CN112724241A - Purification method of abamectin - Google Patents
Purification method of abamectin Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 40
- 238000000746 purification Methods 0.000 title claims abstract description 29
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- 239000005660 Abamectin Substances 0.000 title claims abstract description 24
- 229950008167 abamectin Drugs 0.000 title claims abstract description 24
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- 238000010828 elution Methods 0.000 claims abstract description 27
- 238000010829 isocratic elution Methods 0.000 claims abstract description 24
- 239000007864 aqueous solution Substances 0.000 claims abstract description 14
- 239000012264 purified product Substances 0.000 claims abstract description 12
- 238000004108 freeze drying Methods 0.000 claims abstract description 10
- 239000011259 mixed solution Substances 0.000 claims abstract description 8
- 239000000047 product Substances 0.000 claims abstract description 6
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- 229910002027 silica gel Inorganic materials 0.000 claims description 7
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- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 2
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- 238000010612 desalination reaction Methods 0.000 description 3
- 208000010392 Bone Fractures Diseases 0.000 description 2
- 206010017076 Fracture Diseases 0.000 description 2
- 108700020797 Parathyroid Hormone-Related Proteins 0.000 description 2
- 102000003982 Parathyroid hormone Human genes 0.000 description 2
- 108090000445 Parathyroid hormone Proteins 0.000 description 2
- 102000043299 Parathyroid hormone-related Human genes 0.000 description 2
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- 238000009777 vacuum freeze-drying Methods 0.000 description 2
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- 102400000107 C-terminal peptide Human genes 0.000 description 1
- 101800000655 C-terminal peptide Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 102000015731 Peptide Hormones Human genes 0.000 description 1
- 108010038988 Peptide Hormones Proteins 0.000 description 1
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- 108010038051 abaloparatide Proteins 0.000 description 1
- BVISQZFBLRSESR-XSCWXTNMSA-N abaloparatide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NC(C)(C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](C)N)C(C)C)C1=CN=CN1 BVISQZFBLRSESR-XSCWXTNMSA-N 0.000 description 1
- 229950001959 abaloparatide Drugs 0.000 description 1
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- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000037182 bone density Effects 0.000 description 1
- 230000010072 bone remodeling Effects 0.000 description 1
- 230000037118 bone strength Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
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- 239000011707 mineral Substances 0.000 description 1
- 230000011164 ossification Effects 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Endocrinology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a purification method of abamectin, belonging to the technical field of polypeptide drug purification. The method comprises the steps of pretreating the abapa peptide crude peptide to dissolve the abapa peptide crude peptide into acetonitrile aqueous solution to obtain a crude peptide solution. Then taking the crude peptide solution, carrying out isocratic and gradient elution on a mixed solution of the A1 solution, the A2 solution and the B solution, and collecting elution components to obtain a first purified substance; taking the first purified product, carrying out salt conversion isocratic elution on the mixed solution of the A3 and the B solution, and collecting the eluted components to obtain the final purified product; finally, the final purified product is subjected to reduced pressure rotary steaming concentration and freeze drying to obtain an abapatatin finished product; the preparation method of the abapatatin prepared by the purification method has the advantages of simple steps, low raw material consumption, high product yield and high purity, and is suitable for large-scale industrial production.
Description
Technical Field
The invention relates to the technical field of polypeptide drug purification, and particularly relates to a purification method of abamectin.
Background
Abapa peptide, english name abalopatide.
The abamectin is polypeptide consisting of 34 amino acid residues, the molecular formula is C174H300N56O49, the molecular weight is 3960.64g/mol, the CAS number is 247062-33-5, and the structure is as follows:
Ala1-Val2-Ser3-Glu4-His5-Gln6-Leu7-Leu8-His9-Asp10-Lys11-Gly12-Lys13Ser14-Ile15-Gln16-Asp17-Leu18-Arg19-Arg20-Arg21-Glu22-Leu23-Leu24-Glu25Lys26-Leu27-Leu28-Aib29-Lys30-Leu31-His32-Thr33-Ala34-NH2。
parathyroid hormone (PTH) is a polypeptide hormone secreted from the parathyroid chief cell and regulating calcium and phosphorus metabolism in vivo, and has a C-terminal peptide chain which binds to PTH-II receptor to promote bone apoptosis and an N-terminal peptide chain which binds to PTH-I receptor to promote bone remodeling.
Abapatide (Abalopratide), a novel parathyroid hormone-related peptide (PTHrP) developed by Radius Health, is a potent selective activator of PTH-I receptor, increases bone mineral content, bone density and bone strength, promotes bone formation, has been approved by the FDA on 28/4.2017 and is marketed under the name Tymlos.
Abapatide is injected subcutaneously to treat osteoporosis in postmenopausal women at risk of fracture or ineffective to other therapeutic agents, and is effective in reducing fracture rate of new vertebral body and non-vertebral body.
