A method for crystallizing vancomycin with calcium

Abstract

This invention relates to the field of antibiotic production separation and purification, specifically to a method for vancomycin crystallization with calcium addition, comprising the following steps: dissolving crude vancomycin powder and adjusting the pH to 2.0-4.0 to obtain a solution; filtering the obtained solution through a membrane and adjusting the pH to 2.0-4.0 to obtain a pre-crystallization solution; adding a calcium salt solution to the pre-crystallization solution, then adding ethanol, allowing it to stand, and filtering to obtain crystalline powder; dissolving the obtained crystalline powder in deionized water, removing ethanol using a small membrane apparatus until it meets the requirements, and then obtaining a removed ethanol solution; lyophilizing the obtained removed ethanol solution to obtain the desired vancomycin finished powder. The beneficial effects of this invention are: improving the existing process of crystallization under alkaline conditions, improving the flocculation of vancomycin by using acidic crystallization and the addition of calcium ions, utilizing the effect of ethanol on flocculation for crystallization, and finally lyophilizing the obtained crystalline powder to obtain the required vancomycin finished powder, thus extending the flocculation time of 25% concentration vancomycin.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibiotic production separation and purification, and particularly to a method for crystallizing vancomycin with calcium. BACKGROUND

[0002] Vancomycin is a glycopeptide antibiotic isolated from the fermentation broth of a strain of Amycolatopsis orientalis by Micormick et al. in 1956. It was developed by Eli Lilly and Company and approved by FDA in 1958. In the 1980s, with the extensive use of β-lactam antibiotics, infections caused by methicillin-resistant Staphylococcus aureus (MRSA) gradually became popular. Vancomycin is an important drug for the treatment of serious infections caused by MRSA in clinical practice, and is increasingly attracting attention.

[0003] In the prior art, high-purity crystals of vancomycin are obtained by crystallization, but under high unit conditions, vancomycin will appear to be flocculated into a gel state, which will cause the quality of the product to decrease. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a crystallization method to replace the existing production crystallization process, so as to prolong the flocculation time of vancomycin.

[0005] To solve the above technical problems, the technical scheme adopted by the present application is as follows: a method for crystallizing vancomycin with calcium, comprising the following steps:

[0006] Step (1) dissolving vancomycin coarse powder with deionized water, the vancomycin dissolution unit is 10-15 million, adjusting the pH to 2.0-4.0 to obtain a dissolution solution;

[0007] Step (2) filtering the dissolution solution obtained in step (1), adjusting the pH to 2.0-4.0 to obtain a pre-crystallization solution;

[0008] Step (3) adding the pre-crystallization solution obtained in step (2) to a calcium salt solution in which calcium salt is dissolved with deionized water, adjusting the pH to 4.0-6.0, adding ethanol, standing for 10-24 h, and then filtering to obtain a crystalline powder;

[0009] Step (4) dissolving the crystalline powder obtained in step (3) with deionized water, and removing calcium by column HZ16 resin to obtain a calcium-removed solution;

[0010] Step (5) removing ethanol from the calcium-removed solution obtained in step (4) by using a small membrane machine to obtain an ethanol-removed solution;

[0011] Step (6) freeze-drying the ethanol-removed solution obtained in step (5) to obtain the desired vancomycin finished powder.

[0012] Preferably, in the method for crystallizing vancomycin with calcium as described above, the pH of the solution obtained in step (1) is adjusted to 2.0-4.0 with concentrated hydrochloric acid or 4-10% sodium hydroxide solution, and the temperature of the deionized water is 2-8°C.

[0013] Preferably, in the method for crystallizing vancomycin with calcium as described above, step (2) is specifically as follows: the solution obtained in step (1) is filtered through a 0.22-um nylon mixed filter membrane, and the pH of the filtrate is adjusted to 2.0-4.0 with concentrated hydrochloric acid or 4-10% sodium hydroxide solution.

[0014] Preferably, in the method for crystallizing vancomycin with calcium as described above, the calcium salt is calcium acetate, calcium chloride, or calcium oxide.

