Preparation method for stable crystal form of daptomycin

Abstract

A preparation method for a stable crystal form of daptomycin, comprising the following steps: (1) dissolving a daptomycin raw material in water and mixing evenly to obtain a daptomycin aqueous solutio

Description

TECHNICAL FIELD The present invention belongs to the technical field of fractional crystallization, and relates to a preparation method for a stable crystal form of daptomycin. BACKGROUND Daptomycin (English name Daptomycin, CAS: 103060-53-3, molecular formula: C72H101N17O26, molecular weight: 1620.67 g / mol) contains a cyclodecapeptide formed by linking ten amino acids (3-alanine-S-lactone, 3-methyl-L-glutamyl, D-seryl, L-threonyl, L-aspartylglycyl, D-alanyl, L-aspartyl, L-omithyl and glycyl), and further contains a branched chain formed by linking decanoyl and three amino acids (L-aspartyl, L-asparaginyl and L-tryptophyl), thereby containing a total of 13 amino acids. Daptomycin The daptomycin has a complex structure, is difficult to synthesize chemically, and is usually formed by fermentation of S.roseosporus. In 1961, Falcao and Dailia produced the daptomycin by fermentation of S. roseosporus isolated and screened from Ararat mountain soil samples in Turkey, wherein the daptomycin has a complex molecular structure, and contained not only hydrophilic groups such as carboxyl and amino, but also hydrophobic groups such as a benzene ring and a saturated alkane chain, so that the daptomycin has both hydrophilic and lipophilic properties. Daptomycin is a water-soluble antibiotic, and a pH value ofits aqueous solution is generally about 3.5; and when the pH value is maintained at 3.5-9.0, the daptomycin may be fused in a good state. At room temperature, the daptomycin aqueous solution can remain relatively stable for more than one week. However, if the pH value is changed extremely (the pH value is greater than 11.0 or less than 2.0), the daptomycin may be denatured, and such denaturation is irreversible. Compared with other organic solvents, the daptomycin has relatively poor solubility in acetone and chloroform, and is almost insoluble in acetonitrile. The acquisition of a stable crystal form of the daptomycin has a remarkable patent value in the field of research and development of drugs and the field of preparations. An efficacy of the daptomycin serving as a glycopeptide antibiotic is affected by the stability, solubility and bioavailability of the solid form. By acquiring the stable solid form, the chemical stability can be effectively improved, which avoids degradation or a quality loss during storage and transportation; and meanwhile, the solubility and bioavailability are improved, thereby improving a therapeutic effect of the drug. The optimization of the stable solid form further provides a foundation for dosage form innovation of the daptomycin, especially in an oral preparation and an injection, and better dosage accuracy and duration of action can be achieved through crystal form control. In addition, the stable solid form technology involved in the present invention may not only be applied to the daptomycin, but also provide a valuable reference for crystal form improvement of other glycopeptide antibiotics, thereby having broad application prospects. However, daptomycin presents a significant crystallization challenge, persistently forming amorphous solids under conventional conditions. This difficulty stems from its intricate molecular architecture, which features substantial conformational flexibility due to numerous rotatable bonds, and a highly pH-dependent ionization profile. Specifically, for effective crystallization, daptomycin needs to be in its neutral form. However, as illustrated in Fig. 2, even at the optimal pH of approximately 3.5 where the proportion of the neutral species is maximized, it accounts for no more than 40% of the total population. This results in a low effective concentration of the neutral species available for crystallization. The inherent conformational diversity, coupled with the limited availability of the neutral species, significantly impedes the formation of a well-ordered crystal lattice. This explains why, to date, no well-diffracting single crystals of daptomycin have been reported. SUMMARY The technical problem to be solved by the present invention is that, aiming at the problems such as poor orderliness, poor stability and easy hygroscopicity at room temperature of daptomycin powder in the prior art, the present invention provides a preparation method for a stable crystal form of daptomycin. In order to solve the above technical problem, the following