CN113563426B - Separation and purification method of oritavancin mother nucleus A82846B - Google Patents
Separation and purification method of oritavancin mother nucleus A82846B Download PDFInfo
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- CN113563426B CN113563426B CN202110896123.8A CN202110896123A CN113563426B CN 113563426 B CN113563426 B CN 113563426B CN 202110896123 A CN202110896123 A CN 202110896123A CN 113563426 B CN113563426 B CN 113563426B
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- C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
- C07K9/006—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure
- C07K9/008—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure directly attached to a hetero atom of the saccharide radical, e.g. actaplanin, avoparcin, ristomycin, vancomycin
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Abstract
The invention discloses a separation and purification method of Oritavancin mother nucleus A82846B, which belongs to the technical field of microbial fermentation, and the separation and purification method comprises the steps of adjusting the pH of A82846B fermentation liquor by using liquid alkali, then carrying out ceramic membrane filtration, and collecting filtrate; then adsorbing by ion exchange resin, washing with water until the leaked liquid is nearly colorless, eluting with ammonia water solution, and collecting eluent A; adsorbing with decolorizing resin, washing with water until the eluate is nearly colorless, eluting with ethanol solution, and collecting eluate B; adsorbing with polymer chromatographic packing, eluting with purified water, gradient eluting with ammonium chloride-ethanol solution, and collecting A82846B eluate with purity greater than 98%. Compared with the common separation and purification method, the separation and purification method is simpler, has low requirement on equipment, does not use toxic reagents in the production process, and is safer.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a separation and purification method of oritavancin mother nucleus A82846B.
Background
Oritavancin (Oritavancin) is a second-generation novel semisynthetic antibiotic developed on the basis of vancomycin and teicoplanin, which is a first-generation glycopeptide antibiotic, and is mainly obtained by chemical semisynthesis of a secondary metabolite A82846B derived from microorganisms. Oritavancin is an antibiotic that can be administered in a single dose regimen and was the first antibiotic Drug approved by the united states Food and Drug Administration (FDA) for clinical use in the treatment of acute bacterial skin and skin structure infections (abssi). The antibacterial mechanism is similar to that of vancomycin, the formation of bacterial cell walls is inhibited by transglycosylation during peptidoglycan biosynthesis, and the antibacterial agent has obvious effect on killing methicillin-resistant staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).
At present, in order to obtain a high-purity oritavancin mother nucleus product, the oritavancin mother nucleus product needs to be prepared and separated through high-pressure reverse phase C18 after decoloration is finished, the requirement on equipment is high, a solvent used for preparation usually needs to be separated by using high-toxicity acetonitrile, the whole route is complex, the cost is high, and toxic substances are used to potentially threaten operating personnel.
Disclosure of Invention
In order to overcome the defects of the prior art, the technical problems to be solved by the invention are as follows: provides a simple and safe separation and purification method capable of obtaining high-purity oritavancin mother nucleus A82846B.
In order to solve the technical problems, the invention adopts the technical scheme that:
the separation and purification method of the oritavancin mother nucleus A82846B comprises the following steps:
step 1, adjusting the pH of the A82846B fermentation liquor by using liquid alkali, then filtering by using a ceramic membrane, and collecting filtrate;
step 2, adsorbing the filtrate obtained in the step 1 by ion exchange resin, then washing with water until the leaked liquid is nearly colorless, eluting with ammonia water solution, and collecting eluent A;
adsorbing the eluent A obtained in the step (3) and the step (2) by decolorizing resin, then washing by water until the leaked liquid is nearly colorless, eluting by using ethanol solution, and collecting an eluent B;
and 4, adsorbing the eluent B obtained in the step 3 by using a polymer chromatographic packing, carrying out top washing by using purified water, carrying out gradient elution by using an ammonium chloride-ethanol solution, and collecting the eluent A82846B with the purity of more than 98%.
The invention has the beneficial effects that: according to the method for separating and purifying the oritavancin mother nucleus A82846B, provided by the invention, the polymer chromatographic packing is adopted and is eluted under an ammonium chloride-ethanol water elution system, so that the main impurity A82846A in the oritavancin mother nucleus can be effectively removed, and compared with a common separation and purification method, the oritavancin mother nucleus A82846B with the purity of more than 98% is obtained.
