CN107629116B - Purification method of telavancin - Google Patents

Purification method of telavancin Download PDF

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CN107629116B
CN107629116B CN201710806819.0A CN201710806819A CN107629116B CN 107629116 B CN107629116 B CN 107629116B CN 201710806819 A CN201710806819 A CN 201710806819A CN 107629116 B CN107629116 B CN 107629116B
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telavancin
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乐占线
黄楷
庄鸿
陈秀明
陈宏�
江红
连云阳
乐雨银
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Fujian Institute of Microbiology
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Abstract

The invention provides a purification method of telavancin, which comprises the following steps: 1) and (3) mixing the crude mixture containing telavancin with methanol-formic acid-water solution according to the weight ratio of 1: 5 to 1: 10, mixing and dissolving; 2) purifying the filtrate by a chromatographic medium with well balanced eluent; 3) concentrating the chromatography liquid by nanofiltration membrane, and supplementing hydrochloric acid aqueous solution with pH of 4-5 to obtain concentrated solution under hydrochloric acid; 4) heating the concentrated solution to 40-60 deg.C, and mixing with solvent B selected from acetonitrile, methanol, ethanol, and acetone; 5) then mixing with solvent C of ether or methyl tert-butyl ether, stirring and cooling to 4-10 deg.C, filtering to obtain solid of telavancin hydrochloride. The chromatographic medium naphthyl bonded silica gel or pyrenyl ethyl bonded silica gel selected by the scheme is combined with a proper acid solution or salt solvent system to have a tighter effect on the performance of the telavancin compound and a material medium in the separation process, so that high-purity telavancin hydrochloride can be obtained through separation.

Description

Purification method of telavancin
Technical Field
The invention relates to the field of antibiotic medicines, and particularly relates to a method for purifying telavancin.
Background
The telavancin is the first semi-synthetic novel glycolipid peptide antibiotic, has unique action mechanism and good clinical treatment effect, and has very wide market prospect. The activity of telavancin on MRSA, MRSE and the like is higher than that of vancomycin and teicoplanin, the half-life period is longer than that of vancomycin, and 1-time administration can be carried out clinically. The half-life of Daiwanxing is as long as 174h, and the medicine can be clinically administered 1 time a week. Oritavancin has outstanding pharmacodynamic characteristics, the half-life period of the oritavancin is 393 hours, and the oritavancin can be administrated once per treatment course.
At present, the research on the synthesis process of telavancin mainly focuses on synthesizing hydrophobic side chain N- (9-fluorenylmethoxy cluster) -decylamino technical acid by adopting different methods, and then the telavancin is obtained by the reductive amination reaction of the telavancin and vancomycin, and then the Fmoc deprotection and the Mannich reaction. Because the side reaction product and the target product telavancin in the multi-reaction of the active sites of the glycolipid peptide antibiotics are structural analogues, the purity of the target product is difficult to improve by the conventional production technical processes such as extraction, crystallization and the like to reach the pharmaceutical qualified standard.
The telavancin produced by the processes reported in some documents at present has the defects of insufficient purity, complex production process, easy product formation of Dimer (Dimer) due to long operation time and unstable product quality. If stable crystal salt is formed, good raw medicine can be provided for later preparation.
Disclosure of Invention
In order to solve the technical problem of insufficient purity in a method for preparing telavancin, the invention provides a method for purifying telavancin, which is realized by the following steps:
a method for purifying telavancin, comprising the steps of:
1) and (3) mixing the crude mixture containing telavancin with methanol-formic acid-water solution according to the weight ratio of 1: 5 to 1: 10, mixing to dissolve the crude product mixture, and filtering to remove solid insoluble substances to obtain filtrate; the methanol-formic acid-water solution is prepared according to the volume ratio of 2:1: 1;
one of the process routes for reaction mixtures containing telavancin:
the synthesis method of the novel lipopeptide antibiotic hydrochloride telavancin comprises the following steps: vancomycin hydrochloride (compound I) and N- (9-fluorenylmethoxycarbonyl) -decylaminoacetaldehyde undergo a reductive amination reaction, then 9-fluorenylmethoxycarbonyl protecting groups are removed, and a Mannich reaction is carried out on the obtained product and aminomethylphosphonic acid to synthesize a target product of a crude product of telavancin. (Compound III in the following scheme).