The solid-phase synthesis of the abapatulin generates a large amount of impurities, and the existing purification method has low yield and is complex and difficult to operate.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides a novel purification method of the abapa peptide, which can effectively remove various impurities generated by synthesis to obtain the high-purity abapa peptide.
The object of the present invention is achieved by the following means. The invention relates to a purification method of abamectin, which comprises the following specific technical scheme:
the method comprises the following steps: the abapatatin crude peptide is taken to be pretreated and dissolved in acetonitrile water solution to obtain crude peptide solution.
Step two: taking the crude peptide solution, carrying out isocratic and gradient elution on a mixed solution of the A1 solution, the A2 solution and the B solution, and collecting elution components to obtain a first purified substance;
step three: taking the first purified product, desalting and isocratic eluting with a mixed solution of A3 and B solution, and collecting eluted components to obtain a final purified product;
step four: carrying out reduced pressure rotary steaming, concentration and freeze drying on the final purified product to obtain an abapatulin finished product;
the A1 solution is 10% acetic acid aqueous solution;
the a2 solution was 0.1% TFA in water;
the A3 solution is pure water;
the solution B is 100% acetonitrile.
Preferably, the method for purifying abamectin comprises the step of pretreating abamectin, which comprises:
dissolving the abamectin in acetonitrile water solution to obtain a crude abamectin peptide water solution;
insoluble particles in the crude abapatatin peptide aqueous solution are removed by a filter membrane, and the filtrate is collected.
Preferably, the purification method of the abapatulin comprises the following specific steps of:
taking an octaalkylsilane bonded silica chromatographic column as a stationary phase, 10% acetic acid as a mobile phase A1 and acetonitrile as a mobile phase B, carrying out isocratic elution at a detection wavelength of 260nm, then taking a 0.1% TFA aqueous solution as a mobile phase A2 and acetonitrile as a mobile phase B, carrying out gradient elution at a detection wavelength of 225nm, and collecting fractions containing the abamectin.
The preferable purification method of the abapatulin comprises the following three specific steps:
performing isocratic elution by using a tetraalkylsilane bonded silica gel chromatographic column as a stationary phase, pure water as a mobile phase A3 and acetonitrile as a mobile phase B, wherein the detection wavelength is 225nm, the volume fraction ratio of the pure water to the acetonitrile solution is 40% to 60%, and the isocratic elution time is 30-40 min;
preferably, the method for purifying abapatulin comprises the following specific steps:
the water bath temperature of the rotary evaporator is 30 ℃, the vacuum degree is-0.008 MP, and the freeze-drying is finished in a vacuum freeze-drying machine after the acetonitrile is removed.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for purifying abamectin, which comprises the steps of dissolving crude abamectin in acetonitrile water, filtering to obtain a crude peptide solution, and carrying out one-step purification and one-step desalination to obtain the abamectin. The yield is about 70 percent, the purity is more than 99.0 percent, and the single impurity content is less than 0.15 percent. The invention can effectively solve the problems of low purity and low yield of the abapa peptide finished product.
Detailed Description
The invention discloses a purification method of abamectin. The method may be carried out by those skilled in the art with reference to the disclosure herein, and it is specifically intended that all such alterations and modifications as are obvious to those skilled in the art are deemed to be included in the invention. While the purification methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods and applications described herein, as well as appropriate variations and combinations thereof, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
Embodiment 1, a purification method of abapa peptide, the specific technical scheme includes:
the method comprises the following steps: taking the abapa peptide crude peptide for pretreatment, and dissolving the abapa peptide crude peptide into acetonitrile aqueous solution to obtain crude peptide solution; the method comprises the following specific steps:
dissolving the abamectin in acetonitrile water solution, and fully stirring until the abamectin is completely dissolved to obtain a crude abamectin solution;
insoluble particles in the crude abapatatin peptide aqueous solution are removed by a filter membrane, and the filtrate is collected.
Step two: taking the crude peptide solution, carrying out isocratic and gradient elution on a mixed solution of the A1 solution, the A2 solution and the B solution, and collecting elution components to obtain a first purified substance; the method comprises the following specific steps:
taking an octaalkylsilane bonded silica chromatographic column as a stationary phase, 10% acetic acid as a mobile phase A1 and acetonitrile as a mobile phase B, carrying out isocratic elution at a detection wavelength of 260nm, then taking a 0.1% TFA aqueous solution as a mobile phase A2 and acetonitrile as a mobile phase B, carrying out gradient elution at a detection wavelength of 225nm, and collecting fractions containing the abamectin.
Step three: taking the first purified product, carrying out salt transfer gradient elution by using a mixed solution of A3 and B solution, and collecting elution components to obtain a final purified product; the method comprises the following specific steps:
and (2) performing isocratic elution by taking a tetraalkylsilane bonded silica gel chromatographic column as a stationary phase, a pure water mobile phase A3 and acetonitrile as a mobile phase B, wherein the detection wavelength is 225nm, the volume fraction ratio of pure water to acetonitrile is 40% to 60%, and the isocratic elution time is 30-40 min, so as to complete the desalting treatment.
