CN113637050B - Decoloration method of teicoplanin - Google Patents
Decoloration method of teicoplanin Download PDFInfo
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- CN113637050B CN113637050B CN202111078703.2A CN202111078703A CN113637050B CN 113637050 B CN113637050 B CN 113637050B CN 202111078703 A CN202111078703 A CN 202111078703A CN 113637050 B CN113637050 B CN 113637050B
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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 belongs to the technical field of antibiotic decoloration, and particularly relates to a method for decoloring teicoplanin, which comprises the steps of using MONO HZ20 macroporous adsorption resin as a stationary phase and using an alcoholic solution with pH = 10-11 as a mobile phase to decolor teicoplanin for the first time; and a step of carrying out secondary decolorization on teicoplanin by using Unigel beads Xl ion exchange resin as a stationary phase and using a salt solution with the concentration of 450-500 mmol/L as a mobile phase. The method for decoloring teicoplanin provided by the invention can reduce the decoloring frequency of active carbon, meet the chromaticity standard of teicoplanin and simultaneously improve the purity and yield of teicoplanin.
Description
Technical Field
The invention belongs to the technical field of antibiotic decoloration, and particularly relates to a method for decoloring teicoplanin.
Background
In the prior art, teicoplanin, also known as vancomycin, has a similar antimicrobial spectrum to vancomycin and has antimicrobial activity against both anaerobic and aerobic gram-positive bacteria. The teicoplanin is used as a fermentation product of specific actinoplanes, and the chromaticity of a finished product is difficult to meet the standard due to a large amount of pigment impurities. The conventional teicoplanin decolorizing method mainly utilizes active carbon, and utilizes variable active carbon twice to decolorize teicoplanin, but because the addition of the active carbon is not fixed every time, the active carbon decolorizing method is difficult to meet the technological requirements of standardized and batch production.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to solve the technical problems that: solves the problem that the traditional teicoplanin decolorizing method can not meet the process requirements of standardized and batch production.
In order to solve the technical problems, the invention provides a method for decoloring teicoplanin, which comprises the steps of using MONO HZ20 macroporous adsorption resin as a stationary phase and using an alcohol solution with pH = 10-11 as a mobile phase to decolor teicoplanin for the first time;
and performing secondary decolorization on teicoplanin by using UniGel beads Xl ion exchange resin as a stationary phase and using a salt solution with the concentration of 450-500 mmol/L as a mobile phase.
The invention has the beneficial effects that: because the molecular weight of teicoplanin molecules is different from that of pigment impurities, part of the pigment impurities in teicoplanin fermentation liquor can be adsorbed by specific macroporous adsorption resin, and teicoplanin can be resolved by applying an alcohol solution with high pH, so that the first decolorization treatment on teicoplanin is realized; because the quantity, distribution, variety and other characteristics of the charges on the surfaces of teicoplanin molecules are different from those of pigment impurities, the teicoplanin can be separated through ion exchange resin, and the teicoplanin can be resolved by applying a low-concentration salt solution, so that secondary decolorization treatment on the teicoplanin is realized. By carrying out twice decoloring treatment on teicoplanin, the requirement that the chromaticity standard of teicoplanin can be met only by carrying out one activated carbon decoloring step on teicoplanin can be met, and the purity and the yield of teicoplanin are improved at the same time.
Drawings
FIG. 1 is a photograph showing comparative products obtained by treating teicoplanin fermentation broth in test example 1, example 1 and example 2 according to the present invention, respectively.
Detailed Description
In order to explain the technical contents, the objects and the effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.
It should be noted that, in this document, uniHR Butyl-30L and Unigel beads Xl are available from Nawei technologies, suzhou, and MONO HZ20 and HZ-816 are available from Shanghai Huasha technologies, inc.
A method for decoloring teicoplanin comprises the steps of carrying out primary decoloring on teicoplanin by using MONO HZ20 macroporous adsorption resin as a stationary phase and using an alcoholic solution with pH = 10-11 as a mobile phase;
and a step of carrying out secondary decolorization on teicoplanin by using Unigel beads Xl ion exchange resin as a stationary phase and using a salt solution with the concentration of 450-500 mmol/L as a mobile phase.
Because the molecular weight of teicoplanin molecules is different from that of pigment impurities, part of the pigment impurities in teicoplanin fermentation liquor can be adsorbed through macroporous adsorption resin, and teicoplanin can be resolved by applying high-pH alcoholic solution, so that the first decolorization treatment of teicoplanin is realized.