Figure BDA0001402907550000021
2) Passing the filtrate through a chromatographic medium with well-balanced eluent, wherein the weight ratio of the sample filtrate to the chromatographic medium is 1: 50 to 1: 100, respectively; the chromatography medium is one of naphthyl bonded silica gel and pyrenyl ethyl bonded silica gel; the eluent is 3-5 column volumes of solvent A-phosphate buffer solution, the phosphate buffer solution is: 0.01mol/L-0.05mol/L ammonium dihydrogen phosphate or diammonium hydrogen phosphate, and a buffer solution with pH adjusted to 2.0-2.5 by phosphoric acid: the solvent A is one of acetonitrile, methanol, ethanol and acetone, gradient elution is adopted, the mass fraction of the solvent A in the eluent is changed from 10% to 50% in a gradient manner, and the elution flow rate is 1BV/h to 5 BV/h; collecting a chromatographic solution with the HPLC purity of telavancin of more than 95%;
principle of selecting chromatography media:
Figure BDA0001402907550000022
telavancin is a hydrophobic decaaminoethyl side chain obtained by alkylating and acylating a sugar amine side chain on a 4-position ring (shown in figure 5) of vancomycin, and a hydrophilic phosphonic acid aminomethyl group is introduced at the para position of a 7-position amino acid aromatic ring, wherein the pharmacological hydrophobic group is favorable for increasing the interaction of cell membranes and the antibacterial activity to enterococci, and the hydrophilic group promotes the metabolism and clearance of tissues in vivo, reduces the renal toxicity and improves the pharmacokinetic property of the drug in vivo. In terms of synthesis, separation and purification, due to the introduction of a new group and the large property difference of the vancomycin serving as a raw material in a spatial arrangement structure, new problems are brought to the selection of a separation medium and the operation of a purification route;
in "a method for purifying crude telavancin product CN 201510294070.7", a crude product purification method is disclosed, the process steps in the material are: acidifying and dissolving the crude telavancin product, filtering, extracting the obtained filtrate with an organic solvent, back-extracting with a hydrochloric acid solution, separating and extracting by an ion exchange method to obtain a telavancin hydrochloride solution, and then ultrafiltering, crystallizing, washing and drying the telavancin hydrochloride solution. The document states that the disclosed process realizes effective separation and purification, the effective content reaches 99 percent, the product quality is improved, and the production cost is reduced.
The extraction process and the ion exchange resin separation medium in the reference process are difficult to effectively separate the by-products in the semi-synthetic products of telavancin and the impurities introduced by raw materials of vancomycin and the like. Impurities can be effectively separated by selective chromatography media, the production cost is reduced, and the product purity is improved. The combination of the later crystallization steps can obtain stable raw material of telavancin hydrochloride.
The chromatographic medium selected by the scheme is naphthyl bonded silica gel or pyrenyl ethyl bonded silica gel, the bonded material has specific and selective steric hindrance effect on the telavancin compound in space, and meanwhile, the hydrophobic interaction is stronger.
3) Concentrating the chromatography liquid with nanofiltration membrane to above 1/10, supplementing hydrochloric acid aqueous solution with pH of 4-5 when concentrating to 1/5 of the original volume, repeating for 3-5 times to remove buffer salt, and adding hydrochloric acid to adjust pH to 0.5-2 to obtain concentrated solution;
4) heating the concentrated solution to 40-60 ℃, and mixing the concentrated solution with the concentrated solution in a volume ratio of 1: 4 to 1: 8, mixing the solvent B, wherein the solvent B is one of acetonitrile, methanol, ethanol and acetone to obtain a mixed solution;
5) then mixing with the mixed solution in a volume ratio of 1: 0.3 to 1:1, the solvent C is one of aether and methyl tert-butyl ether, the temperature is reduced to 4 to 10 ℃ by stirring, and the solid of the telavancin hydrochloride is obtained by filtering.
The ether solvent selected by the scheme can effectively perform solubilization on telavancin and impurity removal effects on impurities, ether in later-stage products can be effectively removed at low boiling point and low temperature, and formed crystalline salt can well stabilize the products.
The research in the current situation and development of vancomycin research shows that: the stability of vancomycin is poor, the color gradually turns red after the vancomycin is placed at normal temperature, and a large number of researches show that phenolic groups and diphenol groups exist in the molecular structure of the vancomycin, and the aqueous solution of the phenolic groups is easy to hydrate or oxidize into quinone to be pink when being exposed to light. Through the whole process of the scheme, the purification is rapidly carried out, and the problems of long time and degradation caused by no effective crystallization salt formation can be effectively solved.