[0015] Preferably, in the method for crystallizing vancomycin with calcium as described above, step (3) is specifically as follows: the solution obtained in step (2) is added to a calcium salt solution under stirring, the calcium salt solution is a solution of the calcium salt dissolved in deionized water, the mass ratio of the calcium salt to vancomycin is 0.1-0.9, and the volume ratio of the 90-95% ethanol to the solution obtained in step (2) is 3-10.

[0016] Preferably, in the method for crystallizing vancomycin with calcium as described above, step (4) is specifically as follows: the crystalline powder obtained in step (3) is dissolved in deionized water, and the solution is passed through an HZ16 column to remove calcium, and the HZ16 column is used at a concentration of 100 g / L, and the vancomycin is eluted with water.

[0017] Preferably, in the method for crystallizing vancomycin with calcium as described above, step (5) is specifically as follows: the decalcified solution obtained in step (4) is subjected to ethanol removal using a small membrane machine, the crystalline powder solution is concentrated to a volume of one-fifth, ethanol is removed while water is added, the amount of water added and the flow rate of the dialysis solution are kept constant, until the ethanol content is less than 1000 ppm, and the nanofiltration is stopped, to obtain an ethanol-removed solution.

[0018] Preferably, in the method for crystallizing vancomycin with calcium as described above, step (6) is specifically as follows: the ethanol-removed solution obtained in step (5) is concentrated to contain 100,000-200,000 units of vancomycin, and the vancomycin product powder is obtained after lyophilization.

[0019] The method for crystallizing vancomycin with calcium according to the present application improves the crystallization under the alkaline condition of the existing process, improves the flocculation of vancomycin by using acid crystallization and adding calcium ions, crystallizes by using the influence of ethanol on flocculation, and finally freeze-dries the obtained crystalline powder to obtain a vancomycin finished powder meeting the requirements, which can prolong the flocculation time of vancomycin with a concentration of 25%; effectively improve the flocculation of vancomycin; and meet the production requirements. The process can obtain a vancomycin finished powder meeting the requirements. DETAILED DESCRIPTION

[0020] To illustrate the technical content, purposes and effects of the present application, the following embodiments are described.

[0021] Example 1

[0022] A method for crystallizing vancomycin with calcium, comprising the following steps:

[0023] Step (1), 100 g of vancomycin crude powder is adjusted to pH 2.8 with 8% sodium hydroxide by mass percentage, dissolved in 150,000 units of deionized water to obtain a dissolved solution, and the temperature of the deionized water is 2 degrees.

[0024] Step (2), the dissolved solution obtained in step (1) is filtered through a 0.22 um nylon mixed filter membrane, and the pH is adjusted to 2.8 with 8% sodium hydroxide by mass percentage to obtain a pre-crystallization solution.

[0025] Step (3), the pre-crystallization solution obtained in step (2) is added to a calcium salt solution under stirring, the calcium salt solution is 10 g of calcium acetate dissolved in deionized water, the pH is adjusted to 5.0 with 8% sodium hydroxide by mass percentage, and 95% ethanol by mass percentage is added and filtered after standing for 28 h, obtaining 158 g of crystalline powder; the amount of 95% ethanol by mass percentage added is 10 times the volume of the pre-crystallization solution.

[0026] Step (4), the crystalline powder obtained in step (3) is dissolved in 2 L of deionized water, and then 100 g / L HZ16 resin is used for calcium removal, and then 2 L of deionized water is used to wash the HZ16 resin to obtain a decalcified solution.

[0027] Step (5), the decalcified solution obtained in step (4) is used to chase ethanol by using a small membrane machine, the decalcified solution is concentrated to a volume of 600 ml, water is added while chasing ethanol, the amount of water added and the flow rate of the dialysis solution are kept consistent, until the ethanol content is less than 1000 ppm, the nanofiltration is stopped, and the chased ethanol solution is obtained.

