CN112430256A - Method for separating and purifying anidulafungin mother nucleus - Google Patents
Method for separating and purifying anidulafungin mother nucleus Download PDFInfo
- Publication number
- CN112430256A CN112430256A CN202011324533.7A CN202011324533A CN112430256A CN 112430256 A CN112430256 A CN 112430256A CN 202011324533 A CN202011324533 A CN 202011324533A CN 112430256 A CN112430256 A CN 112430256A
- Authority
- CN
- China
- Prior art keywords
- anidulafungin
- mother nucleus
- purifying
- separating
- column
- 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.)
- Pending
Links
- 108010064760 Anidulafungin Proteins 0.000 title claims abstract description 85
- JHVAMHSQVVQIOT-MFAJLEFUSA-N anidulafungin Chemical compound C1=CC(OCCCCC)=CC=C1C1=CC=C(C=2C=CC(=CC=2)C(=O)N[C@@H]2C(N[C@H](C(=O)N3C[C@H](O)C[C@H]3C(=O)N[C@H](C(=O)N[C@H](C(=O)N3C[C@H](C)[C@H](O)[C@H]3C(=O)N[C@H](O)[C@H](O)C2)[C@@H](C)O)[C@H](O)[C@@H](O)C=2C=CC(O)=CC=2)[C@@H](C)O)=O)C=C1 JHVAMHSQVVQIOT-MFAJLEFUSA-N 0.000 title claims abstract description 85
- 229960003348 anidulafungin Drugs 0.000 title claims abstract description 85
- 238000000034 method Methods 0.000 title claims abstract description 46
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 29
- 238000004440 column chromatography Methods 0.000 claims abstract description 21
- 238000000746 purification Methods 0.000 claims abstract description 19
- 239000000706 filtrate Substances 0.000 claims abstract description 18
- 238000001914 filtration Methods 0.000 claims abstract description 11
- 238000002013 hydrophilic interaction chromatography Methods 0.000 claims abstract description 11
- 239000003960 organic solvent Substances 0.000 claims abstract description 10
- 238000000926 separation method Methods 0.000 claims abstract description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 84
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 239000000945 filler Substances 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 7
- 238000010829 isocratic elution Methods 0.000 claims description 7
- 239000012528 membrane Substances 0.000 claims description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- 239000000741 silica gel Substances 0.000 claims description 6
- 229910002027 silica gel Inorganic materials 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 2
- 238000011067 equilibration Methods 0.000 claims 3
- 238000004090 dissolution Methods 0.000 claims 1
- 230000002829 reductive effect Effects 0.000 abstract description 5
- 238000011097 chromatography purification Methods 0.000 abstract description 4
- 230000005526 G1 to G0 transition Effects 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 22
- 238000004128 high performance liquid chromatography Methods 0.000 description 10
- 239000004005 microsphere Substances 0.000 description 9
- 239000003480 eluent Substances 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 239000007864 aqueous solution Substances 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 238000011068 loading method Methods 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 108010028921 Lipopeptides Proteins 0.000 description 4
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 4
- 125000004122 cyclic group Chemical group 0.000 description 4
- 230000001172 regenerating effect Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 238000012856 packing Methods 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 108010021062 Micafungin Proteins 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- PIEUQSKUWLMALL-YABMTYFHSA-N micafungin Chemical compound C1=CC(OCCCCC)=CC=C1C1=CC(C=2C=CC(=CC=2)C(=O)N[C@@H]2C(N[C@H](C(=O)N3C[C@H](O)C[C@H]3C(=O)N[C@H](C(=O)N[C@H](C(=O)N3C[C@H](C)[C@H](O)[C@H]3C(=O)N[C@H](O)[C@H](O)C2)[C@H](O)CC(N)=O)[C@H](O)[C@@H](O)C=2C=C(OS(O)(=O)=O)C(O)=CC=2)[C@@H](C)O)=O)=NO1 PIEUQSKUWLMALL-YABMTYFHSA-N 0.000 description 2
- 229960002159 micafungin Drugs 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 238000001878 scanning electron micrograph Methods 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- 108010070892 1,3-beta-glucan synthase Proteins 0.000 description 1
- 108010049047 Echinocandins Proteins 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 230000006959 non-competitive inhibition Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
Images
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
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
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 relates to a separation and purification method of anidulafungin mother nucleus, which comprises the following steps: (1) dissolving anidulafungin mother nucleus, and filtering to obtain anidulafungin mother nucleus filtrate; (2) adopting water and organic solvent as mobile phase, subjecting the anidulafungin mother nucleus filtrate to column chromatography with homogeneous HILIC chromatographic column, and collecting eluate of target peak to obtain purified anidulafungin mother nucleus. The method for separating and purifying the anidulafungin mother nucleus can meet the requirements that the purity of the anidulafungin mother nucleus is more than 99 percent and the yield is more than 80 percent only by one-step chromatographic purification, and has high purity, high yield and stability. The used stationary phase can be recycled, the used mobile phase can be recycled, and the cost is also greatly reduced.
