Preparation method of polymyxin B sulfate for injection

Abstract

The invention discloses a preparation method of polymyxin B sulfate for injection, and relates to the technical field of medicines. The auxiliary material of the prepared polymyxin B sulfate for injection is only water for injection, the specification of the polymyxin B sulfate for injection is 0.5 million units, the process comprises the following steps: sub-packaging the polymyxin B sulfate liquid and then freeze-drying, and pressurizing and depressurizing a product box by using inert gas nitrogen in a pre-freezing stage, so that the serious overcooling of the solution can be avoided, and the product quality is improved. According to the freeze-dried product, the formed ice nucleus is large, then the first-stage drying stage and the desorption drying stage are carried out, in the drying stage, more and larger water vapor channels can be formed in the freeze-dried product, and the drying time is shortened. According to the preparation method disclosed by the invention, the freeze-drying time is remarkably shortened, the energy consumption is reduced, the cost is reduced, meanwhile, the obtained polymyxin B sulfate for injection has the advantages that the quality and the stability are remarkably improved, the number of insoluble particles is remarkably reduced, single impurities and total impurities are few, and adverse reactions can be avoided.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for preparing polymyxin B sulfate for injection. Background Art

[0002] Polymyxin is produced by Bacillus polymyxa, which is a general term for a family of alkaline polypeptide antibiotics. The sulfate of polymyxin is also called colistin sulfate, which is composed of a variety of amino acids and fatty acids. According to the different amino acids and fatty acids, polymyxins are mainly divided into six categories: polymyxin A, B, C, D, E, and M. Due to the unstable physical and chemical properties of polymyxin B, polymyxin B is often used in the form of sulfate in clinical practice. In clinical practice, polymyxin B sulfate is mainly used to treat a variety of diseases caused by Gram-negative bacteria, including dysentery, meningitis, etc., and can also be used to prevent Gram-negative bacteria infections without developing drug resistance. Polymyxin B sulfate, as a sulfate form of polymyxin B, is a white or similar crystalline powder with a bitter taste, no odor, and hygroscopicity. Polymyxin B sulfate is a water-soluble substance that is easily soluble in aqueous solutions but not easily soluble in commonly used organic solvents such as ether, ketones, esters and chloroform. Polymyxin B is a polypeptide composed of ten amino acids, seven of which are linked to each other at the N-terminus to form a cyclic fatty chain. The amino acids are linked to form a cyclic fatty chain at the N-terminus, while the amino acids at the tail are scattered and flexible, so there is no fixed structure in the solution.

[0003] Currently, polymyxin B sulfate freeze-dried powder suffers from product shrinkage and cracking, and the reconstitution effect is poor. After reconstitution, the number of insoluble particles is high, which increases the risk of medication.

[0004] Patent CN 108309945 B discloses a method for preparing polymyxin B sulfate for injection. The excipient of polymyxin B sulfate for injection prepared in the invention is only water for injection. The quality and stability of polymyxin B sulfate for injection prepared by the process of the present invention are significantly improved, and the number of insoluble particles is significantly reduced. However, the drying temperature selected in the patent is relatively high at 38°C, and polymyxin B sulfate is a polypeptide, and a high temperature is not conducive to the stability of the polypeptide.

[0005] Patent CN 111420027 A discloses a preparation process of a lyophilized powder containing polymyxin B sulfate, using 3g / L to 16g / L tert-butyl alcohol to increase the solubility of polymyxin B sulfate, and the prepared lyophilized sample is well formed, without shrinkage and cracking, and has a good re-dissolution effect. In this patent, the organic solvent tert-butyl alcohol is introduced into the preparation of polymyxin B sulfate for injection. The temperature of the first drying stage in the patent is -20°C, the time is 4h to 6h, preferably 5h, the time of the first drying is short, and the analytical drying temperature selected in the invention to remove tert-butyl alcohol is 35°C. The tert-butyl alcohol in the final product is still 0.01%, which increases the risk of medication. Summary of the invention

[0006] In view of the above-mentioned defects of the prior art, the present invention proposes a method for preparing polymyxin B sulfate for injection to solve the problems raised by the above-mentioned background technology.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] A method for preparing polymyxin B sulfate for injection comprises the following steps:

[0009] S1. Prepare polymyxin B sulfate solution with a mass concentration of 15-19 g / L;

[0010] S2, filtering the polymyxin B sulfate liquid, packaging it in the filling amount determined according to the specifications, and sending the packaged liquid into the freeze dryer;

[0011] S3. Freeze-drying

[0012] (1) Pre-freezing

[0013] (1)-1 Under standard atmospheric pressure, reduce the plate temperature to 1-8°C and maintain for 50-70 minutes. The cooling time is 20-30 minutes.

