CN115260294A - Separation and purification method of teicoplanin - Google Patents
Separation and purification method of teicoplanin Download PDFInfo
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- CN115260294A CN115260294A CN202211014494.XA CN202211014494A CN115260294A CN 115260294 A CN115260294 A CN 115260294A CN 202211014494 A CN202211014494 A CN 202211014494A CN 115260294 A CN115260294 A CN 115260294A
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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 provides a separation and purification method of teicoplanin. The method sequentially carries out ceramic membrane filtration treatment, anion-cation exchange resin adsorption treatment, ultra-nanofiltration treatment, extraction and back-extraction treatment, polyacrylamide resin chromatography separation treatment and concentration crystallization treatment on teicoplanin fermentation liquor under acidic conditions and alkaline conditions, and obtains teicoplanin pure products through separation and purification with high yield and purity. The separation and purification method avoids the process of carrying out chromatographic separation on the fermentation liquor by using macroporous adsorption resin, does not need to use a large amount of organic solvent in the chromatographic separation process, and reduces the separation cost of teicoplanin.
Description
Technical Field
The invention belongs to the technical field of medicine purification, and relates to a separation and purification method of teicoplanin.
Background
Teicoplanin, also known as vancomycin, was first discovered in 1975. The vancomycin glycopeptide antibiotic is obtained by fermenting and extracting specific actinoplanes, is an antibiotic mixture consisting of a plurality of compounds with very similar chemical structures, has the molecular weight of 1879.66000, is a new generation glycopeptide antibiotic similar to vancomycin, and has the antibacterial spectrum and the antibacterial activity similar to vancomycin. Has stronger effect on staphylococcus aureus than vancomycin and less adverse reaction. Teicoplanin is sensitive to gram-positive bacteria such as staphylococci, streptococci, enterococci and most anaerobic positive bacteria. The preparation is mainly used for treating severe infection caused by sensitive bacteria such as staphylococcus aureus, streptococcus and the like, such as endocarditis, osteomyelitis, septicemia and infection of respiratory tract, urinary tract, skin, soft tissue and the like.
Teicoplanin is represented by A 2-1 、A 2-2 、A 2-3 、A 2-4 、A 2-5 Five glycopeptide compounds with similar structures and deacylated glucosamine A 3-1 A mixture of compositions having the molecular structure shown below:
the existing technology for separating and purifying teicoplanin usually comprises the steps of adsorbing and concentrating biological fermentation broth through macroporous resin, desorbing and crystallizing by adopting an organic solvent to obtain a teicoplanin crude product, and purifying to obtain a teicoplanin pure product. However, macroporous resin is difficult to regenerate after adsorbing fermentation liquor, and macroporous resin is used for chromatographic separation of fermentation liquor, so that a large amount of organic solvent is required for elution, and the production cost is high.
Disclosure of Invention
The invention provides a separation and purification method of teicoplanin, which avoids the process of chromatographic separation by using macroporous adsorption resin, does not need to use a large amount of organic solvent for elution in the process of chromatographic separation, and reduces the separation cost.
The invention provides a separation and purification method of teicoplanin, which comprises the following steps:
1) The pH value of teicoplanin fermentation liquor is adjusted to be acidic, a ceramic membrane is adopted for primary filtration, and a ceramic membrane concentrated solution is collected; adjusting the pH value of the ceramic membrane concentrated solution to be alkaline, performing secondary filtration by adopting a ceramic membrane, and collecting filtrate;
2) Adjusting the pH value of the filtrate to 4.5-6.5, performing adsorption separation treatment on the filtrate by using cation exchange resin and anion exchange resin, and collecting resin effluent liquid;
3) Sequentially carrying out ultrafiltration treatment and nanofiltration treatment on the resin effluent liquid to obtain an ultrafiltration concentrated solution; adjusting the acidity of the nanofiltration concentrated solution to ensure that the pH value of the nanofiltration concentrated solution is not more than 4, and extracting the nanofiltration concentrated solution by using an alcohol solvent to obtain an extract liquid; carrying out back extraction treatment on the extraction liquid by adopting a first alkaline buffer salt solution to obtain a back extraction liquid;
4) Performing chromatographic separation treatment on the strip liquor by adopting polyamide resin and taking a second alkaline buffer salt solution as an eluent, and collecting an eluent containing teicoplanin; and concentrating and crystallizing the eluent to obtain a pure teicoplanin product.