Description
Technical Field
The invention belongs to the technical field of drug purification, and particularly relates to a separation and purification method of anidulafungin mother nucleus, in particular to a separation and purification method of anidulafungin mother nucleus with high purification, high yield and good stability.
Background
Anidulafungin (CAS number 79411-15-7, molecular formula C34H51N7O15), lipopeptide, a naturally occurring cyclic hexapeptide with acyl side chains. It belongs to a class of antifungal drugs called echinocandins, and is a major component of inhibition of fungal cell wall glucan synthesis by a key enzyme, β - (1,3) -D-glucan synthase, which is a non-competitive inhibition. The molecular formula is shown as follows:
at present, most reports of anidulafungin parent nucleus in the prior art are synthetic method patents, and separation and purification methods are only reported, for example, CN105154424B discloses a preparation method of immobilized cyclic lipopeptide deacylase, wherein a cyclic lipopeptide deacylase solution is directly mixed with a porous hydrophilic enzyme carrier without purification, and one or more organic solutions and inorganic salts are added to obtain the immobilized cyclic lipopeptide deacylase; simultaneously discloses that the method can be used for industrial production of micafungin mother nucleus and anidulafungin mother nucleus. The preparation method disclosed by the invention has a simple process, not only saves enzyme purification equipment, but also obviously improves the recovery rate of enzyme activity in the immobilization process, and the immobilized enzyme can be repeatedly used. Compared with a fermentation conversion method, the method for converting the micafungin and anidulafungin intermediates by the enzyme method has the advantages of high reaction substrate concentration, less product impurities and high conversion efficiency.
Disclosure of Invention
In view of the disadvantages of the prior art, the present invention aims to provide a method for separating and purifying anidulafungin mother nucleus, and particularly to provide a method for separating and purifying anidulafungin mother nucleus with high purification, high yield and good stability.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a separation and purification method of anidulafungin mother nucleus, which comprises the following steps:
(1) dissolving anidulafungin mother nucleus, and filtering to obtain anidulafungin mother nucleus filtrate;
(2) adopting water and organic solvent as mobile phase, subjecting the anidulafungin mother nucleus filtrate to column chromatography with homogeneous HILIC chromatographic column, and collecting eluate of target peak to obtain purified anidulafungin mother nucleus.
The method for separating and purifying the anidulafungin mother nucleus can meet the requirements that the purity of the anidulafungin mother nucleus is more than 99 percent and the yield is more than 80 percent only by one-step chromatographic purification, and has high purity, high yield and stability. The used stationary phase can be recycled, the used mobile phase can be recycled, and the cost is also greatly reduced.
The eluent for collecting the target peak is the anidulafungin mother nucleus solution for collecting the target peak in a sectional way, and the component solutions meeting the requirements are gathered.
Preferably, the organic solvent comprises acetonitrile.
Compared with other organic solvents such as ethanol, methanol and the like, the acetonitrile is specially selected as the mobile phase in the invention because other mobile phases have poor purification effect and certain impurities cannot be removed.
Preferably, the volume ratio of the water to the organic solvent is 10:90-20:80, such as 10:90, 11:89, 12:88, 13:87, 14:86, 14.8:85.2, 14.9:85.1, 15:85, 15.1:84.9, 15.2:84.8, 16:84, 17:83, 18:82, 19:81, 20:80, and the like, and other specific values in the above numerical range can be selected, so that the detailed description is omitted. Preferably 16:84-14:86, and more preferably 15.2:84.8-14.8: 85.2.