Detailed Description
In order to explain the technical contents, the objects and effects of the present invention in detail, the following description is given with reference to the embodiments.
The separation and purification method of Oritavancin mother nucleus A82846B of the invention comprises the following steps:
step 1, adjusting the pH of the A82846B fermentation liquor by using liquid alkali, then filtering by using a ceramic membrane, and collecting filtrate;
step 2, adsorbing the filtrate obtained in the step 1 by ion exchange resin, then washing with water until the leaked liquid is nearly colorless, eluting with ammonia water solution, and collecting eluent A;
adsorbing the eluent A obtained in the step (3) and the step (2) by decolorizing resin, then washing by water until the leaked liquid is nearly colorless, eluting by using ethanol solution, and collecting an eluent B;
and 4, adsorbing the eluent B obtained in the step 3 by using a polymer chromatographic packing, carrying out top washing by using purified water, carrying out gradient elution by using an ammonium chloride-ethanol solution, and collecting the eluent A82846B with the purity of more than 98%.
From the above description, the beneficial effects of the present invention are: according to the separation and purification method of oritavancin mother nucleus A82846B, provided by the invention, ceramic membrane filtration is adopted firstly, water-soluble protein, pigment and other macromolecular substances in fermentation liquor are effectively removed, then polymer chromatographic packing is used for eluting under an ammonium chloride-ethanol water elution system after decoloration, main impurities A82846A in oritavancin mother nucleus can be effectively removed, and compared with a common separation and purification method, the oritavancin mother nucleus A82846B with the purity of more than 98% is obtained.
Further, the method for separating and purifying the oritavancin mother nucleus A82846B further comprises the steps of 5 and 3, wherein the A82846B eluent obtained in the step is subjected to nanofiltration and concentration until the ethanol degree is less than 0.5 percent and the unit is more than 20000, and then the oritavancin mother nucleus A82846B is obtained by freeze-drying.
Further, the polymer chromatographic packing in the step 3 is NM-100 polymer chromatographic packing (Somi-Chi-Tech Co., ltd., the same applies hereinafter).
Further, the pH of the fermentation liquor A82846B in the step 1 is adjusted to 10.5-11.5 by liquid alkali.
Further, the step 2 is eluted by 0.2-0.5% (volume percentage) ammonia water solution.
Further, the step 3 adopts 2-5% (volume percent) of ethanol solution for elution.
Further, the step 4 is carried out by using an ammonium chloride-ethanol solution gradient elution method as follows: gradient elution is carried out by using 50mmol/L ammonium chloride-2% ethanol solution and 50mmol/L ammonium chloride-5% ethanol solution in sequence.
Example 1:
the separation and purification method of oritavancin mother nucleus A82846B comprises the following steps:
step 1, adjusting the pH of A82846B fermentation liquor to 10.5 by using liquid alkali, filtering the fermentation liquor by using a ceramic membrane with the aperture of 0.01 mu m, circularly washing the ceramic membrane by using water with the volume of 3 times of that of the fermentation liquor, and collecting clear and transparent filtrate;
adjusting the pH value of the filtrate obtained in the step 1 to 2.5 by glacial acetic acid, adsorbing by ion exchange resin PT151 (Shanghai 26107Yongshilimited), washing with water until the leaked liquid is nearly colorless, eluting with 0.2% (volume percent) of ammonia water solution, and collecting eluent A;
adsorbing the eluent A obtained in the step (2) by decolorizing resin PAD900 (Shanghai 261073 Yongshilimited), then washing with water until the leaked liquid is nearly colorless, eluting with 5 percent (volume percentage) of ethanol solution, and collecting eluent B;
adsorbing the eluent B obtained in the step (4) and the step (3) by using NM-100 polymer chromatographic packing, after top washing by using purified water, sequentially carrying out gradient elution by using 50mmol/L ammonium chloride-2% ethanol solution and 50mmol/L ammonium chloride-5% ethanol solution, collecting the eluent A82846B, and testing the purity of the eluent A82846B to be 98.5%;
and (5) nano-filtering and concentrating the A82846B eluent obtained in the step (4) until the ethanol degree is less than 0.5 percent and the unit is more than 20000, and freeze-drying to obtain the oritavancin mother nucleus A82846B.
