Preparation method of stable crystal form of daptomycin

Abstract

The invention belongs to the technical field of separation and crystallization, and relates to a preparation method of a stable crystal form of daptomycin. The preparation method comprises the following steps: dissolving a daptomycin raw material in water, uniformly mixing, adding an acetic acid buffer pair into an obtained daptomycin aqueous solution, uniformly mixing to obtain a mixed solution, and then carrying out vacuum concentration to obtain concentrated feed liquid; heating and dissolving the concentrated feed liquid to obtain a saturated solution system; cooling the saturated solution system until solids are separated out, and stirring at the current crystallization temperature for crystallization; after crystallization is finished, continuously cooling the system to obtain crystallization mother liquor containing daptomycin crystals; and finally, washing the daptomycin crystal at low temperature, centrifugally collecting a daptomycin crystal crude product, and drying the crude product to obtain the daptomycin crystal. The orderliness of the daptomycin crystal prepared by the method disclosed by the invention is obviously superior to that of the daptomycin crystal prepared by a crystallization method reported in the prior art.

Description

Technical Field

[0001] The invention belongs to the technical field of separation crystallization and relates to a method for preparing a stable crystal form of daptomycin. Background Art

[0002] Daptomycin (English name Daptomycin, CAS: 103060-53-3, molecular formula: C 72 H 101 N 17 O 26 , molecular weight: 1620.67g / mol), daptomycin contains a cyclic decapeptide 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), and also contains a decanoyl and three amino acids (L-asparagyl, L-asparagine, L-tryptophanyl) connected to the side chain, containing a total of 13 amino acids.

[0003]

[0004] Daptomycin has a complex structure and is difficult to chemically synthesize. It is usually fermented by Streptomyces (S. roseosporus). In 1961, Falcao and Dailia isolated and screened S. roseosporus from soil samples from the Ararat Mountains in Turkey and obtained it by fermentation. The molecular structure of daptomycin is relatively complex. It has both hydrophilic carboxyl and amino groups, as well as hydrophobic benzene rings, saturated alkane chains and other groups, which give it both hydrophilic and lipophilic properties. Daptomycin is a water-soluble antibiotic, and the pH value of its aqueous solution is generally around 3.5; when the pH value is maintained at 3.5-9.0, daptomycin can be fused in a good state. At room temperature, the daptomycin aqueous solution can remain relatively stable for more than a week. However, if the pH value changes more extreme (the pH value is greater than 9.0 or less than 2.0), daptomycin will denature and this denaturation cannot be reversed. Daptomycin is relatively less soluble in acetone and chloroform than in other organic solvents and is almost insoluble in acetonitrile.

[0005] Obtaining a stable crystalline form of daptomycin has significant patent value in the field of drug development and formulation. As a glycopeptide antibiotic, the efficacy of daptomycin is affected by the stability, solubility and bioavailability of the crystal form. By obtaining a stable crystal form, its chemical stability can be effectively improved to avoid degradation or quality loss during storage and transportation; at the same time, solubility and bioavailability are improved, thereby improving the therapeutic effect of the drug. The optimization of the stable crystal form also provides a basis for the dosage form innovation of daptomycin, especially in oral preparations and injection formulations, where better dosage accuracy and drug efficacy persistence can be achieved through crystal form control. In addition, the stable crystal form technology involved in the present invention can not only be applied to daptomycin, but also provides a valuable reference for the crystal form improvement of other glycopeptide antibiotics, and has broad application prospects. Summary of the invention

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

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

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

[0009] (1) dissolving a daptomycin raw material in water and mixing well to obtain a daptomycin aqueous solution;

[0010] (2) adding acetate buffer to the daptomycin aqueous solution obtained in step (1), mixing, and obtaining a mixed solution;

[0011] (3) vacuum concentrating the mixed solution obtained in step (2) until solid precipitates, then stopping the vacuum concentrating to obtain a concentrated feed solution;

[0012] (4) heating and dissolving the concentrated liquid obtained in step (3), wherein the solid part in the concentrated liquid is completely dissolved to obtain a saturated solution system;

[0013] (5) cooling the saturated solution system obtained in step (4) while stirring during the cooling process, cooling to a temperature where solids precipitate, and stirring at the current crystallization temperature for crystallization;

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

[0015] (7) washing the crystallization mother liquor containing daptomycin crystals obtained in step (6) at low temperature, centrifuging, and drying to obtain daptomycin crystals.