Because the quantity, distribution, variety and other characteristics of the charges on the surfaces of the teicoplanin molecules are different from those of pigment impurities, the teicoplanin molecules can be separated through ion exchange resin, and the teicoplanin can be resolved by applying a low-concentration salt solution, so that the second decolorization treatment of the teicoplanin is realized.
Preferably, the method for decoloring teicoplanin comprises the following steps:
s1, filtering teicoplanin fermentation liquor by using a ceramic membrane to obtain filtrate;
s2, performing column chromatography on the filtrate through a chromatographic column filled with macroporous adsorption resin, prewashing the chromatographic column with deionized water until the leaked liquid is light yellow, and then analyzing teicoplanin with an alcohol solution with the pH = 10-11 to obtain an analysis liquid;
s3, adjusting the pH = 7.0-7.5 of the analysis solution, loading the analysis solution onto a chromatographic column filled with ion exchange resin, pre-washing the chromatographic column by using a 60% ethanol solution, and then eluting the chromatographic column by using a sodium chloride-ethanol solution to obtain a collection solution;
and S4, carrying out nanofiltration on the collected liquid for alcohol removal, freeze-drying, and heating after freeze-drying to obtain teicoplanin finished product powder.
In S1, the teicoplanin fermentation liquor passes through a ceramic membrane so as to separate solid impurities and high-molecular-weight impurities in the teicoplanin fermentation liquor through the ceramic membrane.
Preferably, the specific method of ceramic membrane filtration is as follows: and (2) carrying out ceramic membrane filtration on the teicoplanin fermentation liquor, concentrating to half of the volume, adding water while filtering, keeping the water addition amount consistent with the filtration flow, and stopping filtration and collection when the total filtrate volume is 2-5 times of the original volume of the fermentation liquor to obtain a filtrate.
In S2, firstly, deionized water is used for prewashing the chromatographic column to reduce bubbles in the chromatographic column, pigment impurities with molecular weight lower than that of teicoplanin in the filtrate are eluted in the prewashing process, when the leakage is light yellow, the main component in the leakage is teicoplanin, the teicoplanin can be eluted from the macroporous adsorption resin by applying an alcohol solution with high pH, and the retention capacity of part of the pigment impurities or the rest of the impurities in the macroporous adsorption resin can be effectively improved by applying the alcohol solution with high pH, so that the purpose of separating the teicoplanin from part of the impurities or the rest of the impurities is achieved.
In S3, neutral or weakly alkaline analytic solution passes through a chromatographic column filled with ion exchange resin for elution, and based on the fact that the quantity, the type and the distribution of the surface charges of the teicoplanin are different from those of pigment impurities or other impurities with approximate molecular weights, ionization of the pigment impurities or other impurities can be inhibited by applying low-concentration salt solution, so that the purpose of separating the teicoplanin from the pigment impurities and the other impurities is achieved.
In general, 1mol/L NaOH or 1mol/LHCl is used for adjusting the pH of the analysis solution.
It should be noted that, in this context, various reagents, such as ethanol, sodium hydroxide or hydrochloric acid, etc., are at least analytically pure.
Further, the alcoholic solution is a 60% alcoholic solution.
Further, the sodium chloride-ethanol solution is 450-500 mmol/L sodium chloride-60% ethanol solution.
Preferably, the sodium chloride-ethanol solution is 500mmol/L sodium chloride-60% ethanol solution.
Furthermore, the nanofiltration membrane with the molecular weight cut-off of 300 is utilized to carry out nanofiltration, and water is added into the collected liquid to drive the alcohol until the conductivity of the water outlet end of the nanofiltration membrane is less than 100.
Wherein, the step of adding water and removing alcohol is to add water and remove alcohol simultaneously, and the water adding amount is kept consistent with the filtering flow.
Further, the method also comprises a concentration step, wherein the concentration step is arranged between the nanofiltration ethanol removal and the freeze-drying, and the concentration step is to concentrate the collected liquid after the nanofiltration ethanol removal until the concentration of teicoplanin is 10maU/mL.
Preferably, the heating temperature is 50 ℃.
Wherein the freeze-drying condition comprises pre-freezing at-70 deg.C for 2h, freeze-drying at 20 deg.C for 20h, and vacuum degree less than 100Pa.
