Method for purifying norvancomycin by using multi-mode filler

Abstract

The invention provides a method for purifying norvancomycin by using a multi-mode filler, and belongs to the technical field of medicine preparation and purification, the method comprises the following treatment steps of: filtering, decolorizing, finely purifying, centrifugally precipitating, redissolving and freeze-drying norvancomycin fermentation liquor, and the finely purifying step is finely purifying by using the multi-mode filler. According to the method for purifying norvancomycin by using the multi-mode filler, the production period can be effectively shortened, and the product purity can be effectively improved.

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 using a multimodal filler to improve the purification efficiency and product purity of norvancomycin.

[0005] To achieve the above-mentioned object, the technical solution of the present invention is: a method for purifying norvancomycin using a multimodal filler, comprising subjecting the fermentation broth of norvancomycin to the following treatment steps in sequence: filtration, decolorization, purification, centrifugal precipitation, reconstitution, and freeze-drying, wherein the purification step is purification using a multimodal filler.

[0006] Furthermore, the matrix of the multi-mode filler is 6% highly cross-linked agarose or high-rigidity agarose, and the ligand is any one of N-benzoyl-homocysteine ​​or complex weak cationic groups.

[0007] Furthermore, the multimodal filler is a filler Capto MMC (matrix 6% highly cross-linked agarose, ligand N-benzoyl-homocysteine), Capto MMC ImpRes (matrix high rigidity agarose, ligand N-benzoyl-homocysteine), MMC Bestarose FF (matrix 6% highly cross-linked agarose, ligand complex weak cationic group), Diamond MMC (matrix high rigidity agarose, ligand complex weak cationic group).

[0008] Furthermore, the purification comprises the following steps: flushing the filler, loading the sample, flushing the filler again, washing impurities, eluting, and collecting the sample.

[0009] Further, the step of washing the filler is to use 10mM-100mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, acetic acid-sodium acetate buffer, acetic acid-ammonium acetate buffer, glycine-hydrochloric acid buffer, citric acid-disodium hydrogen phosphate buffer solution or citric acid-sodium citrate buffer with a pH of 3.0-6.0 to wash the filler for 1-10CV.

[0010] Furthermore, the loading is to adjust the pH of the decolorized norvancomycin fermentation broth to 3.0-6.0 with 1M HCl or 1M NaOH, and load it on the chromatography column, with a loading amount of 10-70 g / L gel.

[0011] Further, the re-rinsing of the filler is to use 10mM-100mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer, acetic acid-ammonium acetate buffer, glycine-hydrochloric acid buffer, citric acid-disodium hydrogen phosphate buffer or citric acid-sodium citrate buffer with a pH of 3.0-6.0 to rinse the filler for 1-10CV.

[0012] Further, the washing is performed using 10mM-100mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer, acetic acid-ammonium acetate buffer, glycine-hydrochloric acid buffer, citric acid-disodium hydrogen phosphate buffer or citric acid-sodium citrate buffer with a pH of 3.0-6.0, and adding 10mM-100mM NaCl or KCl buffer for 1-5CV.

[0013] Furthermore, the elution is performed using 10mM-100mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer, acetic acid-ammonium acetate buffer, glycine-hydrochloric acid buffer, citric acid-disodium hydrogen phosphate buffer or citric acid-sodium citrate buffer with a pH of 3.0-6.0, and eluting with 100mM-500mM NaCl or KCl buffer.

[0014] Furthermore, the sample collection starts when the UV280 absorption value is greater than 500 mAu and ends when the UV280 absorption value is less than 1000 mAu, and the collected sample is the purified norvancomycin.

[0015] The beneficial effects of the method for purifying norvancomycin using a multimodal filler of the present invention are:

[0016] The method for purifying norvancomycin using a multi-mode filler of the present invention can effectively shorten the production cycle and effectively improve the purity of the product.

[0017] The purification method of the present invention uses a multi-mode filler as a purification, the matrix of which is 6% highly cross-linked agarose or high-rigidity agarose, and the ligand is N-benzoyl-homocysteine ​​or other complex weak cationic groups. In the prior art, since macroporous resins usually have poor selectivity and low resolution, the purity of the product of the traditional process is low and can only meet the pharmacopoeia standard; the multi-mode filler used in the present invention has high resolution and less non-specific adsorption, and can significantly improve the purity of the product.

