CN116903703A - Purification method of daptomycin - Google Patents
Purification method of daptomycin Download PDFInfo
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- CN116903703A CN116903703A CN202310661443.4A CN202310661443A CN116903703A CN 116903703 A CN116903703 A CN 116903703A CN 202310661443 A CN202310661443 A CN 202310661443A CN 116903703 A CN116903703 A CN 116903703A
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- Prior art keywords
- solution
- daptomycin
- eluent
- sample solution
- purification method
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- 108010013198 Daptomycin Proteins 0.000 title claims abstract description 59
- DOAKLVKFURWEDJ-QCMAZARJSA-N daptomycin Chemical compound C([C@H]1C(=O)O[C@H](C)[C@@H](C(NCC(=O)N[C@@H](CCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@H](CO)C(=O)N[C@H](C(=O)N1)[C@H](C)CC(O)=O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](CC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CCCCCCCCC)C(=O)C1=CC=CC=C1N DOAKLVKFURWEDJ-QCMAZARJSA-N 0.000 title claims abstract description 59
- 229960005484 daptomycin Drugs 0.000 title claims abstract description 59
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000000746 purification Methods 0.000 title claims abstract description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 67
- 239000003480 eluent Substances 0.000 claims abstract description 27
- 239000012488 sample solution Substances 0.000 claims abstract description 19
- 239000011347 resin Substances 0.000 claims abstract description 16
- 229920005989 resin Polymers 0.000 claims abstract description 16
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims abstract description 14
- 229910052938 sodium sulfate Inorganic materials 0.000 claims abstract description 14
- 235000011152 sodium sulphate Nutrition 0.000 claims abstract description 14
- 159000000000 sodium salts Chemical class 0.000 claims abstract description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 8
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 8
- 238000011067 equilibration Methods 0.000 claims abstract description 6
- 238000011068 loading method Methods 0.000 claims abstract description 6
- 239000011780 sodium chloride Substances 0.000 claims abstract description 4
- 239000001488 sodium phosphate Substances 0.000 claims abstract description 4
- 229910000162 sodium phosphate Inorganic materials 0.000 claims abstract description 4
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims abstract description 4
- 238000001728 nano-filtration Methods 0.000 claims description 24
- 239000012528 membrane Substances 0.000 claims description 14
- 238000005406 washing Methods 0.000 claims description 2
- 238000010612 desalination reaction Methods 0.000 claims 2
- 235000012054 meals Nutrition 0.000 claims 2
- 239000012535 impurity Substances 0.000 abstract description 30
- 239000003814 drug Substances 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 description 30
- 239000000243 solution Substances 0.000 description 25
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 239000000843 powder Substances 0.000 description 11
- 239000000047 product Substances 0.000 description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 150000003839 salts Chemical class 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 239000000706 filtrate Substances 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 3
- 230000001276 controlling effect Effects 0.000 description 3
- 239000012266 salt solution Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 3
- 230000001476 alcoholic effect Effects 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012264 purified product Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012468 concentrated sample Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
Abstract
The invention belongs to the field of biological medicine, and in particular relates to a method for purifying daptomycin. The purification method comprises the steps of loading a sample solution in a C18 resin chromatography column equilibrated with an equilibration solution, and eluting the C18 resin chromatography column with an eluent; wherein the sample solution, the balance solution and the eluent contain sodium salt with the concentration of 0.1-1.0 (W/V)%, and the pH values of the sample solution, the balance solution and the eluent are 6-8; the ethanol degree of the sample solution, the balance solution and the eluent is 5-40%; the sodium salt is selected from one of sodium sulfate, sodium phosphate and sodium chloride. The purification method provided by the invention can realize the efficient removal of the impurity 4 and unknown impurities in the daptomycin, and the obtained product meets the market demand.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a method for purifying daptomycin.
Background
With the development of the market, the competition for daptomycin products is increasing. The quality of the daptomycin product is also higher and higher, and the impurity removal rate is the key point for measuring the quality of the daptomycin product, however, the prior art can only produce products with the impurity 4 of 0.37 percent and the unknown impurity of 0.3 percent, and the quality requirements of the daptomycin product that the impurity 4 is less than 0.25 percent and the unknown impurity is less than 0.1 percent can not be met.