The volume ratio of the water to the organic solvent is selected to be 10:90-20:80, the peak-out time of a target peak is greatly prolonged along with the increase of the proportion of the organic solvent, more mobile phases are consumed, but the purity of a product is slightly improved, in order to balance the relation between cost and purity requirements, the range of 16:84-14:86 is more preferable, the range of 15.2:84.8-14.8:85.2 is most preferable, the elution time can be controlled within 200min, and meanwhile, the purity of more than 99% is guaranteed.
Preferably, the dissolved solvent comprises methanol.
Preferably, the filtration is performed using a 0.45 μm filter membrane.
The filtration operation here is to remove insoluble impurities.
Preferably, the concentration of the anidulafungin mother-nucleus filtrate is 25-35mg/mL, such as 25mg/mL, 26mg/mL, 27mg/mL, 28mg/mL, 29mg/mL, 30mg/mL, 31mg/mL, 32mg/mL, 33mg/mL, 34mg/mL, 35mg/mL, and the like, and other specific points within the above numerical range can be selected, and are not repeated herein.
Preferably, the flow rate of the column chromatography is controlled to be 0.5-1.0mL/min, such as 0.5mL/min, 0.6mL/min, 0.7mL/min, 0.8mL/min, 0.9mL/min, 1.0mL/min, and the like, and other specific values within the above numerical range can be selected, which is not repeated herein.
The flow rate of the column chromatography is specifically controlled within the range of 0.5-1.0mL/min, and as the flow rate decreases, the purification time increases accordingly.
Preferably, the filler of the homogeneous HILIC chromatographic column is a silica gel filler of UniSil-Amide type.
The silica gel filler of the UniSil-Amide type has the particle size of 10 mu m and the pore diameter
The polymer microsphere has strictly controlled particle size and pore diameter structure, is monodisperse and has a pore channel structure, and has good pertinence when used as chromatographic packing. Manufactured by Suzhou Na micro-technology Limited.
Preferably, the elution mode of the column chromatography is isocratic elution.
Preferably, the homogeneous HILIC chromatography column is equilibrated prior to use, comprising: the chromatographic column is regenerated by using 45-55% (e.g. 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, etc., and other specific values within the numerical range can be selected, and are not described herein again), and then the chromatographic column is equilibrated by using 80-90% (e.g. 90%, 89%, 88%, 87%, 86%, 85.2%, 85.1%, 85%, 84.9%, 84.8%, 84%, 83%, 82%, 81%, 80%, etc., and other specific values within the numerical range can be selected, and are not described herein again).
More preferably, the homogeneous HILIC chromatography column is equilibrated prior to use, comprising: the column is regenerated with 45-55% acetonitrile in water and equilibrated with 84-86% acetonitrile in water.
Further preferably, the homogeneous HILIC chromatography column is equilibrated before use, and comprises: the column is regenerated with 45-55% acetonitrile in water and equilibrated with 84.8-85.2% acetonitrile in water.
As a preferred technical scheme of the present invention, the separation and purification method specifically comprises the following steps:
(1) dissolving anidulafungin mother nucleus with methanol, and filtering with 0.45 μm filter membrane to obtain anidulafungin mother nucleus filtrate with concentration of 25-35 mg/mL;
(2) carrying out balance treatment on a chromatographic column taking the silica gel microspheres of the UniSil-Amide type as fillers, wherein the balance treatment comprises the following steps: regenerating the chromatographic column with 45-55% acetonitrile water solution, and balancing the chromatographic column with 80-90% acetonitrile water solution;
(3) taking water and acetonitrile with the volume ratio of 10:90-20:80 as mobile phases, carrying out column chromatography on the anidulafungin mother nucleus filtrate, carrying out isocratic elution with the flow rate controlled at 0.5-1.0mL/min, and collecting eluent of a target peak to obtain purified anidulafungin mother nucleus.
Compared with the prior art, the invention has the following beneficial effects:
the method for separating and purifying the anidulafungin mother nucleus can meet the requirements that the purity of the anidulafungin mother nucleus is more than 99 percent and the yield is more than 80 percent only by one-step chromatographic purification, and has high purity, high yield and stability. The used stationary phase can be recycled, the used mobile phase can be recycled, and the cost is also greatly reduced.