The separation and purification method as described above, wherein the adjusting the pH of the fermentation broth to acidity in step 1) comprises: adjusting the pH value of the fermentation liquor to 1-4; adjusting the pH of the ceramic membrane concentrate to alkaline comprises: and adjusting the pH value of the ceramic membrane concentrated solution to 8-10.
The separation and purification method as described above, wherein, in the step 1), before the pH of the teicoplanin fermentation broth is adjusted to be acidic, the method further comprises a step of pretreating the teicoplanin fermentation broth, wherein the pretreatment comprises: adding inorganic salt into teicoplanin fermentation liquor.
The separation and purification method as described above, wherein, in the step 2), the cation exchange resin is selected from HZ-3B resin, and the anion exchange resin is selected from FPDA-30 resin.
The separation and purification method as described above, wherein, in the step 3), the cut-off molecular weight of the ultrafiltration treatment is 5000-10000 Da.
The separation and purification method as described above, wherein, in the step 3), the alcohol solvent is selected from n-butanol or isobutanol.
The separation and purification method as described above, wherein the first basic buffer salt solution and the second basic buffer salt solution are each independently selected from one of ammonium bicarbonate solution, sodium bicarbonate solution, and disodium hydrogen phosphate solution.
The separation and purification method as described above, wherein the pH of the first alkaline buffered salt solution and/or the second alkaline buffered salt solution is 8 to 10.
The separation and purification method as described above, wherein, in step 4), before the stripping solution is subjected to the chromatographic separation treatment, the method further comprises a step of pretreating the stripping solution, and the pretreatment comprises: and adjusting the pH value of the stripping solution to 4-6.
The separation and purification method as described above, wherein, in the step 4), the crystallization comprises: concentrating the eluent until the concentration of the teicoplanin is 80-150 mg/mL, dropwise adding ethanol or acetone into the concentrated solution to crystallize the concentrated solution, and filtering to obtain the pure teicoplanin.
According to the separation and purification method, the teicoplanin fermentation liquor is subjected to ceramic membrane filtration treatment, anion-cation exchange resin adsorption treatment, ultra-nanofiltration treatment, extraction and back-extraction treatment, polyacrylamide resin chromatography separation treatment and crystallization treatment in sequence under acidic and alkaline conditions, so that the teicoplanin in the fermentation liquor is separated and purified with high yield and purity. The separation and purification method avoids the process of chromatographic separation by using macroporous adsorption resin, does not need to use a large amount of organic solvent for elution, and reduces the separation cost.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a separation and purification method of teicoplanin, which comprises the following steps:
1) Adjusting the pH value of teicoplanin fermentation liquor to be acidic, performing primary filtration by adopting a ceramic membrane, and collecting a ceramic membrane concentrated solution; adjusting the pH value of the ceramic membrane concentrated solution to be alkaline, carrying out secondary filtration by adopting a ceramic membrane, and collecting filtrate;
2) Adjusting the pH value of the filtrate to 4.5-6.5, performing adsorption separation treatment on the filtrate by using cation exchange resin and anion exchange resin, and collecting resin effluent liquid;
3) Sequentially carrying out ultrafiltration treatment and nanofiltration treatment on the resin effluent to obtain an ultrafiltration concentrated solution; adjusting the acidity of the nanofiltration concentrated solution to ensure that the pH value of the ultrafiltration concentrated solution is not more than 4, and extracting the ultrafiltration concentrated solution by adopting an alcohol solvent to obtain an extract liquid; carrying out back extraction treatment on the extraction liquid by adopting a first alkaline buffer salt solution to obtain a back extraction liquid;
4) Performing chromatographic separation treatment on the strip liquor by adopting polyamide resin and taking a first alkaline buffer salt solution as an eluent, and collecting an eluent containing teicoplanin; concentrating the eluent, and crystallizing to obtain a pure teicoplanin product.