Disclosure of Invention
In order to overcome the defects in the prior art, the technical problems to be solved by the invention are as follows: a method for purifying daptomycin can improve the quality of daptomycin, in particular to a method for purifying daptomycin which satisfies the impurity 4 < 0.25% and the unknown impurity < 0.1%.
In order to solve the technical problems, the invention provides a purification method of daptomycin, which comprises the steps of loading a sample solution in a C18 resin chromatographic column balanced by a balance liquid, and eluting the C18 resin chromatographic column by an eluent;
wherein the sample solution, the balance solution and the eluent contain sodium salt with the concentration of 0.1-1.0 (W/V)%, the pH values of the sample solution, the balance solution and the eluent are 6-8, and the ethanol degree of the balance solution and the eluent is 5% -40%;
the sodium salt is selected from one of sodium sulfate, sodium phosphate and sodium chloride.
The invention has the beneficial effects that: the method for purifying the daptomycin can realize the efficient purification of the daptomycin, and the purified product meets the requirement that the impurity is less than 0.25 percent and the unknown impurity is less than 0.1 percent.
Detailed Description
In order to describe the technical contents, the achieved objects and effects of the present invention in detail, the following description will be made with reference to the embodiments.
A method of purification of daptomycin comprising the steps of loading a sample solution in a C18 resin chromatography column equilibrated with an equilibration solution, and eluting the C18 resin chromatography column with an elution solution; wherein the sample solution, the balance solution and the eluent contain sodium salt with the concentration of 0.1-1.0 (W/V)%, and the pH values of the sample solution, the balance solution and the eluent are 6-8; the ethanol degree of the balance liquid and the eluent is 5% -20%; the sodium salt includes, but is not limited to, one of sodium salts such as sodium sulfate, sodium phosphate, sodium chloride, and the like.
The method comprises the steps of adding a certain amount of salt into a sample solution, a balance solution and an eluent to prepare a salt-containing solution, so that the whole chromatographic system is changed, the separation degree of daptomycin and impurities is improved, the daptomycin is eluted from a C18 resin chromatographic column through the eluent, and the impurities can be effectively removed in the analysis process, so that the requirements of 4 < 0.25% of the impurities and less than 0.1% of unknown impurities are met. Compared with the prior art, the production cost of the whole chromatography process is reduced, and meanwhile, the product meets the market requirements.
In one embodiment, the ethanol degree of the sample solution, the balance solution and the eluent is 5-40%. That is, in this embodiment, the equilibration and elution solutions are both ethanol solutions. The ethanol degree is defined generally herein, that is, the volume concentration of ethanol.
In one embodiment, the C18 resin column effluent after equilibration has an ethanol content of 5 to 40%.
In one embodiment, the purification method further comprises the step of removing alcohol from the daptomycin powder solution and concentrating to obtain the sample solution. Wherein the content of daptomycin in the concentrated sample solution is more than or equal to 10g/L. In this embodiment, the daptomycin powder solution is prepared as follows.
In one embodiment, the alcohol removal is performed to remove ethanol from the daptomycin powder solution to an alcohol meter measurement of 0.
In an alternative embodiment, the alcohol expelling and the concentration are performed with nanofiltration membranes. Preferably, the nanofiltration membrane has a molecular weight cut-off of 300Da.
Specifically, the method for purifying daptomycin comprises the following steps:
s1, removing alcohol from a daptomycin powder solution by using a nanofiltration membrane until the measured value of an alcohol meter is 0, and obtaining alcohol removing liquid;
s2, concentrating the alcohol expelling liquid by a nanofiltration membrane until the content of daptomycin is more than or equal to 10g/L, so as to obtain nanofiltration concentrated liquid;
s3, adding a certain amount of salt solution or solid salt into the nanofiltration concentrated solution, controlling the salt concentration to be 0.1-1.0 (W/V), regulating the pH value to be 6-8 by using sodium hydroxide or hydrochloric acid, adding ethanol, and preparing ethanol degree to be 5-40%, thus obtaining upper column liquid;
s4, adding a certain amount of salt solution or solid salt into the balance liquid, controlling the salt concentration to be 0.1-1.0 (W/V)%, regulating the pH value to be 6-8 by using sodium hydroxide or hydrochloric acid, and balancing the balance liquid by using a C18 resin chromatographic column until the ethanol degree of the effluent of the C18 resin chromatographic column is 5-40%;
s5, preparing an eluent, adding a certain amount of salt solution or solid salt into the eluent, controlling the salt concentration to be 0.1-1.0 (W/V), and regulating the pH value to be 6-8 by using sodium hydroxide or hydrochloric acid, wherein the ethanol degree is 5-40%;
s6, eluting the upper column liquid on a C18 resin chromatographic column, synchronously detecting the content of daptomycin and the content of related impurities in filtrate by UPLC, and collecting an effective collection liquid of the daptomycin intermediate with the purity reaching the standard (impurity 4 is less than 0.25 percent and unknown impurity is less than 0.1 percent);
and S7, removing alcohol from the effective collection liquid of the daptomycin intermediate by using a nanofiltration membrane, concentrating until the unit of feed liquid is 100g/L, and then freeze-drying to obtain a daptomycin product.