[0014] (1) -2 Nitrogen is pressurized to 23-28.5 psi, the plate temperature is reduced to -7--1°C, maintained for 50-70 minutes, and the cooling time is 5-20 minutes;

[0015] (1) -3 Depressurize to 1-3 psi, lower the plate temperature to -35-29°C, maintain for 100-150 minutes, and the cooling time is 20-35 minutes;

[0016] (2) Primary drying

[0017] (2) After the pre-freezing is completed, the plate temperature is raised to -11 to -8°C and maintained for 80 to 100 minutes, the vacuum degree is 0.001 to 0.005 psi, and the heating time is 35 to 45 minutes;

[0018] (2)-2 Raise the plate temperature to 3-10°C, maintain for 1200-1800 min, vacuum degree 0.001-0.005 psi, heating time 35-45 min;

[0019] (3) Analytical drying

[0020] (3)-1 Raise the plate temperature to 20-28°C, maintain for 50-70 minutes, vacuum degree 0.001-0.005 psi, heating time 35-45 minutes;

[0021] S4. After freeze-drying, nitrogen degassing, backfilling, plugging, capping and packaging are carried out, and after passing the inspection, the polymyxin B sulfate for injection is obtained.

[0022] Preferably, a method for preparing polymyxin B sulfate for injection comprises the following steps:

[0023] S1. Prepare polymyxin B sulfate solution with a mass concentration of 15.36 g / L;

[0024] S2, filtering the polymyxin B sulfate liquid, packaging it in the filling amount determined according to the specifications, and sending the packaged liquid into the freeze dryer;

[0025] S3. Freeze-drying

[0026] (1) Pre-freezing

[0027] (1)-1 Under standard atmospheric pressure, reduce the plate temperature to 5°C and maintain for 60 minutes, with a cooling time of 25 minutes;

[0028] (1) -2 Nitrogen pressure is increased to 26 psi, the plate temperature is lowered to -5°C, maintained for 60 min, and the cooling time is 10 min;

[0029] (1) -3 Depressurize to 2 psi, lower the plate temperature to -30°C, maintain for 120 min, and cool down for 25 min;

[0030] (2) Primary drying

[0031] (2) After the prefreezing is completed, the plate temperature is raised to -9°C and maintained for 90 min, the vacuum degree is 0.004 psi, and the heating time is 44 min;

[0032] (2)-2 Raise the plate temperature to 5°C and maintain for 1600 min, with a vacuum degree of 0.004 psi and a heating time of 40 min;

[0033] (3) Analytical drying

[0034] (3)-1 Raise the plate temperature to 25°C and maintain for 60 minutes, with a vacuum degree of 0.004 psi and a heating time of 40 minutes;

[0035] S4. After freeze-drying, nitrogen degassing, backfilling, plugging, capping and packaging are carried out, and after passing the inspection, the polymyxin B sulfate for injection is obtained.

[0036] Preferably, the present invention adopts the Controlyo controllable nucleation technology of the SP Lyostar 3 freeze dryer. The SP Lyostar 3 freeze dryer is a pilot-scale freeze dryer, a high-end equipment integrating process analysis technology (PAT) tools, Controlyo controllable nucleation technology, and Auto MTM / SMART technology. The process analysis technology (PAT) tool can ensure the accuracy, reliability and uniformity of the process; the Controlyo controllable nucleation technology can effectively control the freezing point, effectively improve the product appearance, improve the product uniformity, and shorten the freeze-drying cycle. The Controlyo controllable nucleation technology is an innovative technology. During the freezing stage of the freeze dryer, the product box is pressurized and depressurized by an inert gas, so that the product nucleates almost simultaneously and the nucleation is uniform.

[0037] Preferably, the microporous filter membrane in step S2 is a polyvinylidene fluoride microporous filter membrane.

[0038] Preferably, the specification in step S2 is 500,000 units.

[0039] Preferably, the filling volume in step S2 is 4 mL.

