CN110606844B - Mupirocin purification method - Google Patents
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- C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
- C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
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Abstract
The invention provides a mupirocin purification method, which comprises the following steps: dissolving the crude mupirocin product in water, adding salt, adjusting the pH value to 6.0-7.0, and performing chromatographic separation by using medium-polarity macroporous resin to obtain a qualified mupirocin desorption solution; carrying out nanofiltration concentration on the mupirocin desorption qualified liquid; crystallizing and drying the obtained concentrated solution to obtain the purified mupirocin. The method adopts macroporous resin chromatography to separate the aqueous solution of the crude mupirocin product, and then carries out nanofiltration, crystallization and drying, so that the purity of the mupirocin can reach more than 99 percent.
Description
Technical Field
The invention relates to the field of biological fermentation pharmacy, and in particular relates to a mupirocin purification method.
Background
Mupirocin, formula C26H44O9The structural formula is shown as follows, the molecular weight is 500.63, also called pseudomonic acid A, and the pseudomonic acid A is a natural broad-spectrum antibiotic produced by the fermentation of pseudomonas fluorescens.
Mupirocin is highly sensitive to gram-positive cocci, is also effective against some gram-negative cocci, and has no cross-resistance to its antibiotics. Currently, mupirocin is clinically used in China for treating various infectious skin diseases caused by staphylococcus aureus and streptococcus, including: primary skin infections such as impetigo, folliculitis, and scabies; secondary skin infections such as eczema, atopic dermatitis, skin ulcers, surgical wounds, small area burns, skin trauma; others may also be used for infection prevention in small wounds, incisions and other sterile lesions.
Existing methods for purification of mupirocin from mupirocin fermentation broth involve organic solvent extraction, crystallization and desorption on resin using an organic solvent. For example, there is a patent report that mupirocin is purified through the steps of fermentation liquid membrane filtration, filtrate membrane concentration, solvent extraction, dehydrating agent dehydration, concentration under reduced pressure, crystallization and filtration in order, wherein a large amount of extraction organic solvent such as ethyl acetate or acetone is used.
However, the use of a large amount of organic solvent not only increases the cost but also is not favorable for safety and environmental protection.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a mupirocin purification method which does not use organic solvent at all.
The invention provides a mupirocin purification method, which comprises the following steps:
(1) dissolving the crude mupirocin product in water, adding salt, adjusting the pH value to 6.0-7.0, and performing chromatographic separation by using medium-polarity macroporous resin to obtain a qualified mupirocin desorption solution;
(2) carrying out nanofiltration concentration on the mupirocin desorption qualified liquid;
(3) and (3) crystallizing and drying the concentrated solution obtained in the step (2) to obtain purified mupirocin.
The invention discovers that when medium-polarity macroporous resin is used for adsorption in a water phase, a certain amount of salt is added to reduce the solubility of mupirocin and enhance the adsorption capacity of the resin to mupirocin, and the medium-polarity macroporous resin can be selected for both adsorption and desorption, so that the medium-polarity macroporous resin can adsorb the mupirocin well and is easy to desorb, and the use of organic solvent for desorption can be avoided.
The method adopts the medium-polarity macroporous resin to chromatographically separate the aqueous solution of the crude mupirocin product, and then carries out nanofiltration, crystallization and drying, so that the purity of the mupirocin can reach more than 99 percent.
Further, in step (1), the crude mupirocin is obtained by the following method: adjusting the pH value of mupirocin fermentation liquor to 2.5-3.5, then carrying out solid-liquid separation, adding an ammonium bicarbonate solution into the separated slag, carrying out solid-liquid separation to obtain a filtrate, carrying out nanofiltration concentration on the filtrate, and then carrying out coarse crystallization.
When the raw material is mupirocin fermentation broth, the pretreatment is very critical, and if the treatment is relatively extensive or the quality of precipitates is poor, the subsequent purification without an organic solvent is difficult to realize. The invention firstly adjusts the fermentation liquor to be acidic, reduces the solubility of mupirocin, ensures that the mupirocin is basically kept in the mushroom dregs through the first solid-liquid separation, removes a large amount of water-soluble pigment and protein, then uses weak alkaline water for extraction, filters out a large amount of substances with poor alkaline water solubility, can remove a part of micromolecular salt in the concentration process by matching with nanofiltration, and obtains the formed coarse crystal after multiple purifications. And (3) carrying out chromatographic separation on the crude crystals by using medium polarity resin, ensuring the purification effect and obtaining mupirocin with the chromatographic purity of more than 99%.
