a crystallization mother liquor containing daptomycin crystals.

[0072] (5) In order to improve the purity of daptomycin crystals and reduce the influence of impurities in the mother liquor obtained in step (4) on the crystals, the daptomycin crystals were washed with pure chloroform at a low temperature of 4°C, centrifuged at 5000 rpm for 20 min, and the solids were collected.

[0073] (6) The solid obtained in step (5) is vacuum dried at a temperature of 60°C for 4 hours to obtain daptomycin crystals with a purity of 99.1% and a yield of 67%.

[0074] The XRD pattern of the daptomycin crystals prepared in this invention is the same as that of... Figure 1 .

[0075] Example 4:

[0076] (1) Dissolve 10g of amorphous daptomycin raw material in 100mL of aqueous solution, stirring constantly. To accelerate the dissolution process, heat to 50℃ and continue until the solution is clear and free of solids to obtain daptomycin aqueous solution.

[0077] (2) Add 0.8 g of acetate buffer to the daptomycin aqueous solution obtained in step (1) to completely dissolve sodium acetate in the daptomycin aqueous solution to obtain a mixed solution; wherein, the acetate buffer is a mixture of acetic acid and sodium acetate, and the mass ratio of acetic acid to sodium acetate in the mixture is 1:1.

[0078] (3) The mixed solution obtained in step (2) is vacuum concentrated. The vacuum degree is set to -0.097Mpa to -0.1Mpa, and the vacuum evaporation temperature is set to 60℃. Vacuum concentration is stopped when solid daptomycin is precipitated in the concentrated liquid and the mass of the precipitated daptomycin accounts for about 0.3% of the initial amount of daptomycin.

[0079] (4) The concentrated solution obtained in step (3) is heated at 85°C for 0.5 hours to completely dissolve the solid portion of daptomycin in the solution and obtain a saturated solution system. Then the temperature of the system is rapidly reduced to 55°C and then reduced to 45°C at a rate of 3°C / h. During the cooling process, the system is stirred at a stirring rate of 200 rpm to 600 rpm until the solid precipitates. After the solid precipitates, the system is stirred and maintained at the current crystallization temperature of 45°C for 2 hours to crystallize. After the crystallization is completed, the system temperature is reduced to 2°C at a gradient of 3°C / h to obtain a crystallization mother liquor containing daptomycin crystals.

[0080] (5) In order to improve the purity of daptomycin crystals and reduce the influence of impurities in the mother liquor obtained in step (4) on the crystals, the daptomycin crystals were washed with pure acetonitrile at a low temperature of 2°C, centrifuged at 6000 rpm for 15 min, and the solid was collected.

[0081] (6) The solid obtained in step (5) is vacuum dried at a temperature of 65°C for 3 hours to obtain daptomycin crystals with a purity of 99.2% and a yield of 64.3%.

[0082] The XRD pattern of the daptomycin crystals prepared in this invention is the same as that of... Figure 1 .

[0083] Example 5:

[0084] (1) Dissolve 10g of amorphous daptomycin raw material in 100mL of aqueous solution, stirring constantly. To accelerate the dissolution process, heat to 55℃ and continue until the solution is clear and free of solids to obtain daptomycin aqueous solution.

[0085] (2) Add 0.4 g of acetate buffer to the daptomycin aqueous solution obtained in step (1) to completely dissolve sodium acetate in the daptomycin aqueous solution to obtain a mixed solution; wherein, the acetate buffer is a mixture of acetic acid and sodium acetate, and the mass ratio of acetic acid to sodium acetate in the mixture is 1:1.

[0086] (3) The mixed solution obtained in step (2) is vacuum concentrated. The vacuum degree is set to -0.098Mpa to -0.1Mpa, and the vacuum evaporation temperature is set to 65℃. Vacuum concentration is stopped when solid daptomycin is precipitated in the concentrated liquid and the mass of the precipitated daptomycin accounts for about 0.7% of the initial amount of daptomycin.

[0087] (4) The concentrated solution obtained in step (3) is heated at 87°C for 0.5 hours to completely dissolve the solid portion of daptomycin in the solution and obtain a saturated solution system. Then the temperature of the system is rapidly reduced to 55°C and then reduced to 46°C at a rate of 4°C / h. During the cooling process, the system is stirred at a stirring rate of 200 rpm to 600 rpm until the solid precipitates. After the solid precipitates, the system is stirred and maintained at the current crystallization temperature of 46°C for 2 hours to crystallize. After the crystallization is completed, the system temperature is reduced to 7°C at a gradient of 4°C / h to obtain a crystallization mother liquor containing daptomycin crystals.

[0088] (5) In order to improve the purity of daptomycin crystals and reduce the influence of impurities in the mother liquor obtained in step (4) on the crystals, the daptomycin crystals were washed with chloroform at a low temperature of 7°C, centrifuged at 8000 rpm for 18 min, and the solid was collected.