[0040] Preferably, the liquid medicine in step S2 is packaged in medium borosilicate controlled injection bottles.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows: the excipient of the polymyxin B sulfate for injection prepared by the present invention is only water for injection, which reduces the introduction of other excipients in the process of producing the liquid medicine and reduces the safety risks when the medicine is used. The polymyxin B sulfate for injection obtained by the preparation method of the present invention has the advantages of significantly improved quality and stability, significantly reduced number of insoluble particles, less single impurity and total impurity content, and can avoid adverse reactions; and significantly shortens the freeze-drying time, saves energy consumption, reduces costs, and improves the market competitiveness of products. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0043] Example 1

[0044] A method for preparing polymyxin B sulfate for injection comprises the following steps:

[0045] S1. Add 15 g of polymyxin B sulfate into 900 mL of water for injection and stir until completely dissolved. Add water for injection to make up to 1000 mL and mix well to obtain a drug solution.

[0046] S2, filtering the drug solution obtained in step S1 through a 0.22 μm polyvinylidene fluoride microporous filter membrane, determining the filling volume of 4 mL according to the specification of 500,000 units, and filling it into a borosilicate controlled injection bottle, and sending the packaged drug solution into a freeze dryer;

[0047] S3. Freeze-drying

[0048] (1) Pre-freezing

[0049] (1)-1 Under standard atmospheric pressure, reduce the plate temperature to 1°C and maintain for 50 minutes. The cooling time is 20 minutes.

[0050] (1) -2 Nitrogen pressure is increased to 23 psi, the plate temperature is lowered to -7°C, maintained for 50 min, and the cooling time is 5 min;

[0051] (1) -3 Depressurize to 1 psi, lower the plate temperature to -35°C, maintain for 100 min, and cool down for 20 min;

[0052] (2) Primary drying

[0053] (2) After the pre-freezing is completed, the plate temperature is raised to -11°C and maintained for 80 min, the vacuum degree is 0.001 psi, and the heating time is 35 min;

[0054] (2)-2 Raise the plate temperature to 3°C and maintain for 1200 min, with a vacuum degree of 0.001 psi and a heating time of 35 min;

[0055] (3) Analytical drying

[0056] (3)-1 Raise the plate temperature to 20°C and maintain for 50 minutes, with a vacuum degree of 0.001 psi and a heating time of 35 minutes;

[0057] S4. After freeze-drying, nitrogen is backfilled, plugs are pressed, caps are rolled, and packaging is carried out. After passing the inspection, the packaging is packaged and stored, and polymyxin B sulfate for injection is obtained.

[0058] Example 2

[0059] A method for preparing polymyxin B sulfate for injection comprises the following steps:

[0060] S1. Add 19 g of polymyxin B sulfate into 900 mL of water for injection and stir until completely dissolved, add water for injection to make up to 1000 mL, mix well, and obtain a drug solution;

[0061] S2, filtering the drug solution obtained in step S1 through a 0.22 μm polyvinylidene fluoride microporous filter membrane, determining the filling volume of 4 mL according to the specification of 500,000 units, and filling it into a borosilicate controlled injection bottle, and sending the packaged drug solution into a freeze dryer;

[0062] S3. Freeze-drying

[0063] (1) Pre-freezing

[0064] (1)-1 Under standard atmospheric pressure, reduce the plate temperature to 8°C and maintain for 70 minutes, with a cooling time of 30 minutes;

[0065] (1) -2 Nitrogen pressure is increased to 26 psi, the plate temperature is lowered to -1°C, maintained for 70 min, and the cooling time is 20 min;

[0066] (1) -3 Depressurize to 3 psi and lower the plate temperature to -29°C, maintain for 150 min, and the cooling time is 35 min;

[0067] (2) Primary drying

[0068] (2) After the prefreezing is completed, the plate temperature is raised to -8°C and maintained for 100 min, the vacuum degree is 0.005 psi, and the heating time is 45 min;

[0069] (2)-2 Raise the plate temperature to 10°C and maintain for 1800 min, with a vacuum degree of 0.005 psi and a heating time of 45 min;

[0070] (3) Analytical drying

[0071] (3)-1 Raise the plate temperature to 28°C and maintain for 70 minutes, with a vacuum degree of 0.005 psi and a heating time of 45 minutes;

[0072] S4. After freeze-drying, nitrogen is backfilled, plugs are pressed, caps are rolled, and packaging is carried out. After passing the inspection, the packaging is packaged and stored, and polymyxin B sulfate for injection is obtained.