More specifically, the alcohol removal and concentration are as follows: concentrating the feed liquid (daptomycin coarse powder desalted liquid, daptomycin intermediate effective collecting liquid) to half of the volume by using a nanofiltration membrane, adding water, washing while nanofiltration, keeping the water adding amount consistent with nanofiltration flow, and sampling and detecting the gas phase peak area of the filtrate to be less than 10 to obtain the alcohol expelling liquid. Wherein the nanofiltration flow rate is 2.0-2.5L/h, preferably 2.3L/h (alcohol removal and concentration are carried out according to the nanofiltration flow rate parameter in the following examples and comparative examples).
Of course, in order to obtain the final daptomycin powder, the daptomycin product in S7 is preferably lyophilized. Preferably, the lyophilization conditions are: the first stage is pre-frozen for 2h at 70 ℃, the second stage is freeze-dried for 10h at 0 ℃, and the third stage is freeze-dried for 60h at 10 ℃.
Example 1
A method for purifying daptomycin, comprising the following steps:
s1, dissolving 15g of daptomycin powder solution to 1L by deionized water, conducting conductivity removal by a small membrane machine (provided with a nanofiltration membrane with a molecular weight cut-off of 300 Da) after the daptomycin powder solution is completely dissolved, concentrating to 500mL, adding water while concentrating, keeping the water adding speed consistent with the dialysis flow rate, and compressing the volume to 1.2L when the alcoholic strength of filtrate at the filtering end is 0, namely, the daptomycin content is 12.5g/L, so as to obtain nanofiltration concentrate;
s2, taking 240mL of the nanofiltration concentrated solution (namely 3g of daptomycin), adding 1.92g of sodium sulfate until the concentration is 0.8%, adjusting the pH to 7.26 by using 2mol/L NaOH or 2mol/L HCl, and adding a certain amount of ethanol to prepare an upper column liquid containing 14% ethanol degree;
preparing 1L of balance liquid, wherein the ethanol degree of the balance liquid is 14%, and the concentration of sodium sulfate is 0.8% (namely, 8g of sodium sulfate is dissolved);
preparing 3L eluent, wherein the ethanol degree of the eluent is 14%, and the concentration of sodium sulfate is 0.8% (namely dissolving 24g of sodium sulfate);
s3, loading the upper column liquid on 200mL of C18 resin chromatographic column, eluting with the eluent, and introducing filtrate into UPLC for analysis, and collecting daptomycin intermediate effective collection liquid with impurity content of 4 < 0.25% and unknown impurity content of < 0.1%;
s4, concentrating 0.9L of the daptomycin intermediate effective collecting liquid by using a nanofiltration membrane (with the molecular weight cut-off of 300 Da) until the gas phase peak area is less than 10, and freeze-drying after nanofiltration is finished to obtain a daptomycin product.
Comparative example 1
A purification method of daptomycin, which is different from example 1 in that: the ethanol degree in the upper column liquid, the balance liquid and the eluent is inconsistent.
