A dalbavancin intermediate A40926 and its separation and purification method

Abstract

The present invention relates to the technical field of antibiotic production, and in particular to a dalbavancin intermediate A40926 and a separation and purification method thereof, comprising the following steps: S1, filtering a fermentation broth containing the dalbavancin intermediate A40926 to obtain a collected liquid; S2, adsorbing the collected liquid through a macroporous adsorption resin, eluting with ethanol, and collecting an eluate a having an A40926 unit greater than 100 mg / L; S3, ultrafiltrating and concentrating the eluate a to obtain a concentrated liquid having an A40926 unit of 15,000 to 16,000 mg / L; S4, subjecting the concentrated liquid to a chromatography exchange resin, top-washing with pure water, and gradient eluting with an ammonium acetate-ethanol solution, to collect an eluate b having an A40926 purity greater than 95%; S5, adjusting the pH of the eluate b to 8.0±0.2, adding acetone, adjusting the feed liquid temperature to 2 to 8°C, stirring for 6 to 7 hours, performing crystallization, and filtering and drying to obtain the dalbavancin intermediate A40926.

Description

Technical Field

[0001] The present invention relates to the technical field of antibiotic production, and in particular to a dalbavancin intermediate A40926 and a separation and purification method thereof. Background Art

[0002] Existing methods for separating and purifying the dalbavancin intermediate A40926, such as the preparation method disclosed in patent CN112480214A, disclose a method for preparing the key dalbavancin intermediate A40926. This method discloses sequentially subjecting the fermentation broth of the dalbavancin intermediate A40926 to deacylation, plate-and-frame filtration, alkaline adsorption onto a macroporous resin, acid washing, adsorption onto a polymer microsphere packing, concentration, precipitation, and drying to obtain the dalbavancin intermediate A40926. This preparation method involves a complex separation and purification process involving repeated use of alkaline and acidic solutions, and the eluent contains a large amount of salt, which can be corrosive to equipment. Furthermore, since dalbavancin is sensitive to sodium chloride, elution using a low-concentration sodium chloride elution system can easily lead to turbidity in the eluent. Summary of the Invention

[0003] In order to overcome the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide a separation and purification method of dalbavancin intermediate A40926 with a purity of more than 97.45%, and dalbavancin intermediate A40926 separated and purified by the separation and purification method.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for separating and purifying the dalbavancin intermediate A40926, comprising the following steps:

[0005] S1. Filtering the fermentation broth containing the dalbavancin intermediate A40926 to obtain a collection liquid;

[0006] S2. adsorbing the collected solution through a macroporous adsorption resin, washing with water until the leaked solution is nearly colorless, eluting with ethanol, and collecting the eluate a containing more than 100 mg / L of the dalbavancin intermediate A40926;

[0007] S3, concentrating the eluate a by ultrafiltration to a concentrate having a dalbavancin intermediate A40926 unit of 15,000 to 16,000 mg / L;

[0008] S4. The concentrated solution is subjected to chromatography exchange resin, top washed with pure water, and then gradient eluted with ammonium acetate-ethanol solution to collect eluate b having a purity of greater than 95% of dalbavancin intermediate A40926;

[0009] S5. Adjust the pH of the eluent b to 8.0±0.2, add acetone, adjust the temperature of the feed liquid to 2-8° C., stir for 6-7 hours, and then crystallize. After suction filtration and drying, obtain dalbavancin intermediate A40926.

[0010] Further provided is a dalbavancin intermediate A40926, which is obtained by separation and purification using the aforementioned separation and purification method.

[0011] The beneficial effect of the present invention is that by replacing the traditional sodium chloride eluent system with an ammonium acetate-ethanol elution system during the separation and purification process, the present invention can effectively avoid the corrosion of the eluent to the equipment while effectively improving the purity of the dalbavancin intermediate A40926. After testing, the purity of the dalbavancin intermediate A40926 separated and purified by the separation and purification method provided by the present invention is greater than 97.45%. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Shown is the spectrum of the dalbavancin intermediate A40926 separated and purified in Example 1 of the present invention. DETAILED DESCRIPTION

[0013] To illustrate the technical content, achieved objectives and effects of the present invention in detail, the following description is given in conjunction with the embodiments and accompanying drawings.