[0073] Example 3

[0074] A method for preparing polymyxin B sulfate for injection comprises the following steps:

[0075] S1. Add 15.36 g of polymyxin B sulfate into 900 mL of water for injection and stir until completely dissolved. Add water for injection to make up to 1000 mL and mix well to obtain a drug solution.

[0076] S2, filtering the drug solution obtained in step S1 through a 0.22 μm polyvinylidene fluoride microporous filter membrane, determining the filling volume of 4 mL according to the specification of 500,000 units, and filling it into a borosilicate controlled injection bottle, and sending the packaged drug solution into a freeze dryer;

[0077] S3. Freeze-drying

[0078] (1) Pre-freezing

[0079] (1)-1 Under standard atmospheric pressure, reduce the plate temperature to 5°C and maintain for 60 minutes, with a cooling time of 25 minutes;

[0080] (1) -2 Nitrogen pressure is increased to 28.5 psi, the plate temperature is lowered to -5°C, maintained for 60 min, and the cooling time is 10 min;

[0081] (1) -3 Release the pressure to 2 psi and lower the plate temperature to -30°C, maintain for 120 min, and the cooling time is 25 min;

[0082] (2) Primary drying

[0083] (2) After the prefreezing is completed, the plate temperature is raised to -9°C and maintained for 90 min, the vacuum degree is 0.004 psi, and the heating time is 44 min;

[0084] (2)-2 Raise the plate temperature to 5°C and maintain for 1600 min, with a vacuum degree of 0.004 psi and a heating time of 40 min;

[0085] (3) Analytical drying

[0086] (3)-1 Raise the plate temperature to 25°C and maintain for 60 minutes, with a vacuum degree of 0.004 psi and a heating time of 40 minutes;

[0087] S4. After freeze-drying, nitrogen is backfilled, plugs are pressed, caps are rolled, and packaging is carried out. After passing the inspection, the packaging is packaged and stored, and polymyxin B sulfate for injection is obtained.

[0088] Comparative Example 1

[0089] The difference between this comparative example and Example 3 is that in (1)-2 and (1)-3, there is no nitrogen pressurization and pressure relief, that is, there is no Controlyo controlled nucleation technology, and the other ingredients and steps remain unchanged.

[0090] A method for preparing polymyxin B sulfate for injection comprises the following steps:

[0091] S1. Add 15.36 g of polymyxin B sulfate into 900 mL of water for injection and stir until completely dissolved. Add water for injection to make up to 1000 mL and mix well to obtain a drug solution.

[0092] S2, filtering the drug solution obtained in step S1 through a 0.22 μm polyvinylidene fluoride microporous filter membrane, determining the filling volume of 4 mL according to the specification of 500,000 units, and filling it into a borosilicate controlled injection bottle, and sending the packaged drug solution into a freeze dryer;

[0093] S3. Freeze-drying

[0094] (1) Pre-freezing

[0095] (1)-1 Under standard atmospheric pressure, reduce the plate temperature to 5°C and maintain for 60 minutes, with a cooling time of 25 minutes;

[0096] (1)-2 Under standard atmospheric pressure, reduce the plate temperature to -5°C and maintain for 60 minutes. The cooling time is 10 minutes.

[0097] (1)-3 Under standard atmospheric pressure, reduce the plate temperature to -30°C and maintain for 120 minutes, with a cooling time of 25 minutes;

[0098] (2) Primary drying

[0099] (2) After the prefreezing is completed, the plate temperature is raised to -9°C and maintained for 90 min, the vacuum degree is 0.004 psi, and the heating time is 44 min;

[0100] (2)-2 Raise the plate temperature to 5°C and maintain for 1600 min, with a vacuum degree of 0.004 psi and a heating time of 40 min;

[0101] (3) Analytical drying

[0102] (3)-1 Raise the plate temperature to 25°C and maintain for 60 minutes, with a vacuum degree of 0.004 psi and a heating time of 40 minutes;

[0103] S4. After freeze-drying, nitrogen is backfilled, plugs are pressed, caps are rolled, and packaging is carried out. After passing the inspection, the packaging is packaged and stored, and polymyxin B sulfate for injection is obtained.

[0104] Comparative Example 2

[0105] The difference between this comparative example and Example 3 is that the temperature of the primary drying stage is -20°C, the drying time is 300 min, and the other ingredients and steps remain unchanged.

[0106] A method for preparing polymyxin B sulfate for injection comprises the following steps:

[0107] S1. Add 15.36 g of polymyxin B sulfate into 900 mL of water for injection and stir until completely dissolved. Add water for injection to make up to 1000 mL and mix well to obtain a drug solution.