technical solutions are used in the present invention. The present invention discloses a preparation method for a stable crystal form of daptomycin, which comprises the following steps: (1) dissolving a daptomycin raw material in water and mixing evenly to obtain a daptomycin aqueous solution; (2) adding an acetate buffer pair into the daptomycin aqueous solution obtained in the step (1) and mixing evenly to obtain a mixed solution; (3) carrying out vacuum concentration on the mixed solution obtained in the step (2) until a solid is precipitated, and then stopping the vacuum concentration to obtain a concentrated feed solution; (4) heating and dissolving the concentrated feed solution obtained in the step (3), and completely dissolving the solid in the concentrated feed solution to obtain a saturated solution system; (5) cooling the saturated solution system obtained in the step (4), stirring during cooling, and after a solid is precipitated during cooling, stirring for crystallization at a current crystallization temperature; (6) after ending the crystallization in the step (5), continuously cooling the system to obtain a daptomycin crystal-containing mother liquor of crystallization; and (7) washing the daptomycin crystal-containing mother liquor of crystallization obtained in the step (6) at a low temperature, and centrifuging and drying to obtain the daptomycin crystal. In some embodiments, the daptomycin raw material is amorphous daptomycin. In some embodiments, in the step (1), a mass-volume ratio of the daptomycin raw material to the water is 1 g: 10 mL-3 g: 10 mL. A concentration of the daptomycin in the daptomycin aqueous solution is not less than 100 g / L, so as to reduce an evaporation volume of the solvent. In some embodiments, preferably, in the step (1), the mass-volume ratio of the daptomycin raw material to the water is 1 g: 10 mL. In the step (1), in the process of dissolving the daptomycin raw material in the water and mixing evenly, in order to speed up the dissolution process of the daptomycin raw material, continuous stirring can be performed, and meanwhile, the dissolution of the daptomycin raw material is assisted by heating at 40°C~60°C until the solution is clear and solid-free. In some embodiments, the acetate buffer pair is a mixture of sodium acetate and acetic acid; in the acetate buffer pair, a mass ratio of the sodium acetate to the acetic acid is 1.0: (1.0-1.2); and amass of the acetate buffer pair accounts for l%-10%, preferably l%-8%, and further preferably 4%~8%, of a mass of the daptomycin raw material. In some embodiments, preferably, in the acetate buffer pair, the mass ratio of the sodium acetate to the acetic acid is 1.0: 1.0. In some embodiments, in the step (3), the vacuum concentration is carried out at 40°C-70°C, preferably 40°C-60°C; and the vacuum concentration is carried out until the solid is precipitated, a mass of precipitated daptomycin accounts for 0.2%-1.0%, preferably 0.2%-0.8%, of a mass of the daptomycin raw material, and then the vacuum concentration is stopped to obtain the concentrated feed solution. In some embodiments, further preferably, in the step (3), the vacuum concentration is carried out at 50°C-60°C. In the step (3), a vacuum degree for the vacuum concentration is -0.099 to -0.1 MPa. In some embodiments, in the step (3), further preferably, the vacuum concentration is carried out until the solid is precipitated, the mass of the precipitated daptomycin accounts for 0.2%-0.5% of the mass of the daptomycin raw material, and then the vacuum concentration is stopped to obtain the concentrated feed solution. In some embodiments, in the step (4), the concentrated feed solution is heated and dissolved at a heating temperature of 80°C-90°C, preferably 85°C-90°C. In the step (4), the concentrated feed solution is heated and dissolved, and the heating is continued for the period required to completely dissolve the solid daptomycin in the feed solution. In some embodiments, in the step (5), the cooling refers to cooling the saturated solution system to 50°C-58°C first, and then cooling the saturated solution system to 35°C-50°C via gradient cooling, preferably 35°C-46°C; the stirring is carried out at a stirring speed 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 stirring for crystallization refers to lasting the stirring for 1-4 hours, preferably 1-3 hours, for crystallization. In the step (5), the saturated solution system is cooled to 50°C-58°C first, gradient cooling is not needed in the cooling process, a circulating water bath is directly set at a required temperature, and there is no crystallization at the