A method for purifying daptomycin

Abstract

This invention belongs to the field of biomedicine, and specifically relates to a purification method for daptomycin. The purification method includes loading a sample solution onto a C18 resin chromatography column equilibrated with an equilibration buffer, and eluting the C18 resin chromatography column with an eluent; wherein the sample solution, the equilibration buffer, and the eluent contain a sodium salt at a concentration of 0.1–1.0 (w / v)%, and the pH of the sample solution, the equilibration buffer, and the eluent is 6–8; the ethanol content of the sample solution, the equilibration buffer, and the eluent is 5–40%; and the sodium salt is selected from sodium sulfate, sodium phosphate, and sodium chloride. The purification method provided by this invention can achieve efficient removal of impurity 4 and unknown impurities from daptomycin, and the obtained product meets market demand.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a daptomycin purification method. BACKGROUND

[0002] With the development of the market, the competition for daptomycin products is becoming more and more fierce. The quality of daptomycin products is also becoming higher and higher, and the impurity removal rate is the key to measuring the quality of daptomycin products. However, the existing technology can only produce products with impurity 4 of 0.37% and unknown impurities of 0.3%, which cannot meet the quality requirements of impurity 4 < 0.25% and unknown impurities < 0.1% in daptomycin products. SUMMARY

[0003] In order to overcome the defects of the above-mentioned prior art, the technical problem to be solved by the present application is to provide a daptomycin purification method which can improve the quality of daptomycin, specifically to meet the requirements of impurity 4 < 0.25% and unknown impurities < 0.1%.

[0004] In order to solve the above technical problems, the present application provides a daptomycin purification method, comprising the steps of loading a sample solution into a C18 resin chromatographic column equilibrated with an equilibration liquid, and eluting the C18 resin chromatographic column with an eluent.

[0005] Among them, the sample solution, the equilibration liquid and the eluent contain sodium salt with a concentration of 0.1-1.0(W / V)%, the pH of the sample solution, the equilibration liquid and the eluent is 6-8, and the ethanol degree of the equilibration liquid and the eluent is 5%-40%.

[0006] The sodium salt is selected from one of sodium sulfate, sodium phosphate and sodium chloride.

[0007] The daptomycin purification method provided by the present application can realize efficient purification of daptomycin, and the purified product meets the requirements of impurity 4 < 0.25% and unknown impurities < 0.1%. DETAILED DESCRIPTION

[0008] In order to explain the technical content, purposes and effects of the present application in detail, the following embodiments are described.

[0009] A daptomycin purification method, comprising the steps of loading a sample solution into a C18 resin chromatographic column equilibrated with an equilibration liquid, and eluting the C18 resin chromatographic column with an eluent. Among them, the sample solution, the equilibration liquid and the eluent contain sodium salt with a concentration of 0.1-1.0(W / V)%, the pH of the sample solution, the equilibration liquid and the eluent is 6-8, and the ethanol degree of the equilibration liquid and the eluent is 5%-20%. The sodium salt includes but is not limited to one of sodium sulfate, sodium phosphate, sodium chloride and other sodium salts.

[0010] This method involves preparing a salt-containing solution by adding a certain amount of salt to the sample solution, equilibration buffer, and eluent. This alters the entire chromatography system, improving the separation of daptomycin from impurities and facilitating the elution of daptomycin from the C18 resin column. Simultaneously, it effectively removes impurities during the elution process, meeting the requirements of impurity 4 < 0.25% and unknown impurities < 0.1%. Compared to existing technologies, this chromatography process reduces production costs while ensuring the product meets market demands.

[0011] In one embodiment, the ethanol content of the sample solution, the equilibration solution, and the eluent is 5% to 40%. That is, in this embodiment, both the equilibration solution and the eluent are ethanol solutions. It should be noted that the ethanol content mentioned herein is a general definition, i.e., the volume concentration of ethanol.

[0012] In one embodiment, the ethanol content of the effluent from the C18 resin chromatography column after equilibration is 5-40%.

[0013] In one embodiment, the purification method further includes the step of removing alcohol from the daptomycin powder solution and concentrating it to obtain the sample solution. The concentrated sample solution contains daptomycin at a concentration ≥10 g / L. In this embodiment, the daptomycin powder solution is prepared as follows.

[0014] In one embodiment, the alcohol removal is to remove ethanol from the daptomycin powder solution until the alcohol meter reading is 0.

[0015] In one optional embodiment, the alcohol removal and concentration are performed using a nanofiltration membrane. Preferably, the nanofiltration membrane has a molecular weight cutoff of 300 Da.

[0016] Specifically, the purification method for daptomycin includes the following steps:

[0017] S1. After using a nanofiltration membrane to remove alcohol from the daptomycin powder solution until the alcohol meter reading is 0, the alcohol-removed liquid is obtained.