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

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

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

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

[0020] In the step (1), in the process of dissolving the daptomycin raw material in water and mixing, in order to accelerate the dissolution process of the daptomycin raw material, stirring can be performed continuously, and heating at 40° C. to 60° C. can be performed to assist the dissolution of the daptomycin raw material until the solution is clear and free of solid matter.

[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%-10% of the mass of the daptomycin raw material, preferably 1%-8%, and more preferably 4%-8%.

[0022] In some embodiments, preferably, in the acetate buffer pair, the mass ratio of sodium acetate to acetic acid 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 is precipitated, 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%, and then the vacuum concentration is stopped to obtain a concentrated liquid.

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

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

[0026] In some embodiments, in step (3), it is further preferred that the vacuum concentration is stopped until solid is precipitated and the mass of the precipitated daptomycin accounts for 0.2% to 0.5% of the mass of the daptomycin raw material, thereby obtaining a concentrated liquid.

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

[0028] Wherein, in step (4), the concentrated liquid is heated to dissolve, and the heating time is sufficient to allow the solid part of daptomycin in the liquid to be completely dissolved.

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

[0030] In step (5), the temperature of the saturated solution system is first cooled to 50°C to 58°C. During this cooling process, no gradient cooling is required. The circulating water bath can be directly set to the required temperature, and crystallization will not occur within the temperature range of 50°C to 58°C.

[0031] In some embodiments, in step (6), the cooling is to reduce the system temperature to 1°C to 10°C in a gradient manner.

[0032] In some embodiments, in step (5) or step (6), the gradient cooling has a cooling rate of 0.5°C / h to 5°C / h, preferably 2°C / h to 5°C / h.

[0033] In some embodiments, in step (7), the low temperature is 0°C to 10°C; and 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 performed at a centrifugal speed of 3000 rpm to 10000 rpm for 10 min to 30 min; and the drying is performed by forced air drying or vacuum drying at 40° C. to 80° C. for 2 h to 8 h.

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

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

[0037] Beneficial effects:

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

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

[0040] (2) The daptomycin crystals prepared by the present invention have higher storage stability, are not easy to absorb moisture, and have higher stability.

[0041] (3) The orderliness of daptomycin crystals prepared by the method of the present invention is significantly better than that of daptomycin crystals prepared by the crystallization method reported in the prior art.

[0042] (4) The process for preparing a 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 crystals obtained by the present invention are more stable than those prepared by the prior art method. The preparation method of the stable daptomycin crystal form of the present invention has important technical innovation and market application potential. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0045] Figure 2 This is the XRD pattern of daptomycin crystals prepared in Comparative Example 1 using ethanol as the anti-solvent.

[0046] Figure 3 This is the XRD pattern of daptomycin crystals prepared in Comparative Example 2 using isopropanol as the anti-solvent. DETAILED DESCRIPTION

[0047] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

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

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

[0050] In this powder example, daptomycin with a purity of more than 99% is used as a standard product, and a standard curve of daptomycin concentration and absorbance with an absorbance between 0.2 and 0.8 at 268 nm is drawn to calibrate the purity of daptomycin.

[0051] Embodiment 1:

[0052] (1) Dissolve 10 g of amorphous daptomycin raw material in 100 mL of aqueous solution, stir continuously, and heat to 40° C. to accelerate the dissolution process until the solution is clear and free of solid matter, thereby obtaining a daptomycin aqueous solution.

[0053] (2) adding 0.1 g of acetate buffer solution 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 solution 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 subjected to vacuum concentration, the vacuum degree is set to -0.095 MPa to -0.1 MPa, the vacuum evaporation temperature is set to 40° C., and the vacuum concentration is performed until solid daptomycin is precipitated in the obtained concentrated liquid, and the mass of the precipitated daptomycin accounts for about 0.2% of the initial amount of daptomycin charged, and the vacuum concentration is stopped.

[0055] (4) The concentrated liquid obtained in step (3) is heated at a constant temperature of 80° C. for 0.5 hour to completely dissolve the solid part of daptomycin in the liquid to obtain a saturated solution system; then the temperature of the system is rapidly cooled to 55° C., and then cooled to 35° C. at a cooling rate of 5° C. / h. During the cooling process, the system is stirred at a stirring rate of 200 rpm to 600 rpm until solids are precipitated. After the solids are precipitated, the system is stirred at the current crystallization temperature of 35° C. for 1 hour to crystallize; after the stirring crystallization is completed, the system temperature is cooled 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 a centrifugal speed of 3000 rpm for 10 min, and the solids were collected.