[0014] The method for separating and purifying the dalbavancin intermediate A40926 comprises the following steps:

[0015] S1. Filtering the fermentation broth containing the dalbavancin intermediate A40926 to obtain a collection liquid;

[0016] S2. adsorbing the collected solution through a macroporous adsorption resin, washing with water until the leaked solution is nearly colorless, eluting with ethanol, and collecting the eluate a containing more than 100 mg / L of the dalbavancin intermediate A40926;

[0017] S3, concentrating the eluate a by ultrafiltration to a concentrate having a dalbavancin intermediate A40926 unit of 15,000 to 16,000 mg / L;

[0018] S4. The concentrated solution is subjected to chromatography exchange resin, top washed with pure water, and then gradient eluted with ammonium acetate-ethanol solution to collect eluate b having a purity of greater than 95% of dalbavancin intermediate A40926;

[0019] S5. Adjust the pH of the eluent b to 8.0±0.2, add acetone, adjust the temperature of the feed liquid to 2-8° C., stir for 6-7 hours, and then crystallize. After suction filtration and drying, obtain dalbavancin intermediate A40926.

[0020] In S1, the filtration is performed using a ceramic membrane. Preferably, the pore size of the ceramic membrane is 0.01 μm. The fermentation broth is filtered using a ceramic membrane with a pore size of 0.01 μm to remove macromolecular substances such as water-soluble proteins and pigments in the fermentation broth.

[0021] Preferably, S1 comprises filtering the fermentation broth containing the dalbavancin intermediate A40926 through a ceramic membrane, concentrating it to half the volume of the fermentation broth, then filtering while adding water for top-washing until the total volume of the filtrate is 2 to 5 times the volume of the fermentation broth, and then collecting the collected filtrate. The amount of water added during top-washing and filtering should be consistent with the filtration flow rate. In one embodiment, the amount of water added is 5 to 10 L / h.

[0022] In one embodiment, S2 is: adsorbing the collected liquid through a macroporous adsorption resin at a flow rate of 0.8 BV / h, top-washing with purified water after adsorption until the effluent is almost colorless, top-washing with 20% (v / v) ethanol, and then eluting with 60% (v / v) ethanol to collect the eluate a with a concentration of dalbavancin intermediate A40926 greater than 100 mg / L.

[0023] In one embodiment, S3 comprises ultrafiltration of the eluate a through an ultrafiltration membrane with a molecular weight cutoff of 20,000 D, followed by nanofiltration concentration, wherein the temperature of the ultrafiltration and nanofiltration is 15-20° C., and the concentration is to a concentrate having a concentration of 15,000-16,000 mg / L of the dalbavancin intermediate A40926. The nanofiltration membrane used for the nanofiltration is NF-4040.

[0024] In one embodiment, in S4, the gradient elution is: gradient elution using 100 mmol / L ammonium acetate-20% ethanol solution and 200 mmol / L ammonium acetate-50% ethanol solution in sequence.

[0025] Preferably, the gradient elution is: first flushing 10 BV with 100 mmol / L ammonium acetate-20% ethanol solution at a flow rate of 10 BV / h, and then flushing 10 BV with 200 mmol / L ammonium acetate-50% ethanol solution at a flow rate of 10 BV / h.

[0026] In one embodiment, S5 is: concentrating the eluate b to a unit of 20,000 to 80,000 mg / L of dalbavancin intermediate A40926, adjusting the pH to 8.0±0.2, adding acetone and adjusting the feed liquid temperature to 2 to 8°C, stirring for 6 to 7 hours, and then crystallizing, filtering and drying to obtain dalbavancin intermediate A40926.

[0027] Preferably, the macroporous adsorption resin is HZ806, and the chromatography exchange resin is Unips40-500, which are purchased from Nano-Tech.

[0028] The dalbavancin intermediate A40926 was isolated and purified by the aforementioned separation and purification method.

[0029] It should be noted that the unit described in this article is concentration.