Further, in the preparation process of the crude mupirocin product, the solid-liquid separation preferably adopts plate-and-frame filter pressing.
The concentration of the ammonium bicarbonate solution is 5-6 wt%.
The dosage ratio of the ammonium bicarbonate solution to the slag is 1-2 ml:1 g.
The nanofiltration membrane with the molecular weight cutoff of 200-300 is adopted. The nanofiltration membrane in the range can ensure higher yield and prevent the filtering speed from being too slow.
And concentrating after nanofiltration until the content of mupirocin in the concentrated solution is 40-60 g/L.
And the coarse crystallization is implemented by adjusting the pH of the concentrated solution to 3.0-4.0 and then crystallizing, wherein hydrochloric acid, oxalic acid, acetic acid, sulfuric acid or phosphoric acid is adopted for adjusting the pH, and hydrochloric acid is preferably adopted.
Further, in the step (1), the medium-polarity macroporous resin is HZ-806 of Shanghai Huazhen.
And during chromatographic separation, 0.01-0.03 wt% of ammonium bicarbonate is used as a desorption solution.
Mupirocin is poorly water soluble under acidic conditions and well water soluble under alkaline conditions. The ammonia bicarbonate can enhance the water solubility of mupirocin, thereby weakening the combination of the mupirocin and the macroporous resin with medium polarity and desorbing the mupirocin; experiments show that the ammonium bicarbonate solution with a certain concentration can perform good effect on chromatographic separation of products, other alkali chromatographic separation methods have the disadvantages of too fast desorption or too slow desorption, and the separated chromatographic purification is poor, so that the ideal effect is not achieved.
The qualified mupirocin desorption liquid is desorption liquid with the chromatographic purity of more than 90 percent.
In a preferred embodiment of the invention, the step (1) is specifically that the crude mupirocin is dissolved in water to 10 g-20 g/L, 2-3 wt% of sodium chloride is added, the pH value is adjusted to 6.0-7.0 to be used as a column loading solution, column loading is carried out, ammonium bicarbonate is used for desorption after the column loading is finished with water washing, and a desorption solution with the chromatographic purity of more than 90% is collected.
Further, in the step (2), a nanofiltration membrane with the molecular weight cutoff of 200-300 is adopted for nanofiltration.
And concentrating after nanofiltration until the content of mupirocin in the concentrated solution is 40-60 g/L.
Further, in the step (3), the crystallization is performed by adjusting the pH of the concentrated solution to 3.0-4.0, and hydrochloric acid, oxalic acid, acetic acid, sulfuric acid or phosphoric acid is used for adjusting the pH, preferably hydrochloric acid.
Here, adjusting the concentrate to acidic first may reduce the solubility of mupirocin, facilitating crystallization. The experimental results show that the acid crystallization is less than the conventional direct cooling crystallization (not necessarily impurities which can be detected in the traditional sense, but also a plurality of impurities which can not be quantitatively detected without ultraviolet absorption value) in the method. Of course, the final purification of the product to 99% is not necessary with each step, which is achieved by the mutual synergy of the steps.
On the basis of the common knowledge in the field, the above preferred conditions can be combined with each other to obtain the preferred embodiments of the invention.
As a preferred embodiment of the present invention, the purification method comprises the steps of:
(1) adjusting the pH value of mupirocin fermentation liquor with the concentration of 2000-3000 mug/ml to 2.5-3.5, carrying out plate-frame pressure filtration to obtain slag, adding 5-6 wt% ammonium bicarbonate solution into the obtained slag, stirring for 2-4 hours, and carrying out plate-frame pressure filtration to obtain filtrate;
(2) carrying out nanofiltration concentration on the filtrate obtained in the step (1) through a nanofiltration membrane with the molecular weight cutoff of 200-300 until the mupirocin content is 40-60 g/L;
(3) adjusting the pH value of the concentrated solution obtained in the step (2) to 3.0-4.0 for coarse crystallization;
(4) dissolving the crude mupirocin product obtained in the step (3) in water to 10-20 g/L, adding 2-3 wt% of sodium chloride, adjusting the pH value to 6.0-7.0 to be used as a column loading solution, loading the column, desorbing the column after the column loading is finished with water washing by using 0.01-0.03 wt% of ammonium bicarbonate, and collecting a desorption solution with the chromatographic purity of more than 90%;
(5) carrying out nanofiltration concentration on the mupirocin desorption qualified liquid obtained in the step (4) through a nanofiltration membrane with the molecular weight cutoff of 200-300 until the mupirocin content is 40-60 g/L;
(6) adjusting the pH value of the concentrated solution obtained in the step (5) to 3.0-4.0 for crystallization;
(7) and (4) drying the crystals obtained in the step (6) in vacuum.
