CN113717253B - Purification method of daptomycin - Google Patents
Purification method of daptomycin Download PDFInfo
- Publication number
- CN113717253B CN113717253B CN202111078696.6A CN202111078696A CN113717253B CN 113717253 B CN113717253 B CN 113717253B CN 202111078696 A CN202111078696 A CN 202111078696A CN 113717253 B CN113717253 B CN 113717253B
- Authority
- CN
- China
- Prior art keywords
- daptomycin
- solution
- liquid
- ethanol
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the technical field of antibiotic production, and particularly relates to a daptomycin purification method, which comprises the step of pre-reacting daptomycin with the concentration of 3.5-4 maU/mL with a salt solution to enhance the polarity of daptomycin; and a step of gradient elution of daptomycin in a reverse-phase chromatography column using a low-concentration ethanol solution. The purification method of daptomycin provided by the invention can effectively improve the quality of daptomycin products and reduce the usage amount of ethanol in the purification process.
Description
Technical Field
The invention belongs to the technical field of antibiotic production, and particularly relates to a daptomycin purification method.
Background
Daptomycin is a cyclic lipopeptide antibiotic produced by fermentation of streptomyces (S.Reseosporus), is a pharmaceutical intermediate, and can be used for treating concurrent skin and skin structure infections caused by some gram-positive sensitive strains. The specific pharmacological action is that the antibacterial and bactericidal effects can be realized by destroying bacterial cell membranes, disturbing cell membrane to transport amino acid, changing cell membrane potential and the like.
In the prior art, the traditional daptomycin purification method cannot meet the requirement of the market on the quality of daptomycin. Specifically, the conventional daptomycin purification method adopts ammonium acetate as a buffer salt, so that the use amount of ethanol as a solvent in the purification process is large, and the removal rate of known or unknown impurities is relatively low, so that the purification cost of daptomycin is increased, and the purity of daptomycin finished powder is reduced.
Disclosure of Invention
In order to overcome the defects of the prior art, the technical problems to be solved by the invention are as follows: solves the problem of large ethanol consumption in the traditional daptomycin purification method.
In order to solve the technical problems, the invention provides a daptomycin purification method, which comprises the step of pre-reacting daptomycin with the concentration of 3.5-4 maU/mL with a salt solution to enhance the polarity of daptomycin;
and a step of gradient elution of daptomycin in a reverse chromatography column using a low concentration ethanol solution;
the low-concentration ethanol solution comprises 14vol% ethanol solution and 20vol% ethanol solution which are sequentially applied.
The invention has the beneficial effects that: the polarity of daptomycin is increased by utilizing the pre-reaction of a salt solution and daptomycin, and the daptomycin is intensively adsorbed on the same chromatographic layer in the sample loading process due to the fact that the concentration of the daptomycin is 3.5-4 maU/mL, so that the daptomycin can be eluted by utilizing a low-concentration ethanol solution in the elution process, the separation rate and the yield of daptomycin and impurities are improved, and the using amount of ethanol is effectively reduced.
Detailed Description
In order to explain the technical content, the objects and the effects of the present invention in detail, the following description will be given with reference to the embodiments.
It should be noted that, in this document, the reagents used, such as sodium acetate, ammonium acetate, etc., are analytically pure, and ethanol is medical ethanol.
It is also noted that the UniSil 10-120C18 chromatography resin is commercially available from Nawei technologies, suzhou, inc.
A method for purifying daptomycin comprises the steps of pre-reacting daptomycin with the concentration of 3.5-4 maU/mL with a salt solution to enhance the polarity of daptomycin;
and eluting daptomycin in the reverse chromatography column with a low-concentration ethanol solution in a gradient manner;
the low-concentration ethanol solution comprises 14vol% ethanol solution and 20vol% ethanol solution which are sequentially applied.
Specifically, daptomycin and a salt solution are pre-reacted to increase the polarity of daptomycin, namely, on the premise of ensuring the biological activity of daptomycin, a group which is easy to hydrolyze or decompose and contains N element or O element, such as acetic acid group and the like, is introduced to the side chain of daptomycin to increase the polarity of daptomycin, so that the adsorption capacity of a reverse phase chromatographic column on daptomycin is reduced, and the purposes of quickly separating daptomycin from impurities and improving the purity and yield of daptomycin are achieved.
The loading concentration of the daptomycin is set to be 3.5-4 maU/mL, namely the daptomycin is easy to be adsorbed on the same chromatographic layer in the reverse chromatographic column under the condition of high loading concentration, so that the daptomycin can be eluted from the reverse chromatographic column in a gradient elution mode through low-concentration ethanol solution, and the using amount of ethanol in the purification process is reduced.