[0057] (6) The solid obtained in step (5) was subjected to forced air drying at a drying temperature of 40° C. for a drying time of 8 h to obtain daptomycin crystals with a purity of 99.5% and a yield of 62%.

[0058] The XRD pattern of the daptomycin crystal prepared by the present invention is as follows: Figure 1 shown.

[0059] Embodiment 2:

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

[0061] (2) adding 1.0 g of acetate buffer solution 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 solution 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 subjected to vacuum concentration, the vacuum degree is set to -0.099 MPa to -0.1 MPa, the vacuum evaporation temperature is set to 70° C., and the vacuum concentration is performed until solid daptomycin is precipitated from the obtained concentrated liquid, and the mass of the precipitated daptomycin accounts for about 1% of the initial amount of daptomycin charged, and the vacuum concentration is stopped.

[0063] (4) The concentrated liquid obtained in step (3) is heated at a constant temperature of 90° C. for 0.5 hour to completely dissolve the solid part of daptomycin in the liquid to obtain a saturated solution system; then the temperature of the system is rapidly cooled to 55° C., and then cooled to 50° C. at a cooling 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 solids are precipitated. After the solids are precipitated, the system is stirred and maintained at the current crystallization temperature of 50° C. for 4 hours to crystallize; after the stirring crystallization is completed, the system temperature is cooled 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 crystallization mother liquor obtained in step (4) on the crystals, the daptomycin crystals were washed with acetone at a low temperature of 8° C., centrifuged at a centrifugal speed of 10,000 rpm for 30 min, and the solids were collected.

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

[0066] The XRD pattern of the daptomycin crystal prepared by the present invention is the same as Figure 1 .

[0067] Embodiment 3:

[0068] (1) Dissolve 10 g of amorphous daptomycin raw material in 100 mL of aqueous solution, stir continuously, heat to 50° C. to accelerate the dissolution process, and stir until the solution is clear and free of solid matter, to obtain a daptomycin aqueous solution.

[0069] (2) adding 0.5 g of acetate buffer solution 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 solution 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 subjected to vacuum concentration, the vacuum degree is set to -0.098 MPa to -0.1 MPa, the vacuum evaporation temperature is set to 50° C., and the vacuum concentration is performed until solid daptomycin is precipitated from the obtained concentrated liquid, and the mass of the precipitated daptomycin accounts for about 0.5% of the initial amount of daptomycin fed, and the vacuum concentration is stopped.

[0071] (4) The concentrated liquid obtained in step (3) is heated at a constant temperature of 90° C. for 0.5 hour to completely dissolve the solid part of daptomycin in the liquid to obtain a saturated solution system; then the temperature of the system is rapidly cooled to 55° C., and then cooled to 50° C. at a cooling rate of 2° C. / h. During the cooling process, the system is stirred at a stirring rate of 200 rpm to 600 rpm until solids are precipitated. After the solids are precipitated, the system is stirred at the current crystallization temperature of 50° C. for 3 hours to crystallize; after the stirring crystallization is completed, the system temperature is cooled 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 crystallization 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 a centrifugal speed of 5000 rpm for 20 min, and the solids were collected.

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

[0074] The XRD pattern of the daptomycin crystal prepared by the present invention is the same as Figure 1 .

[0075] Embodiment 4:

[0076] (1) Dissolve 10 g of amorphous daptomycin raw material in 100 mL of aqueous solution, stir continuously, heat to 50° C. to accelerate the dissolution process, and stir until the solution is clear and free of solid matter, to obtain a daptomycin aqueous solution.

[0077] (2) adding 0.8 g of acetate buffer solution 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 solution 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 subjected to vacuum concentration, the vacuum degree is set to -0.097 MPa to -0.1 MPa, the vacuum evaporation temperature is set to 60° C., and the vacuum concentration is performed until solid daptomycin is precipitated from the obtained concentrated liquid, and the mass of the precipitated daptomycin accounts for about 0.3% of the initial amount of daptomycin charged, and the vacuum concentration is stopped.

