A method for separating and purifying teicoplanin

Abstract

The present invention provides a method for separating and purifying teicoplanin. This method sequentially subjects a teicoplanin fermentation broth to ceramic membrane filtration under acidic and alkaline conditions, adsorption with anion and cation exchange resins, ultra-nanofiltration, extraction and stripping, chromatographic separation with polyacrylamide resins, and concentration and crystallization, resulting in purified teicoplanin with high yield and purity. This separation and purification method avoids the use of macroporous adsorption resins for chromatographic separation of the fermentation broth, eliminating the need for large amounts of organic solvents during the chromatographic separation process and reducing the cost of separating teicoplanin.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug purification and relates to a method for separating and purifying teicoplanin. Background Art

[0002] Teicoplanin, also known as teicoplanin, was first discovered in 1975. It is a vancomycin-family glycopeptide antibiotic obtained through fermentation and extraction from specific motile actinomycetes. It is a mixture of multiple chemically similar compounds with a molecular weight of 1879.66000. It is a new-generation glycopeptide antibiotic similar to vancomycin, with an antimicrobial spectrum and activity similar to those of vancomycin. It is more potent than vancomycin against Staphylococcus aureus and has fewer adverse reactions. Teicoplanin is sensitive to Gram-positive bacteria such as Staphylococci, Streptococci, Enterococci, and most anaerobic Gram-positive bacteria. It is primarily used clinically to treat serious infections caused by sensitive bacteria such as Staphylococci and Streptococci, such as endocarditis, osteomyelitis, sepsis, and infections of the respiratory tract, urinary tract, skin, and soft tissue.

[0003] Teicoplanin is produced by A 2-1 、A 2-2 、A 2-3 、A 2-4 、A 2-5 Five structurally similar glycopeptides and desacyl glucosamine A 3-1 The molecular structure of the mixture is as follows:

[0004]

[0005] Existing teicoplanin separation and purification technologies typically involve concentrating the fermentation broth through adsorption on a macroporous resin, then desorbing and crystallizing it with an organic solvent to obtain crude teicoplanin, which is then purified to obtain pure teicoplanin. However, macroporous resins are difficult to regenerate after adsorption, and chromatographic separation of the fermentation broth using macroporous resins requires large amounts of organic solvent for elution, resulting in high production costs. Summary of the Invention

[0006] The present invention provides a method for separating and purifying teicoplanin, which avoids the process of using a macroporous adsorption resin for chromatographic separation, does not require the use of a large amount of organic solvent for elution during the chromatographic separation process, and reduces separation costs.

[0007] The present invention provides a method for separating and purifying teicoplanin, comprising the following steps:

[0008] 1) adjusting the pH of the teicoplanin fermentation broth to acidic, performing a primary filtration using a ceramic membrane, and collecting a ceramic membrane concentrate; adjusting the pH of the ceramic membrane concentrate to alkaline, performing a secondary filtration using a ceramic membrane, and collecting a filtrate;

[0009] 2) adjusting the pH value of the filtrate to 4.5 to 6.5, performing adsorption separation treatment on the filtrate using a cation exchange resin and an anion exchange resin, and collecting the resin effluent;

[0010] 3) subjecting the resin effluent to ultrafiltration and nanofiltration in sequence to obtain an ultrafiltration concentrate; adjusting the acidity of the ultrafiltration concentrate so that its pH value does not exceed 4, and extracting the concentrate with an alcohol solvent to obtain an extract; and back-extracting the extract with a first alkaline buffer salt solution to obtain a back-extract;

[0011] 4) performing chromatography separation on the stripping solution using a polyamide resin and a second alkaline buffered salt solution as an eluent, collecting the eluate containing teicoplanin; concentrating the eluate and then crystallizing it to obtain pure teicoplanin.