[0030] Example 1

[0031] The separation, purification and distribution of the dalbavancin intermediate A40926 comprises the following steps:

[0032] S1. Filter 1 L of fermentation broth containing the dalbavancin intermediate A40926 through a ceramic membrane with a pore size of 0.01 μm. Concentrate to half the volume of the fermentation broth, then add water (purified water) for top washing while filtering. The amount of water added and the filtration flow rate are both 5 L / h. When the total volume of the filtrate is 3 times the volume of the fermentation broth, collect the clear and transparent collected solution.

[0033] S2. Adjust the pH of the collected solution to 8.0 with hydrochloric acid, pass it through an HZ806 column at a flow rate of 0.8 BV / h, top-wash the column with purified water until the effluent is nearly colorless, pre-wash 6 BV with 20% (v / v) ethanol at a flow rate of 5 BV / h, and then elute with 2 BV of 60% (v / v) ethanol at a flow rate of 5 BV / h to collect eluate a containing more than 100 mg / L of dalbavancin intermediate A40926 units;

[0034] S3. Take 500 mL of the eluate a and perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 20,000 D, followed by nanofiltration concentration. During the ultrafiltration and nanofiltration processes, control the liquid temperature at 20° C. and concentrate to 15,000 mg / L of the dalbavancin intermediate A40926. Adjust the pH of the concentrate to 8 and apply it to a ps40-500 column at a flow rate of 5 BV / h. Top-wash 1 BV with purified water, then rinse 10 BV with a 100 mmol / L ammonium acetate-20% ethanol solution at a flow rate of 10 BV / h, and then rinse 10 BV with a 200 mmol / L ammonium acetate-50% ethanol solution at a flow rate of 10 BV / h. Collect the eluate b with a purity of greater than 95% of the dalbavancin intermediate A40926.

[0035] S4. Concentrate the eluate b to 60,000 mg / L of dalbavancin intermediate A40926, adjust the pH to 8.0±0.2 with 1 mol / L sodium hydroxide, add 2.0 L of acetone, control the feed liquid temperature to 6°C, stir for 6 h until no more crystals precipitate, filter, and dry to obtain dalbavancin intermediate A40926.

[0036] Example 2

[0037] The separation, purification and distribution of the dalbavancin intermediate A40926 comprises the following steps:

[0038] S1. Filter 1 L of fermentation broth containing the dalbavancin intermediate A40926 through a ceramic membrane with a pore size of 0.01 μm. Concentrate to half the volume of the fermentation broth, then add water (purified water) for top washing while filtering. The amount of water added and the filtration flow rate are both 5 L / h. When the total volume of the filtrate is 3 times the volume of the fermentation broth, collect the clear and transparent collected solution.

[0039] S2. Adjust the pH of the collected solution to 8.0 with hydrochloric acid, pass it through an HZ806 column at a flow rate of 0.8 BV / h, top-wash the column with purified water until the effluent is nearly colorless, pre-wash 6 BV with 20% (v / v) ethanol at a flow rate of 5 BV / h, and then elute with 2 BV of 60% (v / v) ethanol at a flow rate of 5 BV / h to collect eluate a containing more than 100 mg / L of dalbavancin intermediate A40926 units;

[0040] S3. Take 500 mL of the eluate a and perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 20,000 D, followed by nanofiltration concentration. During the ultrafiltration and nanofiltration processes, control the liquid temperature at 15° C. and concentrate to 15,000 mg / L of the dalbavancin intermediate A40926. Adjust the pH of the concentrate to 9 and apply it to a ps40-500 column at a flow rate of 5 BV / h. Top-wash 1 BV with purified water, then rinse 10 BV with a 100 mmol / L ammonium acetate-20% ethanol solution at a flow rate of 10 BV / h, and then rinse 10 BV with a 200 mmol / L ammonium acetate-50% ethanol solution at a flow rate of 10 BV / h. Collect the eluate b with a purity of greater than 95% of the dalbavancin intermediate A40926.