[0018] The purification steps of the present invention are filtration, decolorization, purification, precipitation, redissolution, and freeze drying, and the composite filler is used for purification instead of the macroporous resin adsorption in the original process. Agarose fillers are usually used for the purification of macromolecules, and are rarely used for the purification of antibiotic products (because small molecules have less charge and are difficult to adsorb to the filler through charge, and the agarose pore size is larger, which further increases the difficulty of purification); the multi-mode filler used in the present invention allows norvancomycin to be bound to the filler, and then the norvancomycin can be eluted by increasing the conductivity, which significantly improves the purity of the product.

[0019] 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

[0020] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0021] Figure 1 is the HPLC chart of the final product in Example 1 of the present invention;

[0022] Figure 2 is the HPLC chart of the final product in Example 2 of the present invention;

[0023] Figure 3 It is the HPLC chart of the final product in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0024] 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.

[0025] A method for purifying norvancomycin using a multi-mode filler comprises subjecting a fermentation broth of norvancomycin to the following treatment steps in sequence: filtration, decolorization, purification, centrifugal precipitation, reconstitution, and freeze drying, wherein the purification step is performed using a multi-mode filler.

[0026] The matrix of the multi-mode filler is 6% highly cross-linked agarose or high-rigidity agarose, and the ligand is N-benzoyl-homocysteine ​​or other complex weak cationic groups.

[0027] Furthermore, the multimodal filler is a filler Capto MMC (matrix 6% highly cross-linked agarose, ligand N-benzoyl-homocysteine), Capto MMC ImpRes (matrix high rigidity agarose, ligand N-benzoyl-homocysteine), MMC Bestarose FF (matrix 6% highly cross-linked agarose, ligand complex weak cationic group), Diamond MMC (matrix high rigidity agarose, ligand complex weak cationic group).

[0028] The purification comprises the following steps: flushing the filler, loading the sample, flushing the filler again, washing impurities, eluting, and collecting the sample.

[0029] Further, the step of washing the filler is to use 10mM-100mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer, acetic acid-ammonium acetate buffer, glycine-hydrochloric acid buffer, citric acid-disodium hydrogen phosphate buffer or citric acid-sodium citrate buffer with a pH of 3.0-6.0 to wash the filler for 1-10CV. The loading is to adjust the pH of the decolorized norvancomycin fermentation broth to 3.0-6.0 with 1M HCl or 1M NaOH, and load it on the chromatography column, with a loading amount of 10-70g / L gel. The second washing of the filler is to use 10mM-100mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer, acetic acid-ammonium acetate buffer, glycine-hydrochloric acid buffer, citric acid-disodium hydrogen phosphate buffer or citric acid-sodium citrate buffer with a pH of 3.0-6.0 to wash the filler for 1-10CV. The washing is performed by using 10mM-100mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer, acetic acid-ammonium acetate buffer, glycine-hydrochloric acid buffer, citric acid-disodium hydrogen phosphate buffer or citric acid-sodium citrate buffer at a pH of 3.0-6.0, and adding 10mM-100mM NaCl or KCl buffer for 1-5CV. The elution is performed by using 10mM-100mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer, acetic acid-ammonium acetate buffer, glycine-hydrochloric acid buffer, citric acid-disodium hydrogen phosphate buffer or citric acid-sodium citrate buffer at a pH of 3.0-6.0, and adding 100mM-500mM NaCl or KCl buffer for elution. The sample collection starts when the UV280 absorption value is greater than 500 mAu and ends when the UV280 absorption value is less than 1000 mAu. The collected sample is the purified norvancomycin.

[0030] Example 1

[0031] A method for purifying norvancomycin using a multimodal filler comprises subjecting a fermentation broth of norvancomycin to the following treatment steps in sequence: filtration, decolorization, purification, precipitation, reconstitution, and freeze drying. The specific steps are as follows:

[0032] 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.