In the step 1), by utilizing the characteristic that teicoplanin is insoluble in alkali and acid, firstly enabling teicoplanin to form micelles under an acidic condition, carrying out primary filtration by adopting a ceramic membrane to enable the teicoplanin to remain in a ceramic membrane concentrated solution, filtering out a large amount of pigments and partial proteins through the primary filtration, then adjusting the pH of the ceramic membrane concentrated solution to be alkaline to enable the teicoplanin to be released and dissolved in fermentation liquor, and obtaining a filtrate containing the teicoplanin through secondary filtration;
the isoelectric point of teicoplanin is 5.1, in the step 2), the pH value of the filtrate is adjusted to be close to the isoelectric point of teicoplanin, then cation exchange resin and anion exchange resin are used for carrying out adsorption separation on the filtrate, the cation exchange resin can adsorb protein impurities of polyamino and metal salt ions, the anion exchange resin can adsorb protein impurities of polycarboxyl, and the purification of teicoplanin is further completed;
in the step 3), the resin effluent is subjected to ultrafiltration and nanofiltration treatment, macromolecular protein and pigment can be further removed, the resin effluent is concentrated, an alcohol solvent is adopted to extract the concentrated solution, and an alkaline buffer salt solution is adopted to carry out back extraction on the extract liquor, so that inorganic salt impurities and organic micromolecular impurities can be respectively removed;
teicoplanin contains an amino group and a carboxyl group, belongs to an amphoteric compound, contains an alkyl side chain in a molecule, has the amino acid characteristics very suitable for chromatography on polyacrylamide resin, is subjected to initial purification in the previous steps to remove part of impurities crosslinked with teicoplanin, is adsorbed on the polyacrylamide resin in a fixed phase, does not need to use a large amount of organic solvent, can finish the elution of teicoplanin only by using a small amount of alkaline buffer salt solution, and in the step 4), after the back extraction solution is subjected to chromatographic separation by adopting the polyacrylamide resin, the eluent is concentrated and crystallized to obtain a pure teicoplanin product.
According to the separation and purification method, the teicoplanin fermentation liquor is sequentially subjected to ceramic membrane filtration treatment, anion-cation exchange resin adsorption treatment, ultra-nanofiltration treatment, extraction and back-extraction treatment, polyacrylamide resin chromatography separation treatment and crystallization treatment under acidic conditions and alkaline conditions, and the separation and purification of teicoplanin in the fermentation liquor are realized with high yield and purity. The method avoids the process of chromatographic separation by using macroporous adsorption resin, and does not need to use a large amount of organic solvent for elution in the process of chromatographic separation, thereby reducing the separation cost.
In a specific embodiment, the step 1) of adjusting the pH of the fermentation broth to acidity comprises: the pH value of the fermentation liquor is adjusted to 1-4, preferably 2-3, under the strong acid condition, the solubility of teicoplanin is lower, and loss caused by dissolution of teicoplanin into the filtrate obtained by primary filtration can be avoided.
In a specific embodiment, the step 1) of adjusting the pH of the ceramic membrane concentrate to alkaline comprises: the pH of the ceramic membrane concentrate is adjusted to 8 to 10, preferably 9 to 10. The stronger the alkalinity is, the more favorable the dissolution of teicoplanin in the ceramic membrane concentrated solution is, but the stability of teicoplanin under the strong alkaline condition is poorer. According to the invention, the pH value of the ceramic membrane concentrated solution is adjusted to be within the range, so that the teicoplanin has good stability, and the problems of increased cooling cost and operation difficulty caused by strong alkaline conditions can be solved.
In the step 1), before adjusting the pH of the teicoplanin fermentation liquid to be acidic, the method further comprises a step of pretreating the teicoplanin fermentation liquid, wherein the pretreatment comprises the following steps: inorganic salt is added into the teicoplanin fermentation liquor, and the addition of the inorganic salt can further increase the indissolvable degree of teicoplanin under an acidic condition, so that the condition that the teicoplanin enters the filtrate obtained by one-time filtration to cause product loss is avoided.