The method adopts macroporous resin chromatography to separate the aqueous solution of the crude mupirocin product, and then carries out nanofiltration, crystallization and drying, so that the purity of the mupirocin can reach more than 99 percent.
Detailed Description
The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
The embodiment provides a mupirocin purification method, which specifically comprises the following steps:
(1) adjusting 60L of 2275ug/ml mupirocin fermentation broth to pH2.5 with dilute hydrochloric acid, and filtering with plate frame to remove water soluble protein, pigment and other macromolecular substances to obtain mupirocin bacterial residue; weighing the mushroom dregs, adding an ammonium bicarbonate solution with the concentration of 5 percent, wherein the volume (ml) of the ammonium bicarbonate solution is 1.5 times of the weight (g) of the mushroom dregs, stirring for 4 hours, then carrying out plate-and-frame filtration, and further removing impurities such as pigment and the like to obtain 40L of mupirocin filtrate with the content of 3345.72 ug/ml;
(2) carrying out primary nanofiltration concentration on the mupirocin filtrate until the mupirocin content in the concentrated solution is 40 g/L;
(3) adjusting the pH of the concentrated solution to 3.0 by using dilute hydrochloric acid, performing primary crystallization, and centrifuging to obtain 115.34g of crude mupirocin;
(4) dissolving crude mupirocin in water to 10g/L, adding 2% sodium chloride, adjusting pH to 6.0, loading onto column at flow rate of 1 times of column bed, wherein the column is HZ-806 of Shanghai Huazhen, washing with water (volume of 3 times of column bed), desorbing with 0.025% ammonium bicarbonate after washing, collecting desorption solution with chromatographic purity of more than 90%, and containing 103.00g of mupirocin;
(5) carrying out secondary nanofiltration concentration on the mupirocin desorption qualified liquid until the mupirocin content in the concentrated liquid is 40 g/L;
(6) adjusting the pH of the concentrated solution to 4.0 by using dilute hydrochloric acid, performing secondary crystallization, and centrifuging to obtain mupirocin tide crystals;
(7) the wet crystals were dried under vacuum at 55 ℃ under-0.092 MPa to yield 88.61g of mupirocin final product with a chromatographic purity of 99.4%.
Example 2
The embodiment provides a mupirocin purification method, which specifically comprises the following steps:
(1) adjusting pH of 2870.22ug/ml mupirocin fermentation broth 60L with dilute hydrochloric acid to 3.5, and filtering with plate frame to remove water soluble protein, pigment and other macromolecular substances to obtain mupirocin bacterial residue; weighing the mushroom dregs, adding an ammonium bicarbonate solution with the concentration of 5 percent, wherein the volume of the ammonium bicarbonate solution is 2 times of the weight of the mushroom dregs, stirring for 2 hours, then carrying out plate-and-frame filtration, and further removing impurities such as pigment and the like to obtain 60L of mupirocin filtrate with the content of 2812.96 ug/ml;
(2) carrying out primary nanofiltration concentration on the mupirocin filtrate until the mupirocin content in the concentrated solution is 60 g/L;
(3) adjusting the pH of the concentrated solution to 4.0 by using dilute hydrochloric acid, performing primary crystallization, and centrifuging to obtain 148.74g of crude mupirocin;
(4) dissolving crude mupirocin in water to 20g/L, adding 3% sodium chloride, adjusting pH to 7.0, loading onto column at flow rate of 1 times of column bed, wherein the column is HZ-806 of Shanghai Huazhen, washing with water (volume of 3 times of column bed), desorbing with 0.025% ammonium bicarbonate after washing, collecting desorption solution with chromatographic purity of more than 90%, and containing 131.19g of mupirocin;
(5) carrying out secondary nanofiltration concentration on the mupirocin desorption qualified liquid until the mupirocin content in the concentrated liquid is 60 g/L;
(6) adjusting the pH of the concentrated solution to 3.0 by using dilute hydrochloric acid, performing secondary crystallization, and centrifuging to obtain mupirocin tide crystals;
(7) the wet crystals were dried under vacuum at 60 ℃ under-0.092 MPa to yield 114.50g of mupirocin final product with a chromatographic purity of 99.1%.