temperature of 50°C-58°C. In some embodiments, in the step (6), the cooling refers to cooling the system to l°C-10°C via gradient cooling. In some embodiments, in the step (5) or the step (6), the gradient cooling is carried out at a cooling rate of 0.5°C / h-5°C / h, preferably 2°C / h-5°C / h. In some embodiments, in the step (7), the low temperature refers to a temperature of 0°C-10°C; and as for the washing, a solvent used in washing the daptomycin crystal is any one or a combination of several of acetonitrile, acetone, and chloroform, preferably the acetonitrile. In some embodiments, in the step (7), the centrifuging refers to centrifuging at a centrifugation speed of 3000 rpm-10000 rpm for 10 minutes~30 minutes; and the drying refers to forced-air drying or vacuum drying, and the drying is carried out at 40°C-80°C for 2 hours-8 hours. In some embodiments, preferably, in the step (7), the centrifuging refers to centrifuging at a centrifugation speed of 3000 rpm-7000 rpm for 10 minutes-30 minutes; and the drying refers to forced-air drying or vacuum drying, and the drying is carried out at 40°C-65°C for 2 hours-8 hours. The daptomycin crystal prepared by the above method has a purity of not less than 99.0% and a yield of not less than 60.0%. Beneficial effects: Compared with the prior art, the present invention has the following advantages. (1) The daptomycin of the present invention adopts a crystal obtained by a water system, thereby avoiding the use of a large amount of organic solvent. (2) The daptomycin crystal prepared by the present invention has higher storage stability, less hygroscopicity and higher stability. (3) An orderliness of the daptomycin crystal prepared by the method of the present invention is obviously better than that of the daptomycin crystal prepared by the crystallization method reported in the prior art. (4) In the present invention, the technology for preparing the stable crystal form of the daptomycin is simple, the separation process is simple to operate, the crystal product has the purity not less than 99.0% and the yield more than 60.0%, and compared with the crystal prepared by the prior art, the crystal obtained by the present invention is more stable, and the preparation method for the stable crystal form of the daptomycin of the present invention has important technical innovation and market application potential. BRIEF DESCRIPTION OF THE DRAWINGS The present invention is further described in detail hereinafter with reference to the drawings and specific embodiments, and the advantages of the above and / or other aspects of the present invention will be clearer. FIG. 1 is an XRD pattern of a daptomycin crystalline powder prepared in an embodiment of the present invention. FIG. 2 is an XRD pattern of a daptomycin powder prepared by taking ethanol as an anti solvent in Comparative Example 1. FIG. 3 is an XRD pattern of a daptomycin powder prepared by taking isopropanol as an anti-solvent in Comparative Example 2. DETAILED DESCRIPTION The present invention can be better understood according to the following embodiments. However, those skilled in the art easily understand that the contents described in the embodiments are only used to illustrate the present invention, and shall not and will not limit the present invention described in detail in the claims. The experimental methods described in the following embodiments are all conventional methods unless otherwise specified. The reagents and materials can all be obtained from commercial sources unless otherwise specified. A daptomycin raw material used in the embodiments of the present invention is amorphous powder with a purity above 98% and a batch number HZP202209 from Hubei Huizepu Pharmaceutical Technology Co., Ltd. In the powder embodiments, daptomycin with a purity above 99% is used as a standard product to draw a standard curve of concentration and absorbance of the daptomycin when the absorbance ranges from 0.2 to 0.8 at 268 nm, which is used to calibrate the purity of the daptomycin. Embodiment 1: (1) 10 g of amorphous daptomycin raw material was dissolved in 100 mL of aqueous solution and continuously stirred. In order to speed up the dissolution process, the daptomycin raw material was heated to 40°C until the solution was clear and solid-free, so as to obtain a daptomycin aqueous solution. (2) 0.1 g of acetate buffer pair was added into the daptomycin aqueous solution obtained in the step (1) to completely dissolve sodium acetate in the daptomycin aqueous solution, so as to obtain a mixed solution, wherein the acetate buffer pair was a mixture of acetic acid and sodium acetate, and a mass