Drawings
FIG. 1 is a scanning electron micrograph of a silica gel microsphere of type UniSil-Amide;
FIG. 2 is a high performance liquid chromatogram of the parent nucleus of anidulafungin before purification in example 1;
FIG. 3 is a high performance liquid chromatogram of the parent nucleus of anidulafungin purified in example 1;
FIG. 4 is a high performance liquid chromatogram of the parent nucleus of anidulafungin purified in example 4;
FIG. 5 is a high performance liquid chromatogram of the parent nucleus of anidulafungin purified in example 5.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The following examples and comparative examples relate to silica gel microspheres of type UniSil-Amide manufactured by Sovium nano technologies, Inc., and their scanning electron micrographs are shown in FIG. 1. The related filler with the model RPC-30SCX is produced by Suzhou nano micro-technology GmbH; other starting materials of interest are available commercially.
The conditions of the HPLC analysis in the following examples were as follows: the chromatographic column model is Waters Xbridge Amide 4.6 x 150mm 3.5 μm, the mobile phase is acetonitrile and water, the column temperature is 40 deg.C, the flow rate is 1mL/min, and the elution mode is isocratic elution. The following comparative examples relate to the conditions of the HPLC analysis: the chromatographic column model is Waters Xbridge Amide 4.6 x 250mm 3.5 μm, the mobile phase is ethanol and water, the column temperature is 40 deg.C, the flow rate is 1mL/min, and the elution mode is gradient elution.
Example 1
This example provides a method for separating and purifying anidulafungin mother nucleus, which comprises the following steps:
(1) dissolving anidulafungin mother nucleus with purity of 92.445% with pure methanol, wherein the concentration of anidulafungin mother nucleus in the solution is 30 mg/mL; filtering with 0.45 μm filter membrane, and collecting filtrate;
(2) A4.6X 250mm chromatographic column is adopted, and UniSil-Amide microspheres are used as a chromatographic column filler for column loading, and the column loading volume is 4.2 mL. Then carrying out equilibrium treatment, namely regenerating a chromatographic column by using 50% acetonitrile aqueous solution, and then balancing the chromatographic column by using 85% acetonitrile aqueous solution;
(3) taking water and acetonitrile with a volume ratio of 15:85 as mobile phases, carrying out column chromatography and isocratic elution on the anidulafungin mother nucleus filtrate, controlling the flow rate at 1.0mL/min, collecting solutions of target peak values in sections, and summarizing component solutions meeting the requirements to obtain purified anidulafungin mother nuclei. The column chromatography process takes 180 min.
The high performance liquid chromatograms of the parent nucleus of anidulafungin before and after purification in this example are shown in fig. 2 and fig. 3, respectively. The purity of the anidulafungin mother nucleus in the eluent is 99.5 percent and the yield is 82 percent by high performance liquid chromatography analysis.
Example 2
This example provides a method for separating and purifying anidulafungin mother nucleus, which comprises the following steps:
(1) dissolving anidulafungin mother nucleus with purity of 92.445% with pure methanol, wherein the concentration of anidulafungin mother nucleus in the solution is 30 mg/mL; filtering with 0.45 μm filter membrane, and collecting filtrate;
(2) A4.6X 250mm chromatographic column is adopted, and UniSil-Amide microspheres are used as a chromatographic column filler for column loading, and the column loading volume is 4.2 mL. Then carrying out equilibrium treatment, namely regenerating a chromatographic column by using 45% acetonitrile aqueous solution, and then balancing the chromatographic column by using 85.2% acetonitrile aqueous solution;
(3) and (2) adopting water and acetonitrile with a volume ratio of 14.8:85.2 as mobile phases, carrying out column chromatography on the anidulafungin mother nucleus filtrate, carrying out isocratic elution, controlling the flow rate at 0.5mL/min, collecting solutions of target peak values in sections, and summarizing component solutions meeting the requirements to obtain the purified anidulafungin mother nucleus. The column chromatography process takes 400 min.
The purity of the anidulafungin mother nucleus in the eluent is 99.65 percent and the yield is 85 percent by high performance liquid chromatography analysis.