Example 3
The embodiment provides a mupirocin purification method, which specifically comprises the following steps:
(1) adjusting pH of 2091.56ug/ml mupirocin fermentation broth 60L with dilute hydrochloric acid to 3.5, and filtering with plate frame to remove water soluble protein, pigment and other macromolecular substances to obtain mupirocin bacterial residue; weighing the mushroom dregs, adding 6% ammonium bicarbonate solution with the volume 1 time of the weight of the mushroom dregs, stirring for 3 hours, then performing plate-and-frame filtration, and further removing impurities such as pigment and the like to obtain 32L of mupirocin filtrate with the content of 3831.94 ug/ml;
(2) carrying out primary nanofiltration concentration on the mupirocin filtrate until the mupirocin content in the concentrated solution is 40 g/L;
(3) adjusting the pH of the concentrated solution to 3.53 by using dilute hydrochloric acid, performing primary crystallization, and centrifuging to obtain 106.37g of crude mupirocin;
(4) dissolving crude mupirocin in water to 15g/L, adding 2.5% sodium chloride, adjusting pH to 6.5, loading onto column at flow rate of 1 times of column bed, wherein the column is HZ-806 of Shanghai Huazhen, washing with water (volume of 3 times of column bed), desorbing with 0.025% ammonium bicarbonate, and collecting desorption solution with chromatographic purity of more than 90% containing 93.93g of mupirocin;
(5) carrying out secondary nanofiltration concentration on the mupirocin desorption qualified liquid until the mupirocin content in the concentrated liquid is 50 g/L;
(6) adjusting the pH of the concentrated solution to 3.50 with dilute hydrochloric acid for secondary crystallization, and centrifuging to obtain mupirocin tide crystals;
(7) the wet crystals were dried under vacuum at 55 deg.C under-0.092 MPa to yield 82.28g of mupirocin product with a chromatographic purity of 99.2%.
Finally, the examples are only preferred embodiments and are not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A mupirocin purification method is characterized by comprising the following steps:
(1) adjusting the pH value of mupirocin fermentation liquor with the concentration of 2000-3000 mug/ml to 2.5-3.5, carrying out plate-frame pressure filtration to obtain slag, adding 5-6 wt% ammonium bicarbonate solution into the obtained slag, stirring for 2-4 hours, and carrying out plate-frame pressure filtration to obtain filtrate;
(2) carrying out nanofiltration concentration on the filtrate obtained in the step (1) through a nanofiltration membrane with the molecular weight cutoff of 200-300 until the mupirocin content is 40-60 g/L;
(3) adjusting the pH value of the concentrated solution obtained in the step (2) to 3.0-4.0 for coarse crystallization;
(4) dissolving the crude mupirocin product obtained in the step (3) in water to 10-20 g/L, adding 2-3 wt% of sodium chloride, adjusting the pH value to 6.0-7.0 to be used as column loading liquid, loading the column on an HZ-806 column, desorbing the column after washing the column with water by using 0.01-0.03 wt% of ammonium bicarbonate, and collecting desorption liquid with the chromatographic purity of more than 90%;
(5) carrying out nanofiltration concentration on the mupirocin desorption qualified liquid obtained in the step (4) through a nanofiltration membrane with the molecular weight cutoff of 200-300 until the mupirocin content is 40-60 g/L;
(6) adjusting the pH value of the concentrated solution obtained in the step (5) to 3.0-4.0 for crystallization;
(7) and (4) drying the crystals obtained in the step (6) in vacuum.
2. The purification method according to claim 1, wherein in the step (1), the ratio of the ammonium bicarbonate solution to the slag is 1-2 ml:1 g.
3. The purification method according to claim 1, wherein in the step (3), hydrochloric acid, oxalic acid, acetic acid, sulfuric acid or phosphoric acid is used for pH adjustment.
4. The purification method according to claim 3, wherein hydrochloric acid is used for pH adjustment in the step (3).
5. The purification method according to claim 1, wherein in the step (6), hydrochloric acid, oxalic acid, acetic acid, sulfuric acid or phosphoric acid is used for pH adjustment.
6. The purification method according to claim 5, wherein in the step (6), hydrochloric acid is used for pH adjustment.
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Effective date of registration: 20230609 Address after: 3007, Hengqin international financial center building, No. 58, Huajin street, Hengqin new area, Zhuhai, Guangdong 519031 Patentee after: New founder holdings development Co.,Ltd. Patentee after: CHONGQING DAXIN PHARMACEUTICAL Co.,Ltd. Patentee after: Peking University Medical Management Co.,Ltd. Address before: 100871 8th floor, founder building, 298 Chengfu Road, Haidian District, Beijing Patentee before: PEKING UNIVERSITY FOUNDER GROUP Co.,Ltd. Patentee before: CHONGQING DAXIN PHARMACEUTICAL Co.,Ltd. Patentee before: PKU HEALTHCARE INDUSTRY Group |