[0108] S2, filtering the drug solution obtained in step S1 through a 0.22 μm polyvinylidene fluoride microporous filter membrane, determining the filling volume of 4 mL according to the specification of 500,000 units, and filling it into a borosilicate controlled injection bottle, and sending the packaged drug solution into a freeze dryer;

[0109] S3. Freeze-drying

[0110] (1) Pre-freezing

[0111] (1)-1 Under standard atmospheric pressure, reduce the plate temperature to 5°C and maintain for 60 minutes, with a cooling time of 25 minutes;

[0112] (1) -2 Nitrogen pressure is increased to 28.5 psi, the plate temperature is lowered to -5°C, maintained for 60 min, and the cooling time is 10 min;

[0113] (1) -3 Depressurize to 2 psi, lower the plate temperature to -30°C, maintain for 120 min, and cool down for 25 min;

[0114] (2) Primary drying

[0115] (2) After the prefreezing is completed, the plate temperature is raised to -20°C and maintained for 300 min, the vacuum degree is 0.004 psi, and the heating time is 44 min;

[0116] (3) Analytical drying

[0117] (3)-1 Raise the plate temperature to 25°C and maintain for 60 minutes, with a vacuum degree of 0.004 psi and a heating time of 40 minutes;

[0118] S4. After freeze-drying, samples were collected. The obtained samples were in a solution state, and no freeze-dried cake-like samples were obtained.

[0119] Comparative Example 3

[0120] The difference between this comparative example and Example 3 is that the primary drying (2)-2 stage drying time is 2400 min, and the other ingredients and steps remain unchanged.

[0121] A method for preparing polymyxin B sulfate for injection comprises the following steps:

[0122] S1. Add 15.36 g of polymyxin B sulfate into 900 mL of water for injection and stir until completely dissolved. Add water for injection to make up to 1000 mL and mix well to obtain a drug solution.

[0123] S2, filtering the drug solution obtained in step S1 through a 0.22 μm polyvinylidene fluoride microporous filter membrane, determining the filling volume of 4 mL according to the specification of 500,000 units, and filling it into a borosilicate controlled injection bottle, and sending the packaged drug solution into a freeze dryer;

[0124] S3. Freeze-drying

[0125] (1) Pre-freezing

[0126] (1)-1 Under standard atmospheric pressure, reduce the plate temperature to 5°C and maintain for 60 minutes, with a cooling time of 25 minutes;

[0127] (1) -2 Nitrogen pressure is increased to 28.5 psi, the plate temperature is lowered to -5°C, maintained for 60 min, and the cooling time is 10 min;

[0128] (1) -3 Depressurize to 2 psi, lower the plate temperature to -30°C, maintain for 120 min, and cool down for 25 min;

[0129] (2) Primary drying

[0130] (2) After the prefreezing is completed, the plate temperature is raised to -9°C and maintained for 90 min, the vacuum degree is 0.004 psi, and the heating time is 44 min;

[0131] (2)-2 Raise the plate temperature to 5°C and maintain for 2400 min, with a vacuum degree of 0.004 psi and a heating time of 40 min;

[0132] (3) Analytical drying

[0133] (3)-1 Raise the plate temperature to 25°C and maintain for 60 minutes, with a vacuum degree of 0.004 psi and a heating time of 40 minutes;

[0134] S4. After freeze-drying, nitrogen is backfilled, plugs are pressed, caps are rolled, and packaging is carried out. After passing the inspection, the packaging is packaged and stored, and polymyxin B sulfate for injection is obtained.

[0135] Comparative Example 4

[0136] The difference between this comparative example and Example 3 is that: (1) the pre-freezing does not involve staged cooling, and there is no nitrogen pressurization and pressure relief step in (1)-2 and (1)-3, and the other components and steps remain unchanged.

[0137] A method for preparing polymyxin B sulfate for injection comprises the following steps:

[0138] S1. Add 15.36 g of polymyxin B sulfate into 900 mL of water for injection and stir until completely dissolved. Add water for injection to make up to 1000 mL and mix well to obtain a drug solution.

[0139] S2, filtering the drug solution obtained in step S1 through a 0.22 μm polyvinylidene fluoride microporous filter membrane, determining the filling volume of 4 mL according to the specification of 500,000 units, and filling it into a borosilicate controlled injection bottle, and sending the packaged drug solution into a freeze dryer;

[0140] S3. Freeze-drying

[0141] (1) Pre-freezing

[0142] (1)-1 Under standard atmospheric pressure, reduce the plate temperature to -30℃ and maintain it for 240min. The cooling time is 60min.