[0028] Step (6), the alcohol-removed liquid obtained in step (5) is concentrated to 100,000 units, the pH of the liquid is adjusted to 2.8 by using 1 mol / L NaOH, and then freeze-drying is performed, so that the desired product is obtained after the freeze-drying is completed.

[0029] Example 2

[0030] A method for crystallizing vancomycin with calcium, comprising the following steps:

[0031] Step (1), 100 g of vancomycin crude powder is adjusted to pH 3.0 by using 8% sodium hydroxide by mass percentage, and is dissolved to 80,000 units by using deionized water, wherein the temperature of the deionized water is 2-8 degrees.

[0032] Step (2), the dissolved liquid obtained in step (1) is filtered through a 0.22 um nylon mixed filter membrane, and the pH is adjusted to 3 by using 8% sodium hydroxide by mass percentage to obtain a pre-crystallization liquid.

[0033] Step (3), the pre-crystallization liquid obtained in step (2) is added to a calcium salt solution under stirring, wherein the calcium salt solution is 50 g of calcium acetate dissolved in deionized water, the pH is adjusted to 5.5 by using 8% sodium hydroxide by mass percentage, and the crystalline powder is obtained after being placed for 18 h and filtered after adding 90-95% ethanol by mass percentage; the amount of 90-95% ethanol by mass percentage added is 7 times the volume of the pre-crystallization liquid.

[0034] Step (4), the crystalline powder obtained in step (3) is dissolved to 2 L by using deionized water, and then decalcification is performed by using 100 g / L HZ16 resin, and then the HZ16 resin is washed with 2 L of deionized water, so that a decalcified liquid is obtained.

[0035] Step (5), the decalcified liquid obtained in step (4) is subjected to ethanol removal by using a small membrane machine, the decalcified liquid is concentrated to a volume of 600 ml, water is added while ethanol is removed, the amount of water added and the flow rate of the dialysis liquid are kept consistent, until the ethanol content is less than 1,000 ppm, the nanofiltration is stopped, and an alcohol-removed liquid is obtained.

[0036] Step (6), the alcohol-removed liquid obtained in step (5) is concentrated to 100,000-200,000 units, the pH of the liquid is adjusted to 2.8 by using 1 mol / L NaOH or 1 mol / L HCl, and then freeze-drying is performed, so that the desired product is obtained after the freeze-drying is completed.

[0037] Example 3

[0038] A method for crystallizing vancomycin with calcium, comprising the following steps:

[0039] Step (1), 100g of crude vancomycin powder is adjusted to pH 4.0 with 10% sodium hydroxide by mass percentage, and dissolved in 150,000 units of deionized water, and the temperature of the deionized water is 8 degrees;

[0040] Step (2), the dissolved solution obtained in step (1) is filtered through a 0.22um nylon mixed filter membrane, and the pH is adjusted to 4.0 with 10% sodium hydroxide by mass percentage to obtain a crystallization solution.

[0041] Step (3), the crystallization solution obtained in step (2) is added to a calcium salt solution under stirring, the calcium salt solution is 90g of calcium acetate dissolved in deionized water, and the pH is adjusted to 6.0 with concentrated hydrochloric acid or 4-10% sodium hydroxide by mass percentage, and then filtered after standing for 12h with 90-95% ethanol by mass percentage, to obtain a crystalline powder; the amount of 90-95% ethanol by mass percentage added is 4 times the volume of the crystallization solution.

[0042] Step (4), the crystalline powder obtained in step (3) is dissolved in 2L of deionized water, and then 100g / L HZ16 resin is used to remove calcium, and then 2L of deionized water is used to wash the HZ16 resin, to obtain a decalcification solution.

[0043] Step (5), the decalcification solution obtained in step (4) is concentrated to 600ml by using a small membrane machine, and the ethanol is removed while adding water, and the water addition amount and the dialysis solution flow rate are kept consistent, until the ethanol content is less than 1000ppm, and the nanofiltration is stopped, to obtain an ethanol removal solution.