[0089] (6) The solid obtained in step (5) is vacuum dried at a temperature of 50°C for 6 hours to obtain daptomycin crystals with a purity of 99.1% and a yield of 63.6%.

[0090] The XRD pattern of the daptomycin crystals prepared in this invention is the same as that of... Figure 1 .

[0091] Comparative Example 1:

[0092] (1) Dissolve 10g of amorphous daptomycin raw material in 100mL of aqueous solution and stir continuously. To accelerate the dissolution process, heat to 40℃ until the solution is clear and free of solids to obtain daptomycin aqueous solution.

[0093] (2) Following the experimental methods of steps (5), (6), (7), and (8) in Example 1 of Patent CN 110117310 A, nanofiltration concentration, crystallization (using 95% industrial ethanol as the antisolvent), vacuum filtration, and freeze-drying were performed sequentially to prepare daptomycin crystals with a purity of 99.4%. The XRD pattern of the crystals is shown in [reference needed]. Figure 2 .

[0094] Comparative Example 2:

[0095] (1) Dissolve 10g of amorphous daptomycin raw material in 100mL of aqueous solution and stir continuously. To accelerate the dissolution process, heat to 40℃ until the solution is clear and free of solids to obtain daptomycin aqueous solution.

[0096] (2) Following the experimental methods in steps g and h of Example 1 of patent CN 103224547 A, the product was concentrated and purified by crystallization (using isopropanol as the antisolvent) to prepare daptomycin crystals with a purity of 99.6%. The XRD pattern of the crystals is shown in [Figure number missing]. Figure 3 .

[0097] Example 6:

[0098] (1) Orderliness of crystal structure

[0099] from Figure 1 It can be seen that the daptomycin crystals obtained using the crystallization process of this invention have richer characteristic peaks; daptomycin crystals prepared by repeating different antisolvent crystallization processes of other existing technologies (prepared in Comparative Examples 1 and 2, respectively) show that... Figures 2-3 As can be seen, the XRD powder spectrum shows only one bulge and no characteristic peaks, indicating that the crystal has poor order and is closer to an amorphous powder. In contrast, the XRD spectrum of the daptomycin crystal obtained in this invention has obvious characteristic peaks at more 2-theta angles, indicating that the daptomycin crystal structure prepared in this invention has higher order.

[0100] (2) Hygroscopicity of crystals

[0101] The stability of daptomycin crystals prepared in Example 1, Comparative Example 1, and Comparative Example 2 at room temperature was compared. Approximately 1.5 g of daptomycin sample was added to a petri dish dried to constant weight and placed in a sealed desiccator with relative humidity of RH 43% (K2CO3 aqueous solution), RH 67% (CuCl2 aqueous solution), and RH 98% (saturated K2SO4) at room temperature for 72 h. The weight changes during the process were recorded, and the results are shown in Table 1.

[0102] The experimental results are shown in Table 1:

[0103] Crystals not prepared under this patent (Comparative Example 1) showed a 3.2% weight gain at 43% RH, exhibiting hygroscopicity but with limited hygroscopicity; an 8.4% weight gain at 76% RH, also exhibiting hygroscopicity; and a 25.2% weight gain at 98% RH, exhibiting strong hygroscopicity. Crystals not prepared under this patent (Comparative Example 2) showed a 4.2% weight gain at 43% RH, exhibiting hygroscopicity but with limited hygroscopicity; an 9.7% weight gain at 76% RH, also exhibiting hygroscopicity; and a 26.3% weight gain at 98% RH, exhibiting strong hygroscopicity. The crystals prepared under this invention (Example 1) showed a 0.1% weight gain at 43% RH, a 0.95% weight gain at 76% RH, and a 4.9% weight gain at 98% RH, exhibiting hygroscopicity but with limited hygroscopicity.

[0104] All the above data were obtained by adding approximately 1.5 g of daptomycin sample to a culture dish that had been dried to constant weight, placing it in a high-temperature oven at 50°C for 12 hours, recording the weight every hour, and observing the weight change throughout the experiment. The final results are shown in Table 1.

[0105] Table 1. Water absorption of different crystals under different water-containing environments.

[0106]

[0107]

[0108] (3) High temperature stability of crystal

[0109] The purity of daptomycin crystals prepared in Examples 1, 1, and 2 was determined by spectrophotometry after being placed in desiccants at 90°C and 43% humidity for 6 h, 12 h, 24 h, and 48 h. The results are shown in Table 2. The results indicate that the stability of daptomycin crystals obtained in Example 1 is significantly better than that of crystals in Comparative Examples 1 and 2.