Specifically, the inorganic salt may be selected from inorganic sodium salt or inorganic ammonium salt, and the mass volume concentration of the inorganic salt in the fermentation liquid is preferably 0.3-3%.
In a specific embodiment, the cation exchange resin of step 2) is selected from the group consisting of HZ-3B resins and the anion exchange resin is selected from the group consisting of FPDA-30 resins. Anion exchange resins and cation exchange resins may be connected in series to increase the efficiency of the separation.
Furthermore, the molecular weight cut-off in the ultrafiltration treatment is 5000-100000 Da.
Before extraction treatment, the acidity of the concentrated solution of the ultrananofiltration is adjusted to ensure that the pH value of the concentrated solution is not more than 4, so that the solubility of teicoplanin in an aqueous phase system can be reduced, and the extraction of teicoplanin by using an alcohol solvent is facilitated. Further, before the extraction treatment, the acidity of the ultrafiltration concentrated solution is adjusted to 1 to 4, and further adjusted to 2 to 3.
The alcohol solvent of the invention is selected from n-butyl alcohol or isobutyl alcohol, and both the two solvents have better extraction effect on teicoplanin.
The first and second alkaline buffered salt solutions are not particularly limited in kind, and may be alkaline buffered salt solutions conventionally used in the chemical field, for example, the first and second alkaline buffered salt solutions may be each independently selected from one of an ammonium bicarbonate solution, a sodium bicarbonate solution, and a sodium dihydrogen phosphate solution.
Further, the pH value of the first alkaline buffer salt solution and/or the second alkaline buffer salt solution is 8-10, and under the alkaline condition, the dissolution of teicoplanin in the buffer salt solution is facilitated, and the stable existence of teicoplanin in the separation process can be ensured.
Further, before the stripping solution is subjected to chromatographic separation treatment, the method also comprises a pretreatment process of the stripping solution, wherein the pretreatment process comprises the following steps: the pH value of the stripping solution is adjusted to 4-6. In the vicinity of isoelectric points of teicoplanin, the adsorption capacity of teicoplanin on resin is strongest, most other impurities on resin are weaker than that of teicoplanin, so that the teicoplanin can be separated from the teicoplanin without being adsorbed on polyacrylamide resin, and other impurities with stronger adsorption capacity than that of teicoplanin can be separated from the teicoplanin during elution. In the chromatographic separation treatment, before elution with the second basic buffer salt solution, a small amount of water is used for further removing the pigment in the stripping solution.
The crystallization after the concentration of the eluent can adopt the conventional crystallization treatment operation in the field, for example, the eluent can be concentrated until the concentration of the teicoplanin is 80-150 mg/mL, then ethanol or acetone concentrated solution is dripped into the concentrated solution for crystallization, and the pure teicoplanin can be obtained after filtration.
The method for separating and purifying teicoplanin provided by the present invention will be further described with reference to the following specific examples. In the following examples, all starting materials were prepared by either commercially available or conventional methods, unless otherwise specified.
It should be noted that, in step 1) of the following examples, adding 0.5% (w/v) of sodium chloride means adding 5g of sodium chloride per liter of teicoplanin fermentation broth; in the step 3), adding 10 percent (mass volume ratio) of sodium chloride refers to adding 100g of sodium chloride into each liter of the ultra-nanofiltration concentrated solution, and performing back extraction on the extract by adopting 2 percent (mass volume ratio) of ammonium bicarbonate solution refers to adding 20g of ammonium bicarbonate solution into each liter of the extract.
Example 1
The separation and purification method of teicoplanin of this embodiment includes the following steps:
1) After adding 0.5% (weight to volume) of sodium chloride into 29L of teicoplanin fermentation liquor (containing 24g of teicoplanin), adjusting the pH of the system to 2.0 by oxalic acid, carrying out primary filtration by using a ceramic membrane, washing by using 60L of water, and collecting concentrated ceramic membrane concentrated solution until the volume is 11L. The pH of the ceramic membrane concentrate was adjusted to 9.5 using sodium hydroxide solution, and the ceramic membrane was subjected to secondary filtration and top water (top water in the present invention means washing with water), and 90L of the filtrate (containing 22.1g of teicoplanin) was collected.