Example 3
This example provides a method for separating and purifying anidulafungin mother nucleus, which comprises the following steps:
(1) dissolving anidulafungin mother nucleus with purity of 92.445% with pure methanol, wherein the concentration of anidulafungin mother nucleus in the solution is 30 mg/mL; filtering with 0.45 μm filter membrane, and collecting filtrate;
(2) A4.6X 250mm chromatographic column is adopted, and UniSil-Amide microspheres are used as a chromatographic column filler for column loading, and the column loading volume is 4.2 mL. Then carrying out equilibrium treatment, namely regenerating the chromatographic column by using 55% acetonitrile water solution and then balancing the chromatographic column by using 84.8% acetonitrile water solution;
(3) taking water and acetonitrile with a volume ratio of 15.2:84.8 as mobile phases, carrying out column chromatography on the anidulafungin mother nucleus filtrate, carrying out isocratic elution with the flow rate controlled at 0.7mL/min, collecting solutions of target peak values in sections, and summarizing component solutions meeting requirements to obtain purified anidulafungin mother nuclei. The column chromatography process takes 240 min.
The purity of the anidulafungin mother nucleus in the eluent is 99.6 percent and the yield is 83 percent by high performance liquid chromatography analysis.
Example 4
This example provides a method for separating and purifying anidulafungin mother nucleus, which is different from example 1 only in that the chromatographic column is equilibrated with 87% acetonitrile in water in step (2), water and acetonitrile in a volume ratio of 13:87 are used as mobile phase in step (3), and other conditions are kept unchanged. The column chromatography process takes 360 min.
The HPLC chromatogram of the purified parent nucleus of anidulafungin in this example is shown in FIG. 4. The purity of the anidulafungin mother nucleus in the eluent is 99.7 percent and the yield is 85 percent by high performance liquid chromatography analysis.
Example 5
This example provides a method for separating and purifying anidulafungin mother nucleus, which is different from example 1 only in that the chromatographic column is equilibrated with 83% acetonitrile in water in step (2), water and acetonitrile in a volume ratio of 17:83 are used as mobile phase in step (3), and other conditions are kept unchanged. The column chromatography process takes 80 min.
The HPLC chromatogram of the purified parent nucleus of anidulafungin in this example is shown in FIG. 5. The purity of the anidulafungin mother nucleus in the eluent is 98.6 percent and the yield is 80 percent by high performance liquid chromatography analysis.
Comparative example 1
The comparative example provides a separation and purification method of anidulafungin mother nucleus, which comprises the following steps:
(1) dissolving anidulafungin mother nucleus with purity of 92.445% with pure water, wherein the concentration of anidulafungin mother nucleus in the solution is 30 mg/mL; filtering with 0.45 μm filter membrane, and collecting filtrate;
(2) the column is filled by adopting a chromatographic column with the size of 4.6 multiplied by 250mm and RPC-30SCX microspheres as the packing of the chromatographic column, and the volume of the filled column is 4.2 mL. Then carrying out balance treatment, and balancing the chromatographic column by using pure water;
(3) taking pure water as an A phase and 100% ethanol as a B phase, carrying out column chromatography on the anidulafungin mother nucleus filtrate, setting 0% B-100% B for 0-60 min to carry out gradient elution, controlling the flow rate at 1.0mL/min, collecting solutions of target peak values in sections, and summarizing component solutions meeting the requirements to obtain the purified anidulafungin mother nucleus. The column chromatography process takes 80 min.
The purity of the anidulafungin mother nucleus in the eluent is 99.1 percent and the yield is 20.5 percent by high performance liquid chromatography analysis.
Thus, it can be seen that: the method for separating and purifying the anidulafungin parent nucleus can meet the requirements that the purity of the anidulafungin parent nucleus is more than 99 percent and the yield is more than 80 percent only by one-step chromatographic purification, has high and stable purification yield, and is simple, convenient and short in time consumption.
As can be seen from the results of comparative example 1 and examples 2 to 3, when the flow rate is decreased, the time for the column chromatography process is increased, but the purity and yield are also maintained at a high level. Comparing example 1 and example 4, when the concentration of the acetonitrile aqueous solution is increased from 85% to 87%, the purity and yield are also maintained at a high level, but the column chromatography process takes a significantly prolonged time; comparing example 1 and example 5, when the concentration of the acetonitrile aqueous solution is reduced from 85% to 83%, the purity and yield are both significantly reduced although the time consumption is shortened; as can be seen from the comparative examples, the UniSil-Amide microspheres in the separation and purification method provided by the invention are used as the packing of the chromatographic column, and the high purity and the high yield are ensured.