Preferably, the purification method of daptomycin comprises the following steps:
s1, dissolving daptomycin coarse powder by using deionized water to obtain a daptomycin solution;
s2, performing nanofiltration on the daptomycin solution until the conductivity of the water outlet end of the nanofiltration membrane is less than 100 to obtain a conductive driving liquid;
s3, carrying out nanofiltration concentration on the electric conduction driving liquid until the concentration of daptomycin in the electric conduction driving liquid is 3.5-4 maU/mL to obtain a concentrated solution;
s4, adding a sodium acetate solution and an ethanol solution into the concentrated solution, and adjusting the pH = 6.5-7.0 to obtain a column feeding solution;
s5, putting the upper column liquid into a chromatographic column filled with C18 chromatographic resin, pre-washing the chromatographic column by using an ethanol solution, carrying out gradient elution by using a low-concentration ethanol solution, and collecting by taking the known impurities less than 0.3% and the unknown impurities less than 0.2% as standards to obtain a collecting liquid;
s6, carrying out nanofiltration on the collected liquid to remove alcohol, concentrating the collected liquid until the concentration of the daptomycin is 5-6 maU/mL, and obtaining a daptomycin product liquid.
Specifically, in S2, S3 and S5, the nanofiltration membrane with the molecular weight cutoff of 300 is utilized, alcohol is removed while water is added, the water addition amount is kept consistent with the nanofiltration flow, and the nanofiltration operation is finished when the conductivity of the water outlet end of the nanofiltration membrane is less than 100, namely the ethanol degree in the solution is 0; except that both S2 and S5 included concentrating the solution to half volume and then performing alcohol removal with addition of water.
In S5, the known impurities are less than 0.3% and the unknown impurities are less than 0.2%, specifically, samples are separately collected at each resin volume and subjected to UPLC detection to detect the impurities in the collected liquid, and the collected liquid is collected at the known impurities of less than 0.3% and the unknown impurities of less than 0.2%. Wherein the judgment standards of the known impurities and the unknown impurities are as follows: the known impurities are defined by a reference preparation, the reference preparation is preferably daptomycin Kexin for injection, and particularly the retention time of the unknown impurities is about 19.093min; unknown impurities are defined by the antibiotic guidelines of EMA, and are specifically guidelines on setting specifications for related antibiotics in antibiotics.
The sodium acetate is a salt solution for increasing the polarity of daptomycin, acetate ions in the sodium acetate can be combined with hydroxyl, acylamino or amino on a side chain of daptomycin through hydrogen bonds to increase the polarity of daptomycin, and the acetate ions have strong hydrolyzation and can be removed in the purification process, so that acetate impurities are prevented from being mixed in a daptomycin finished product.
Preferably, in the upper column liquid, the content of sodium acetate is 2vol%.
Preferably, the ethanol content in the upper column liquid is 8vol%.
Further, the gradient elution is to elute 5BV with 14vol% ethanol solution at a flow rate of 3BV/h, and then elute 10BV with 20vol% ethanol solution.
Example 1
S1, dissolving 20g of daptomycin coarse powder into 1L of deionized water, after the daptomycin coarse powder is dissolved, concentrating the solution by using a small membrane machine (model JMD1812-1, dalian east membrane engineering equipment Co., ltd., 220V, 50HZ), after the volume of the solution is one half of the original volume, adding water into the solution to drive alcohol, keeping the water adding speed (5L/h) consistent with the dialysis solution speed, and when the conductivity of the water outlet end of a nanofiltration membrane (molecular weight cut-off 300) is less than 100, compressing the volume of the solution to 0.55L, namely the concentrated solution with the daptomycin content of 3.6 maU/mL;
s2, taking 61mL of concentrated solution, namely containing 2.2g of daptomycin, adding 2vol% of sodium acetate, namely 0.04g, adjusting the pH to be =6.71, and adding a certain amount of ethanol to prepare upper column solution containing 8vol% of ethanol;
s3, putting the upper column liquid on a column 200mLUNISil 10-120C18 chromatographic resin, pre-washing with 8vol% ethanol solution, eluting with 14% ethanol solution for 5BV at the flow rate of 3BV/h, eluting with 20% ethanol solution for 10BV, collecting the eluted daptomycin, separately collecting and sampling by volume per time of the resin, detecting the purity by UPLC, and collecting the eluent when the known impurities in the eluent are less than 0.3% and the unknown impurities are less than 0.2% to obtain a collected liquid;
and S4, concentrating the collected liquid by 1.1L by using a nanofiltration membrane (with the molecular weight cut-off of 300) until the ethanol degree is zero (the step of concentrating and removing the alcohol is shown as S1), and concentrating the collected liquid to 40mL to obtain the daptomycin product.
The detection proves that the purity of the product in the daptomycin is 96.72 percent, and the yield is 74.18 percent.