[0012] In the separation and purification method as described above, in step 1), adjusting the pH of the fermentation broth to acidic includes adjusting the pH of the fermentation broth to 1-4; and adjusting the pH of the ceramic membrane concentrate to alkaline includes adjusting the pH of the ceramic membrane concentrate to 8-10.

[0013] The separation and purification method as described above, wherein, in step 1), before adjusting the pH of the teicoplanin fermentation broth to acidic, further comprises a process of pretreating the teicoplanin fermentation broth, wherein the pretreatment comprises: adding an inorganic salt to the teicoplanin fermentation broth.

[0014] The separation and purification method as described above, wherein, in step 2), the cation exchange resin is selected from HZ-3B resin, and the anion exchange resin is selected from FPDA-30 resin.

[0015] In the separation and purification method as described above, in step 3), the molecular weight cut-off of the ultrafiltration treatment is 5000 to 10000 Da.

[0016] The separation and purification method as described above, wherein, in step 3), the alcohol solvent is selected from n-butanol or isobutanol.

[0017] In the separation and purification method as described above, the first alkaline buffer saline solution and the second alkaline buffer saline solution are each independently selected from one of ammonium bicarbonate solution, sodium bicarbonate solution, and disodium hydrogen phosphate solution.

[0018] In the separation and purification method as described above, the pH value of the first alkaline buffer salt solution and / or the second alkaline buffer salt solution is 8-10.

[0019] The separation and purification method as described above, wherein, in step 4), before the stripping solution is subjected to chromatographic separation treatment, the process further includes pre-treating the stripping solution, wherein the pre-treatment includes adjusting the pH of the stripping solution to 4-6.

[0020] The separation and purification method as described above, wherein in step 4), the crystallization comprises: concentrating the eluate to a teicoplanin concentration of 80 to 150 mg / mL, dropwise adding ethanol or acetone to the concentrated solution to crystallize the concentrated solution, and filtering to obtain the pure teicoplanin.

[0021] The separation and purification method of the present invention achieves the separation and purification of teicoplanin from the fermentation broth with high yield and purity by sequentially subjecting the teicoplanin fermentation broth to ceramic membrane filtration under acidic and alkaline conditions, adsorption with anion and cation exchange resins, ultra-nanofiltration, extraction and stripping, chromatography with polyacrylamide resins, and crystallization. This method avoids the use of macroporous adsorption resins for chromatographic separation, eliminates the need for large amounts of organic solvents for elution, and reduces separation costs. DETAILED DESCRIPTION

[0022] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0023] The present invention provides a method for separating and purifying teicoplanin, comprising the following steps:

[0024] 1) adjusting the pH of the teicoplanin fermentation broth to acidic, performing a primary filtration using a ceramic membrane, and collecting the ceramic membrane concentrate; adjusting the pH of the ceramic membrane concentrate to alkaline, performing a secondary filtration using a ceramic membrane, and collecting the filtrate;

[0025] 2) adjusting the pH value of the filtrate to 4.5 to 6.5, performing adsorption separation treatment on the filtrate using a cation exchange resin and an anion exchange resin, and collecting the resin effluent;

[0026] 3) subjecting the resin effluent to ultrafiltration and nanofiltration in sequence to obtain an ultrafiltration concentrate; adjusting the acidity of the ultrafiltration concentrate so that its pH value does not exceed 4, extracting the concentrate with an alcohol solvent to obtain an extract; and back-extracting the extract with a first alkaline buffer salt solution to obtain a back-extract;

[0027] 4) using a polyamide resin and a first alkaline buffer salt solution as an eluent to perform chromatography separation on the stripping solution, collecting the eluate containing teicoplanin; concentrating the eluate and then crystallizing it to obtain pure teicoplanin.