The applicant states that the present invention is illustrated by the above examples to describe a method for separating and purifying anidulafungin mother nucleus of the present invention, but the present invention is not limited to the above examples, i.e. it is not meant that the present invention is implemented only by relying on the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (10)
1. A method for separating and purifying anidulafungin mother nucleus is characterized by comprising the following steps:
(1) dissolving anidulafungin mother nucleus, and filtering to obtain anidulafungin mother nucleus filtrate;
(2) adopting water and organic solvent as mobile phase, subjecting the anidulafungin mother nucleus filtrate to column chromatography with homogeneous HILIC chromatographic column, and collecting eluate of target peak to obtain purified anidulafungin mother nucleus.
2. The method for separating and purifying anidulafungin mother nucleus according to claim 1, wherein the organic solvent comprises acetonitrile.
3. The method for separating and purifying anidulafungin mother nucleus according to claim 1 or 2, wherein the volume ratio of water to the organic solvent is 10:90 to 20:80, preferably 16:84 to 14:86, and more preferably 15.2:84.8 to 14.8: 85.2.
4. The method for the separation and purification of anidulafungin mother nucleus according to any one of claims 1 to 3, wherein the solvent for dissolution comprises methanol;
preferably, the filtration is performed with a 0.45 μm filter membrane;
preferably, the concentration of the anidulafungin mother-nucleus filtrate is 25-35 mg/mL.
5. The method for separating and purifying anidulafungin mother nucleus according to any one of claims 1 to 4, wherein the flow rate of the column chromatography is controlled to 0.5 to 1.0 mL/min.
6. The method for separating and purifying anidulafungin mother nucleus according to any one of claims 1 to 5, wherein the filler of the homogeneous HILIC column is silica gel filler of UniSil-Amide type.
7. The method for separating and purifying anidulafungin mother nucleus according to any one of claims 1 to 6, wherein the elution manner of the column chromatography is isocratic elution.
8. The method for separating and purifying anidulafungin mother nucleus according to any one of claims 1 to 7, wherein the homogeneous HILIC column is subjected to an equilibration treatment before use, comprising: the column is regenerated with 45-55% acetonitrile in water and equilibrated with 80-90% acetonitrile in water.
9. The method for separating and purifying anidulafungin mother nucleus according to any one of claims 1 to 7, wherein the homogeneous HILIC column is subjected to an equilibration treatment before use, comprising: the column is regenerated with 45-55% acetonitrile in water and equilibrated with 84-86% acetonitrile in water.
10. The method for separating and purifying anidulafungin mother nucleus according to any one of claims 1 to 7, wherein the homogeneous HILIC column is subjected to an equilibration treatment before use, comprising: the column is regenerated with 45-55% acetonitrile in water and equilibrated with 84.8-85.2% acetonitrile in water.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011324533.7A CN112430256A (en) | 2020-11-23 | 2020-11-23 | Method for separating and purifying anidulafungin mother nucleus |
| PCT/CN2020/132200 WO2022104880A1 (en) | 2020-11-23 | 2020-11-27 | Method for separation and purification of echinocandin b nucleus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011324533.7A CN112430256A (en) | 2020-11-23 | 2020-11-23 | Method for separating and purifying anidulafungin mother nucleus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112430256A true CN112430256A (en) | 2021-03-02 |
Family
ID=74693751
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011324533.7A Pending CN112430256A (en) | 2020-11-23 | 2020-11-23 | Method for separating and purifying anidulafungin mother nucleus |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN112430256A (en) |
| WO (1) | WO2022104880A1 (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102481336A (en) * | 2009-08-14 | 2012-05-30 | 克塞里尔制药公司 | Separation and/or purification of pneumocandin b0 from c0 |