Preferably, when the solvent a is acetonitrile in step 3), the mass fraction gradient of acetonitrile in the eluent is selected to be 15%, 18%, 20%, 50%; when the solvent A is methanol, the mass fraction gradient of the methanol in the eluent is selected to be 20%, 23%, 25% and 50%; when the solvent A is ethanol, the mass fraction gradient of the ethanol in the eluent is selected to be 15%, 18%, 20% and 50%; when the solvent A is acetone, the gradient of the mass fraction of acetone in the eluent is selected to be 15%, 18%, 20% and 50%.
Preferably, the nanofiltration membrane in the step 3) is a polyethersulfone organic solvent-resistant nanofiltration membrane, and the molecular weight cutoff is 200-1000 daltons.
The range can effectively remove degraded micromolecular impurities, simultaneously dephosphorize buffer salt, achieve the effect of low-temperature concentration of dilute sample elution solution, and avoid the thermal damage caused by overhigh freeze-drying cost or heating, decompressing and concentrating.
Preferably, the chromatography in step 3) is followed by HPLC to track the purity of telavancin in the chromatography liquid.
Preferably, the crude mixture of telavancin in step 1) may be a crude product of telavancin synthesized by a reductive amination reaction between vancomycin hydrochloride and N- (9-fluorenylmethoxycarbonyl) -decylaminoacetaldehyde, removal of a 9-fluorenylmethoxycarbonyl protecting group, and a mannich reaction with aminomethylphosphonic acid, or a sample with purity of 95% or more after one-time purification.
Preferably, the method further comprises a step 6) of washing the solid of telavancin hydrochloride obtained in the step 5) with a washing solution, wherein the washing solution is prepared by mixing the solvent B, the solvent C and water according to a volume ratio of 4: 2:1, washing and filtering the mixed solution to obtain a final product solid.
The invention has the beneficial effects that:
the chromatographic medium selected by the scheme is naphthyl bonded silica gel or pyrenyl ethyl bonded silica gel, the bonded material has specific and selective steric hindrance effect on the telavancin compound in space, meanwhile, the hydrophobic interaction is stronger, the scheme is combined with a proper acid solution or salt solvent system to enable the telavancin compound to have tighter effect on the performance of the material medium in the separation process, and high-purity telavancin can be obtained through separation.
Drawings
FIG. 1 is a Telavancin DAD detector UV spectrum;
FIG. 2 raw sample HPLC of the reaction;
FIG. 3 is a HPLC chart after the purification method of example 5;
FIG. 4 is a mass spectrum analysis of Telavancin;
FIG. 5 is a structural diagram of vancomycin.
Detailed Description
In order to explain technical contents, structural features, and objects and effects of the present invention in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.
Example 1
A method for purifying telavancin, comprising the steps of:
1) and (3) mixing the crude mixture containing telavancin with methanol-formic acid-water solution according to the weight ratio of 1: 5, mixing to dissolve the crude product mixture, and filtering to remove solid insoluble substances to obtain filtrate; the methanol-formic acid-water solution is prepared according to the volume ratio of 2:1: 1; the crude product mixture of the telavancin in the step 1) is a crude product of the telavancin synthesized by the reductive amination reaction of vancomycin hydrochloride and N- (9-fluorenylmethoxycarbonyl) -decylaminoacetaldehyde, then the 9-fluorenylmethoxycarbonyl protecting group is removed, and the Mannich reaction of the crude product mixture of the telavancin and aminomethylphosphonic acid is carried out;
2) passing the filtrate through a chromatographic medium with well-balanced eluent, wherein the weight ratio of the sample filtrate to the chromatographic medium is 1: 50; the chromatography medium is naphthyl bonded silica gel; the eluent is 3-5 column volumes of solvent A-phosphate buffer solution, the phosphate buffer solution is: ammonium dihydrogen phosphate 0.01mol/L, buffer solution with phosphoric acid to adjust pH at 2.0: when the solvent A is acetonitrile, the mass fraction gradient of the acetonitrile in the eluent is selected to be 15%, 18%, 20% and 50%, and the elution flow rate is 1 BV/h; collecting chromatographic liquid with the HPLC purity of telavancin of more than 95% (when the eluent contains telavancin with the cross purity of less than 95%, combining, adsorbing with macroporous adsorbent resin HP20, recovering, and further purifying);