ratio of the acetic acid to the sodium acetate in the mixture was 1: 1. (3) Vacuum concentration was carried out on the mixed solution obtained in the step (2), wherein a vacuum degree was set to be -0.095 MPa—0.1 MPa, and a vacuum evaporation temperature was set to be 40°C. The vacuum concentration was lasted until solid daptomycin was precipitated in a concentrated feed solution and a mass of the precipitated daptomycin accounted for about 0.2% of an initial dosage of the daptomycin, and then the vacuum concentration was stopped. (4) The concentrated feed solution obtained in the step (3) was heated at a constant temperature of 80°C for 0.5 hour, so that the solid daptomycin in the feed solution was completely dissolved to obtain a saturated solution system. Subsequently, the system was quickly cooled to 55°C, then cooled to 35°C at a cooling rate of 5°C / h, and stirred at a stirring rate of 200 rpm~600 rpm during the cooling process until a solid was precipitated. After the solid was precipitated, the system was stirred at a current crystallization temperature of 3 5 °C for 1 hour for crystallization. After ending the stirring crystallization, the system was cooled to 1 °C at a rate of 5°C / h via gradient cooling, so as to obtain a daptomycin crystal-containing mother liquor of crystallization. (5) In order to improve a purity of the daptomycin crystal and reduce an influence of impurities in the mother liquor of crystallization obtained in the step (4) on the crystal, the daptomycin crystal was washed with pure acetonitrile at a low temperature of 0°C and centrifuged at a centrifugal speed of 3000 rpm for 10 minutes, and a solid was collected. (6) The solid obtained in the step (5) was subjected to forced air drying at a drying temperature of 40°C for 8 hours, so as to obtain a daptomycin crystal with a purity of 99.5% and a yield of 62%. An XRD pattern of the daptomycin crystal prepared by the present invention was as shown in FIG. 1. Embodiment 2: (1) 10 g of amorphous daptomycin raw material was dissolved in 100 mL of aqueous solution and continuously stirred. In order to speed up the dissolution process, the daptomycin raw material was heated to 60°C until the solution was clear and solid-free, so as to obtain a daptomycin aqueous solution. (2) 1.0 g of acetate buffer pair was added into the daptomycin aqueous solution obtained in the step (1) to completely dissolve sodium acetate in the daptomycin aqueous solution, so as to obtain a mixed solution, wherein the acetate buffer pair was a mixture of acetic acid and sodium acetate, and a mass ratio of the acetic acid to the sodium acetate in the mixture was 1: 1. (3) Vacuum concentration was carried out on the mixed solution obtained in the step (2), wherein a vacuum degree was set to be -0.099 to -0.1 Mpa, and a vacuum evaporation temperature was set to be 70°C. The vacuum concentration was lasted until solid daptomycin was precipitated in a concentrated feed solution and a mass of the precipitated daptomycin accounted for about 1% of an initial dosage of the daptomycin, and then the vacuum concentration was stopped. (4) The concentrated feed solution obtained in the step (3) was heated at a constant temperature of 90°C for 0.5 hour, so that the solid daptomycin in the feed solution was completely dissolved to obtain a saturated solution system. Subsequently, the system was quickly cooled to 55°C, then cooled to 50°C at a cooling rate of 0.5°C / h, and stirred at a stirring rate of 200 rpm~600 rpm during the cooling process until a solid was precipitated. After the solid was precipitated, the system was stirred at a current crystallization temperature of 50°C for 4 hour for crystallization. After ending the stirring crystallization, the system was cooled to 10°C at a rate of 0.5°C / h via gradient cooling, so as to obtain a daptomycin crystalcontaining mother liquor of crystallization. (5) In order to improve a purity of the daptomycin crystal and reduce an influence of impurities in the mother liquor of crystallization obtained in the step (4) on the crystal, the daptomycin crystal was washed with acetone at a low temperature of 8 °C and centrifuged at a centrifugal speed of 10000 rpm for 30 minutes, and a solid was collected. (6) The solid obtained in the step (5) was subjected to vacuum drying at a drying temperature of 80°C for 2 hours, so as to obtain a daptomycin crystal with a purity of 99.0% and a yield of 70.2%. An XRD pattern of the daptomycin crystal prepared by the present invention was the same as FIG. 1. Embodiment 3: (1) 10 g of amorphous daptomycin raw material was dissolved in 100 mL of aqueous solution and