[0033] The decolorizing resin flow-through containing 6 g was purified with 200 ml of MMC Bestrose FF filler; 15 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer with a pH of 5.0 was used for balancing for 3CV; the sample was loaded with 1 M HCl to adjust the pH to 5.0; the sample was washed with 15 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer with a pH of 5.0 for 3CV; the impurities were washed with 15 mM sodium dihydrogen phosphate-disodium hydrogen phosphate with a pH of 5.0 plus 30 mM NaCl buffer, and washed for 3CV; the impurities were eluted with 15 mM sodium dihydrogen phosphate-disodium hydrogen phosphate with a pH of 5.0 plus 150 mM NaCl buffer; the starting and ending points of the collection were 1000 mAu-200 mAu; 5.14 g of norvancomycin with a purity of 86.58% was obtained, and the yield was 85.7%.

[0034] Centrifugal precipitation, reconstitution, and freeze drying: adjust the pH to 8.5 with 1M NaOH, then centrifuge at 8000rpm for 20min, remove the supernatant and collect the precipitate; dissolve the precipitate in purified water adjusted to pH 3.5 with HCl, with a concentration of 25g / L; then freeze dry under the following conditions: pre-freeze at -40°C for 2h, then evacuate 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 4.8g of norvancomycin powder with a purity of 94.72% (see the results). Figure 1 ).

[0035] 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.

[0036] Table 1

[0037] 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

[0038] Example 2

[0039] The same as Example 1, except that the purification conditions are as follows: 10 g of the decolorizing resin flow-through is purified with 200 ml of Diamond MMC filler; 15 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer with a pH of 5.0 is used for equilibration for 3CV; the sample is adjusted to pH 5.0 with 1 M HCl and loaded; 20 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer with a pH of 5.0 is used for washing for 3CV; 20 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate with a pH of 5.0 plus 30 mM NaCl buffer is used for washing, and washing for 3CV; 15 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate with a pH of 5.0 plus 100 mM NaCl buffer is used for elution; the collection start and end points are 700 mAu-50 mAu; 8.83 g of norvancomycin with a purity of 89.12% is obtained, and the yield is 88.3%.

[0040] After freeze-drying, 7.9 g of norvancomycin powder with a purity of 95.12% was obtained (see Figure 2 ).

[0041] Example 3

[0042] The same as Example 2, except that the conditions for purification are as follows: 10 g of the decolorizing resin flow-through is purified with 200 ml of Capto MMC filler; 15 mM citric acid-disodium hydrogen phosphate buffer solution with a pH of 5.0 is used for equilibration for 3CV; the sample is adjusted to pH 5.0 with 1 M HCl and loaded; 20 mM citric acid-disodium hydrogen phosphate buffer solution with a pH of 5.0 is used for washing for 3CV; 20 mM citric acid-disodium hydrogen phosphate buffer solution with a pH of 5.0 plus 30 mM KCl buffer is used for washing, and washing for 3CV; 15 mM citric acid-disodium hydrogen phosphate buffer solution with a pH of 5.0 plus 100 mM KCl buffer is used for elution; the collection start and end points are 700 mAu-50 mAu; 8.77 g of norvancomycin with a purity of 89.26% is obtained, and the yield is 87.7%.

[0043] After freeze-drying, 7.8 g of norvancomycin powder with a purity of 95.17% was obtained.

[0044] Example 4

[0045] The same as Example 2, except that the purification conditions are as follows: 10 g of the decolorizing resin flow-through is purified with 200 ml of Capto MMC ImpRes filler; 15 mM acetic acid-sodium acetate buffer solution with a pH of 5.0 is used for equilibration for 3CV; the sample is adjusted to pH 5.0 with 1 M HCl and loaded; 20 mM acetic acid-sodium acetate buffer solution with a pH of 5.0 is used for washing for 3CV; 20 mM acetic acid-sodium acetate buffer solution with a pH of 5.0 plus 30 mM KCl buffer is used for washing, and washing for 3CV; 15 mM acetic acid-sodium acetate buffer solution with a pH of 5.0 plus 100 mM KCl buffer is used for elution; the collection start and end points are 700 mAu-50 mAu; 8.72 g of norvancomycin with a purity of 89.26% is obtained, and the yield is 87.2%.

[0046] After freeze-drying, 7.9 g of norvancomycin powder with a purity of 95.11% was obtained.

[0047] Comparative Example 1

[0048] Comparative Example 1 is a comparative test of Example 1. Comparative Example 1 uses the purification method of the prior art to purify the fermentation broth of norvancomycin, including filtration, decolorization, macroporous resin adsorption, desorption, precipitation, re-dissolution, and freeze-drying.

[0049] 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.