[0041] S4. Concentrate the eluate b to 60,000 mg / L of dalbavancin intermediate A40926, adjust the pH to 8.0±0.2 with 1 mol / L acetone, add 2.0 L acetone, control the feed liquid temperature to 8°C, stir for 7 h until no more crystals precipitate, filter, and dry to obtain dalbavancin intermediate A40926.

[0042] Test example

[0043] The purity of the dalbavancin intermediate A40926 isolated and purified in Example 1 was tested. Figure 1 As shown. Among them, Figure 1 The upper graph is the chromatogram of Dalbavancin intermediate A40926, and the lower graph is the graph corresponding to the chromatogram. Figure 1 It can be seen that the purity (A0+A1+B0+B1+B2) of the dalbavancin intermediate A40926 separated and purified in Example 1 is 97.45%.

[0044] In summary, the present invention uses an ammonium acetate-ethanol elution system instead of a traditional sodium chloride eluent system during the separation and purification process, thereby effectively avoiding corrosion of the eluent to the equipment while effectively improving the purity of the dalbavancin intermediate A40926. Testing shows that the purity of the dalbavancin intermediate A40926 separated and purified by the separation and purification method provided by the present invention is greater than 97.45%.

[0045] 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's description and drawings, 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 separating and purifying the dalbavancin intermediate A40926, characterized in that: The steps include: S1. Filtering the fermentation broth containing the dalbavancin intermediate A40926 to obtain a collection liquid; S2. Passing the collected liquid through a macroporous adsorption resin at a flow rate of 0.8 BV / h for adsorption. After adsorption, top-washing with purified water until the effluent is nearly colorless, top-washing with 20% (v / v) ethanol, and then eluting with 60% (v / v) ethanol to collect the eluate a having a concentration of dalbavancin intermediate A40926 greater than 100 mg / L; S3, concentrating the eluate a by ultrafiltration to a concentrate having a dalbavancin intermediate A40926 unit of 15,000 to 16,000 mg / L; S4. The concentrated solution was subjected to a chromatography exchange resin, and after a top wash with pure water, 10 BV of the concentrated solution was first rinsed with a 100 mmol / L ammonium acetate-20% ethanol solution at a flow rate of 10 BV / h, and then 10 BV of the concentrated solution was rinsed with a 200 mmol / L ammonium acetate-50% ethanol solution at a flow rate of 10 BV / h, and the eluate b having a purity of greater than 95% of the dalbavancin intermediate A40926 was collected; S5. Adjust the pH of the eluent b to 8.0±0.2, add acetone, adjust the temperature of the feed liquid to 2-8° C., stir for 6-7 hours, and then crystallize. After suction filtration and drying, obtain dalbavancin intermediate A40926.

2. The separation and purification method according to claim 1, wherein The filtration in S1 is carried out using a ceramic membrane filtration.

3. The separation and purification method according to claim 2, wherein The S1 comprises: filtering the fermentation broth containing the dalbavancin intermediate A40926 through a ceramic membrane, concentrating it to half the volume of the fermentation broth, adding water for top washing while filtering, and collecting the collected liquid after the total volume of the filtrate is 2 to 5 times the volume of the fermentation broth.

4. The separation and purification method according to claim 1, wherein The S3 is: ultrafiltration of the eluate a using an ultrafiltration membrane with a molecular weight cutoff of 20,000D, followed by nanofiltration concentration, wherein the liquid temperature during the ultrafiltration and nanofiltration is 15-20°C, and the eluate is concentrated to a concentrate having a dalbavancin intermediate A40926 unit of 15,000-16,000 mg / L.

5. The separation and purification method according to claim 1, wherein The S5 is as follows: concentrating the eluate b to a concentration of 20,000 to 80,000 mg / L of the dalbavancin intermediate A40926, adjusting the pH to 8.0±0.2, adding acetone and adjusting the feed liquid temperature to 2 to 8° C., stirring for 6 to 7 hours, and then crystallizing. After filtration and drying, the dalbavancin intermediate A40926 is obtained.

6. The separation and purification method according to claim 1, wherein The macroporous adsorption resin is HZ806, and the chromatography exchange resin is ps40-500.

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