continuously stirred. In order to speed up the dissolution process, the daptomycin raw material was heated to 50°C until the solution was clear and solid-free, so as to obtain a daptomycin aqueous solution. (2) 0.5 g of acetate buffer pair was added into the daptomycin aqueous solution obtained in the step (1) to completely dissolve sodium acetate in the daptomycin aqueous solution, so as to obtain a mixed solution, wherein the acetate buffer pair was a mixture of acetic acid and sodium acetate, and a mass ratio of the acetic acid to the sodium acetate in the mixture was 1: 1. (3) Vacuum concentration was carried out on the mixed solution obtained in the step (2), wherein a vacuum degree was set to be -0.098 MPa to -0.1 MPa, and a vacuum evaporation temperature was set to be 50°C. The vacuum concentration was lasted until solid daptomycin was precipitated in a concentrated feed solution and a mass of the precipitated daptomycin accounted for about 0.5% of an initial dosage of the daptomycin, and then the vacuum concentration was stopped. (4) The concentrated feed solution obtained in the step (3) was heated at a constant temperature of 90°C for 0.5 hour, so that the solid daptomycin in the feed solution was completely dissolved to obtain a saturated solution system. Subsequently, the system was quickly cooled to 55°C, then cooled to 50°C at a cooling rate of 2°C / h, and stirred at a stirring rate of 200 rpm-600 rpm during the cooling process until a solid was precipitated. After the solid was precipitated, the system was stirred at a current crystallization temperature of 50°C for 3 hour for crystallization. After ending the stirring crystallization, the system was cooled to 4°C at a rate of 2°C / h via gradient cooling, so as to obtain a daptomycin crystal-containing mother liquor of crystallization. (5) In order to improve a purity of the daptomycin crystal and reduce an influence of impurities in the mother liquor of crystallization obtained in the step (4) on the crystal, the daptomycin crystal was washed with pure chloroform at a low temperature of 4°C and centrifuged at a centrifugal speed of 5000 rpm for 20 minutes, and a solid was collected. (6) The solid obtained in the step (5) was subjected to vacuum drying at a drying temperature of 60°C for 4 hours, so as to obtain a daptomycin crystal with a purity of 99.1% and a yield of 67%. An XRD pattern of the daptomycin crystal prepared by the present invention was the same as FIG. 1. Embodiment 4: (1) 10 g of amorphous daptomycin raw material was dissolved in 100 mL of aqueous solution and continuously stirred. In order to speed up the dissolution process, the daptomycin raw material was heated to 50°C until the solution was clear and solid-free, so as to obtain a daptomycin aqueous solution. (2) 0.8 g of acetate buffer pair was added into the daptomycin aqueous solution obtained in the step (1) to completely dissolve sodium acetate in the daptomycin aqueous solution, so as to obtain a mixed solution, wherein the acetate buffer pair was a mixture of acetic acid and sodium acetate, and a mass ratio of the acetic acid to the sodium acetate in the mixture was 1: 1. (3) Vacuum concentration was carried out on the mixed solution obtained in the step (2), wherein a vacuum degree was set to be -0.097 Mpa—0.1 Mpa, and a vacuum evaporation temperature was set to be 60°C. The vacuum concentration was lasted until solid daptomycin was precipitated in a concentrated feed solution and a mass of the precipitated daptomycin accounted for about 0.3% of an initial dosage of the daptomycin, and then the vacuum concentration was stopped. (4) The concentrated feed solution obtained in the step (3) was heated at a constant temperature of 85°C for 0.5 hour, so that the solid daptomycin in the feed solution was completely dissolved to obtain a saturated solution system. Subsequently, the system was quickly cooled to 55°C, then cooled to 45°C at a cooling rate of 3°C / h, and stirred at a stirring rate of 200 rpm-600 rpm during the cooling process until a solid was precipitated. After the solid was precipitated, the system was stirred at a current crystallization temperature of 45 °C for 2 hour for crystallization. After ending the stirring crystallization, the system was cooled to 2°C at a rate of 3°C / h via gradient cooling, so as to obtain a daptomycin crystal-containing mother liquor of crystallization. (5) In order to improve a purity of the daptomycin crystal and reduce an influence of impurities in the mother liquor of crystallization obtained in the step (4) on the crystal, the daptomycin crystal was washed with pure acetonitrile at a low temperature of 2°C and centrifuged at a centrifugal speed of 6000 rpm for 15 minutes, and a solid was collected. (6) The solid obtained in the step (5) was subjected to vacuum drying at a drying temperature of 65°C for 3 hours, so as to obtain a daptomycin crystal with a purity of 99.2% and a yield of 64.3%. An XRD pattern of the daptomycin crystal prepared by the present invention was the same as FIG. 1. Embodiment 5: (1) 10 g of amorphous daptomycin raw material was dissolved in 100 mL of aqueous solution and continuously stirred. In order to speed up the dissolution process, the daptomycin raw material was heated to 5 5 °C until the solution was clear and solid-free, so as to obtain a daptomycin aqueous solution. (2) 0.4 g of acetate buffer pair was added into the daptomycin aqueous solution obtained in the step (1) to completely dissolve sodium acetate in the daptomycin aqueous solution, so as to obtain a mixed solution, wherein the acetate buffer pair was a mixture of acetic acid and sodium acetate, and a mass ratio of the acetic acid to the sodium acetate in the mixture was 1: 1. (3) Vacuum concentration was carried out on the mixed solution obtained in the step (2), wherein a vacuum degree was set to be -0.098 Mpa—0.1 Mpa, and a vacuum evaporation temperature was set to be 65°C. The vacuum concentration was lasted until solid daptomycin was precipitated in a concentrated feed solution and a mass of the precipitated daptomycin accounted for about 0.7% of an initial dosage of the daptomycin, and then the vacuum concentration was stopped. (4) The concentrated feed solution obtained in the step (3) was heated at a constant temperature of 87°C for 0.5 hour, so that the solid daptomycin in the feed solution was completely dissolved to obtain a saturated solution system. Subsequently, the system was quickly cooled to 55°C, then cooled to 46°C at a cooling rate of 4°C / h, and stirred at a stirring rate of 200 rpm~600 rpm during the cooling process until a solid was precipitated. After the solid was precipitated, the system was stirred at a current crystallization temperature of 46°C for 2 hour for crystallization. After ending the stirring crystallization, the system was cooled to 7°C at a rate of 4°C / h via gradient cooling, so as to obtain a daptomycin crystal-containing mother liquor of crystallization. (5) In order to improve a purity of the daptomycin crystal and reduce an influence of impurities in the mother liquor of crystallization obtained in the step (4) on the crystal, the daptomycin crystal was washed with chloroform at a low temperature of 7°C and centrifuged at a centrifugal speed of 8000 rpm for 18 minutes, and a solid was collected. (6) The solid obtained in the step (5) was subjected to vacuum drying at a drying temperature of 50°C for 6 hours, so as to obtain a daptomycin crystal with a purity of 99.1% and a yield of 63.6%. An XRD pattern of the daptomycin crystal prepared by the present invention was the same as FIG. 1. Comparative Example 1: (1) 10 g of amorphous daptomycin raw material was dissolved in 100 mL of aqueous solution and continuously stirred. In order to speed up the dissolution process, the daptomycin raw material was heated to 40°C until the solution was clear and solid-free, so as to obtain a daptomycin aqueous solution. (2) According to experimental methods of step (5), step (6), step (7) and step (8) in Embodiment 1 in Specification of the patent CN 110117310 A, nanofiltration concentration, crystallization (with 95% industrial ethanol serving as an anti-solvent), suction filtration and freeze-drying were carried out in sequence to prepare a daptomycin powder with a purity of 99.4%. An XRD pattern of the resulting powder was as shown in FIG. 2. Comparative Example 2: (1) 10 g of amorphous daptomycin raw material was dissolved in 100 mL of aqueous solution and continuously stirred. In order to speed up the dissolution process, the daptomycin raw material was heated to 40°C until the solution was clear and solid-free, so as to obtain a daptomycin aqueous solution. (2) According to experimental methods of step g and step h in Embodiment 1 in Specification of the patent CN 103224547 A, concentration and crystallization purification (with isopropanol serving as an anti-solvent) were carried out in sequence to prepare a daptomycin crystal with a purity of 99.6%. An XRD pattern of the resulting powder was as shown in FIG. 3. Embodiment 6: (1) Structural orderliness of crystal It could be seen from FIG. 1 that a daptomycin crystal obtained by a crystallization technology in the present invention had more abundant characteristic peaks. As for daptomycin powders (prepared in Comparative Example 1 and Comparative Example 2 respectively) prepared by repeating other crystallization technologies adopting different antisolvents in the prior arts, it could be seen from FIG. 2-FIG. 3 that there was only one bulge on the powder XRD pattern, and there was no characteristic peak. The XRD pattern indicated that the powder had poor orderliness and was closer to amorphous powder. However, the XRD pattern of the daptomycin crystal obtained by the present invention showed obvious characteristic peaks at more 2-theta angles, which indicated that the daptomycin crystal prepared by the present invention had higher orderliness in structure. (2) Hygroscopicity of crystal Stabilities of the daptomycin powders prepared in Embodiment 1, Comparative Example 1 and Comparative Example 2 were compared at room temperature. About 1.5 g of daptomycin sample was added into a culture dish dried to a constant weight, and placed in a sealed dryer with environmental relative humidities comprising RH43%(K2CO3 aqueous solution), RH67% (CuCh aqueous solution) and RH98% (saturated K2SO4) respectively at room temperature for 72 hours, and weight changes during the process were recorded. Results were as shown in Table 1. The experimental results were as shown in Table 1: The powder (Comparative Example 1), which was not prepared by this patent, had a weight gain of 3.2% in an environment at RH43%, with low hygroscopicity; had a weight gain of 8.4% in an environment at a humidity of 76%, with hygroscopicity; and had a weight gain of 25.2% in an environment at RH98%, with high hygroscopicity. The powder (Comparative Example 2), which was not prepared by this patent, had a weight gain of 4.2% in an environment at RH43%, with low hygroscopicity; had a weight gain of 9.7% in an environment at a humidity of 76%, with hygroscopicity; and had a weight gain of 26.3% in an environment at RH98%, with high hygroscopicity. The crystal (crystalline powder, Embodiment 1), which was prepared by this patent, had a weight gain of 0.1% in an environment at RH43%, a weight gain of 0.95% in an environment at a humidity of 76%, and a weight gain of 4.9% in an environment at RH98%, with low hygroscopicity. As for the above data, about 1.5 g of daptomycin sample was added into a culture dish dried to a constant weight and placed in a high-temperature oven at 50°C to stand for 12 hours, a weight was recorded every hour, and weight changes in the whole experiment were recorded. Final results recorded were as shown in Table 1. Table 1 Water absorption of different powders in different water-containing environments Serial number Embodiment Environmental humidity Placement time Weight Increase 1 Embodiment 1 RH43% 72 hours 0.1% 2 Embodiment 1 RH76% 72 hours 0.95% 3 Embodiment 1 RH98% 72 hours 4.9% 4 Comparative Example 1 RH43% 72 hours 3.2% 5 Comparative Example 1 RH76% 72 hours 8.4% 6 Comparative Example 1 RH98% 72 hours 25.2% 7 Comparative Example 2 RH43% 72 hours 4.2% 8 Comparative Example 2 RH76% 72 hours 9.7% 9 Comparative Example 2 RH98% 72 hours 26.3% (3) High temperature stability of different powders Purity results of the daptomycin powders prepared in Embodiment 1, Comparative Example 1 and Comparative Example 2 after being placed in a dried dish at 90°C and a humidity of 43% for 6 hours, 12 hours, 24 hours and 48 hours were determined by spectrophotometry. The results were as shown in Table 2. The results indicated that the stability of the daptomycin powder obtained in Embodiment 1 was obviously better than those of the daptomycin powders obtained in Comparative Example 1 and Comparative Example 2. Table 2 High temperature stabilities of daptomycin solid powders obtained by different methods Embodiment Initial Purity after 6 Purity after 12 Purity after 24 Purity after 48 purity hours hours hours hours Comparative Example 1 99.4% 98.2% 96.7% 92.3% 76.1% Comparative Example 2 99.6% 97.9% 95% 93.9% 90% Embodiment 1 99.5% 99.4% 99.1% 99% 98.8% The present invention provides an idea and a method for a preparation method for a stable crystal form of daptomycin, with many methods and ways to realize the technical solution specifically. Those described above are merely the preferred embodiments of the present invention, and it should be pointed out that those of ordinary skills in the art may further make improvements and decorations without departing from the principle of the present invention, and these improvements and decorations should also be regarded as falling within the scope of protection of the present invention. All the unspecified components in the embodiments can be realized by the prior art.

