Production method of daptomycin

Abstract

The invention relates to the technical field of medicine synthesis, in particular to a daptomycin production method which comprises the following steps: conducting a daptomycin crude product by using a nanofiltration membrane, and performing nanofiltration concentration to obtain a nanofiltration concentrated solution; loading the obtained column loading liquid on NMQ-K resin, washing with water, carrying out gradient elution with a sodium chloride solution, and collecting an effective daptomycin production intermediate collection liquid with the purity of more than 75%; and concentrating by using a nanofiltration membrane until the unit of feed liquid is 50,000-160,000, so as to obtain a purified daptomycin product. The method disclosed by the invention has the beneficial effects that daptomycin is combined with NMQ-K ion exchange resin for adsorption by utilizing the characteristic that daptomycin contains quaternary amine groups, then gradient analysis is performed through a salt solution, and the part meeting the requirements is collected. The yield can be improved, and the production requirement can be met. By using the process disclosed by the invention, a daptomycin product meeting the requirement can be obtained, the sample loading amount is increased to several times of the original sample loading amount, and the impurity removal rate is also increased while the productivity is improved.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical synthesis, and in particular to a method for producing daptomycin. Background Art

[0002] Daptomycin is a light yellow or yellow crystalline powder with a melting point between 202-204°C. It is naturally present in the soil saprophytic Streptomyces roseosporus. Clinically, it is a lipoprotein antibiotic used to treat infections caused by Gram-positive bacteria that threaten the system and life.

[0003] The purification level of daptomycin in the prior art is still low, and the operation is complicated. CN102718839A discloses a method for separating and purifying daptomycin, comprising the following steps: ceramic membrane filtration of daptomycin fermentation broth, macroporous adsorption resin chromatography, nanofiltration concentration, hydrophobic resin column chromatography first step rough separation; ion exchange resin column chromatography, hydrophobic resin column chromatography second step rough separation, ultrafiltration, freeze drying to obtain daptomycin with a purity of more than 99.2%. The disadvantages of this method are long operation steps, low production capacity, and low yield. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for producing daptomycin with improved production capacity, yield and purity.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: to provide a method for producing daptomycin, comprising the steps of: Step (1) using a nanofiltration membrane to conduct the crude daptomycin product to obtain a conduction solution; Step (2) concentrating the electroconductive liquid obtained in step (1) by nanofiltration to obtain a nanofiltration concentrate; Step (3) adjusting the pH of the nanofiltration concentrate obtained in step (2) to obtain a column liquid; Step (4) washing the NMQ-K resin with water, applying the column liquid obtained in step (3) to the NMQ-K resin, washing with water, gradient eluting with a sodium chloride solution, and collecting the effective collected liquid of daptomycin intermediate with a purity of more than 75%; Step (5) The effective collection liquid of the daptomycin intermediate obtained in step (4) is concentrated using a nanofiltration membrane to a feed liquid unit of 50,000 to 160,000 to obtain a purified daptomycin product.

[0006] Preferably, in the above-mentioned method for producing daptomycin, the step (1) is specifically: The crude daptomycin is concentrated to half its volume in the nanofiltration membrane, and then water is added for top washing while nanofiltration is performed, and the amount of water added and the nanofiltration flow rate are kept consistent. When the dialysate is taken and the conductivity is measured to be ≤1000, the conductivity-driving solution is obtained.

[0007] Preferably, in the above-mentioned method for producing daptomycin, the step (2) is specifically: concentrating the electroconductive liquid obtained in step (1) by nanofiltration to 10 g / L-50 g / L to obtain a nanofiltration concentrate.

[0008] Preferably, in the above-mentioned method for producing daptomycin, the step (3) is specifically as follows: adjusting the pH of the nanofiltration concentrate obtained in step (2) to 3-8 with 2 mol / L NaOH or 2 mol / L HCl to obtain a column liquid.

[0009] Preferably, in the above-mentioned method for producing daptomycin, the loading concentration of the column in step (4) is 10-100 g / L.

[0010] Preferably, in the above-mentioned method for producing daptomycin, the "gradient elution with sodium chloride solution" in step (4) is specifically: eluting 10-30bv with 20mmol-90mmol sodium chloride solution and eluting 12-32bv with 90mmol-200mmol sodium chloride solution in sequence, performing gradient elution.

[0011] Preferably, in the above-mentioned method for producing daptomycin, the nanofiltration membrane in step (5) is a nanofiltration membrane with a molecular weight of 300.