[0018] S2. The alcohol-removing solution is concentrated using a nanofiltration membrane until the daptomycin content is ≥10g / L to obtain a nanofiltration concentrate;

[0019] S3. Add a certain amount of salt solution or solid salt to the nanofiltration concentrate, control the salt concentration to 0.1-1.0 (W / V)%, and adjust the pH to 6-8 with sodium hydroxide or hydrochloric acid. Add ethanol to prepare an ethanol content of 5-40% to obtain the column loading solution.

[0020] S4. Add a certain amount of salt solution or solid salt to the equilibrium solution, control the salt concentration to 0.1-1.0 (W / V)%, and adjust the pH to 6-8 with sodium hydroxide or hydrochloric acid. Equilibrate the C18 resin chromatography column with the equilibrium solution until the ethanol content of the C18 resin chromatography column effluent is 5-40%.

[0021] S5. Prepare the eluent by adding a certain amount of salt solution or solid salt to the eluent, controlling the salt concentration to be 0.1-1.0 (W / V)%, and adjusting the pH to 6-8 with sodium hydroxide or hydrochloric acid, and the ethanol content to be 5-40%.

[0022] S6. After loading the above-mentioned column liquid onto a C18 resin chromatography column, elute with the above-mentioned eluent, and simultaneously detect the daptomycin content and related impurity content in the filtrate by UPLC. Collect the effective collection liquid of daptomycin intermediate with the purity meeting the standard (impurity 4 < 0.25%, unknown impurities < 0.1%).

[0023] S7. The effective collection liquid of the daptomycin intermediate is subjected to nanofiltration to remove alcohol and concentrated to a feed unit of 100g / L, and then freeze-dried to obtain the daptomycin product.

[0024] More specifically, the alcohol removal and concentration are as follows: The feed solution (daptomycin crude powder desalting solution, daptomycin intermediate effective collection solution) is concentrated using a nanofiltration membrane to half its volume, and then nanofiltration is performed while adding water for top washing, maintaining the water addition rate consistent with the nanofiltration flow rate. When the gas phase peak area of ​​the filtrate is sampled and found to be <10, the alcohol-removed solution is obtained. The nanofiltration flow rate is 2.0–2.5 L / h, preferably 2.3 L / h (this nanofiltration flow rate parameter is used for alcohol removal and concentration in the following examples and comparative examples).

[0025] Of course, in order to obtain daptomycin powder, it is preferable to freeze-dry the daptomycin product in S7. Preferably, the freeze-drying conditions are: first stage -70°C pre-freezing for 2 hours, second stage freeze-drying at 0°C for 10 hours, and third stage freeze-drying at 10°C for 60 hours.

[0026] Example 1

[0027] A method for purifying daptomycin includes the following steps:

[0028] S1. Dissolve 15g of daptomycin powder solution in deionized water to 1L. After it is completely dissolved, use a small membrane apparatus (equipped with a nanofiltration membrane with a molecular weight cutoff of 300Da) to increase conductivity and concentrate to 500mL. While concentrating, add water and keep the water addition rate consistent with the dialysis flow rate. When the alcohol content of the filtrate at the filter end is 0, compress the volume to 1.2L, that is, the daptomycin content is 12.5g / L, to obtain the nanofiltration concentrate.

[0029] S2. Take 240 mL of the nanofiltration concentrate (containing 3 g of daptomycin), add 1.92 g of sodium sulfate to a concentration of 0.8%, adjust the pH to 7.26 with 2 mol / L NaOH or 2 mol / L HCl, add a certain amount of ethanol, and prepare a column loading solution containing 14% ethanol.

[0030] Prepare 1L of equilibrium solution, wherein the ethanol content of the equilibrium solution is 14% and the sodium sulfate concentration is 0.8% (i.e., 8g of sodium sulfate is dissolved);

[0031] Prepare 3L of eluent, wherein the ethanol content of the eluent is 14% and the sodium sulfate concentration is 0.8% (i.e., 24g of sodium sulfate is dissolved);

[0032] S3. Load the above-mentioned loading solution onto a 200mL C18 resin chromatography column, elute with the above-mentioned eluent, and connect the filtrate to UPLC for analysis. Collect the effective collection solution of daptomycin intermediate with impurities <0.25% and unknown impurities <0.1%.

[0033] S4. The effective collection liquid of 0.9L of the daptomycin intermediate is concentrated and the alcohol is removed by nanofiltration membrane (molecular weight cutoff 300Da) until the gas phase peak area is <10. After nanofiltration, the product is lyophilized to obtain the daptomycin product.

[0034] Comparative Example 1

[0035] A method for purifying daptomycin differs from Example 1 in that the ethanol content in the loading solution, equilibration solution, and eluent is inconsistent.