[0110] Table 2. High-temperature stability of daptomycin crystals obtained by different methods

[0111]

[0112] This invention provides a method for preparing a stable crystal form of daptomycin. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing a stable crystal form of daptomycin, characterized in that, Includes the following steps: (1) Dissolve daptomycin raw material in water and mix well to obtain daptomycin aqueous solution; (2) Add acetate buffer to the daptomycin aqueous solution obtained in step (1), mix well, and obtain a mixed solution; (3) Vacuum concentrate the mixed solution obtained in step (2) until solid precipitates out, then stop vacuum concentration to obtain concentrated liquid. (4) The concentrated liquid obtained in step (3) is heated and dissolved, and the solid part in the concentrated liquid is completely dissolved to obtain a saturated solution system; (5) Cool down the temperature of the saturated solution system obtained in step (4) while stirring. After cooling down to the point where solid precipitates, stir at the current crystallization temperature to crystallize. (6) After crystallization in step (5) is completed, the system temperature is further cooled to obtain a mother liquor containing daptomycin crystals; (7) Wash the mother liquor containing daptomycin crystals obtained in step (6) at low temperature, centrifuge and dry to obtain daptomycin crystals; The acetate buffer is a mixture of sodium acetate and acetic acid; in the acetate buffer, the mass ratio of sodium acetate to acetic acid is 1.0:(1.0~1.2). In step (5), the cooling process involves first cooling the saturated solution system to 50℃~58℃, and then gradually cooling it down to 35℃~50℃; the crystallization temperature is 35℃~50℃; the stirring process is carried out for crystallization, and the stirring is maintained for 1~4 hours. In step (6), the cooling process involves gradually reducing the system temperature to 1°C to 10°C. The gradient cooling has a cooling rate of 0.5℃ / h to 5℃ / h.

2. The preparation method according to claim 1, characterized in that, The daptomycin raw material is amorphous daptomycin.

3. The preparation method according to claim 1, characterized in that, In step (1), the mass-to-volume ratio of the daptomycin raw material to water is 1 g: 10 mL to 3 g: 10 mL.

4. The preparation method according to claim 1, characterized in that, The mass of the acetate buffer pair is 1% to 10% of the mass of the daptomycin raw material.

5. The preparation method according to claim 1, characterized in that, The mass of the acetate buffer pair is 1% to 8% of the mass of the daptomycin raw material.

6. The preparation method according to claim 1, characterized in that, The mass of the acetate buffer pair is 4% to 8% of the mass of the daptomycin raw material.

7. The preparation method according to claim 1, characterized in that, In step (3), the vacuum concentration is carried out at 40℃~70℃; the vacuum concentration is carried out until solid precipitates out, and the mass of the precipitated daptomycin accounts for 0.2%~1.0% of the mass of the daptomycin raw material, then the vacuum concentration is stopped and a concentrated liquid is obtained.

8. The preparation method according to claim 1, characterized in that, In step (3), the vacuum concentration is carried out at 40℃~60℃.

9. The preparation method according to claim 1, characterized in that, In step (3), the vacuum concentration is carried out until solid precipitates out. The mass of the precipitated daptomycin accounts for 0.2% to 0.8% of the mass of the daptomycin raw material. Then, the vacuum concentration is stopped to obtain a concentrated liquid.

10. The preparation method according to claim 1, characterized in that, In step (4), the concentrated liquid is heated and dissolved at a temperature of 80°C to 90°C.

11. The preparation method according to claim 1, characterized in that, In step (4), the concentrated liquid is heated to dissolve at a temperature of 85°C to 90°C.

12. The preparation method according to claim 1, characterized in that, In step (5), the cooling process involves first cooling the saturated solution system to 50℃~58℃, and then gradually cooling it down to 35℃~46℃; the stirring process involves a stirring rate of 100 rpm~800 rpm; the crystallization temperature is 35℃~46℃; and the crystallization process involves stirring for 1~3 hours.

13. The preparation method according to claim 1, characterized in that, In step (5), the stirring speed is 200 rpm to 600 rpm.

14. The preparation method according to claim 1, characterized in that, The gradient cooling has a cooling rate of 2℃ / h to 5℃ / h.

15. The preparation method according to claim 1, characterized in that, In step (7), the low temperature is 0℃~10℃; the solvent used in washing the daptomycin crystals is any one or a combination of several of acetonitrile, acetone and chloroform.

16. The preparation method according to claim 1, characterized in that, In step (7), the washing process involves using acetonitrile as the solvent for washing the daptomycin crystals.

17. The preparation method according to claim 1, characterized in that, In step (7), the centrifugation is carried out at a centrifugation speed of 3000 rpm to 10000 rpm for 10 min to 30 min; the drying is carried out by forced air drying or vacuum drying at 40℃ to 80℃ for 2 h to 8 h.

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