Example 2
S1, dissolving 20g of daptomycin coarse powder into 1L of deionized water, after the daptomycin coarse powder is dissolved, concentrating the solution by using a small membrane machine (model number JMD1812-1, east Longi Membrane engineering Equipment Co., ltd., 220V, 50HZ), after the volume of the solution is one half of the original volume, adding water into the solution to expel alcohol, keeping the water adding speed (5L/h) consistent with the dialysate speed, and when the conductivity of the water outlet end of a nanofiltration membrane (molecular weight cut-off 300) is less than 100, compressing the volume of the solution to 0.61L, namely the concentrated solution with the daptomycin content of 4 maU/mL;
s2, taking 61mL of concentrated solution, namely containing 2.2g of daptomycin, adding 2vol% of sodium acetate, namely 0.04g, adjusting the pH to be =7 by using hydrochloric acid (2 mol/L), and adding a certain amount of ethanol to prepare upper column solution containing 8vol% of ethanol;
s3, putting the upper column liquid on a column 200mLUNISil 10-120C18 chromatographic resin, pre-washing with 8vol% ethanol solution, eluting with 14% ethanol solution for 5BV at the flow rate of 3BV/h, eluting with 20% ethanol solution for 10BV, collecting the eluted daptomycin, separately collecting and sampling by volume per time of the resin, detecting the purity by UPLC, and collecting the eluent when the known impurities in the eluent are less than 0.3% and the unknown impurities are less than 0.2% to obtain a collected liquid;
and S4, concentrating the collected liquid by 1.1L by using a nanofiltration membrane (with the molecular weight cut-off of 300) until the ethanol degree is zero (the step of concentrating and removing the alcohol is shown as S1), and concentrating the collected liquid to 40mL to obtain the daptomycin product.
Through detection, the purity of the product in daptomycin is 96.21%. The yield was 77.54%.
Detection example 1
The removal efficiency and yield of the known impurities and the unknown impurities are compared by the pre-improvement process and the post-improvement process, and the ethanol dosage is compared.
The experimental method comprises the following steps:
the improved process comprises the following steps: as in example 1.
The process before improvement comprises the following steps: the difference from example 1 is that sodium acetate is replaced by ammonium acetate. In particular, the pH value of the upper column liquid is adjusted to 5.5-6. 0, prewashing UniSil 10-120C18 chromatography resin 2BV with 2vol% ammonium acetate-15 vol% ethanol, and then eluting with 2vol% ammonium acetate-40 vol% ethanol.
Taking daptomycin coarse powder for chromatographic analysis to obtain impurity peak areas of known impurities (less than 0.3 percent) and unknown impurities (less than 0.2 percent); respectively carrying out chromatographic analysis on daptomycin finished products obtained by the post-improvement process and the pre-improvement process to obtain impurity peak areas of known impurities (< 0.3%) and unknown impurities (< 0.2%), and obtaining the removal rate and the respective yield of the known impurities (< 0.3%) and the unknown impurities (< 0.2%) by the two processes through calculation, wherein the results are shown in Table 1; and the ethanol usage amount of the two processes is counted (1L column chromatography is taken as a standard), and the statistical result is shown in Table 2.
TABLE 1
TABLE 2
As can be seen from Table 1, the removal rates of the improved process for known impurities (< 0.3%) and unknown impurities (< 0.2%) are 48.48% and 40.6%, respectively, and the yield is 75.86%, while the removal rates of the improved process for known impurities (< 0.3%) and unknown impurities (< 0.2%) are only 24.12% and 21.4%, and the yield is only 65.32%, which indicates that the improved process, i.e., the daptomycin purification method provided by the present application, can effectively improve the removal rates for known impurities (< 0.3%) and unknown impurities (< 0.2%) and improve the yield of daptomycin products compared with the process before the improvement.
As can be seen from Table 2, the total ethanol usage of the pre-modification process is 7968mL, which is much higher than 2911mL of the post-modification process, i.e., indicating that the purification method of daptomycin provided by the present application has a significantly reduced ethanol usage compared to the pre-modification process.
In summary, the daptomycin purification method provided by the invention utilizes the salt solution to carry out pre-reaction with daptomycin so as to increase the polarity of daptomycin, and because the concentration of daptomycin is 3.5-4 maU/mL, daptomycin is intensively adsorbed on the same chromatographic layer in the sample loading process, daptomycin can be eluted by utilizing the ethanol solution with low concentration in the elution process, so that the separation rate and yield of daptomycin and impurities are improved, and the usage amount of ethanol is effectively reduced.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent modifications made by the present invention in the specification or directly or indirectly applied to the related technical field are included in the scope of the present invention.