[0036] A method for purifying daptomycin includes the following steps:

[0037] S1. Dissolve 15g of daptomycin powder solution in deionized water to 1L. After it is completely dissolved, use a small membrane apparatus (equipped with a nanofiltration membrane with a molecular weight cutoff of 300Da) to increase conductivity and concentrate to 500mL. While concentrating, add water and keep the water addition rate consistent with the dialysis flow rate. When the alcohol content of the filtrate at the filter end is 0, compress the volume to 1.2L, that is, the daptomycin content is 12.5g / L, to obtain the nanofiltration concentrate.

[0038] S2. Take 240 mL of the nanofiltration concentrate (containing 3 g of daptomycin), add 1.92 g of sodium sulfate to a concentration of 0.8%, adjust the pH to 7.26 with 2 mol / L NaOH or 2 mol / L HCl, add a certain amount of ethanol, and prepare a column loading solution containing 25% ethanol.

[0039] Prepare 1L of equilibrium solution, wherein the ethanol content of the equilibrium solution is 25% and the sodium sulfate concentration is 0.8% (i.e., 8g of sodium sulfate is dissolved);

[0040] Prepare 3L of eluent, wherein the ethanol content of the eluent is 25% and the sodium sulfate concentration is 0.8% (i.e., 24g of sodium sulfate is dissolved);

[0041] S3. Load the above-mentioned loading solution onto a 200mL C18 resin chromatography column, elute with the above-mentioned eluent, and connect the filtrate to UPLC for analysis. Collect the effective collection solution of daptomycin intermediate with impurities <0.25% and unknown impurities <0.1%.

[0042] S4. The effective collection liquid of 0.9L of the daptomycin intermediate is concentrated and the alcohol is removed by nanofiltration membrane (molecular weight cutoff 300Da) until the gas phase peak area is <10. After nanofiltration, the product is lyophilized to obtain the daptomycin product.

[0043] Detection example

[0044] The composition of the daptomycin crude powder desalting solution and daptomycin product in Example 1 and Comparative Example 1 was analyzed, and the results are shown in Table 1.

[0045] Table 1

[0046]

[0047] Impurity 4 is a known impurity identified and named by our company (relative retention time is 0.89), and unknown impurities are all unknown impurities other than those identified.

[0048] As can be seen from Table 1, the purification method for daptomycin provided by the present invention can achieve efficient removal of unknown impurities and impurity 4, and improve the overall yield.

[0049] In summary, the daptomycin purification method provided by this invention can achieve efficient purification of daptomycin, and the purified product meets the requirements of impurity 4 < 0.25% and unknown impurities < 0.1%.

[0050] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for purifying daptomycin, characterized in that, Includes the following steps: S1. Use a nanofiltration membrane to remove alcohol from the crude daptomycin powder solution to obtain the alcohol-removed solution; S2. The alcohol-removing solution is concentrated using a nanofiltration membrane until the daptomycin content is ≥10g / L to obtain a nanofiltration concentrate; S3. Add salt solution or solid salt to the nanofiltration concentrate, control the salt concentration to 0.1~1.0% (W / V), adjust the pH to 6~8 with sodium hydroxide or hydrochloric acid, add ethanol to prepare an ethanol content of 14% (V / V), and obtain the column loading solution. S4. Add salt solution or solid salt to the equilibration solution, control the salt concentration to 0.1~1.0% (W / V), and adjust the pH to 6~8 with sodium hydroxide or hydrochloric acid. The ethanol content of the equilibration solution is 14% (V / V). Equilibrate the C18 resin chromatography column with the equilibration solution. S5. Prepare the eluent by adding a salt solution or solid salt to the eluent, controlling the salt concentration to be 0.1~1.0% (W / V), and adjusting the pH to 6~8 with sodium hydroxide or hydrochloric acid, and the ethanol content to be 14% (V / V). S6. After loading the above-column solution onto a C18 resin chromatography column, elute with the above-column solution, and simultaneously detect the daptomycin content and related impurity content in the filtrate by UPLC, and collect the effective collection solution of daptomycin intermediate with the required purity. S7. The effective collection liquid of the daptomycin intermediate is subjected to alcohol removal by nanofiltration and concentrated, and then freeze-dried to obtain the daptomycin product. In S3 to S5, the salt in the salt solution or solid salt is selected from sodium sulfate, sodium phosphate, and sodium chloride.

2. The purification method according to claim 1, characterized in that, The nanofiltration membrane has a molecular weight cutoff of 300 Da.

3. The purification method according to claim 1, characterized in that, S1 specifically involves using a nanofiltration membrane to remove alcohol from the crude daptomycin powder solution until the ethanol content reaches 0, thereby obtaining the alcohol-removed solution.

Images