[0028] In step 1), utilizing the characteristics of teicoplanin being alkali-soluble but acid-insoluble, teicoplanin is first formed into micelles under acidic conditions, and then filtered once using a ceramic membrane to allow teicoplanin to remain in the ceramic membrane concentrate. A large amount of pigment and part of the protein are removed by the primary filtration, and then the pH of the ceramic membrane concentrate is adjusted to alkaline to release teicoplanin and dissolve it in the fermentation broth. A filtrate containing teicoplanin is obtained by secondary filtration;

[0029] The isoelectric point of teicoplanin is 5.1. In step 2), the pH of the filtrate is adjusted to near the isoelectric point of teicoplanin, and then the filtrate is adsorbed and separated using a cation exchange resin and an anion exchange resin. The cation exchange resin can adsorb polyamino protein impurities and metal salt ions, and the anion exchange resin can adsorb polycarboxyl protein impurities, thereby further completing the purification of teicoplanin.

[0030] In step 3), the resin effluent is subjected to ultrafiltration and nanofiltration to further remove macromolecular proteins and pigments and to concentrate the resin effluent. The concentrate is then extracted with an alcohol solvent, and the extract is back-extracted with an alkaline buffered salt solution to remove inorganic salt impurities and organic small molecule impurities, respectively.

[0031] Teicoplanin contains an amino group and a carboxyl group, is an amphiphilic compound, and has an alkyl side chain in the molecule. Its amino acid characteristics are very suitable for chromatography on polyacrylamide resin. After the initial purification in the previous steps, some impurities cross-linked with teicoplanin are removed. On the polyacrylamide resin, after teicoplanin is adsorbed on the stationary phase, it can be eluted using only a small amount of alkaline buffered salt solution without the need for a large amount of organic solvent. In step 4), after the stripping solution is chromatographically separated using the polyacrylamide resin, the eluate is concentrated and crystallized to obtain pure teicoplanin.

[0032] The separation and purification method of the present invention sequentially subjects the fermentation broth of teicoplanin to ceramic membrane filtration under acidic and alkaline conditions, adsorption with anion and cation exchange resins, ultra-nanofiltration, extraction and stripping, chromatography with polyacrylamide resin, and crystallization, achieving separation and purification of teicoplanin from the fermentation broth with high yield and purity. This method avoids the use of macroporous adsorption resins for chromatographic separation, eliminates the need for large amounts of organic solvents for elution during the chromatographic separation process, and reduces separation costs.

[0033] In a specific embodiment, in step 1), adjusting the pH of the fermentation broth to acidic includes: adjusting the pH of the fermentation broth to 1-4, preferably to 2-3. Under strongly acidic conditions, the solubility of teicoplanin is lower, which can prevent it from dissolving into the filtrate of the primary filtration and causing loss.

[0034] In a specific embodiment, in step 1), adjusting the pH of the ceramic membrane concentrate to alkaline includes adjusting the pH of the ceramic membrane concentrate to 8-10, preferably to 9-10. Stronger alkalinity facilitates the dissolution of teicoplanin in the ceramic membrane concentrate, but teicoplanin has poor stability under strongly alkaline conditions. Adjusting the pH of the ceramic membrane concentrate to within the above range ensures good stability of teicoplanin and avoids the increased cooling costs and operational difficulty associated with strongly alkaline conditions.

[0035] In step 1), before adjusting the pH of the teicoplanin fermentation broth to acidic, the teicoplanin fermentation broth is also pretreated. The pretreatment includes adding inorganic salts to the teicoplanin fermentation broth. The addition of inorganic salts can further increase the solubility of teicoplanin under acidic conditions, thereby preventing teicoplanin from entering the filtrate of the primary filtration and causing product loss.

[0036] Specifically, the inorganic salt can be selected from inorganic sodium salt or inorganic ammonium salt. The mass volume concentration of the inorganic salt in the fermentation broth is preferably 0.3-3%.

[0037] In a specific embodiment, the cation exchange resin in step 2) is selected from HZ-3B resin, and the anion exchange resin is selected from FPDA-30 resin. The anion exchange resin and the cation exchange resin can be connected in series to improve the separation efficiency.