| CN103193868A (en) * | 2013-04-25 | 2013-07-10 | 华北制药集团新药研究开发有限责任公司 | Purification method of echinocandins antifungal drug anidulafungin |
| CN103724403A (en) * | 2012-10-12 | 2014-04-16 | 重庆乾泰生物医药有限公司 | Separation purification method and use of echinocandin B |
| CN107365353A (en) * | 2017-07-10 | 2017-11-21 | 杭州华东医药集团新药研究院有限公司 | Anidulafungin analogue and its application |
| CN107674116A (en) * | 2016-08-02 | 2018-02-09 | 北大方正集团有限公司 | A kind of purification process of Pneumocandin B0 |
| CN107759668A (en) * | 2017-05-25 | 2018-03-06 | 博瑞生物医药（苏州）股份有限公司 | A kind of high purity echinocandin B parent nucleus or its salt purification process |
| CN110655557A (en) * | 2018-06-28 | 2020-01-07 | 浙江医药股份有限公司新昌制药厂 | Separation and purification method of pneumocandin B0 serine analogue |
-
2020
- 2020-11-23 CN CN202011324533.7A patent/CN112430256A/en active Pending
- 2020-11-27 WO PCT/CN2020/132200 patent/WO2022104880A1/en active Application Filing
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102481336A (en) * | 2009-08-14 | 2012-05-30 | 克塞里尔制药公司 | Separation and/or purification of pneumocandin b0 from c0 |
| CN103724403A (en) * | 2012-10-12 | 2014-04-16 | 重庆乾泰生物医药有限公司 | Separation purification method and use of echinocandin B |
| CN103193868A (en) * | 2013-04-25 | 2013-07-10 | 华北制药集团新药研究开发有限责任公司 | Purification method of echinocandins antifungal drug anidulafungin |
| CN107674116A (en) * | 2016-08-02 | 2018-02-09 | 北大方正集团有限公司 | A kind of purification process of Pneumocandin B0 |
| CN107759668A (en) * | 2017-05-25 | 2018-03-06 | 博瑞生物医药（苏州）股份有限公司 | A kind of high purity echinocandin B parent nucleus or its salt purification process |
| CN107365353A (en) * | 2017-07-10 | 2017-11-21 | 杭州华东医药集团新药研究院有限公司 | Anidulafungin analogue and its application |
| CN110655557A (en) * | 2018-06-28 | 2020-01-07 | 浙江医药股份有限公司新昌制药厂 | Separation and purification method of pneumocandin B0 serine analogue |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2022104880A1 (en) | 2022-05-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101514903B1 (en) | Process for purifying cyclic lipopeptide compounds or salts thereof | |
| CN105001309B (en) | A kind of isolation and purification method of Dalbavancin | |
| CN101372492A (en) | Method for preparing high-purity moxidectin | |
| KR101479389B1 (en) | Purification method of azacyclohexapeptide or its salt | |
| CN111100029B (en) | Purification method of high-load iohexol | |
| CN102993251B (en) | A kind of method of high-efficient liquid phase chromatogram purification TCM B | |
| TWI488862B (en) | Separation and Purification of Cyclohexyl Compounds and Their Salts | |
| CN107759667B (en) | Separation and purification method of cetrorelix | |
| CN108250270A (en) | A kind of method of the enrichment extraction Daptomycin from zymotic fluid | |
| CN112430256A (en) | Method for separating and purifying anidulafungin mother nucleus | |
| EP1558622A1 (en) | Method of purifying macrolides | |
| CN109651301B (en) | Purification method of orlistat | |
| CN108250273A (en) | Knob not Kangding high efficiency separation and purification method | |
| CN101238136A (en) | Purification of tacrolimus on supports of vegetable origin | |
| CN103864862B (en) | A kind of purification process of pentose compound | |
| CN114478750A (en) | Purification method of teriparatide | |
| CN102690333A (en) | Preparation method of high-purity teicoplanin | |
| WO2022110003A1 (en) | Purification method for pneumocandin b0 | |
| CN103570774A (en) | Method for purifying pentose compounds | |
| CN103910783B (en) | A kind of preparation method of high-purity echinocandin B parent nucleus | |
| CN113801203A (en) | Preparation method of caspofungin acetate impurity D | |
| CN105418631A (en) | Method for separating and purifying nemadectin by using HPLC (high performance liquid chromatography) | |
| Qin et al. | Separation of alkaloids from Sophora alopecuroides L. by adsorption using macroporous resins | |
| CN113087774A (en) | Method for removing echinocandin B mother nucleus degradation impurities | |
| CN117683115A (en) | Separation and purification method of somalupeptide |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210302 |