[0050] 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 1000mAu-800mAu; obtain 8.6g of norvancomycin with a purity of 77.83% and a yield of 57.3%.

[0051] 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 7.3g of norvancomycin powder with a yield of 84.9%.

[0052] 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 90.58%.

[0053] Example 5

[0054] The process time of pure or macroporous resin adsorption in the above Examples 1-4 and Comparative Example 1 is summarized as shown in Table 2 below.

[0055] Table 2

[0056] project Technology Flow rate Process time Example 1 MMC Bestrose FF 8CV / h 3.1h Example 2 Diamond MMC 10CV / h 2.3h Example 3 Capto MMC 10CV / h 2.4 Example 4 Capto MMC ImpRes 10CV / h 2.4 Comparative Example 1 LX-38 1CV / h 22h

[0057] 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.

[0058] 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 multimodal filler, characterized in that: The method comprises sequentially subjecting the fermentation broth of norvancomycin to the following treatment steps: filtration, decolorization, purification, centrifugal precipitation, re-dissolution and freeze-drying, wherein the purification step is performed by using a multi-mode filler for purification.

2. A method for purifying norvancomycin using a multimodal filler according to claim 1, characterized in that: The matrix of the multi-mode filler is 6% highly cross-linked agarose or high-rigidity agarose, and the ligand is any one of N-benzoyl-homocysteine ​​or complex weak cationic groups.

3. A method for purifying norvancomycin using a multimodal filler according to claim 2, characterized in that: The multimodal filler is Capto MMC, Capto MMC ImpRes, MMC Bestarose FF or Diamond MMC.

4. A method for purifying norvancomycin using a multimodal filler according to any one of claims 1 to 3, characterized in that: The purification comprises the following steps: flushing the filler, loading the sample, flushing the filler again, washing impurities, eluting, and collecting the sample.

5. A method for purifying norvancomycin using a multimodal filler according to claim 4, characterized in that: The step of washing the filler is to wash the filler for 1-10CV using a 10mM-100mM buffer with a pH of 3.0-6.0, wherein the buffer is selected from any one of the following: potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer, acetic acid-ammonium acetate buffer, glycine-hydrochloric acid buffer, citric acid-disodium hydrogen phosphate buffer or citric acid-sodium citrate buffer.

6. A method for purifying norvancomycin using a multimodal filler according to claim 4, characterized in that: The loading is to adjust the pH of the decolorized norvancomycin fermentation broth to 3.0-6.0 with 1M HCl or 1M NaOH, and load the broth onto a chromatography column with a loading amount of 10-70 g / L gel.

7. A method for purifying norvancomycin using a multimodal filler according to claim 4, characterized in that: The second flushing of the filler is to use a 10mM-100mM buffer with a pH of 3.0-6.0 to flush the filler for 1-10CV, and the buffer is selected from any one of the following: potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer, acetic acid-ammonium acetate buffer, glycine-hydrochloric acid buffer, citric acid-disodium hydrogen phosphate buffer or citric acid-sodium citrate buffer.

8. The method for purifying norvancomycin using a multimodal filler according to claim 4, characterized in that: The washing is performed by using a 10mM-100mM buffer with a pH of 3.0-6.0 and a 10mM-100mM NaCl or KCl buffer for 1-5CV, wherein the buffer with a pH of 3.0-6.0 is selected from any one of the following: potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer, acetic acid-ammonium acetate buffer, glycine-hydrochloric acid buffer, citric acid-disodium hydrogen phosphate buffer or citric acid-sodium citrate buffer.

9. A method for purifying norvancomycin using a multimodal filler according to claim 4, characterized in that: The elution is performed by using a 10mM-100mM buffer with a pH of 3.0-6.0 and a 100mM-500mM NaCl or KCl buffer, wherein the buffer with a pH of 3.0-6.0 is selected from any one of the following: potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer, acetic acid-ammonium acetate buffer, glycine-hydrochloric acid buffer, citric acid-disodium hydrogen phosphate buffer or citric acid-sodium citrate buffer.

10. The method for purifying norvancomycin using a multimodal filler according to claim 4, characterized in that: The sample collection starts when the UV280 absorption value is greater than 500 mAu and ends when the UV280 absorption value is less than 1000 mAu. The collected sample is the purified norvancomycin.

Images