[0079] (4) The concentrated liquid obtained in step (3) is heated at a constant temperature of 85° C. for 0.5 hour to completely dissolve the solid part of daptomycin in the liquid to obtain a saturated solution system; then the temperature of the system is rapidly cooled to 55° C., and then cooled to 45° C. at a cooling rate of 3° C. / h. During the cooling process, the system is stirred at a stirring rate of 200 rpm to 600 rpm until solids are precipitated. After the solids are precipitated, the system is stirred at the current crystallization temperature of 45° C. for 2 hours to crystallize; after the stirring crystallization is completed, the system temperature is cooled 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 crystallization 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 a centrifugal speed of 6000 rpm for 15 min, and the solids were collected.

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

[0082] The XRD pattern of the daptomycin crystal prepared by the present invention is the same as Figure 1 .

[0083] Embodiment 5:

[0084] (1) Dissolve 10 g of amorphous daptomycin raw material in 100 mL of aqueous solution, stir continuously, heat to 55° C. to accelerate the dissolution process, and stir until the solution is clear and free of solid matter, to obtain a daptomycin aqueous solution.

[0085] (2) adding 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 subjected to vacuum concentration, the vacuum degree is set to -0.098 MPa to -0.1 MPa, the vacuum evaporation temperature is set to 65° C., and the vacuum concentration is performed until solid daptomycin is precipitated from the obtained concentrated liquid, and the mass of the precipitated daptomycin accounts for about 0.7% of the initial amount of daptomycin charged, and the vacuum concentration is stopped.

[0087] (4) The concentrated liquid obtained in step (3) is heated at a constant temperature of 87° C. for 0.5 hour to completely dissolve the solid part of daptomycin in the liquid to obtain a saturated solution system; then the temperature of the system is rapidly cooled to 55° C., and then cooled to 46° C. at a cooling rate of 4° C. / h. During the cooling process, the system is stirred at a stirring rate of 200 rpm to 600 rpm until solids are precipitated. After the solids are precipitated, the system is stirred at the current crystallization temperature of 46° C. for 2 hours to crystallize; after the stirring crystallization is completed, the system temperature is cooled 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 crystallization 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 a centrifugal speed of 8000 rpm for 18 min, and the solids were collected.

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

[0090] The XRD pattern of the daptomycin crystal prepared by the present invention is the same as Figure 1 .

[0091] Comparative Example 1:

[0092] (1) Dissolve 10 g of amorphous daptomycin raw material in 100 mL of aqueous solution, stir continuously, and heat to 40° C. to accelerate the dissolution process until the solution is clear and free of solid matter, thereby obtaining a daptomycin aqueous solution.

[0093] (2) According to the experimental method of step (5), step (6), step (7) and step (8) in Example 1 of the specification of patent CN 110117310 A, nanofiltration concentration, crystallization (using 95% industrial ethanol as anti-solvent), suction filtration and freeze-drying were carried out in sequence to prepare daptomycin crystals with a purity of 99.4%. The crystal XRD pattern is shown in Figure 2 .

[0094] Comparative Example 2:

[0095] (1) Dissolve 10 g of amorphous daptomycin raw material in 100 mL of aqueous solution, stir continuously, and heat to 40° C. to accelerate the dissolution process until the solution is clear and free of solid matter, thereby obtaining a daptomycin aqueous solution.

[0096] (2) According to the experimental methods of step g and step h in Example 1 of the specification of patent CN 103224547 A, concentration and crystallization purification (using isopropanol as anti-solvent) were performed in sequence to prepare daptomycin crystals with a purity of 99.6%. The crystal XRD pattern is shown in Figure 3 .

[0097] Embodiment 6:

[0098] (1) Crystal structure order

[0099] from Figure 1 It can be seen that the characteristic peaks of the daptomycin crystals obtained by the crystallization process of the present invention are richer; the daptomycin crystals prepared by repeating the different anti-solvent crystallization processes of other prior arts (prepared in Comparative Example 1 and Comparative Example 2, respectively) are Figure 2-3 It can be seen that there is only one bulge on the XRD powder spectrum and no characteristic peaks. The XRD spectrum shows that the order of the crystal is poor and is closer to an amorphous powder. The XRD spectrum of the daptomycin crystal obtained by the present invention has obvious characteristic peaks at more 2-theta angles, indicating that the daptomycin crystal structure prepared by the present invention has higher order.