2) The pH of the filtrate was adjusted to 5.5 and then passed through an ion exchange resin column containing 5L of HZ-3B and 5L of FPDA-30 in series, and the resin effluent (containing 21.7g of teicoplanin) was collected.
3) Adjusting the pH of the resin effluent liquid to 7-9, sequentially performing ultrafiltration by an ultrafiltration membrane of 10000Da, performing nanofiltration by a nanofiltration membrane of 300-500Da, finally performing nanofiltration to obtain concentrated solution until the concentration of teicoplanin in the concentrated solution is 50mg/mL, adding 10% (mass-volume ratio) sodium chloride, adjusting the pH value of the system to 3.0, adding n-butyl alcohol to perform extraction, performing back extraction on the extract liquid by adopting 2% (mass-volume ratio) ammonium bicarbonate solution, and collecting a back-extraction liquid (containing 18.5g of teicoplanin).
4) After the pH of the back extraction liquid was adjusted to 5.0, the back extraction liquid was chromatographically separated by using 2L polyacrylamide resin column, washed with water and eluted with 0.1% ammonium bicarbonate solution (pH 8.0), and the eluent containing teicoplanin (containing 16.4g of teicoplanin) was collected. And (3) concentrating the eluent by nanofiltration until the concentration of the teicoplanin is 109mg/mL, dropwise adding acetone into the concentrated solution to separate out crystals, and separating to obtain 14.5g of teicoplanin pure product with the purity of 90.83 percent and the separation yield of 60.4 percent.
Example 2
The separation and purification method of teicoplanin of this embodiment includes the following steps:
1) After adding 0.5% (weight to volume) of sodium chloride into 29.7L of teicoplanin fermentation liquor (containing 24.8g of teicoplanin), adjusting the pH of the system to 2.5 by using oxalic acid, carrying out primary filtration by using a ceramic membrane, washing by using 60L of water, and concentrating the ceramic membrane concentrated solution to a volume of 11L. The pH of the ceramic membrane concentrate was adjusted to 9.61 using sodium hydroxide solution, and the ceramic membrane was subjected to secondary filtration to collect 90L filtrate (containing 21.7g of teicoplanin).
2) The pH of the filtrate was adjusted to 5.5 and then passed through an ion exchange resin column containing 5L of HZ-3B and 5L of FPDA-30 in series, and the resin effluent (containing 20.3g of teicoplanin) was collected.
3) Adjusting the pH value of the resin effluent to 7-9, sequentially carrying out ultrafiltration and nanofiltration, finally carrying out nanofiltration to obtain the concentration until the concentration of teicoplanin in the concentrated solution is 50mg/mL, adding 10% (mass to volume ratio of the concentrated solution) of sodium chloride, adjusting the pH value of the system to 2.0, adding n-butanol to carry out extraction, carrying out back extraction on the extract by adopting 2% (mass to volume ratio) of ammonium bicarbonate solution, and collecting the back-extraction solution (containing 18.5g of teicoplanin).
4) After the pH of the back extraction liquid was adjusted to 5.0, the back extraction liquid was chromatographically separated by using 2L polyacrylamide resin column, and eluted with water and 0.1% ammonium bicarbonate solution (pH 8.0), and the eluent containing teicoplanin (containing 16.1g of teicoplanin) was collected. And (3) concentrating the eluent by nanofiltration until the concentration of the teicoplanin is 120mg/mL, dropwise adding acetone into the concentrated solution to separate out crystals, and separating to obtain 14.7g of teicoplanin pure product with the purity of 90.31% and the separation yield of 59.3%.
Example 3
The separation and purification method of teicoplanin of this embodiment includes the following steps:
1) After 0.5% (weight to volume) of sodium chloride is added into 28.3L of teicoplanin fermentation liquor (containing 23.9g of teicoplanin), oxalic acid is adopted to adjust the pH of the system to 2.0, a ceramic membrane is adopted for primary filtration, 60L of water is used for washing, and the concentrated ceramic membrane liquor is concentrated to the volume of 11L. The pH of the ceramic membrane concentrate was adjusted to 9.44 using sodium hydroxide solution and secondary filtration was performed using a ceramic membrane to collect 90L filtrate (containing 21.8g of teicoplanin).