Because teicoplanin is multicomponent, the main peak of A2-2 can be converted into the main peak of A3 by heating the freeze-dried powder after freeze-drying.
Example 1
A method for decoloring teicoplanin comprises the following steps:
s1, filtering 3L of teicoplanin fermentation liquor by 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 that of the fermentation liquor, and keeping the water adding amount (5L/h) consistent with the filtering flow rate to obtain a filtrate, wherein the obtained filtrate is clear and transparent;
s2, adjusting the pH of the filtrate to be =7.0, adsorbing the filtrate by an upper column MONO HZ20 column, prewashing the resin by deionized water at the flow rate of 0.5BV/h until the color of the leaked liquid is light yellow, eluting the resin by using a 60% ethanol solution with the pH of =10.5, and collecting the eluted liquid;
s3, feeding 200ml of decolorizing resin Unigel beads Xl into a column of the analytic solution, prewashing for 2BV by 60 percent ethanol at the flow rate of 1BV/h, then eluting for 5BV by 500mmol/L sodium chloride-60 percent ethanol solution, and collecting leakage with light color to obtain a collecting solution;
s4, putting the collected liquid into a small membrane machine (model JMD1812-1, 220V,50HZ of capital east China Membrane engineering equipment Co., ltd.), performing nanofiltration by using a nanofiltration membrane with the molecular weight cutoff of 300, adding water and simultaneously removing alcohol in the nanofiltration process, keeping the water addition amount (5L/h) consistent with the filtration flow, concentrating teicoplanin to 10maU/L when the conductivity of the water outlet end of the nanofiltration membrane is less than 100, adjusting the pH =7.0 of the effective collected liquid by using 1mol/L NaOH or 1mol/L HCl, and then performing freeze-drying (the freeze-drying condition is shown in the specification); and after the freeze drying is finished, heating teicoplanin by using an oven at 50 ℃ to enable the component A3 to be more than 5.0, and then finishing heating to obtain a teicoplanin product.
The detection proves that the yield of the teicoplanin product is 91%, and the chromatic value is BY3.
Example 2
A method for decoloring teicoplanin comprises the following steps:
s1, filtering 3L of teicoplanin fermentation liquor by 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 the fermentation liquor, and keeping the water adding amount (5L/h) consistent with the filtering flow to obtain a filtrate, wherein the obtained filtrate is clear and transparent;
s2, adjusting the pH of the filtrate to be =7.5, adsorbing the filtrate by an upper column MONO HZ20 column, prewashing the resin by deionized water at the flow rate of 0.5BV/h until the color of the leaked liquid is light yellow, and eluting the resin by using a 60% ethanol solution with the pH of =11 to obtain an analytic solution;
s3, feeding 200ml of decolorizing resin Unigel beads Xl into a column of the analytic solution, prewashing for 2BV by 60 percent ethanol at the flow rate of 1BV/h, then eluting for 5BV by 500mmol/L sodium chloride-60 percent ethanol solution, and collecting leakage with light color to obtain a collecting solution;
s4, putting the collected liquid into a small membrane machine (model JMD1812-1, OTD 220V, 50HZ), performing nanofiltration by using a nanofiltration membrane with the molecular weight cutoff of 300, adding water and alcohol during the nanofiltration process, keeping the water addition amount (5L/h) consistent with the filtration flow, concentrating teicoplanin to 10maU/L when the conductivity of the water outlet end of the nanofiltration membrane is less than 100, adjusting the pH of the effective collected liquid to =7.5 by using 1mol/L NaOH or 1mol/L HCl, and then performing freeze-drying (the freeze-drying conditions are as shown in the above); and after the freeze drying is finished, heating teicoplanin by using an oven at 50 ℃ to enable the component A3 to be more than 5.0, and then finishing heating to obtain a teicoplanin product.
The detection proves that the yield of the teicoplanin product is 93.35%, and the chromatic value is BY3.
Comparative example 1
A method for decoloring teicoplanin is different from the method in example 1 in that: replacing the MONO HZ20 with HZ-816, carrying out twice analysis by using 0.1% ammonia water, and collecting the analysis solution twice without a Unigel beads Xl decoloring step to obtain a collected solution.
And detecting that the colorimetric value of the collection liquid is BY1.
Comparative example 2:
a method for decoloring teicoplanin is different from the method in example 1 in that: replacing the MONO HZ20 with HZ-816, and analyzing by 0.1% ammonia water; the UniGel beads Xl resin was replaced with UniHR Butyl-30L to obtain a harvest.
And detecting that the colorimetric value of the collection liquid is BY2.
It should be noted that the above detection standards for the colorimetric values refer to the european pharmacopoeia.
Detection example 1
The collected liquids obtained in example 1, comparative example 1 and comparative example 2 were subjected to color contrast by photographing, and the photographed image is shown in FIG. 1, wherein FX-06 represents example 1, FX-04 represents comparative example 2, and HZ806 represents comparative example 1.
As can be seen from FIG. 1, the comparison of FX-04 and HZ-806 shows that the content of pigment in the teicoplanin solution can be effectively reduced by chromatography on a macroporous adsorbent resin, and that by applying two types of resins, namely MONO HZ20 and Unigel beads Xl, the teicoplanin solution has a lower color than that obtained by applying HZ-816 and HR UniButyl-30L, and better meets the teicoplanin color standard by comparing FX-04 and FX-06.
In summary, according to the method for decoloring teicoplanin provided by the present invention, because the molecular weights of teicoplanin molecules and pigment impurities are different, part of the pigment impurities in teicoplanin fermentation broth can be adsorbed by macroporous adsorbent resin, and teicoplanin can be resolved by applying high pH alcoholic solution, so as to realize the first decoloring treatment of teicoplanin; because the quantity, distribution, variety and other characteristics of the charges on the surfaces of teicoplanin molecules are different from those of pigment impurities, the teicoplanin can be separated through ion exchange resin, and the teicoplanin can be resolved by applying a low-concentration salt solution, so that secondary decolorization treatment on the teicoplanin is realized. The method has the advantages that the teicoplanin is subjected to two times of decoloring treatment, the chromaticity standard of the teicoplanin can be met only by performing the activated carbon decoloring step on the teicoplanin once, and the purity and the yield of the teicoplanin are improved simultaneously.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.
Claims (5)
1. A method for decoloring teicoplanin is characterized by comprising the steps of carrying out primary decoloring on teicoplanin by using MONO HZ20 macroporous adsorption resin as a stationary phase and using an alcohol solution with pH = 10-11 as a mobile phase;
and a step of carrying out secondary decolorization on teicoplanin by using Unigel beads Xl ion exchange resin as a stationary phase and using a salt solution with the concentration of 450-500 mmol/L as a mobile phase;
wherein the alcoholic solution is 60% ethanol;
the salt solution is sodium chloride-60% ethanol solution.
2. The process for depigmenting teicoplanin according to claim 1, characterized in that it comprises the following steps:
s1, filtering teicoplanin fermentation liquor by using a ceramic membrane to obtain filtrate;
s2, performing column chromatography on the filtrate through a chromatographic column filled with macroporous adsorption resin, prewashing the chromatographic column with deionized water until the leaked liquid is light yellow, and then analyzing teicoplanin with an alcohol solution with the pH = 10-11 to obtain an analysis liquid;
s3, adjusting the pH = 7.0-7.5 of the analysis solution, loading the analysis solution onto a chromatographic column filled with ion exchange resin, pre-washing the chromatographic column with 60% ethanol solution, and then eluting with sodium chloride-ethanol solution to obtain a collection solution;
and S4, carrying out nanofiltration on the collected liquid for alcohol removal, freeze-drying, and heating after freeze-drying to obtain teicoplanin finished product powder.
3. The method for decoloring teicoplanin according to claim 2, wherein the nanofiltration alcohol removal is performed by adding water to the collected liquid to remove alcohol by using a nanofiltration membrane with a molecular weight cut-off of 300 until the conductivity of the outlet end of the nanofiltration membrane is less than 100.
4. The method for decoloring teicoplanin according to claim 2, further comprising a concentration step, wherein the concentration step is arranged between the nanofiltration penthanol and the freeze-drying, and the concentration step is to concentrate the collected liquid after the nanofiltration penthanol completion to a teicoplanin concentration of 10maU/mL.
5. The process for decolorization of teicoplanin according to claim 2, characterized in that the temperature of heating is 50 ℃.
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| CN107365358B (en) * | 2016-05-13 | 2021-10-01 | 鲁南新时代生物技术有限公司 | Method for separating and purifying teicoplanin |
| CN106589074A (en) * | 2016-12-16 | 2017-04-26 | 沁浩膜技术(厦门)有限公司 | Teicoplanin purifying method |
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