[0038] Furthermore, the molecular weight cut-off in the ultrafiltration process is 5000 to 100000 Da.

[0039] Before extraction, adjusting the acidity of the ultra-nanofiltration concentrate to a pH not exceeding 4 can reduce the solubility of teicoplanin in the aqueous phase, facilitating the extraction of teicoplanin using an alcoholic solvent. Furthermore, before extraction, the acidity of the ultra-nanofiltration concentrate is adjusted to a pH of 1 to 4, and further to a pH of 2 to 3.

[0040] The alcohol solvent of the present invention is selected from n-butanol or isobutanol, and both solvents have good extraction effects on teicoplanin.

[0041] The present invention does not particularly limit the types of the first alkaline buffer saline solution and the second alkaline buffer saline solution. Alkaline buffer saline solutions commonly used in the chemical field can be used. For example, the first alkaline buffer saline solution and the second alkaline buffer saline solution can each independently be selected from one of ammonium bicarbonate solution, sodium bicarbonate solution, and sodium dihydrogen phosphate solution.

[0042] Furthermore, the pH value of the first alkaline buffer saline solution and / or the second alkaline buffer saline solution is 8-10. Under such alkaline conditions, it is beneficial for teicoplanin to dissolve in the buffer saline solution and can also ensure the stable existence of teicoplanin during the separation process.

[0043] Furthermore, before the stripping solution is subjected to chromatographic separation, a pretreatment process is also included. The pretreatment includes adjusting the pH of the stripping solution to 4-6. Teicoplanin has the strongest adsorption capacity on the resin near its isoelectric point, while most other impurities have weaker adsorption capacity on the resin than teicoplanin. This allows them to be separated from teicoplanin without adsorption on the polyacrylamide resin. Other impurities with stronger adsorption capacity than teicoplanin will be separated from teicoplanin during elution. During the chromatographic separation process, washing with a small amount of water before eluting with the second alkaline buffered saline solution can further remove pigments from the stripping solution.

[0044] The crystallization after concentration of the eluate can be carried out by conventional crystallization treatment operations in the art. For example, the eluate can be concentrated to a teicoplanin concentration of 80 to 150 mg / mL, and then ethanol or acetone is added dropwise to the concentrate for crystallization. After filtration, pure teicoplanin can be obtained.

[0045] The following is a further introduction to the separation and purification method of teicoplanin provided by the present invention with reference to specific examples. In the following examples, unless otherwise specified, all raw materials can be purchased commercially or prepared by conventional methods.

[0046] It should be noted that in step 1) of the following examples, the addition of 0.5% (weight-to-volume) sodium chloride means adding 5 g of sodium chloride per liter of teicoplanin fermentation broth; in step 3), the addition of 10% (weight-to-volume) sodium chloride means adding 100 g of sodium chloride per liter of ultra-nanofiltration concentrate, and the back extraction of the extract with 2% (weight-to-volume) ammonium bicarbonate solution means adding 20 g of ammonium bicarbonate solution per liter of extract.

[0047] Example 1

[0048] The method for separating and purifying teicoplanin in this embodiment comprises the following steps:

[0049] 1) After adding 0.5% (weight to volume) sodium chloride to 29 L of teicoplanin fermentation broth (containing 24 g of teicoplanin), the pH of the system was adjusted to 2.0 with oxalic acid, filtered once using a ceramic membrane, and washed with 60 L of water. The ceramic membrane concentrate was concentrated to a volume of 11 L. The pH of the ceramic membrane concentrate was adjusted to 9.5 using sodium hydroxide solution, and filtered twice using a ceramic membrane with top-water addition (top-water addition in this invention refers to washing with water), collecting 90 L of filtrate (containing 22.1 g of teicoplanin).

[0050] 2) The pH value of the filtrate was adjusted to 5.5 and then passed through an ion exchange resin column consisting of 5 L of HZ-3B and 5 L of FPDA-30 connected in series, and the resin effluent (containing 21.7 g of teicoplanin) was collected.

[0051] 3) After adjusting the pH of the resin effluent to 7-9, ultrafiltration was performed using a 10,000 Da ultrafiltration membrane, followed by nanofiltration using a 300-500 Da nanofiltration membrane. Finally, the concentrate was concentrated by nanofiltration to a teicoplanin concentration of 50 mg / mL. 10% (mass by volume) sodium chloride was added, and the pH of the system was adjusted to 3.0. Extraction was performed by adding n-butanol, and the extract was back-extracted with a 2% (mass by volume) ammonium bicarbonate solution. The back-extract (containing 18.5 g of teicoplanin) was collected.

[0052] 4) After adjusting the pH of the stripping solution to 5.0, the stripping solution was chromatographed on a 2 L polyacrylamide resin column, washed with water and eluted with a 0.1% ammonium bicarbonate solution (pH 8.0), and the eluate containing teicoplanin (16.4 g teicoplanin) was collected. The eluate was concentrated by nanofiltration to a teicoplanin concentration of 109 mg / mL. Acetone was added dropwise to the concentrated solution to precipitate crystals. After separation, 14.5 g of pure teicoplanin was obtained with a purity of 90.83% and an isolated yield of 60.4%.

[0053] Example 2

[0054] The method for separating and purifying teicoplanin in this embodiment comprises the following steps:

[0055] 1) 0.5% (weight to volume) sodium chloride was added to 29.7 L of teicoplanin fermentation broth (containing 24.8 g of teicoplanin). The pH of the system was adjusted to 2.5 with oxalic acid, filtered once using a ceramic membrane, and washed with 60 L of water. The ceramic membrane concentrate was concentrated to a volume of 11 L. The pH of the ceramic membrane concentrate was adjusted to 9.61 with sodium hydroxide solution, and filtered twice using a ceramic membrane, collecting 90 L of filtrate (containing 21.7 g of teicoplanin).

[0056] 2) The pH value of the filtrate was adjusted to 5.5 and then passed through an ion exchange resin column consisting of 5 L of HZ-3B and 5 L of FPDA-30 connected in series, and the resin effluent (containing 20.3 g of teicoplanin) was collected.

[0057] 3) The pH of the resin effluent was adjusted to 7-9, followed by ultrafiltration and nanofiltration. The concentrate was concentrated by nanofiltration to a concentration of 50 mg / mL of teicoplanin. 10% (mass to volume ratio) sodium chloride was added, and the pH of the system was adjusted to 2.0. N-butanol was added for extraction, and the extract was back-extracted with 2% (mass to volume ratio) ammonium bicarbonate solution. The back-extract (containing 18.5 g of teicoplanin) was collected.

[0058] 4) After adjusting the pH of the stripping solution to 5.0, the stripping solution was chromatographed on a 2 L polyacrylamide resin column, washed with water and eluted with a 0.1% ammonium bicarbonate solution (pH 8.0), and the eluate containing teicoplanin (16.1 g teicoplanin) was collected. The eluate was concentrated by nanofiltration to a teicoplanin concentration of 120 mg / mL. Acetone was added dropwise to the concentrated solution to precipitate crystals. After separation, 14.7 g of pure teicoplanin was obtained with a purity of 90.31% and an isolated yield of 59.3%.

[0059] Example 3

[0060] The method for separating and purifying teicoplanin in this embodiment comprises the following steps:

[0061] 1) 0.5% (weight to volume) sodium chloride was added to 28.3 L of teicoplanin fermentation broth (containing 23.9 g of teicoplanin). The pH of the system was adjusted to 2.0 with oxalic acid, filtered once using a ceramic membrane, and washed with 60 L of water. The ceramic membrane concentrate was concentrated to a volume of 11 L. The pH of the ceramic membrane concentrate was adjusted to 9.44 with sodium hydroxide solution, and filtered twice using a ceramic membrane, collecting 90 L of filtrate (containing 21.8 g of teicoplanin).