3) concentrating the chromatography liquid with nanofiltration membrane to above 1/10, supplementing hydrochloric acid aqueous solution with pH4 when concentrating to 1/5 of the original volume, repeating for 3 times to remove buffer salt, and adding hydrochloric acid to adjust pH to 0.5 to obtain concentrated solution; the nanofiltration membrane is polyether sulfone organic solvent resistant and has a molecular weight cutoff of 200 daltons; the purity of telavancin in a chromatographic solution is tracked by HPLC;
4) heating the concentrated solution to 40 ℃, and mixing the concentrated solution with the concentrated solution in a volume ratio of 1: 4, mixing the solvent B with acetonitrile to obtain a mixed solution;
5) then mixing with the mixed solution in a volume ratio of 1: 0.3 of solvent C, wherein the solvent C is diethyl ether, stirring and cooling to 4 ℃, and filtering to obtain solid of telavancin hydrochloride;
6) washing the solid of telavancin hydrochloride obtained in the step 5) with a washing solution, wherein the washing solution is prepared by mixing a solvent B, a solvent C and water according to a volume ratio of 4: 2:1, washing and filtering the mixed solution to obtain a final product solid.
Example 2
A method for purifying telavancin, comprising the steps of:
1) and (3) mixing the crude mixture containing telavancin with methanol-formic acid-water solution according to the weight ratio of 1: 10, mixing to dissolve the crude product mixture, and filtering to remove solid insoluble substances to obtain filtrate; the methanol-formic acid-water solution is prepared according to the volume ratio of 2:1: 1; the crude product mixture of telavancin in the step 1) is a sample with purity of more than 95 percent after one-time purification;
2) passing the filtrate through a chromatographic medium with well-balanced eluent, wherein the weight ratio of the sample filtrate to the chromatographic medium is 1: 100, respectively; the chromatography medium is pyrenyl ethyl bonded silica gel; the eluent is a solvent A-phosphate buffer solution with 5 column volumes, and the phosphate buffer solution is: diammonium hydrogen phosphate 0.05mol/L, buffer solution with phosphoric acid to adjust pH to 2.5: when the solvent A is methanol, the mass fraction gradient of the methanol in the eluent is selected to be 20%, 23%, 25% and 50%, and the elution flow rate is 1BV/h to 5 BV/h; collecting chromatographic liquid with the HPLC purity of telavancin of more than 95% (when the eluent contains telavancin with the cross purity of less than 95%, combining, adsorbing with macroporous adsorbent resin HP20, recovering, and further purifying);
3) concentrating the chromatography liquid with nanofiltration membrane to above 1/10, supplementing hydrochloric acid aqueous solution with pH5 when concentrating to 1/5 of the original volume, repeating for 5 times to remove buffer salt, and adding hydrochloric acid to adjust pH to 2 to obtain concentrated solution; the nanofiltration membrane is polyether sulfone organic solvent resistant and has a molecular weight cutoff of 1000 daltons; the purity of telavancin in a chromatographic solution is tracked by HPLC;
4) heating the concentrated solution to 60 ℃, and mixing the concentrated solution with the concentrated solution in a volume ratio of 1: 8, mixing the solvent B with methanol to obtain a mixed solution;
5) then mixing with the mixed solution in a volume ratio of 1:1, the solvent C is methyl tert-butyl ether, the mixture is stirred and cooled to 10 ℃, and solid of telavancin hydrochloride is obtained by filtering;
6) washing the solid of telavancin hydrochloride obtained in the step 5) with a washing solution, wherein the washing solution is prepared by mixing a solvent B, a solvent C and water according to a volume ratio of 4: 2:1, washing and filtering the mixed solution to obtain a final product solid.