The preparation method of the A82846B fermentation liquor specifically comprises the following steps:
step 1, inoculating Amycolatopsis orientalis NRRL 18099 into a first seed culture medium for shake flask seed culture; wherein the culture period is 72 + -6 h, the culture temperature is 29 + -1 ℃, the rotation speed of a shaking table is 240-260rpm, and the inoculation amount is 1-1.5%, so as to obtain a seed solution;
the formula of the first seed culture medium is as follows: 2.0wt% of soluble starch, 1.0wt% of glucose, 1.5wt% of soybean cake powder, 0.5wt% of yeast extract and 0.1wt% of calcium carbonate, and the pH value is 7.0 +/-0.2;
step 2, inoculating the seed solution into a second seed culture medium according to the inoculation amount of 0.13-0.20% for seed tank culture; wherein the culture period is 72 +/-6 h, the culture temperature is 30 +/-1 ℃, and the air flow is as follows: 1, 1.8-1, wherein vvm is 1.5vvm, the culture rotation speed is 30-40HZ, and the culture tank pressure is 0.03 +/-0.01 MPa, so as to obtain a seed tank solution;
the formula of the second seed culture medium is as follows: 2.0wt% of soluble starch, 1.0wt% of glucose, 1.5wt% of soybean cake powder, 0.5wt% of yeast extract, 0.1wt% of calcium carbonate and 0.05wt% of natural plant extracts;
step 3, inoculating 10 +/-1% of the inoculation amount of the seed tank liquid into a fermentation medium for fermentation culture to obtain A82846B fermentation liquid; wherein the culture period is 7 +/-1 d, the culture temperature is 32 +/-1 ℃, and the air flow is as follows: 1, 0.6-1, 0.8vvm, a culture rotation speed of 35-50HZ, a culture tank pressure of 0.03 +/-0.01 MPa, and culture dissolved oxygen of more than or equal to 30 percent;
the formula of the fermentation medium is as follows: 3.0wt% of maltodextrin, 1.0wt% of glucose, 3.0wt% of cane sugar, 2.5wt% of soybean meal, 0.5wt% of yeast powder, 0.5wt% of corn protein powder, 0.15wt% of ammonium chloride, 0.2wt% of calcium carbonate, 0.8wt% of anhydrous calcium chloride 10g/L and 0.05wt% of natural killer.
Example 2:
the separation and purification method of the oritavancin mother nucleus A82846B comprises the following steps:
step 1, adjusting the pH of the A82846B fermentation liquor to 11.5 by using liquid alkali, filtering the fermentation liquor by using a ceramic membrane with the aperture of 0.01 mu m, circularly washing the ceramic membrane by using water with the volume of 3 times of that of the fermentation liquor, and collecting filtrate;
step 2, adjusting the pH of the filtrate obtained in the step 1 to 3.5 by glacial acetic acid, adsorbing by ion exchange resin (same as the example 1), then washing by water until the leaked liquid is nearly colorless, eluting by 0.2 percent (volume percentage) of ammonia water solution, and collecting eluent A;
adsorbing the eluent A obtained in the step (3) and the step (2) by decolorizing resin (same as the example 1), then washing by water until the leaked liquid is nearly colorless, eluting by 5 percent (volume percentage) of ethanol solution, and collecting eluent B;
adsorbing the eluent B obtained in the step (4) and the step (3) by using NM-100 polymer chromatographic packing, after top washing by using purified water, sequentially carrying out gradient elution by using 50mmol/L ammonium chloride-2% ethanol solution and 50mmol/L ammonium chloride-5% ethanol solution, collecting the eluent A82846B, and testing the purity of the eluent A82846B to be 98.9%;
nanofiltration and concentration are carried out on the A82846B eluent obtained in the step 5 and the step 4 until the ethanol degree is less than 0.5 percent and the unit is more than 20000, and then the oritavancin mother nucleus A82846B is obtained by freeze-drying;
the preparation method of the A82846B fermentation broth is the same as that in example 1.
Example 3:
the separation and purification method of the oritavancin mother nucleus A82846B comprises the following steps:
step 1, adjusting the pH of the A82846B fermentation liquor to 11.5 by using liquid alkali, filtering the fermentation liquor by using a ceramic membrane with the aperture of 0.01 mu m, circularly washing the ceramic membrane by using water with the volume of 3 times of that of the fermentation liquor, and collecting filtrate;
adjusting the pH value of the filtrate obtained in the step 1 to 3 by glacial acetic acid, adsorbing by ion exchange resin (same as the example 1), washing by water until the leaked liquid is nearly colorless, eluting by 0.5 percent (volume percentage) of ammonia water solution, and collecting the eluent A;
adsorbing the eluent A obtained in the step (3) and the step (2) by decolorizing resin (same as the example 1), then washing by water until the leaked liquid is nearly colorless, eluting by 2 percent (volume percentage) of ethanol solution, and collecting eluent B;
adsorbing the eluent B obtained in the step (4) and the step (3) by using NM-100 polymer chromatographic packing, after top washing by using purified water, sequentially carrying out gradient elution by using 50mmol/L ammonium chloride-2% ethanol solution and 50mmol/L ammonium chloride-5% ethanol solution, collecting the eluent A82846B, and testing the purity of the eluent A82846B to be 98.2%;
nanofiltration and concentration are carried out on the A82846B eluent obtained in the step 5 and the step 4 until the ethanol degree is less than 0.5 percent and the unit is more than 20000, and then the oritavancin mother nucleus A82846B is obtained by freeze-drying;
wherein, the preparation method of the A82846B fermentation liquor is the same as that of the example 1.
Comparative example 1:
comparative example 1 differs from example 1 only in that the eluate B of step 4 of comparative example 1 is adsorbed onto PS-40 polymer chromatography packing (UniPS 40-300 polymer matrix chromatography packing, sovium Nano Microbiol science Limited).
The purity of the eluate in test a82846B was 96.4%.
In conclusion, the method for separating and purifying the oritavancin mother nucleus A82846B provided by the invention can effectively remove the main impurity A82846A in the oritavancin mother nucleus by adopting the specific polymer chromatographic packing and eluting under an ammonium chloride-ethanol water elution system, and compared with the common separation and purification method, the method for separating and purifying the oritavancin mother nucleus A82846B with the purity of more than 98% is obtained.
The above description is only an example of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by the present specification, or directly or indirectly applied to the related technical field are included in the scope of the present invention.
Claims (2)
1. The separation and purification method of Oritavancin mother nucleus A82846B is characterized by comprising the following steps:
step 1, adjusting the pH of the A82846B fermentation liquor by using liquid alkali, then filtering by using a ceramic membrane, and collecting filtrate;
adsorbing the filtrate obtained in the step 1 by using ion exchange resin PT151, washing with water, eluting with an ammonia solution, and collecting eluent A;
adsorbing the eluent A obtained in the step (3) and the step (2) by decolorizing resin PAD900, eluting by using ethanol solution after water washing, and collecting eluent B;
adsorbing the eluent B obtained in the step (4) and the step (3) by using a polymer chromatographic packing, carrying out top washing by using purified water, carrying out gradient elution by using an ammonium chloride-ethanol solution, and collecting an A82846B eluent with the purity of more than 98%;
the polymer chromatographic packing in the step 4 is NM-100 polymer chromatographic packing;
in the step 1, the pH value of the A82846B fermentation liquor is adjusted to 10.5-11.5 by liquid alkali;
eluting with 0.2-0.5% ammonia water solution in the step 2;
eluting by using 2-5% ethanol solution in the step 3;
the step 4 of gradient elution by using an ammonium chloride-ethanol solution comprises the following steps: gradient elution is carried out by using 50mmol/L ammonium chloride-2% ethanol solution and 50mmol/L ammonium chloride-5% ethanol solution in sequence.
2. The separation and purification method of oritavancin mother nucleus A82846B as claimed in claim 1, further comprising a step 5, a step 4, wherein the A82846B eluent obtained is subjected to nanofiltration concentration until the ethanol degree is less than 0.5% and the unit is more than 20000, and then the oritavancin mother nucleus A82846B is obtained by freeze-drying.
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| CN107434823B (en) * | 2016-05-26 | 2021-11-16 | 江苏恒瑞医药股份有限公司 | Purification method of oritavancin intermediate A82846B |
| CN106928323B (en) * | 2017-03-02 | 2021-08-20 | 重庆乾泰生物医药有限公司 | Preparation method of high-purity oritavancin key intermediate A82846B |
| CN109988226A (en) * | 2017-12-29 | 2019-07-09 | 上海来益生物药物研究开发中心有限责任公司 | A kind of purification process of oritavancin |
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