[0062] 2) The pH value of the filtrate was adjusted to 4.5 and then passed through an ion exchange resin column consisting of 5 L of HZ-3B and 5 L of FPDA-30 connected in series, and the resin effluent (containing 19.1 g of teicoplanin) was collected.

[0063] 3) After adjusting the pH of the resin effluent to 7-9, ultrafiltration and nanofiltration were performed sequentially. Finally, the concentrate was concentrated by nanofiltration to a concentration of 50 mg / mL of teicoplanin. 10% (mass to volume ratio of the concentrate) sodium chloride was added, and the pH of the system was adjusted to 3.0. N-butanol was added for extraction, and the extract was back-extracted with 2% (mass to volume ratio) ammonium bicarbonate solution. The back-extract (containing 16.6 g of teicoplanin) was collected.

[0064] 4) After adjusting the pH of the stripping solution to 5.0, the stripping solution was chromatographed on a 2 L polyacrylamide resin column, washed with water and eluted with a 0.1% ammonium bicarbonate solution (pH 8.0), collecting the eluate containing teicoplanin (14.7 g). The eluate was concentrated by nanofiltration to a teicoplanin concentration of 117 mg / mL. Acetone was added dropwise to the concentrated solution to precipitate crystals. After separation, 13.1 g of pure teicoplanin was obtained with a purity of 90.26% and an isolated yield of 54.8%.

[0065] Example 4

[0066] The method for separating and purifying teicoplanin in this embodiment comprises the following steps:

[0067] 1) 0.5% (weight to volume) sodium chloride was added to 28.8 L of teicoplanin fermentation broth (containing 24.5 g of teicoplanin). The pH of the system was adjusted to 2.0 with oxalic acid, filtered once using a ceramic membrane, and washed with 60 L of water. The ceramic membrane concentrate was concentrated to a volume of 11 L. The pH of the ceramic membrane concentrate was adjusted to 9.59 with sodium hydroxide solution, and filtered a second time using a ceramic membrane, collecting 90 L of filtrate (containing 22.4 g of teicoplanin).

[0068] 2) The pH value of the filtrate was adjusted to 6.5 and then passed through an ion exchange resin column consisting of 5 L of HZ-3B and 5 L of FPDA-30 connected in series, and the resin effluent (containing 20.6 g of teicoplanin) was collected.

[0069] 3) The pH of the resin effluent was adjusted to 7-9, followed by ultrafiltration and nanofiltration. The concentrate was concentrated by nanofiltration to a concentration of 50 mg / mL of teicoplanin. 10% (mass to volume ratio) sodium chloride was added, and the pH of the system was adjusted to 2.0. N-butanol was added for extraction, and the extract was back-extracted with 2% (mass to volume ratio) ammonium bicarbonate solution. The back-extract (containing 18.6 g of teicoplanin) was collected.

[0070] 4) After adjusting the pH of the stripping solution to 5.0, the stripping solution was chromatographed on a 2 L polyacrylamide resin column, washed with water and eluted with a 0.1% ammonium bicarbonate solution (pH 8.0), and the eluate containing teicoplanin (16.5 g teicoplanin) was collected. The eluate was concentrated by nanofiltration to a teicoplanin concentration of 109 mg / mL. Acetone was added dropwise to the concentrated solution to precipitate crystals. After separation, 14.8 g of pure teicoplanin was obtained with a purity of 90.78% and an isolated yield of 60.4%.

[0071] Example 5

[0072] The pH value of the ceramic membrane concentrate after the first filtration was changed to determine its effect on the separation and purification of teicoplanin in the second filtration.

[0073] In the following experiments of Group A and Group B, the single filtration process was as follows: 0.5% (mass-to-volume ratio) of sodium chloride was added to 30 L of teicoplanin fermentation broth (containing 25.4 g of teicoplanin), the pH of the system was adjusted to 2.0 with oxalic acid, and the filtration was performed using a ceramic membrane. The filtration was then washed with 60 L of water, and the ceramic membrane concentrate was concentrated to a volume of 10 L (containing 24.4 g of teicoplanin).

[0074] A. The pH value of 10 L of ceramic membrane concentrate obtained after the primary filtration was adjusted to 9.56, and a secondary filtration was performed using a ceramic membrane. Water was added to collect 50 L of filtrate. The purity of teicoplanin in the filtrate was determined to be 63.25%, and the yield of the secondary filtration was 93.70%.

[0075] B. The pH value of 10 L of ceramic membrane concentrate obtained after the primary filtration was adjusted to 12.45, and a secondary filtration was performed using a ceramic membrane. Water was added to collect 50 L of filtrate. The purity of teicoplanin in the filtrate was determined to be 58.77%, and the yield of the secondary filtration was 92.01%.

[0076] From the comparison of the experiments of Group A and Group B, it can be seen that when the pH value of the ceramic membrane concentrate is adjusted to above 12 before the secondary filtration, the purity of teicoplanin in the secondary filtrate and the yield of the secondary filtration are reduced compared with when the pH value is adjusted to 9.56. This shows that adjusting the pH value of the ceramic membrane concentrate to 8-10 before the secondary filtration can ensure the stability of teicoplanin during the separation process.

[0077] Example 6

[0078] The effect of adding inorganic salts to the teicoplanin fermentation broth before the primary filtration on the separation and purification of teicoplanin in the primary filtration was determined.

[0079] A. After adding 0.5% (weight to volume) sodium chloride to 10 L of teicoplanin fermentation broth (containing 10.1 g of teicoplanin), the pH of the system was adjusted to 3.0 with oxalic acid. The filtrate was filtered once using a ceramic membrane, and 60 L of the filtrate was collected. The filtrate was determined to contain 0.6 g of teicoplanin. Calculation showed that 5.9% of teicoplanin was lost after the single filtration.

[0080] B. The pH of 10 L of teicoplanin fermentation broth (containing 10.1 g of teicoplanin) was adjusted to 3.0 with oxalic acid, and filtered once using a ceramic membrane. Water was added to yield 60 L of the filtrate from the first filtration. The filtrate from the first filtration was determined to contain 1.05 g of teicoplanin. Calculation showed that 10.4% of teicoplanin was lost after the first filtration.

[0081] The experimental comparison between Group A and Group B showed that adding inorganic salts to the fermentation broth could reduce the solubility of teicoplanin under acidic conditions and reduce the loss of teicoplanin during the single filtration, separation and purification process.

[0082] Comparative Example 1

[0083] The separation and purification method of teicoplanin in this comparative example comprises the following steps:

[0084] 1) 0.5% (weight to volume) sodium chloride was added to 30 L of teicoplanin fermentation broth (containing 25.6 g of teicoplanin). The pH of the system was adjusted to 2.0 with oxalic acid, filtered once using a ceramic membrane, and washed with 60 L of water. The ceramic membrane concentrate was concentrated to a volume of 11 L. The pH of the ceramic membrane concentrate was adjusted to 9.78 with sodium hydroxide solution, and filtered twice using a ceramic membrane, collecting 90 L of filtrate (containing 23.6 g of teicoplanin).

[0085] 2) The pH value of the filtrate was adjusted to 5.0, and the filtrate was subjected to chromatography analysis using a 2 L polyacrylamide resin column. After washing with water, the filtrate was eluted once with a 0.1% (mass volume ratio) ammonium bicarbonate solution (pH 8.0). The obtained primary eluate contained 3.7 g of teicoplanin (isolation yield calculated from the fermentation broth: 14.5%). The secondary eluate was eluted twice with a 0.1% (mass volume ratio) ammonium bicarbonate solution (pH 9.0). The obtained secondary eluate was combined with the primary eluate, and the combined solution contained a total of 10.1 g of teicoplanin (isolation yield calculated from the fermentation broth: 39.4%). The elution was performed three times using a mixed solution of the fermentation solution (pH 9.0) and 30% ethanol (30% ethanol means that 1 L of the eluent contains 300 ml of ethanol). The resulting three eluates were combined with the combined solution of the first eluate and the second eluate. The combined solution contained 16.8 g of teicoplanin (the separation yield was 65.6% calculated from the fermentation broth). The combined solution of the three eluates was concentrated by nanofiltration to a teicoplanin concentration of 109 mg / mL. Acetone was added dropwise to the concentrated solution to precipitate crystals. After solid-liquid separation, 13.2 g of pure teicoplanin (crystallization yield was 78.6%) was obtained with a purity of 88.5%. The total separation yield was 51.5%.

[0086] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for separating and purifying teicoplanin, characterized in that: The following steps are involved: 1) adjusting the pH of the teicoplanin fermentation broth to acidic, performing a primary filtration using a ceramic membrane, and collecting the ceramic membrane concentrate; The pH of the ceramic membrane concentrate is adjusted to alkaline, secondary filtration is performed using a ceramic membrane, and the filtrate is collected; 2) adjusting the pH value of the filtrate to 4.5-6.5, performing adsorption separation treatment on the filtrate using a cation exchange resin and an anion exchange resin, and collecting the resin effluent; 3) subjecting the resin effluent to ultrafiltration and nanofiltration in sequence to obtain an ultrafiltration concentrate; adjusting the acidity of the ultrafiltration concentrate so that its pH value does not exceed 4, extracting the concentrate with an alcohol solvent to obtain an extract; and back-extracting the extract with a first alkaline buffer salt solution to obtain a back-extract; 4) performing chromatography on the stripping solution using a polyacrylamide resin and a second alkaline buffered salt solution as an eluent, and collecting the eluate containing teicoplanin; The eluate is concentrated and then crystallized to obtain pure teicoplanin; In step 1), adjusting the pH of the fermentation broth to acidic includes: adjusting the pH of the fermentation broth to 1-4; Adjusting the pH of the ceramic membrane concentrated solution to alkaline includes: adjusting the pH of the ceramic membrane concentrated solution to 8-10; In step 1), before adjusting the pH of the teicoplanin fermentation broth to acidic, the process of pretreating the teicoplanin fermentation broth is also included. The pretreatment comprises: adding sodium chloride to the teicoplanin fermentation broth.

2. The separation and purification method according to claim 1, wherein In step 2), the cation exchange resin is selected from HZ-3B resin, and the anion exchange resin is selected from FPDA-30 resin.

3. The separation and purification method according to claim 1, wherein In step 3), the molecular weight cut-off of the ultrafiltration treatment is 5000-10000 Da.

4. The separation and purification method according to claim 1, wherein In step 3), the alcohol solvent is selected from n-butanol or isobutanol.

5. The separation and purification method according to claim 1, wherein The first alkaline buffer saline solution and the second alkaline buffer saline solution are each independently selected from one of ammonium bicarbonate solution, sodium bicarbonate solution, and disodium hydrogen phosphate solution.

6. The separation and purification method according to claim 5, wherein The pH value of the first alkaline buffered saline solution and / or the second alkaline buffered saline solution is 8-10.

7. The separation and purification method according to claim 1, wherein In step 4), before the stripping solution is subjected to chromatographic separation, the stripping solution is pretreated. The pretreatment includes adjusting the pH of the stripping solution to 4-6.

8. The separation and purification method according to claim 1, wherein In step 4), the crystallization comprises: concentrating the eluate to a teicoplanin concentration of 80-150 mg / mL, dropwise adding ethanol or acetone to the concentrate to crystallize the concentrate, and filtering to obtain the pure teicoplanin.

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