Example 3
A method for purifying telavancin, comprising the steps of:
1) and (3) mixing the crude mixture containing telavancin with methanol-formic acid-water solution according to the weight ratio of 1: 6, mixing to dissolve the crude product mixture, and filtering to remove solid insoluble substances to obtain filtrate; the methanol-formic acid-water solution is prepared according to the volume ratio of 2:1: 1; the crude product mixture of telavancin in the step 1) is a sample with purity of more than 95% or after one-time purification;
2) passing the filtrate through a chromatographic medium with well-balanced eluent, wherein the weight ratio of the sample filtrate to the chromatographic medium is 1: 80; the chromatography medium is pyrenyl ethyl bonded silica gel; the eluent is a 4 column volumes of solvent a-phosphate buffer solution, the phosphate buffer solution is: diammonium hydrogen phosphate 0.03mol/L, buffer solution with pH adjusted to 2.3 by phosphoric acid: when the solvent A is ethanol, the mass fraction gradient of the ethanol in the eluent is selected to be 15%, 18%, 20% and 50%, and the elution flow rate is 1BV/h to 5 BV/h; collecting chromatographic liquid with the HPLC purity of telavancin of more than 95% (when the eluent contains telavancin with the cross purity of less than 95%, combining, adsorbing with macroporous adsorbent resin HP20, recovering, and further purifying);
3) concentrating the chromatography liquid with nanofiltration membrane to above 1/10, supplementing hydrochloric acid aqueous solution with pH of 4.5 when concentrating to 1/5 of the original volume, repeating for 4 times to remove buffer salt, and adding hydrochloric acid to adjust pH to 1.5 to obtain concentrated solution; the nanofiltration membrane is polyether sulfone organic solvent resistant and has a molecular weight cutoff of 1000 daltons; the purity of telavancin in a chromatographic solution is tracked by HPLC;
4) heating the concentrated solution to 50 ℃, and mixing the concentrated solution with the concentrated solution in a volume ratio of 1: 6, mixing the solvent B with ethanol to obtain a mixed solution;
5) then mixing with the mixed solution in a volume ratio of 1: 0.7 of solvent C, wherein the solvent C is diethyl ether, stirring and cooling to 8 ℃, and filtering to obtain solid of telavancin hydrochloride;
6) washing the solid of telavancin hydrochloride obtained in the step 5) with a washing solution, wherein the washing solution is prepared by mixing a solvent B, a solvent C and water according to a volume ratio of 4: 2:1, washing and filtering the mixed solution to obtain a final product solid.
Example 4
A method for purifying telavancin, comprising the steps of:
1) and (3) mixing the crude mixture containing telavancin with methanol-formic acid-water solution according to the weight ratio of 1: 9, mixing to dissolve the crude product mixture, and filtering to remove solid insoluble substances to obtain filtrate; the methanol-formic acid-water solution is prepared according to the volume ratio of 2:1: 1; the crude product mixture of the telavancin in the step 1) is a crude product of the telavancin synthesized by the reductive amination reaction of vancomycin hydrochloride and N- (9-fluorenylmethoxycarbonyl) -decylaminoacetaldehyde, then the 9-fluorenylmethoxycarbonyl protecting group is removed, and the Mannich reaction of the crude product mixture of the telavancin and aminomethylphosphonic acid is carried out;
2) passing the filtrate through a chromatographic medium with well-balanced eluent, wherein the weight ratio of the sample filtrate to the chromatographic medium is 1: 85 parts by weight; the chromatography medium is naphthyl bonded silica gel; the eluent is 3 column volumes of solvent a-phosphate buffer solution, the phosphate buffer solution is: ammonium dihydrogen phosphate 0.02mol/L, buffer solution with phosphoric acid to adjust pH to 2.1: when the solvent A is acetone, the mass fraction gradient of the acetone in the eluent is selected to be 15%, 18%, 20% and 50%, and the elution flow rate is 4 BV/h; collecting chromatographic liquid with the HPLC purity of telavancin of more than 95% (when the eluent contains telavancin with the cross purity of less than 95%, combining, adsorbing with macroporous adsorbent resin HP20, recovering, and further purifying);
3) concentrating the chromatography liquid with nanofiltration membrane to above 1/10, supplementing hydrochloric acid aqueous solution with pH5 when concentrating to 1/5 of the original volume, repeating for 3 times to remove buffer salt, and adding hydrochloric acid to adjust pH to 1 to obtain concentrated solution; the nanofiltration membrane is polyether sulfone organic solvent resistant and has a molecular weight cutoff of 200 daltons; the purity of telavancin in a chromatographic solution is tracked by HPLC;
4) heating the concentrated solution to 55 ℃, and mixing the concentrated solution with the concentrated solution in a volume ratio of 1: 7, mixing the solvent B with acetone to obtain a mixed solution;
5) then mixing with the mixed solution in a volume ratio of 1: 0.9 of solvent C, namely methyl tert-butyl ether, stirring, cooling to 5 ℃, and filtering to obtain solid of telavancin hydrochloride;
6) washing the solid of telavancin hydrochloride obtained in the step 5) with a washing solution, wherein the washing solution is prepared by mixing a solvent B, a solvent C and water according to a volume ratio of 4: 2:1, washing and filtering the mixed solution to obtain a final product solid.