[0012] The beneficial effect of the present invention is that: with the development of the market, the demand for daptomycin products is increasing. In order to improve production efficiency while ensuring quality, the present invention utilizes the characteristics of quaternary amine groups in daptomycin, so that it is combined with NMQ-K ion exchange resin for adsorption, and then gradient analysis is performed by saline solution to collect the parts that meet the requirements. Both the yield can be improved and the requirements of production can be met. Using the process of the present invention, a daptomycin product that meets the requirements can be obtained, and the sample loading is increased to several times of the original, and impurity removal is also improved while increasing production capacity. DETAILED DESCRIPTION

[0013] In order to explain the technical content, achieved objectives and effects of the present invention in detail, the following is an explanation in conjunction with the implementation modes.

[0014] The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0015] Example 1 Step (1) (2) Take 800 ml of crude daptomycin, with a content of 50 g / L, a total of 40 g, use a small membrane machine to conduction, add water while concentrating, keep the water addition speed consistent with the dialysate speed, and stop concentrating when the dialysate conductivity is ≤1000 to obtain a nanofiltration concentrate; the concentration of the nanofiltration concentrate is 10 g / L; Step (3) Take 4 L of the nanofiltration concentrate obtained in the above step, i.e. 40 g of daptomycin, and adjust the pH to 3.0 with 2 mol / L NaOH or 2 mol / L HCl to obtain a column liquid; Step (4) The NMQ-K resin is washed with water, and the column liquid obtained in step (3) is applied to the NMQ-K resin at a concentration of 10-100 g / L. After washing with water, 10-30 bv is eluted with a 20 mmol-90 mmol sodium chloride solution, and 12-32 bv is eluted with a 90 mmol-200 mmol sodium chloride solution. 33.64 g of the effective collected liquid of daptomycin intermediate with a purity of ≥75% is collected; the column is applied at a concentration of 100 g / L.

[0016] Step (5) A total of 56 L of effective collected liquid was obtained in Example 2, which was concentrated using a nanofiltration membrane to a feed liquid unit of 50 g / L-160 g / L. The concentration was stopped to obtain an improved product of daptomycin.

[0017] The yield of daptomycin in the method for producing daptomycin implemented herein is 84.1% and the purity is 83.2%.

[0018] Example 2 Steps (1) + (2) Take 800 ml of crude daptomycin, with a content of 50 g / L, a total of 40 g, use a small membrane machine to conduction, add water while concentrating, keep the water addition speed consistent with the dialysate speed, and stop concentrating when the dialysate conductivity is ≤1000 to obtain a nanofiltration concentrate; the concentration of the nanofiltration concentrate is 50 g / L; Step (3) Take 800 ml of the nanofiltration concentrate obtained in the above step, i.e. 40 g of daptomycin, and adjust the pH to 3.0 with 2 mol / L NaOH or 2 mol / L HCl to obtain a column liquid; Step (4) The NMQ-K resin is washed with water, and the upper column liquid obtained in step (3) is applied to the NMQ-K resin column. After washing with water, 10-30 bv is eluted with 20 mmol-90 mmol sodium chloride solution, and 12-32 bv is eluted with 90 mmol-200 mmol sodium chloride solution. 34.76 g of effective collected liquid of daptomycin intermediate with a purity of ≥75% is collected; the upper column is loaded at 10 g / L.

[0019] Step (5) A total of 56 L of effective collected liquid was obtained in Example 2, which was concentrated using a nanofiltration membrane to a feed liquid unit of 50 g / L-160 g / L. The concentration was stopped to obtain an improved product of daptomycin.

[0020] The yield of daptomycin in the method for producing daptomycin is 86.9% and the purity is 86.1%.

[0021] Example 3 Steps (1) + (2) Take 800 ml of crude daptomycin, with a content of 50 g / L, a total of 40 g, use a small membrane machine to conduction, add water while concentrating, keep the water addition speed consistent with the dialysate speed, and stop concentrating when the dialysate conductivity is ≤1000 to obtain a nanofiltration concentrate; the concentration of the nanofiltration concentrate is 25 g / L; Step (3) Take 1.6L of the nanofiltration concentrate obtained in the above step, i.e. 40g of daptomycin, and adjust the pH to 3.0 with 2mol / L NaOH or 2mol / L HCl to obtain a column liquid; Step (4) The NMQ-K resin is washed with water, and the upper column liquid obtained in step (3) is applied to the NMQ-K resin column. After washing with water, 10-30 bv is eluted with 20 mmol-90 mmol sodium chloride solution, and 12-32 bv is eluted with 90 mmol-200 mmol sodium chloride solution. 35.7 g of effective collected liquid of daptomycin intermediate with a purity of ≥75% is collected; the upper column is loaded at 50 g / L.