Claims

1. A preparation method for a stable crystal form of daptomycin, comprising the following steps:(1) dissolving a daptomycin raw material in water and mixing evenly to obtain a daptomycin aqueous solution;(2) adding an acetate buffer pair into the daptomycin aqueous solution obtained in the step (1) and mixing evenly to obtain a mixed solution;(3) carrying out vacuum concentration on the mixed solution obtained in the step (2) until a solid is precipitated, and then stopping the vacuum concentration to obtain a concentrated feed solution;(4) heating and dissolving the concentrated feed solution obtained in the step (3), and completely dissolving the solid in the concentrated feed solution to obtain a saturated solution system;(5) cooling the saturated solution system obtained in the step (4), stirring during cooling, and after a solid is precipitated during cooling, stirring for crystallization at a current crystallization temperature;(6) after ending the crystallization in the step (5), continuously cooling the system to obtain a daptomycin crystal-containing mother liquor of crystallization; and(7) washing the daptomycin crystal-containing mother liquor of crystallization obtained in the step (6) at a low temperature, and centrifuging and drying to obtain the daptomycin crystal.

2. The preparation method according to claim 1, wherein the daptomycin raw material is amorphous daptomycin.

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

4. The preparation method according to claim 1, wherein, the acetate buffer pair is a mixture of sodium acetate and acetic acid; in the acetate buffer pair, a mass ratio of the sodium acetate to the acetic acid is 1.0: (1.0-1.2); and a mass of the acetate buffer pair accounts for 1 %-10%, preferably 1 %-8%, and further preferably 4%-8%, of a mass of the daptomycin rawmaterial.

5. The preparation method according to claim 1, wherein, in the step (3), the vacuum concentration is carried out at 40°C-70°C, preferably 40°C-60°C; and the vacuum concentration is carried out until the solid is precipitated, a mass of precipitated daptomycin accounts for 0.2%-1.0%, preferably 0.2%-0.8%, of a mass of the daptomycin raw material, and then the vacuum concentration is stopped to obtain the concentrated feed solution.

6. The preparation method according to claim 1, wherein, in the step (4), the concentrated feed solution is heated and dissolved at a heating temperature of 80°C-90°C, preferably 85°C-90°C.

7. The preparation method according to claim 1, wherein, in the step (5), the cooling refers to cooling the saturated solution system to 50°C-58°C first, and then g cooling the saturated solution system to 35°C-50°C via gradient cooling, preferably 35°C-46°C; the stirring is carried out at a stirring speed 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 stirring for crystallization refers to lasting the stirring for 1-4 hours, preferably 1-3 hours, for crystallization.

8. The preparation method according to claim 1, wherein, in the step (6), the cooling refers to gradiently cooling the system to l°C-10°C.

9. The preparation method according to claim 7 or 8, wherein the gradient cooling is carried out at a cooling rate of 0.5 °C / h-5°C / h, preferably 2 °C / h-5°C / h.

10. The preparation method according to claim 1, wherein, in the step (7), the low temperature refers to a temperature of 0°C-10°C; and as for the washing, a solvent used in washing the daptomycin crystal is any one or a combination of several of acetonitrile, acetone, and chloroform, preferably the acetonitrile.

11. The preparation method according to claim 1, wherein, in the step (7), the centrifuging refers to the centrifugation is performed at a speed of 3000 rpm-10000 rpm for 10 minutes~30 minutes; and the drying refers to forced-air drying or vacuum drying, and the drying is carried out at 40°C-80°C for 2 hours-8 hours.T +44(0)30 0300 2000A

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