[0143] (2) Primary drying

[0144] (2) After the prefreezing is completed, the plate temperature is raised to -9°C and maintained for 90 min, the vacuum degree is 0.004 psi, and the heating time is 44 min;

[0145] (2)-2 Raise the plate temperature to 5°C and maintain for 1600 min, with a vacuum degree of 0.004 psi and a heating time of 40 min;

[0146] (3) Analytical drying

[0147] (3)-1 Raise the plate temperature to 25°C and maintain for 60 minutes, with a vacuum degree of 0.004 psi and a heating time of 40 minutes;

[0148] S4. After freeze-drying, nitrogen is backfilled, plugs are pressed, caps are rolled, and packaging is carried out. After passing the inspection, the packaging is packaged and stored, and polymyxin B sulfate for injection is obtained.

[0149] Comparative Example 5

[0150] The difference between this comparative example and Example 3 is that: (2) the primary drying is not heated in stages, and the other components and steps remain unchanged.

[0151] A method for preparing polymyxin B sulfate for injection comprises the following steps:

[0152] S1. Add 15.36 g of polymyxin B sulfate into 900 mL of water for injection and stir until completely dissolved. Add water for injection to make up to 1000 mL and mix well to obtain a drug solution.

[0153] S2, filtering the drug solution obtained in step S1 through a 0.22 μm polyvinylidene fluoride microporous filter membrane, determining the filling volume of 4 mL according to the specification of 500,000 units, and filling it into a borosilicate controlled injection bottle, and sending the packaged drug solution into a freeze dryer;

[0154] S3. Freeze-drying

[0155] (1) Pre-freezing

[0156] (1)-1 Under standard atmospheric pressure, reduce the plate temperature to 5°C and maintain for 60 minutes, with a cooling time of 25 minutes;

[0157] (1) -2 Nitrogen pressure is increased to 28.5 psi, the plate temperature is lowered to -5°C, maintained for 60 min, and the cooling time is 10 min;

[0158] (1) -3 Depressurize to 2 psi, lower the plate temperature to -30°C, maintain for 120 min, and cool down for 25 min;

[0159] (2) Primary drying

[0160] (2)-1 After the prefreezing is completed, the plate temperature is raised to 5°C and maintained for 1690 min, the vacuum degree is 0.004 psi, and the heating time is 84 min;

[0161] (3) Analytical drying

[0162] (3)-1 Raise the plate temperature to 25°C and maintain for 60 minutes, with a vacuum degree of 0.004 psi and a heating time of 40 minutes;

[0163] S4. After freeze-drying, nitrogen is backfilled, plugs are pressed, caps are rolled, and packaging is carried out. After passing the inspection, the packaging is packaged and stored, and polymyxin B sulfate for injection is obtained.

[0164] Experimental Example 1: Investigation of properties, re-dissolution time, insoluble particles and loss on drying

[0165] 1.1 Experimental Materials

[0166] Polymyxin B sulfate for injection prepared in Examples 1 to 3 and Comparative Examples 1 to 5;

[0167] 1.2 Experimental methods

[0168] 1.2.1 Reconstitution time: Reconstitute polymyxin B sulfate with an equal volume of water for injection, and measure the time it takes for it to completely dissolve in water, which is the reconstitution time;

[0169] 1.2.2 Insoluble particles: Measure the number of insoluble particles in the solution according to the light obscuration method in Part 4 0903 Insoluble Particles Test Method of the 2020 Edition of the Chinese Pharmacopoeia;

[0170] 1.2.3 Loss on drying: in accordance with the 0831 Loss on drying method in Part 4 of the 2020 edition of the Chinese Pharmacopoeia;

[0171] 1.2.4 According to the 1201 Antibiotic Microbiological Test Method of Part IV of the 2020 Edition of the Chinese Pharmacopoeia, the content percentage of polymyxin B sulfate in the prepared polymyxin B sulfate for injection was measured.