Example 5
A method for purifying telavancin, comprising the steps of:
1) and (3) mixing the crude mixture containing telavancin with methanol-formic acid-water solution according to the weight ratio of 1: 6, mixing to dissolve the crude product mixture, and filtering to remove solid insoluble substances to obtain filtrate; the methanol-formic acid-water solution is prepared according to the volume ratio of 2:1: 1; the crude product mixture of telavancin in the step 1) is a sample with purity of more than 95 percent after one-time purification;
2) passing the filtrate through a chromatographic medium with well-balanced eluent, wherein the weight ratio of the sample filtrate to the chromatographic medium is 1: 65; the chromatography medium is naphthyl bonded silica gel; the eluent is a solvent A-phosphate buffer solution with 5 column volumes, and the phosphate buffer solution is: ammonium dihydrogen phosphate 0.04mol/L, buffer solution with pH adjusted by phosphoric acid at 2.4: when the solvent A is ethanol, the mass fraction gradient of the ethanol in the eluent is selected to be 15%, 18%, 20% and 50%, and the elution flow rate is 2 BV/h; collecting chromatographic liquid with the HPLC purity of telavancin of more than 95% (when the eluent contains telavancin with the cross purity of less than 95%, combining, adsorbing with macroporous adsorbent resin HP20, recovering, and further purifying);
3) concentrating the chromatography liquid with nanofiltration membrane to above 1/10, supplementing hydrochloric acid aqueous solution with pH4 when concentrating to 1/5 of the original volume, repeating for 4 times to remove buffer salt, and adding hydrochloric acid to adjust pH to 2 to obtain concentrated solution; the nanofiltration membrane is polyether sulfone organic solvent resistant and has a molecular weight cutoff of 200 daltons; the purity of telavancin in a chromatographic solution is tracked by HPLC;
4) heating the concentrated solution to 45 ℃, and mixing the concentrated solution with the concentrated solution in a volume ratio of 1: 7, mixing the solvent B with ethanol to obtain a mixed solution;
5) then mixing with the mixed solution in a volume ratio of 1: 0.9 of solvent C, wherein the solvent C is diethyl ether, stirring and cooling to 5 ℃, and filtering to obtain solid of telavancin hydrochloride;
6) washing the solid of telavancin hydrochloride obtained in the step 5) with a washing solution, wherein the washing solution is prepared by mixing a solvent B, a solvent C and water according to a volume ratio of 4: 2:1, washing and filtering the mixed solution to obtain a final product solid.
And (4) HPLC detection:
1. detection conditions are as follows:
mobile phase: ammonium dihydrogen phosphate water (0.05mol/L, pH2.0 adjusted with phosphoric acid): acetonitrile 80: 20,
a chromatographic column: kromasil 100-5-C184.6 × 250mm, flow rate: 1.0ml/min, column temperature: 40 ℃, detection wavelength: diode Array Detector (DAD), 210nm tracking wavelength; and Rt of Telavancin is 23.2 min.