A method for purifying daptomycin, comprising the following steps:
s1, dissolving 15g of daptomycin powder solution to 1L by deionized water, conducting conductivity removal by a small membrane machine (provided with a nanofiltration membrane with a molecular weight cut-off of 300 Da) after the daptomycin powder solution is completely dissolved, concentrating to 500mL, adding water while concentrating, keeping the water adding speed consistent with the dialysis flow rate, and compressing the volume to 1.2L when the alcoholic strength of filtrate at the filtering end is 0, namely, the daptomycin content is 12.5g/L, so as to obtain nanofiltration concentrate;
s2, taking 240mL of the nanofiltration concentrated solution (namely 3g of daptomycin), adding 1.92g of sodium sulfate until the concentration is 0.8%, regulating the pH to 7.26 by using 2mol/L NaOH or 2mol/L HCl, and adding a certain amount of ethanol to prepare an upper column liquid containing 25% ethanol degree;
preparing 1L of balance liquid, wherein the ethanol degree of the balance liquid is 25%, and the concentration of sodium sulfate is 0.8% (namely, 8g of sodium sulfate is dissolved);
preparing 3L eluent, wherein the ethanol degree of the eluent is 25%, and the concentration of sodium sulfate is 0.8% (namely dissolving 24g of sodium sulfate);
s3, loading the upper column liquid on 200mL of C18 resin chromatographic column, eluting with the eluent, and introducing filtrate into UPLC for analysis, and collecting daptomycin intermediate effective collection liquid with impurity content of 4 < 0.25% and unknown impurity content of < 0.1%;
s4, concentrating 0.9L of the daptomycin intermediate effective collecting liquid by using a nanofiltration membrane (with the molecular weight cut-off of 300 Da) until the gas phase peak area is less than 10, and freeze-drying after nanofiltration is finished to obtain a daptomycin product.
Test case
The composition analysis of the daptomycin crude powder desalted solution and daptomycin product of example 1 and comparative example 1 was performed, and the results are shown in Table 1.
TABLE 1
Impurity 4 is a known impurity (relative retention time of 0.89) that i am identified and named, and the unknown impurity is all unknown impurities except identified.
From table 1, it can be seen that the purification method of daptomycin provided by the invention can realize efficient removal of unknown impurities and impurity 4 and improve the overall yield.
In conclusion, the daptomycin purification method provided by the invention can realize the efficient purification of daptomycin, and the purified product meets the requirements of impurity 4 < 0.25% and unknown impurity < 0.1%.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent modifications made by the teachings of the present invention, or direct or indirect application in the relevant art, are intended to be included within the scope of the present invention.
Claims (9)
1. A method for purifying daptomycin, comprising the steps of loading a sample solution into a C18 resin chromatography column equilibrated with an equilibration solution, and eluting with an eluent after washing the C18 resin chromatography column with a pre-wash;
wherein the sample solution, the balance solution and the eluent contain sodium salt with the concentration of 0.1-1.0 (W/V)%, and the pH values of the sample solution, the balance solution and the eluent are 6-8;
the sodium salt is selected from one of sodium sulfate, sodium phosphate and sodium chloride.
2. The method according to claim 1, wherein the ethanol content of the sample solution and the equilibrium solution is 5 to 40%.
3. The purification method according to claim 1, wherein the ethanol content of the eluent is 5 to 40%.
4. The purification method according to claim 1, wherein the ethanol content of the effluent of the C18 resin chromatography column after equilibration is 5 to 40%.
5. The method according to claim 1, wherein the daptomycin content in the sample solution is not less than 10g/L.
6. The method of claim 1, further comprising the step of removing alcohol from the daptomycin meal desalination solution and concentrating the solution to obtain the sample solution.
7. The purification method according to claim 6, wherein the alcohol removal is performed by removing ethanol from the daptomycin meal desalination solution and detecting a gas phase peak area of < 10.
8. The purification method according to claim 6, wherein said driving alcohol and said concentrating are performed with nanofiltration membranes.
9. The purification method according to claim 8, wherein the nanofiltration membrane has a molecular weight cut-off of 300Da.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310661443.4A CN116903703A (en) | 2023-06-06 | 2023-06-06 | Purification method of daptomycin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310661443.4A CN116903703A (en) | 2023-06-06 | 2023-06-06 | Purification method of daptomycin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116903703A true CN116903703A (en) | 2023-10-20 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310661443.4A Pending CN116903703A (en) | 2023-06-06 | 2023-06-06 | Purification method of daptomycin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116903703A (en) |
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2023
- 2023-06-06 CN CN202310661443.4A patent/CN116903703A/en active Pending
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