[0172] 1.2.5 The area percentage of related substances (total impurities and single impurities) was measured by HPLC area normalization method; the sum of the area percentages of total impurities and polymyxin B components was 100%;

[0173] 1.3 Experimental Results

[0174] As shown in Table 1:

[0175] Table 1 Properties, re-dissolution time, insoluble particles and drying loss

[0176]

[0177]

[0178] As can be seen from Table 1, the polymyxin B sulfate for injection prepared in Examples 1 to 3 has good product properties, short reconstitution time, few insoluble particles and low drying loss, especially Example 3; Comparison between Example 3 and Comparative Example 1 shows that the drug solution is not pressurized and depressurized with nitrogen in the pre-freezing stage, and the controllable nucleation technology is not used, and the insoluble particles, properties and drying loss of the prepared sample are poor; Comparison between Example 3 and Comparative Examples 2 to 3 shows that the primary drying time affects the quality of the final product. If the time is too long, it is easy to cause the product reconstitution time to be prolonged, the number of insoluble particles is high, and the content of related substances is high; if the time is too short, a large amount of water still remains in the product, and the sample dissolves after being taken out of the box, and finally no freeze-dried product is obtained. Comparison between Example 3 and Comparative Examples 4 to 5 shows that the pre-freezing stage and the primary drying stage need to be cooled and heated in stages, and the freeze-dried product obtained by cooling and heating in stages has good properties, fewer insoluble particles, and lower content of related substances.

[0179] Experimental Example 2 High temperature, high humidity and thermal cycle investigation

[0180] 2.1 Experimental Materials

[0181] The polymyxin B sulfate for injection prepared in Example 3;

[0182] 2.2 Inspection conditions

[0183] 2.2.1 High temperature (60℃ and 40℃)

[0184] 2.2.2 High humidity (92.5% RH)

[0185] 2.2.3 Thermal cycle (3 days at 40°C, then 2 days at 2-8°C, 3 cycles)

[0186] 2.3 Experimental Results

[0187] As shown in Table 2:

[0188] Table 2 High temperature, high humidity and thermal cycle investigation

[0189]

[0190]

[0191] As can be seen from Table 2, the polymyxin B sulfate for injection prepared in Example 3 was placed under high temperature and high humidity conditions for 10 days each and under thermal cycle conditions for 15 days. The various indicators of the product obtained by the present invention did not change significantly, and the quality was stable.

[0192] Experimental Example 3: Long-term stability study

[0193] 3.1 Experimental materials and test methods

[0194] The long-term stability of the polymyxin B sulfate for injection prepared in Example 3 and the commercially available product (main ingredient: polymyxin B sulfate, excipient: water) was investigated at a temperature of 25±2° C. and a humidity of 60%±5% RH, and the test indicators were all tested using conventional test methods.

[0195] 3.2 Experimental Results

[0196] As shown in Table 3:

[0197] Table 3 Long-term stability study

[0198]

[0199] As can be seen from Table 3, the product prepared by Example 3 of the present invention can be stored for a long time, and its content decrease rate is low, only 0.57%, the number of particles ≥10 μm is small, and the growth rate is slow when placed for a long time; while the content decrease rate of the commercially available product is 4.7%, the number of particles ≥10 μm is large, and the growth rate is fast when placed for a long time. Compared with the commercially available polymyxin B sulfate injection product, the product quality of the present invention is more stable, long-term storage does not affect the product efficacy, and is more suitable for market promotion.

[0200] Experimental Example 4 Safety Test

[0201] 4.1 Experimental methods:

[0202] According to the requirements of the Drug Registration Management Measures of the State Food and Drug Administration, rabbits and guinea pigs were injected with the polymyxin B sulfate product for injection prepared in Example 3, and the conditions of the rabbits and guinea pigs were observed.

[0203] 4.2 Experimental results:

[0204] (1) In the vascular irritation test of polymyxin B sulfate for injection, no congestion, bleeding, degeneration and necrosis occurred in the rabbit ear vein experimental group and the commercial group, and no pathological damage was found, indicating that intravenous administration of polymyxin B sulfate for injection of the present invention did not produce irritation reaction on the rabbit ear blood vessels;

[0205] (2) In the single-dose irritation test of polymyxin B sulfate for injection, no swelling, congestion, degeneration and necrosis occurred in the biceps femoris of rabbit legs in the experimental group and the commercial group, and no pathological damage was found, indicating that a single intramuscular injection of polymyxin B sulfate for injection of the present invention did not produce a stimulating reaction on the biceps femoris of rabbits;

[0206] (3) In the systemic active allergic test of polymyxin B sulfate for injection, the guinea pigs in the test group were stimulated (dorsal vein

[0207] injection) did not cause systemic allergic reaction, indicating that the polymyxin B sulfate for injection of the present invention did not produce systemic active allergic reaction in guinea pigs;

[0208] The polymyxin B sulfate for injection prepared by the present invention did not cause irritation reaction in rabbits in a vascular irritation test and a single muscle administration irritation test, and the polymyxin B sulfate for injection did not cause an allergic reaction in guinea pigs, indicating that the polymyxin B sulfate for injection prepared by the present invention has high safety.