Claims (1)
1. A method for purifying daptomycin, comprising the following steps:
s1, dissolving daptomycin coarse powder by using deionized water to obtain a daptomycin solution;
s2, performing nanofiltration on the daptomycin solution until the conductivity of the water outlet end of the nanofiltration membrane is less than 100 to obtain a conductive driving liquid;
s3, carrying out nanofiltration concentration on the electric conduction driving liquid until the concentration of daptomycin in the electric conduction driving liquid is 3.5-4 maU/mL to obtain a concentrated solution;
s4, adding a sodium acetate solution and an ethanol solution into the concentrated solution, and adjusting the pH = 6.5-7.0 to obtain a column feeding solution;
s5, putting the upper column liquid into UniSil 10-120C18 chromatographic resin, pre-washing a chromatographic column by using an ethanol solution, carrying out gradient elution by using a low-concentration ethanol solution, and collecting by using the standard that the known impurities are less than 0.3 percent and the unknown impurities are less than 0.2 percent to obtain a collecting liquid;
s6, carrying out nanofiltration on the collected liquid to remove alcohol, concentrating the collected liquid until the concentration of daptomycin is 5-6 maU/mL, and obtaining a daptomycin product liquid;
wherein, in the upper column liquid, the content of the sodium acetate is 2vol%;
in the upper column liquid, the content of the ethanol is 8vol%;
the gradient elution is to elute 5BV by using 14vol% ethanol solution at the flow rate of 3BV/h, and then elute 10BV by using 20vol% ethanol solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111078696.6A CN113717253B (en) | 2021-09-15 | 2021-09-15 | Purification method of daptomycin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111078696.6A CN113717253B (en) | 2021-09-15 | 2021-09-15 | Purification method of daptomycin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113717253A CN113717253A (en) | 2021-11-30 |
| CN113717253B true CN113717253B (en) | 2023-03-21 |
Family
ID=78683790
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111078696.6A Active CN113717253B (en) | 2021-09-15 | 2021-09-15 | Purification method of daptomycin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113717253B (en) |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060014674A1 (en) * | 2000-12-18 | 2006-01-19 | Dennis Keith | Methods for preparing purified lipopeptides |
| CN102492024A (en) * | 2011-12-09 | 2012-06-13 | 厦门大学 | Method for extracting daptomycin from fermentation broth |
| CN105481950B (en) * | 2016-01-28 | 2019-01-04 | 丽珠集团福州福兴医药有限公司 | A kind of Daptomycin extracting method |
| CN112979756B (en) * | 2019-12-13 | 2023-01-03 | 浙江昌海制药有限公司 | Purification method of daptomycin |
| CN113004373A (en) * | 2019-12-19 | 2021-06-22 | 鲁南制药集团股份有限公司 | Daptomycin purification method |
-
2021
- 2021-09-15 CN CN202111078696.6A patent/CN113717253B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN113717253A (en) | 2021-11-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109721487B (en) | Process for efficiently purifying shikimic acid by using continuous ion exchange technology | |
| CN109593034B (en) | Method for preparing shikimic acid from ginkgo leaf extraction waste liquid | |
| CN102718839A (en) | Method for separating and purifying daptomycin | |
| CN111057140B (en) | Selepress purification method | |
| CN103408639A (en) | Preparation method of vancomycin with high purity | |
| CN101029077A (en) | Method for purifying gene-recombinant insulin precursor | |
| CN104693250B (en) | Method for purifying acarbose from acarbose-containing solution | |
| CN102698602B (en) | The method reclaiming threonine from threonine crystallization mother liquid | |
| CN113717253B (en) | Purification method of daptomycin | |
| CN111635402B (en) | Separation and purification method of pyrroloquinoline quinone | |
| CN101089017A (en) | Process of separating and purifying melittin | |
| CN110256597B (en) | Method for reducing heavy metal residues in ganoderma lucidum polysaccharide by membrane method | |
| CN116425810B (en) | Purification method of 3-fucosyllactose in mixed solution | |
| CN105439847A (en) | Separation purification method for natural abscisic acid | |
| CN111887341A (en) | Method for extracting, separating and purifying miraculin | |
| CN103450295A (en) | Method for purifying gentamicin sulphate | |
| CN111393537A (en) | Method for extracting bioactive substances from sea cucumber | |
| CN107778357B (en) | Extraction and purification method of pneumocandin B0 | |
| CN113563424B (en) | Daptomycin purification method | |
| CN112409426A (en) | Preparation method of sisomicin sulfate | |
| CN110862427B (en) | Purification method of gentamicin C1a | |
| CN109705174B (en) | Method for extracting tobramycin | |
| CN109400743B (en) | Method for extracting sulfated galactan from sea grapes | |
| CN106608908B (en) | Method for extracting glucose tolerance factor from chromium-containing yeast | |
| CN103073622B (en) | A kind of high purity lung sac Kangding B 0preparation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |