Method for purifying norvancomycin by using polymer filler
By using polymer fillers to perform the purification step of norvancomycin, the problems of long process time and low purity in the existing process are solved, and a more efficient purification process and higher product purity are achieved.
Patent Information
- Application Number
- CN202411382850.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-30
- Publication Date
- 2025-02-28
AI Technical Summary
The existing purification process of norvancomycin is long, and the nonspecific adsorption of macroporous resins leads to low product purity.
The polymer filler is used for purification, including filler treatment, sample pretreatment, loading, rinsing, elution and collection of products. The filler is treated with 28-29% ammonia water, the sample pH is adjusted to 3.0-6.0, and eluted with 0.1-1.0M ammonia water or other alkaline eluent.
It effectively shortens the production cycle, improves product purity, and avoids the dangers and costs of using organic solvents, and reduces the cost of factory construction.
Smart Images
Figure CN119529032A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a purification method, in particular to a purification method of norvancomycin, and belongs to the technical field of drug preparation and purification. Background Art
[0002] Norvancomycin is a new antibiotic independently discovered in my country. It has one less methyl group on the terminal amino group than vancomycin. The chemical structure, pharmacological properties and antibacterial spectrum of Norvancomycin are similar to those of vancomycin, but Norvancomycin has slightly stronger antibacterial activity. It mainly has significant antibacterial effects on Gram-positive bacteria, and is more sensitive to methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. Its efficacy on membranous enteritis caused by Bacillus difficile is the same as that of vancomycin. It can act on the bacterial cell wall and inhibit protein synthesis, and is a fast-acting bactericidal drug.
[0003] At present, the purification process of norvancomycin usually includes filtration, decolorization, macroporous resin adsorption, desorption, precipitation, re-dissolution, and freeze-drying. Since the macroporous resin usually requires a low flow rate of 1-2CV / h, the process time of this method is relatively long; and the macroporous resin is purified by the principle of non-specific adsorption, which usually leads to low product purity. Summary of the invention
[0004] In view of the above problems, the present invention provides a method for purifying norvancomycin to improve the purification efficiency and product purity of norvancomycin.
[0005] To achieve the above object, the technical solution of the present invention is: a method for purifying norvancomycin,
[0006] The method comprises the following processing steps in sequence: filtering, decolorizing, purifying, centrifugal precipitation, re-dissolving and freeze-drying the crude product of norvancomycin, wherein the purifying step is to purify with a polymer filler.
[0007] Furthermore, the matrix of the polymer filler is styrene-divinylbenzene, polyacrylate, butyl methacrylate or hydroxyethyl methacrylate, and the ligand is sulfonic acid group, sulfomethyl group, carboxymethyl group or xanthocyanin group.
[0008] Furthermore, the polymer filler is HD-8 resin (matrix styrene, ligand sulfonic acid group), LXMS-30GS resin (matrix styrene-divinylbenzene, ligand sulfonic acid group), LXMS-50S (matrix styrene-divinylbenzene, ligand sulfonic acid group), BestPoly 30S (matrix styrene-divinylbenzene, ligand sulfomethyl group), UniCM 30S (matrix polyacrylate, ligand carboxymethyl group), UniSP 30S (matrix polyacrylate, ligand sulfomethyl group), UniCM 50XS (matrix polyacrylate, ligand carboxymethyl group), UniSP 50XS (matrix polyacrylate, ligand sulfomethyl group).
[0009] Furthermore, the purification includes the following steps: filler treatment, filler pretreatment, sample pretreatment, sample loading, washing, elution, and product collection.
[0010] Furthermore, the filler treatment is to use 28-29% ammonia water to treat filler 3CV.
[0011] Furthermore, the filler pretreatment is to rinse the filler with purified water until it is neutral, and the sample pretreatment is to adjust the pH of the crude norvancomycin to 3.0-6.0 with 1M NaOH or 1M HCl.
[0012] Furthermore, the loading is to load the sample after adjusting the pH, and the loading amount is 10-60g / L gel.
[0013] Furthermore, the elution is performed with 0.1-1.0M ammonia water, 0.01-1M ammonium chloride at pH 8-12, 0.01-1M ammonium carbonate at pH 8-12, 0.01-1M ammonium bicarbonate at pH 8-12, 0.01-1M ammonium formate at pH 8-12 or 0.01-1M ammonium acetate at pH 8-12.
[0014] Furthermore, the collection of the product starts when the UV280 absorption value is greater than 300 mAu and ends when it is less than 1000 mAu.
[0015] Furthermore, the centrifugal precipitation is to centrifuge the product solution obtained after purification at 1000G-10000G for 1min-30min, and remove the supernatant to obtain a precipitate; the re-dissolution is to re-dissolve the precipitate with 0.1%-1.0% hydrochloric acid.
[0016] The beneficial effects of the method for purifying norvancomycin using a polymer filler of the present invention are:
[0017] The method for purifying norvancomycin of the present invention can effectively shorten the production cycle and effectively improve the purity of the product.
[0018] The polymer filler used in the purification method of the present invention is used as a pure material, and its matrix is styrene-divinylbenzene, polyacrylate, butyl methacrylate, hydroxyethyl methacrylate, etc., and its ligand is sulfonic acid group, sulfomethyl group, carboxymethyl group, xanthocyanin group, etc.; the polymer filler used in the present invention has high resolution and less nonspecific adsorption, and can greatly improve the purity of the product.
[0019] Patent CN1113886552A discloses a method for purification using a new type of polymer filler, which uses a reverse phase polymer filler and requires the use of organic solvents such as methanol, ethanol, and isopropanol as a balance solution and eluent. However, the use of organic solvents is costly, and as current regulations become increasingly stringent, organic solvents need to be stored in separate explosion-proof areas; in addition, organic solvents are toxic, and protective measures must be taken to prevent danger when used. The method of using polymer fillers in the present invention can effectively shorten the production cycle and effectively improve the purity of the product. In addition, the present invention does not use organic solvents, and does not require the construction of additional explosion-proof areas during factory production, which reduces factory construction costs and is more environmentally friendly and safe.
[0020] The purification method of the present invention can replace the macroporous resin adsorption process in the traditional process. The macroporous resin adsorption flow rate is relatively low at 1-3CV / h, while the flow rate of the present invention is 5-15CV / h, which can greatly shorten the process time. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.
[0022] Figure 1 is the HPLC chart of the final product in Example 1 of the present invention;
[0023] Figure 2 is the HPLC chart of the final product in Example 2 of the present invention;
[0024] Figure 3 is the HPLC chart of the final product in Example 3 of the present invention;
[0025] Figure 4 It is the HPLC chart of the final product in Comparative Example 1 of the present invention;
[0026] Figure 5 It is the HPLC chart of the final product in Comparative Example 2 of the present invention. DETAILED DESCRIPTION
[0027] The technical scheme in the embodiment of the present invention is described clearly and completely below in conjunction with the accompanying drawings in the embodiment of the present invention. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.
[0028] A method for purifying norvancomycin using a polymer filler comprises sequentially subjecting a crude product of norvancomycin to the following treatment steps: filtration, decolorization, purification, centrifugal precipitation, re-dissolution, and freeze-drying, wherein the purification step is purification using a polymer filler.
[0029] The matrix of the polymer filler is styrene-divinylbenzene, polyacrylate, butyl methacrylate or hydroxyethyl methacrylate, and the ligand is sulfonic acid group, sulfomethyl group, carboxymethyl group or xanthocyanin group.
[0030] Further, the polymer filler is HD-8 resin (matrix styrene, ligand sulfonic acid group), LXMS-30GS resin (matrix styrene-divinylbenzene, ligand sulfonic acid group), LXMS-50S (matrix styrene-divinylbenzene, ligand sulfonic acid group), BestPoly 30S (matrix styrene-divinylbenzene, ligand sulfomethyl group), UniCM 30S (matrix polyacrylate, ligand carboxymethyl group), UniSP 30S (matrix polyacrylate, ligand sulfomethyl group), UniCM 50XS (matrix polyacrylate, ligand carboxymethyl group), UniSP 50XS (matrix polyacrylate, ligand sulfomethyl group). The composite filler purification comprises the following steps:
[0031] Packing treatment, packing pretreatment, sample pretreatment, loading, washing, elution, and product collection.
[0032] Furthermore, the filler treatment is to use 28-29% ammonia water to treat the filler for 3CV; the filler pre-treatment is to rinse the filler with purified water until the sample is neutral, and the sample pre-treatment is to adjust the pH of the crude norvancomycin to 3.0-6.0 with 1M NaOH or 1M HCl; the loading is to load the sample with adjusted pH, and the loading amount is 10-60g / L gel; the elution is to elute with 0.1-1.0M ammonia water, 0.01-1M ammonium chloride with pH 8-12, 0.01-1M ammonium carbonate with pH 8-12, 0.01-1M ammonium bicarbonate with pH 8-12, 0.01-1M ammonium formate with pH 8-12 or 0.01-1M ammonium acetate with pH 8-12; the collection of the product is to start collecting after the UV280 absorption value is greater than 300mAu and end collecting after it is less than 1000mAu.
[0033] Furthermore, the centrifugal precipitation is to centrifuge the product solution obtained after purification at 1000G-10000G for 1min-30min, and remove the supernatant to obtain a precipitate; the re-dissolution is to re-dissolve the precipitate with 0.1%-1.0% hydrochloric acid.
[0034] Example 1
[0035] A method for purifying norvancomycin using a polymer filler comprises subjecting a crude product of norvancomycin to the following treatment steps in sequence: filtration, decolorization, purification, precipitation, reconstitution, and freeze drying. The specific steps are as follows:
[0036] Take 10L of fermentation broth with an expression level of 6.3g / L, add 5% perlite and 10% potassium hematite, and collect the supernatant by plate and frame filtration; adjust the supernatant pH to 5.5 with 1M HCl and load it on 1L of B001 decolorization resin at a flow rate of 1CV / h, and collect the flow-through.
[0037] A chromatography column with 1 L of HD-8 resin as filler was used for chromatography, the pH of the sample was adjusted to 4.5 with 1 M HCl, and eluted with 0.5 M ammonia solution; the loading amount was 20 g / L gel, and the collection start and end points were 600 mAu-500 mAu; the collected samples were centrifuged at 5000 G for 20 min, and the supernatant was removed; the precipitate was re-dissolved with 0.1% hydrochloric acid; and then freeze-dried under the following conditions: pre-freezing at -40°C for 2 h, then vacuumizing to below 20 Pa, and then heating and drying with a program of heating by 20°C for 2 h, and maintaining the temperature at 40°C for 30 h to obtain 12.6 g of norvancomycin crude powder with a yield of 63%.
[0038] The purity of freeze-dried norvancomycin was determined. Figure 1 As shown in the HPLC chart of the final product, the purity of norvancomycin was 96.61%.
[0039] The above analysis method is: using a C18 column; mobile phase A: triethylamine buffer (3 ml of triethylamine, add 1000 ml of water, adjust the pH to 3.2 with phosphoric acid): acetonitrile: tetrahydrofuran = 96:3:1, mobile phase B: triethylamine buffer: acetonitrile: tetrahydrofuran = 70:29:1, linear gradient elution according to Table 1; flow rate 1 ml / min; wavelength 280 nm; injection volume 20 ul.
[0040] Table 1
[0041] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 23 100 0 38 0 100 40 0 100 41 100 0 50 100 0
[0042] Example 2
[0043] The same as Example 1, except that 1 L of HD-8 resin was used as the chromatography column filler for chromatography, and 0.5 M ammonium chloride-sodium hydroxide solution (pH 10.1) was used for elution; the sample load was 20 g / L gel, and the collection start and end points were 800 mAu-400 mAu; the collected samples were centrifuged at 5000 G for 20 min, and the supernatant was removed; the precipitate was re-dissolved with 0.1% hydrochloric acid; and then freeze-dried under the following conditions: pre-freezing at -40°C for 2 h, then vacuumizing to below 20 Pa, and then drying by heating at a rate of 20°C for 2 h, and then maintaining the temperature at 40°C for 30 h to obtain 14.55 g of norvancomycin crude powder with a yield of 73.8%.
[0044] The purity of freeze-dried norvancomycin was determined. Figure 2 As shown in the HPLC chart of the final product, the purity of norvancomycin was 99.61%.
[0045] Example 3
[0046] Same as Example 1, except that the pure filler used is LXMS-30GS.
[0047] A 200 ml LXMS-30GS column was used as a filler for chromatography. The sample was adjusted to pH 5.0 with 1 M HCl, washed with 0.01 M ammonia water for 3CV, and eluted with 0.5 M ammonia solution. The loading amount was 30 g / L gel, and the collection start and end points were 800 mAu-100 mAu. The collected samples were centrifuged at 5000 G for 20 min, and the supernatant was removed. The precipitate was re-dissolved with 0.1% hydrochloric acid, and then freeze-dried under the following conditions: pre-freezing at -40°C for 2 h, then vacuuming to below 20 Pa, and then drying by heating at a rate of 20°C for 2 h, and then maintaining the temperature at 40°C for 30 h to obtain 21.66 g of norvancomycin crude powder with a yield of 72.2%.
[0048] The purity of freeze-dried norvancomycin was determined. Figure 3 As shown in the HPLC chart of the final product, the purity of norvancomycin was 99.23%.
[0049] Example 4
[0050] The same as Example 1, except that on the basis of Example 1, a step of further removing impurities such as pigments and endotoxins is added.
[0051] Take 10 g of the sample prepared by the process of Example 1, re-dissolve it with purified water to a concentration of 20 g / L, ultrafilter it with a membrane package with a pore size of 8 kD, and top wash it with 3 L of purified water. The filtrate is collected and then spray-dried to obtain 9.2 g of norvancomycin powder with a purity of 99.86% and a yield of 92%.
[0052] Comparative Example 1
[0053] Comparative Example 1 is a comparative test of Example 1. Comparative Example 1 uses the purification method of the prior art to purify the crude product of norvancomycin, including filtration, decolorization, macroporous resin adsorption, desorption, precipitation, re-dissolution, and freeze-drying.
[0054] Take 10L of fermentation broth, add 5% perlite and 10% potassium ferrate, collect the supernatant by plate and frame filtration; adjust the pH of the supernatant to 5.5 with 1M HCl, load it on B001 decolorization resin, flow rate 1CV / h, and collect the flow-through.
[0055] Take 15g of the decolorizing resin flow-through liquid and adsorb it with 1L of LX-38; balance with purified water for 3CV; adjust the pH of the sample to 4.5 with 1M HCl and load it; rinse with purified water for 5CV; desorb with 20% ethanol at pH 4.5, and collect the starting and ending points of 200mAu-200mAu; obtain 11.4g of vancomycin with a purity of 77.83% and a yield of 76.0%.
[0056] The desorption liquid of the macroporous resin was adjusted to pH 8.5 with 1M NaOH, and then centrifuged at 8000rpm for 20min, the supernatant was removed and the precipitate was collected; the precipitate was dissolved in purified water with HCl adjusted to pH 3.5, with a concentration of 25g / L; and then freeze-dried under the following conditions: pre-freeze at -40℃ for 2h, then evacuate to below 20Pa, and then heat and dry by increasing the temperature by 20℃ for 2h, and maintain the temperature at 20℃ for 30h to obtain 9.7g of norvancomycin crude powder with a yield of 85.1%.
[0057] The purity of freeze-dried norvancomycin was determined. Figure 4 As shown in the HPLC chart of the final product, the purity of norvancomycin was 82.93%.
[0058] Comparative Example 2
[0059] Comparative Example 2 is a comparative test of Example 1. Comparative Example 2 uses the purification method of the prior art to purify the fermentation broth of norvancomycin, including filtration, decolorization, polymer reverse phase purification, membrane concentration, precipitation, re-dissolution, and freeze-drying.
[0060] Take 10L of fermentation broth, add 5% perlite and 10% potassium ferrate, collect the supernatant by plate and frame filtration; adjust the pH of the supernatant to 5.5 with 1M HCl, load it on B001 decolorization resin, flow rate 1CV / h, and collect the flow-through.
[0061] 200 ml of UniPMM40 was rinsed with purified water for 2CV, and the decolorizing resin flow-through containing 4 g of norvancomycin was loaded, washed with 1% methanol solution for 2CV (pH adjusted to 7.2 with HCl), eluted with 10% methanol solution (pH adjusted to 7.0 with HCl), and the starting and ending points of the collection were 650 mAu-650 mAu; 2.5 g of vancomycin with a purity of 83.38% was obtained, and the yield was 62.5%.
[0062] The eluate from UniPMM40 was concentrated to a concentration of 10 g / L using a membrane and then eluted with 3 volumes of purified water.
[0063] The membrane concentrate was adjusted to pH 8.5 with 1M NaOH, then centrifuged at 8000rpm for 20min, the supernatant was removed and the precipitate was collected; the precipitate was dissolved in purified water with HCl adjusted to pH 3.5, with a concentration of 25g / L; then freeze-dried under the following conditions: pre-freeze at -40°C for 2h, then vacuumize to below 20Pa, then heat and dry by increasing the temperature by 20°C for 2h, and maintain the temperature at 40°C for 30h to obtain 1.9g of norvancomycin crude powder with a purity of 91.56 and a yield of 76%. Figure 5 The final product is shown in HPLC.
[0064] Example 5
[0065] The process time of pure or macroporous resin adsorption in Examples 1-4 and Comparative Examples 1-2 is summarized as shown in Table 2.
[0066] Table 2
[0067] project Technology Flow rate Process time Example 1 HD-8 resin 8CV / h 3.4h Example 2 HD-8 resin 8CV / h 3.4h Example 3 LXMS-30GS 10CV / h 3.2 Example 4 HD-8 resin 8CV / h 3.4 Comparative Example 1 LX-38 1CV / h 22h Comparative Example 2 UniPMM40 2CV / h 7.5h
[0068] It can be seen from the statistical values in the above table that the purification method of the present invention can replace the macroporous resin adsorption process in the traditional process. The macroporous resin adsorption flow rate is relatively low at 1-3CV / h, while the flow rate of the present invention is 5-15CV / h, which can greatly shorten the process time.
[0069] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
Claims
1. A method for purifying norvancomycin using a polymer filler, characterized in that: The method comprises the following processing steps in sequence: filtering, decolorizing, purifying, centrifugal precipitation, re-dissolving and freeze-drying the crude product of norvancomycin, wherein the purifying step is to purify with a polymer filler.
2. The method for purifying norvancomycin using a polymer filler according to claim 1, characterized in that: The matrix of the polymer filler is styrene-divinylbenzene, polyacrylate, butyl methacrylate or hydroxyethyl methacrylate, and the ligand is sulfonic acid group, sulfomethyl group, carboxymethyl group or xanthocyanin group.
3. The method for purifying norvancomycin using a polymer filler according to claim 2, characterized in that: The polymer filler is HD-8 resin or LXMS-30GS resin.
4. A method for purifying norvancomycin using a polymer filler according to any one of claims 1 to 3, characterized in that: The purification process includes the following steps: filler treatment, filler pre-treatment, sample pre-treatment, sample loading, washing, elution, and product collection.
5. The method for purifying norvancomycin using a polymer filler according to claim 4, characterized in that: The filler treatment is to treat the filler 3CV with 28-29% ammonia water.
6. The method for purifying norvancomycin using a polymer filler according to claim 4, characterized in that: The filler pretreatment is to rinse the filler with purified water until it is neutral, and the sample pretreatment is to adjust the pH of the crude norvancomycin to 3.0-6.0 with 1M NaOH or 1M HCl.
7. The method for purifying norvancomycin using a polymer filler according to claim 4, characterized in that: The sample loading is to load the sample after adjusting the pH, and the loading amount is 10-60g / L gel.
8. The method for purifying norvancomycin using a polymer filler according to claim 4, characterized in that: The elution is performed with 0.1-1.0 M ammonia water, 0.01-1 M ammonium chloride at pH 8-12, 0.01-1 M ammonium carbonate at pH 8-12, 0.01-1 M ammonium bicarbonate at pH 8-12, 0.01-1 M ammonium formate at pH 8-12 or 0.01-1 M ammonium acetate at pH 8-12.
9. The method for purifying norvancomycin using a polymer filler according to claim 4, characterized in that: The collected product starts collecting when the UV280 absorption value is greater than 300mAu and ends collecting when it is less than 1000mAu.
10. A method for purifying norvancomycin using a polymer filler according to any one of claims 1 to 3, characterized in that: The centrifugal precipitation is to centrifuge the product solution obtained after purification at 1000G-10000G for 1min-30min, remove the supernatant to obtain the precipitate; the re-dissolution is to re-dissolve the precipitate with 0.1%-1.0% hydrochloric acid.