[0209] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing polymyxin B sulfate for injection, characterized in that: The following steps are involved: S1. Prepare polymyxin B sulfate solution with a mass concentration of 15-19 g / L; S2, filtering the polymyxin B sulfate liquid, packaging it in the filling amount determined according to the specifications, and sending the packaged liquid into the freeze dryer; S3. Freeze-drying (1) Pre-freezing (1)-1 Under standard atmospheric pressure, reduce the plate temperature to 1-8°C and maintain for 50-70 minutes. The cooling time is 20-30 minutes. (1) -2 Nitrogen is pressurized to 23-28.5 psi, the plate temperature is reduced to -7--1°C, maintained for 50-70 minutes, and the cooling time is 5-20 minutes; (1) -3 Depressurize to 1-3 psi, lower the plate temperature to -35-29°C, maintain for 100-150 minutes, and the cooling time is 20-35 minutes; (2) Primary drying (2) After the pre-freezing is completed, the plate temperature is raised to -11 to -8°C and maintained for 80 to 100 minutes, the vacuum degree is 0.001 to 0.005 psi, and the heating time is 35 to 45 minutes; (2)-2 Raise the plate temperature to 3-10°C, maintain for 1200-1800 min, vacuum degree 0.001-0.005 psi, heating time 35-45 min; (3) Analytical drying (3)-1 Raise the plate temperature to 20-28°C, maintain for 50-70 minutes, vacuum degree 0.001-0.005 psi, heating time 35-45 minutes; S4. After freeze-drying, nitrogen is backfilled, plugs are pressed, caps are rolled, and packaging is carried out. After passing the inspection, the packaging is packaged and stored, and polymyxin B sulfate for injection is obtained.

2. The method for preparing polymyxin B sulfate for injection according to claim 1, characterized in that: The following steps are involved: S1. Prepare polymyxin B sulfate solution with a mass concentration of 15.36 g / L; S2, filtering the polymyxin B sulfate liquid, packaging it in the filling amount determined according to the specifications, and sending the packaged liquid into the freeze dryer; S3. Freeze-drying (1) Pre-freezing (1)-1 Under standard atmospheric pressure, reduce the plate temperature to 5°C and maintain for 60 minutes, with a cooling time of 25 minutes; (1) -2 Nitrogen pressure is increased to 26 psi, the plate temperature is lowered to -5°C, maintained for 60 min, and the cooling time is 10 min; (1) -3 Depressurize to 2 psi, lower the plate temperature to -30°C, maintain for 120 min, and cool down for 25 min; (2) Primary drying (2) After the prefreezing is completed, the plate temperature is raised to -9°C and maintained for 90 min, the vacuum degree is 0.004 psi, and the heating time is 44 min; (2)-2 Raise the plate temperature to 5°C and maintain for 1600 min, with a vacuum degree of 0.004 psi and a heating time of 40 min; (3) Analytical drying (3)-1 Raise the plate temperature to 25°C and maintain for 60 minutes, with a vacuum degree of 0.004 psi and a heating time of 40 minutes; S4. After freeze-drying, nitrogen is backfilled, plugs are pressed, caps are rolled, and packaging is carried out. After passing the inspection, the packaging is packaged and stored, and polymyxin B sulfate for injection is obtained.

3. A method for preparing polymyxin B sulfate for injection according to any one of claim 1 or claim 2, characterized in that: The filtration in step S2 is performed using a polyvinylidene fluoride microporous filter membrane.

4. A method for preparing polymyxin B sulfate for injection according to any one of claim 1 or claim 2, characterized in that: The specification in step S2 is 500,000 units.

5. A method for preparing polymyxin B sulfate for injection according to any one of claim 1 or claim 2, characterized in that: The filling volume in step S2 is 4 mL.

6. A method for preparing polymyxin B sulfate for injection according to any one of claim 1 or claim 2, characterized in that: The drug solution described in step S2 is packaged into medium borosilicate controlled injection bottles.

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