[0100] (2) Crystalline hygroscopicity

[0101] The stability of the daptomycin crystals prepared in Example 1, Comparative Example 1 and Comparative Example 2 at room temperature was compared. About 1.5 g of daptomycin sample was added to a culture dish dried to constant weight and placed in an environment with relative humidity (Relative Humidity) of RH43% (K 2 CO 3 aqueous solution), RH67%(CuCl 2 Aqueous solution), RH98% (saturated K 2 SO 4 ) was placed in a closed desiccator at room temperature for 72 h, and the weight change during the process was recorded. The results are shown in Table 1.

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

[0103] The crystals not prepared by the present patent (Comparative Example 1) gained 3.2% in an environment of RH43% and were hygroscopic but less hygroscopic; when the humidity was 76%, they gained 8.4% and were hygroscopic; when the relative humidity was 98%, they gained 25.2% and were extremely hygroscopic. The crystals not prepared by the present patent (Comparative Example 2) gained 4.2% in an environment of RH43% and were hygroscopic but less hygroscopic; when the humidity was 76%, they gained 9.7% and were hygroscopic; when the relative humidity was 98%, they gained 26.3% and were extremely hygroscopic. The crystals prepared by the present invention (Example 1) gained 0.1% in an environment of RH43%, gained 0.95% in an environment of humidity of 76%, and gained 4.9% at a relative humidity of 98%, were hygroscopic but less hygroscopic.

[0104] The above data were obtained by adding about 1.5 grams of daptomycin sample to a culture dish 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 of the entire experiment. The final results recorded are shown in Table 1.

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

[0106]

[0107]

[0108] (3) Crystal high temperature stability

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

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

[0111]

[0112] The present invention provides a method and idea for preparing a stable crystalline form of daptomycin. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing a stable crystalline form of daptomycin, characterized in that: The steps include: (1) dissolving a daptomycin raw material in water and mixing well to obtain a daptomycin aqueous solution; (2) adding acetate buffer to the daptomycin aqueous solution obtained in step (1), mixing, and obtaining a mixed solution; (3) vacuum concentrating the mixed solution obtained in step (2) until solid precipitates, then stopping the vacuum concentrating to obtain a concentrated feed solution; (4) heating and dissolving the concentrated liquid obtained in step (3), wherein the solid part in the concentrated liquid is completely dissolved to obtain a saturated solution system; (5) cooling the saturated solution system obtained in step (4) while stirring during the cooling process, cooling to a temperature where solids precipitate, and stirring at the current crystallization temperature for crystallization; (6) After the crystallization in step (5) is completed, the system temperature is further cooled to obtain a crystallization mother solution containing daptomycin crystals; (7) washing the crystallization mother liquor containing daptomycin crystals obtained in step (6) at low temperature, centrifuging, and drying to obtain daptomycin crystals.

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 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 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%-10% of the mass of the daptomycin raw material, preferably 1%-8%, and more preferably 4%-8%.

5. The preparation method according to claim 1, characterized in that: 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 is precipitated, 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%, and then the vacuum concentration is stopped to obtain a concentrated liquid.

6. The preparation method according to claim 1, characterized in that: In step (4), the concentrated liquid is heated to dissolve, and the heating temperature is 80°C to 90°C, preferably 85°C to 90°C.

7. The preparation method according to claim 1, characterized in that: In step (5), the cooling is to first cool the temperature of the saturated solution system to 50°C to 58°C, and then to 35°C to 50°C, preferably to 35°C to 46°C, by gradient cooling; the stirring is to have a stirring rate of 100rpm to 800rpm, preferably 200rpm to 600rpm; the crystallization temperature is 35°C to 50°C, preferably 35°C to 46°C; the stirring is for crystallization, and the stirring is maintained for 1 to 4 hours for crystallization, preferably for 1 to 3 hours for crystallization.

8. The preparation method according to claim 1, characterized in that: In step (6), the temperature of the system is lowered to 1°C to 10°C in a gradient manner.

9. The preparation method according to claim 7 or 8, characterized in that: The gradient cooling has a cooling rate of 0.5°C / h to 5°C / h, preferably 2°C / h to 5°C / h.

10. The preparation method according to claim 1, characterized in that: 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.

11. The preparation method according to claim 1, characterized in that: In step (7), the centrifugation is performed at a centrifugal speed of 3000 rpm to 10000 rpm for 10 min to 30 min; and the drying is performed by forced air drying or vacuum drying at 40° C. to 80° C. for 2 h to 8 h.

Images