[0044] Step (6), the ethanol removal solution obtained in step (5) is concentrated to 100,000-200,000 units, and the pH of the solution is adjusted to 2.8 with 1mol / L NaOH or 1mol / L HCl, and then freeze-dried, to obtain the desired product.

[0045] The flocculation time of the vancomycin product powder obtained in the above examples 1 to 3 is shown in table 1.

[0046] Table 1

[0047]

[0048] Note: 25% concentration is defined as 250g / L.

[0049] The above is only an embodiment of the present application, and does not limit the patent scope of the present application, and any equivalent transformation or direct or indirect application in related technical fields using the content of the present application is also included in the patent protection scope of the present application.

Claims

1. A process for crystallizing vancomycin plus calcium characterized in that, It comprises the following steps: Step (1) dissolving crude vancomycin with deionized water, the vancomycin dissolving unit is 10-15 million, adjusting pH to 2.0-4.0 to obtain a dissolving solution; Step (2) filtering the dissolving solution obtained in step (1), adjusting pH to 2.0-4.0 to obtain a pre-crystallization solution; Step (3) adding the pre-crystallization solution obtained in step (2) into a calcium salt solution dissolved with deionized water, adjusting pH to 4.0-6.0, adding 90-95% ethanol, standing for 10-24 hours, and then filtering to obtain a crystalline powder; the calcium salt is calcium acetate; Step (4) dissolving the crystalline powder obtained in step (3) with deionized water, and removing calcium by HZ16 resin column to obtain a decalcified solution; Step (5) removing ethanol from the decalcified solution obtained in step (4) by using a small membrane machine to obtain an ethanol-removed solution; Step (6) freeze-drying the ethanol-removed solution obtained in step (5) to obtain the desired vancomycin product powder.

2. The process for crystallization of vancomycin calcium according to claim 1, characterized in that, In step (1), concentrated hydrochloric acid or 4-10% sodium hydroxide is used to adjust pH to 2.0-4.0 to obtain the dissolving solution, and the temperature of the deionized water is 2-8 degrees.

3. The process for crystallization of vancomycin calcium according to claim 1, characterized in that, In step (2), the dissolving solution obtained in step (1) is filtered through a 0.22um nylon mixed filter membrane, and concentrated hydrochloric acid or 4-10% sodium hydroxide is used to adjust pH to 2.0-4.0 to obtain the pre-crystallization solution.

4. The process for crystallizing vancomycin calcium according to claim 1, characterized in that, In step (3), the pre-crystallization solution obtained in step (2) is added into the calcium salt solution under stirring, the calcium salt solution is a solution in which calcium salt is dissolved with deionized water, the mass ratio of calcium salt to vancomycin is 0.1-0.9, concentrated hydrochloric acid or 4-10% sodium hydroxide is used to adjust pH to 4.0-6.0, 90-95% ethanol is added, and then filtered after standing for 10-24 hours to obtain the crystalline powder; the amount of 90-95% ethanol added is 3-10 times the volume of the pre-crystallization solution.

5. The process for crystallization of vancomycin calcium according to claim 1, characterized in that, In step (4), the crystalline powder obtained in step (3) is dissolved with deionized water, and calcium is removed by HZ16 resin column, and 100g / L HZ16 is used to elute vancomycin with water; a decalcified solution is obtained.

6. The process for crystallization of vancomycin calcium according to claim 1, characterized in that, In step (5), the decalcified solution obtained in step (4) is used to remove ethanol by using a small membrane machine, the crystalline powder solution is concentrated to one-fifth of the volume, ethanol is removed while water is added, the amount of water added and the flow rate of the dialysis solution are kept consistent, until the ethanol content is less than 1000ppm, and then the nanofiltration is stopped to obtain an ethanol-removed solution.

7. The process for crystallization of vancomycin calcium according to claim 1, characterized in that, In step (6), the ethanol-removed solution obtained in step (5) is concentrated to 10-20 million units, and the vancomycin product powder is obtained after freeze-drying.

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