2) The pH of the filtrate was adjusted to 4.5 and then passed through a column of 5L of HZ-3B and 5L of FPDA-30 in series on an ion exchange resin, and the resin effluent (containing teicoplanin 19.1 g) was collected.
3) Adjusting the pH of the resin effluent liquid to 7-9, sequentially performing ultrafiltration and nanofiltration, finally performing nanofiltration to obtain the concentration until the concentration of teicoplanin in the concentrated solution is 50mg/mL, adding 10% (mass to volume ratio of the concentrated solution) of sodium chloride, adjusting the pH value of the system to 3.0, adding n-butyl alcohol to perform extraction, performing back extraction on the extract liquid by adopting 2% (mass to volume ratio) of ammonium bicarbonate solution, and collecting the back-extraction liquid (containing 16.6g of teicoplanin).
4) After the pH of the back extraction liquid was adjusted to 5.0, the back extraction liquid was chromatographically separated by using 2L polyacrylamide resin column, and eluted with water and 0.1% ammonium bicarbonate solution (pH 8.0), and the eluent containing teicoplanin (containing 14.7g of teicoplanin) was collected. And (3) concentrating the eluent by nanofiltration until the concentration of the teicoplanin is 117mg/mL, dropwise adding acetone into the concentrated solution to separate out crystals, and separating to obtain 13.1g of teicoplanin pure product with the purity of 90.26% and the separation yield of 54.8%.
Example 4
The separation and purification method of teicoplanin of this embodiment includes the following steps:
1) After 0.5% (weight to volume) of sodium chloride was added to 28.8L of teicoplanin broth (containing 24.5g of teicoplanin), the pH of the system was adjusted to 2.0 with oxalic acid, primary filtration was performed with ceramic membranes, and 60L of water was used to wash the filtrate, and the concentrated ceramic membrane broth was collected to a volume of 11L. The pH of the ceramic membrane concentrate was adjusted to 9.59 with sodium hydroxide solution and secondary filtration was carried out with ceramic membrane, and 90L filtrate (containing 22.4g teicoplanin) was collected.
2) The pH of the filtrate was adjusted to 6.5 and then passed through an ion exchange resin column containing 5L of HZ-3B and 5L of FPDA-30 in series, and the resin effluent (containing 20.6g of teicoplanin) was collected.
3) Adjusting the pH value of the resin effluent to 7-9, sequentially carrying out ultrafiltration and nanofiltration, finally carrying out nanofiltration to obtain the concentration until the concentration of teicoplanin in the concentrated solution is 50mg/mL, adding 10% (mass to volume ratio of the concentrated solution) of sodium chloride, adjusting the pH value of the system to 2.0, adding n-butanol to carry out extraction, carrying out back extraction on the extract by adopting 2% (mass to volume ratio) of ammonium bicarbonate solution, and collecting the back-extraction solution (containing 18.6g of teicoplanin).
4) After the pH of the back extraction liquid was adjusted to 5.0, the back extraction liquid was chromatographically separated by using 2L polyacrylamide resin column, washed with water and eluted with 0.1% ammonium bicarbonate solution (pH 8.0), and the eluent containing teicoplanin (containing 16.5g of teicoplanin) was collected. And (3) concentrating the eluent by nanofiltration until the concentration of the teicoplanin is 109mg/mL, dropwise adding acetone into the concentrated solution to separate out crystals, and separating to obtain 14.8g of teicoplanin pure product with the purity of 90.78% and the separation yield of 60.4%.
Example 5
And changing the pH value of the ceramic membrane concentrated solution after the primary filtration, and determining the influence on the separation and purification effect of teicoplanin in the secondary filtration.
In the following experiments in groups A and B, the process of one filtration was as follows: after 0.5% (mass volume ratio) of sodium chloride is added into 30L of teicoplanin fermentation liquor (containing 25.4g of teicoplanin), the pH of the system is adjusted to 2.0 by oxalic acid, ceramic membrane filtration is adopted, 60L of water is used for washing, and the concentrated ceramic membrane concentrated solution is collected to the volume of 10L (containing 24.4g of teicoplanin).