[0022] Step (5) A total of 56 L of effective collected liquid was obtained in Example 2, which was concentrated using a nanofiltration membrane to a feed liquid unit of 50 g / L-160 g / L. The concentration was stopped to obtain an improved product of daptomycin.

[0023] The yield of daptomycin in the method for producing daptomycin implemented herein is 89.25% and the purity is 92.31%.

[0024] Comparative Example 1 A method for producing daptomycin comprises the following steps: Steps (1) + (2) Take 800 ml of crude daptomycin, with a content of 50 g / L, a total of 40 g, use a small membrane machine to conduction, add water while concentrating, keep the water addition speed consistent with the dialysate speed, and stop concentrating when the dialysate conductivity is ≤1000 to obtain a nanofiltration concentrate; the concentration of the nanofiltration concentrate is 40 g / L; Step (3) Take 1L of the nanofiltration concentrate obtained in the above step, i.e. 40g of daptomycin, and adjust the pH to 3.0 with 2mol / L NaOH or 2mol / L HCl to obtain the column liquid; Step (4) The MQ resin was washed with water, and the column liquid obtained in step (3) was applied to the MQ resin column. After washing with water, it was eluted with 25 mmol ammonium chloride solution and 100 mmol ammonium chloride solution to collect 28.88 g of effective daptomycin intermediate effective collected liquid; the column was loaded at 50 g / L.

[0025] The yield of daptomycin in the production method of daptomycin in this comparative example is 71.3%, and the purity is 72.2%.

[0026] The results of the examples and comparative examples of the present application are analyzed in Tables 1 and 2 below.

[0027] Table 1

[0028] Table 2 Comparison of impurity data

[0029] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for producing daptomycin, characterized in that: Includes steps: Step (1) using a nanofiltration membrane to conduct the crude daptomycin product to obtain a conduction solution; Step (2) concentrating the electroconductive liquid obtained in step (1) by nanofiltration to obtain a nanofiltration concentrate; Step (3) adjusting the pH of the nanofiltration concentrate obtained in step (2) to obtain a column liquid; Step (4) washing the NMQ-K resin with water, applying the column liquid obtained in step (3) to the NMQ-K resin, washing with water, gradient eluting with a sodium chloride solution, and collecting the effective collected liquid of daptomycin intermediate with a purity of more than 75%; Step (5) collecting the effective liquid of the daptomycin intermediate obtained in step (4) and concentrating it using a nanofiltration membrane until the liquid unit is 50,000 to 160,000, thereby obtaining a purified daptomycin product.

2. The method for producing daptomycin according to claim 1, characterized in that: The step (1) is specifically: The crude daptomycin is concentrated to half its volume in the nanofiltration membrane, and then water is added for top washing while nanofiltration is performed, and the amount of water added and the nanofiltration flow rate are kept consistent. When the dialysate is taken and the conductivity is measured to be ≤1000, the conductivity-driving solution is obtained.

3. The method for producing daptomycin according to claim 1, characterized in that: The step (2) specifically comprises: concentrating the electroconductive liquid obtained in the step (1) to 10 g / L-50 g / L by nanofiltration to obtain a nanofiltration concentrated liquid.

4. The method for producing daptomycin according to claim 1, characterized in that: The step (3) specifically comprises: adjusting the pH of the nanofiltration concentrate obtained in step (2) to 3-8 with 2 mol / L NaOH or 2 mol / L HCl to obtain a column liquid.

5. The method for producing daptomycin according to claim 1, characterized in that: The column loading in step (4) is performed at a rate of 10-100 g / L.

6. The method for producing daptomycin according to claim 1, characterized in that: The "gradient elution with sodium chloride solution" in step (4) is specifically: eluting 10-30bv with 20mmol-90mmol sodium chloride solution and eluting 12-32bv with 90mmol-200mmol sodium chloride solution in sequence, performing gradient elution.

7. The method for producing daptomycin according to claim 1, characterized in that: The nanofiltration membrane in step (5) is a nanofiltration membrane with a molecular weight of 300.

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