2. Test sample comparison test
The ultraviolet spectrum of the DAD detector of Telavancin is shown in detail in figure 1;
taking the reactant as an original sample, and detecting the purity of the reactant according to the HPLC detection conditions, wherein the details are shown in figure 2;
taking a sample of the sample obtained by the purification method of example 5, and detecting the purity of the sample according to the HPLC detection conditions, see FIG. 3 in detail;
the sample obtained by the purification method of example 5 was sampled and its mass spectrum was detected, as shown in FIG. 4(HRMS (ESI) m/z: calcd C)80H106Cl2N11O27P[M+2H]2+877.8187，found 877.8249[M+2H]2+，585.5532[M+3H]3+)。
Comparing fig. 2 with fig. 3(HPLC purity above 98%) and fig. 4, it can be seen that the purity of telavancin can be significantly improved by the purification method of the present invention.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by using the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims (6)

1. A method for purifying telavancin is characterized in that: which comprises the following steps:
1) performing Mannich reaction on a crude telavancin product synthesized by performing Mannich reaction or a sample which is subjected to primary purification and has the purity of more than 95%, and performing reaction on the crude telavancin product or the sample with the purity of more than 95% by using methanol-formic acid-aqueous solution according to the weight ratio of 1: 5 to 1: 10, mixing, dissolving the crude telavancin product synthesized by the Mannich reaction or a sample with the purity of more than 95 percent after primary purification, and filtering to remove solid insoluble substances to obtain filtrate; the methanol-formic acid-water solution is prepared according to the volume ratio of 2:1: 1;
2) passing the filtrate through a chromatographic medium with well-balanced eluent, wherein the weight ratio of the sample filtrate to the chromatographic medium is 1: 50 to 1: 100, respectively; the chromatography medium is one of naphthyl bonded silica gel and pyrenyl ethyl bonded silica gel; the eluent is 3-5 column volumes of solvent A-phosphate buffer solution, the phosphate buffer solution is: 0.01mol/L-0.05mol/L ammonium dihydrogen phosphate or diammonium hydrogen phosphate, and phosphoric acid to regulate pH to 2.0-2.5; the solvent A is one of acetonitrile, methanol, ethanol and acetone, gradient elution is adopted, the mass fraction of the solvent A in the eluent is changed from 10% to 50% in a gradient manner, and the elution flow rate is 1BV/h to 5 BV/h; collecting a chromatographic solution with the HPLC purity of telavancin of more than 95%;
3) concentrating the chromatography liquid with nanofiltration membrane to above 1/10, supplementing hydrochloric acid aqueous solution with pH of 4-5 when concentrating to 1/5 of the original volume, repeating for 3-5 times to remove buffer salt, and adding hydrochloric acid to adjust pH to 0.5-2 to obtain concentrated solution;
4) heating the concentrated solution to 40-60 ℃, and mixing the concentrated solution with the concentrated solution in a volume ratio of 1: 4 to 1: 8, mixing the solvent B, wherein the solvent B is one of acetonitrile, methanol, ethanol and acetone to obtain a mixed solution;
5) then mixing with the mixed solution in a volume ratio of 1: 0.3 to 1:1, the solvent C is one of aether and methyl tert-butyl ether, the temperature is reduced to 4 to 10 ℃ by stirring, and the solid of the telavancin hydrochloride is obtained by filtering.
2. A method of purifying telavancin according to claim 1, wherein: when the solvent A is acetonitrile in the step 2), selecting the mass fraction gradient of the acetonitrile in the eluent to be 15%, 18%, 20% and 50%; when the solvent A is methanol, the mass fraction gradient of the methanol in the eluent is selected to be 20%, 23%, 25% and 50%; when the solvent A is ethanol, the mass fraction gradient of the ethanol in the eluent is selected to be 15%, 18%, 20% and 50%; when the solvent A is acetone, the gradient of the mass fraction of acetone in the eluent is selected to be 15%, 18%, 20% and 50%.
3. A method of purifying telavancin according to claim 1, wherein: the nanofiltration membrane in the step 3) is a polyethersulfone organic solvent resistant nanofiltration membrane, and the molecular weight cutoff is 200-1000 daltons.
4. A method of purifying telavancin according to claim 1, wherein: and (3) adopting HPLC to track the purity of the telavancin in the chromatographic solution in the step 2).
5. A method of purifying telavancin according to claim 1, wherein: the crude product mixture of telavancin in the step 1) can be a crude product of telavancin synthesized by the reductive amination reaction of vancomycin hydrochloride and N- (9-fluorenylmethoxycarbonyl) -decylaminoacetaldehyde, then the 9-fluorenylmethoxycarbonyl protecting group is removed, and the Mannich reaction of the crude product and aminomethylphosphonic acid is carried out, or a sample with the purity of more than 95 percent after one-time purification.
6. A method of purifying telavancin according to claim 1, wherein: the method also comprises a step 6) of washing the solid of the telavancin hydrochloride obtained in the step 5) with a washing solution, wherein the washing solution is prepared by mixing a solvent B, a solvent C and water according to a volume ratio of 4: 2:1, washing and filtering the mixed solution to obtain a final product solid.
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