A. Adjusting the pH value of 10L of ceramic membrane concentrated solution obtained after primary filtration to 9.56, carrying out secondary filtration by using a ceramic membrane, carrying out water lifting until 50L of filtrate is collected, and determining that the purity of teicoplanin in the filtrate is 63.25% and the yield of secondary filtration is 93.70%.
B. Adjusting the pH value of 10L of ceramic membrane concentrated solution obtained after primary filtration to 12.45, performing secondary filtration by using a ceramic membrane, carrying out water lifting until 50L of filtrate is collected, and determining that the purity of teicoplanin in the filtrate is 58.77% and the yield of secondary filtration is 92.01%.
It can be seen from the comparison of the experiments in group a and group B that the purity of teicoplanin in the secondary filtrate and the yield of secondary filtration are reduced by adjusting the pH of the ceramic membrane concentrate to 12 or more before the secondary filtration as compared with adjusting the pH to 9.56, which indicates that the stability of teicoplanin in the separation process can be ensured by adjusting the pH of the ceramic membrane concentrate to 8-10 before the secondary filtration.
Example 6
And (3) whether inorganic salt is added into the teicoplanin fermentation liquor before primary filtration is examined, and the influence on the separation and purification effect of teicoplanin in the primary filtration is determined.
A. Adding 0.5 percent (weight volume ratio) of sodium chloride into 10L of teicoplanin fermentation liquor (containing 10.1g of teicoplanin), adjusting the pH of the system to 3.0 by using oxalic acid, performing primary filtration by using a ceramic membrane, carrying out water topping until 60L of primary filtered filtrate is collected, determining that the primary filtered filtrate contains 0.6g of teicoplanin, and calculating to obtain the loss of 5.9 percent of teicoplanin after the primary filtration.
B. Adjusting the pH value of 10L of teicoplanin fermentation liquor (containing 10.1g of teicoplanin) to 3.0 by oxalic acid, carrying out primary filtration by adopting a ceramic membrane, carrying out water jacking until the yield reaches 60L of filtrate obtained by primary filtration, determining that the filtrate obtained by primary filtration contains 1.05g of teicoplanin, and calculating to obtain that 10.4% of teicoplanin is lost after primary filtration.
The experimental comparison of the group A and the group B shows that the addition of the inorganic salt in the fermentation liquor can reduce the solubility of the teicoplanin under the acidic condition and reduce the loss of the teicoplanin in the process of one-time filtration, separation and purification.
Comparative example 1
The separation and purification method of teicoplanin of the comparative example comprises the following steps:
1) After 0.5% (weight to volume) of sodium chloride is added into 30L of teicoplanin fermentation liquor (containing 25.6g of teicoplanin), the pH of the system is adjusted to 2.0 by oxalic acid, a ceramic membrane is used for primary filtration, 60L of water is used for washing, and the concentrated ceramic membrane concentrated solution is collected until the volume is 11L. The pH of the ceramic membrane concentrate was adjusted to 9.78 with sodium hydroxide solution, and the ceramic membrane was used for secondary filtration, and 90L (containing 23.6g of teicoplanin) of the filtrate was collected.
2) The pH of the filtrate was adjusted to 5.0, the filtrate was chromatographed using 2L polyacrylamide resin column, washed with water, and once eluted using 0.1% (mass to volume) ammonium bicarbonate solution (pH 8.0), and the obtained primary eluate contained 3.7g of teicoplanin (separation yield from fermentation broth: 14.5%), performing secondary elution by using 0.1% (mass to volume) ammonium bicarbonate solution (pH is 9.0), obtaining secondary eluent, combining the primary eluent and the secondary eluent, and combining the combined solution to contain 10.1g of teicoplanin (the separation yield is calculated from fermentation liquor: 39.4%) of the total solution of the first eluent and the second eluent was eluted three times using a mixed solution of 0.1% (by mass/volume) of ammonium bicarbonate solution (pH 9.0) and 30% ethanol (30% ethanol means that 300 mL of ethanol was contained in 1L of the eluent) as an eluent to obtain a third eluent, the combined solution of the first eluent and the second eluent was combined with the third eluent to obtain a combined solution containing 16.8g of teicoplanin in total (the separation yield was 65.6% from the fermentation broth), the combined solution of the third eluent was subjected to nanofiltration and concentration until the concentration of teicoplanin was 109mg/mL, acetone was added dropwise to the concentrated solution to precipitate crystals, and after solid-liquid separation, 13.2g of teicoplanin pure product (the crystallization yield was 78.6%), the purity was 88.5%, and the total separation yield was 51.5% was obtained.
The above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A separation and purification method of teicoplanin is characterized by comprising the following steps:
1) Adjusting the pH value of teicoplanin fermentation liquor to be acidic, performing primary filtration by adopting a ceramic membrane, and collecting a ceramic membrane concentrated solution; adjusting the pH value of the ceramic membrane concentrated solution to be alkaline, performing secondary filtration by adopting a ceramic membrane, and collecting filtrate;
2) Adjusting the pH value of the filtrate to 4.5-6.5, performing adsorption separation treatment on the filtrate by using cation exchange resin and anion exchange resin, and collecting resin effluent liquid;
3) Sequentially carrying out ultrafiltration treatment and nanofiltration treatment on the resin effluent to obtain an ultrafiltration concentrated solution; adjusting the acidity of the ultrafiltration concentrated solution to ensure that the pH value of the ultrafiltration concentrated solution is not more than 4, and extracting the ultrafiltration concentrated solution by adopting an alcohol solvent to obtain an extract liquid; carrying out back extraction treatment on the extraction liquid by adopting a first alkaline buffer salt solution to obtain a back extraction liquid;
4) Performing chromatographic separation treatment on the strip liquor by using polyacrylamide resin and a second alkaline buffer salt solution as an eluent, and collecting an eluent containing teicoplanin; and concentrating and crystallizing the eluent to obtain a pure teicoplanin product.
2. The separation and purification method according to claim 1, wherein the step 1), adjusting the pH of the fermentation broth to acidity comprises: adjusting the pH value of the fermentation liquor to 1-4;
and/or adjusting the pH of the ceramic membrane concentrate to alkaline comprises: and adjusting the pH value of the ceramic membrane concentrated solution to 8-10.
3. The separation and purification method according to claim 1 or 2, wherein the step 1) further comprises a process of pretreating the teicoplanin fermentation broth before adjusting the pH of the teicoplanin fermentation broth to acidity, wherein the pretreatment comprises: adding inorganic salt into teicoplanin fermentation liquor.
4. The separation and purification method according to any one of claims 1 to 3, wherein in the step 2), the cation exchange resin is selected from the group consisting of HZ-3B resin, and the anion exchange resin is selected from the group consisting of FPDA-30 resin.
5. The separation and purification method according to any one of claims 1 to 4, wherein the ultrafiltration treatment in step 3) has a molecular weight cut-off of 5000 to 10000Da.
6. The separation and purification method according to any one of claims 1 to 5, wherein the alcohol solvent in step 3) is selected from n-butanol or isobutanol.
7. The separation and purification process according to any one of claims 1 to 6, wherein the first basic buffered salt solution and the second basic buffered salt solution are each independently selected from one of an ammonium bicarbonate solution, a sodium bicarbonate solution, and a disodium hydrogen phosphate solution.
8. The separation and purification process according to claim 7, wherein the pH value of the first alkaline buffered salt solution and/or the second alkaline buffered salt solution is 8 to 10.
9. The separation and purification method according to any one of claims 1 to 8, wherein before the stripping solution is subjected to the chromatographic separation treatment in step 4), a pretreatment process is further included for the stripping solution, and the pretreatment process comprises: and adjusting the pH value of the back extraction solution to 4-6.
10. The separation and purification method according to any one of claims 1 to 9, wherein in the step 4), the crystallization comprises: concentrating the eluent until the concentration of the teicoplanin is 80-150 mg/mL, dropwise adding ethanol or acetone into the concentrated solution to crystallize the concentrated solution, and filtering to obtain the pure teicoplanin.
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