Step four: and (4) carrying out reduced pressure rotary steaming concentration and freeze drying on the final purified product to obtain the abapatatin finished product. The method comprises the following specific steps:
the water bath temperature of the rotary evaporator is 30 ℃, the vacuum degree is below-0.008 MP, and the freeze-drying is finished in a vacuum freeze-drying machine after the acetonitrile is removed.
Example 2, purification of crude abapa peptide experiment 1:
the abapa peptide was synthesized on a solid phase with a crude peptide purity of 69%.
Sample treatment: 3.0g of solid crude peptide was dissolved in acetonitrile water, and after stirring to completely dissolve the sample, the sample was filtered through a 0.45u filter, and the filtrate was collected for use.
First-step purification:
chromatographic conditions are as follows: the chromatographic column using the octaalkylsilane bonded silica as a stationary phase has the following diameters and lengths: 30mm by 250 mm. 10% acetic acid is used as a mobile phase A1, acetonitrile is used as a mobile phase B, the detection wavelength is 260nm, isocratic elution is firstly carried out, then 0.1% TFA aqueous solution is used as a mobile phase A2, acetonitrile is used as a mobile phase B, the detection wavelength is 225nm, gradient elution is carried out, and the maximum sample loading amount of a single needle is 0.8 g.
The specific elution gradients are shown in the following table:
collecting the abapatulin sample fraction with the purity of more than 99.0 percent and the single impurity of less than 0.15 percent.
And a second step of desalting:
the method is characterized in that a tetraalkylsilane bonded silica gel chromatographic column is used as a stationary phase column, and the diameter and the length are as follows: 50 mm. times.260 mm. Pure water as a mobile phase A3 and acetonitrile as a mobile phase B were subjected to isocratic elution at a detection wavelength of 225nm to complete desalting. Wherein the volume fraction ratio of pure water to acetonitrile is 40% to 60%, the isocratic elution time is 30min, and the maximum sample loading amount of a single needle is 2.0 g.
The specific elution gradients are shown in the following table:
all desalted samples were collected. Concentrating the collected target peak fraction by rotary evaporation at water temperature of 30 deg.C under reduced pressure (-below 0.008 MP), and freeze drying to obtain abapatatin with purity of 99.1% and purification yield of 66.7%.
Example 3: purification experiment 2 of crude Abapatide
The abapa peptide was synthesized on a solid phase with a crude peptide purity of 69%.
Sample treatment: 14.0g of solid crude peptide was dissolved in acetonitrile water, and after stirring to completely dissolve the sample, the sample was filtered through a 0.45u filter, and the filtrate was collected for use.
First-step purification:
chromatographic conditions are as follows: the chromatographic column using the octaalkylsilane bonded silica as a stationary phase has the following diameters and lengths: 50mm by 270 mm. 10% acetic acid is used as a mobile phase A1, acetonitrile is used as a mobile phase B, the detection wavelength is 260nm, isocratic elution is firstly carried out, then 0.1% TFA aqueous solution is used as a mobile phase A2, acetonitrile is used as a mobile phase B, the detection wavelength is 225nm, gradient elution is carried out, and the maximum sample loading amount of a single needle is 2.2 g.
The specific elution gradients are shown in the following table:
collecting the abapatulin sample fraction with the purity of more than 99.0 percent and the single impurity of less than 0.15 percent.
And a second step of desalting:
the method is characterized in that a tetraalkylsilane bonded silica gel chromatographic column is used as a stationary phase column, and the diameter and the length are as follows: 50mm multiplied by 260mm, pure water is used as a mobile phase A3, acetonitrile is used as a mobile phase B, the detection wavelength is 225nm, isocratic elution is carried out, wherein the volume fraction ratio of the pure water to the acetonitrile is 40 percent to 60 percent, the isocratic elution time is 30min, and the maximum sample loading amount of a single needle is 4.0 g.
The specific elution gradients are shown in the following table:
all desalted samples were collected. Concentrating the collected target peak fraction by rotary evaporation at water temperature of 30 deg.C under reduced pressure (-below 0.008 MP), and freeze drying to obtain abapatatin with purity of 99.0% and purification yield of 70.3%.
Example 4, abapa peptide crude peptide purification experiment 3:
the abapa peptide was synthesized on a solid phase with a crude peptide purity of 71%.
Sample treatment: 15.0g of solid crude peptide was dissolved in acetonitrile water, and after stirring to completely dissolve the sample, the sample was filtered through a 0.45u filter, and the filtrate was collected for use.
First-step purification:
chromatographic conditions are as follows: the chromatographic column using the octaalkylsilane bonded silica as a stationary phase has the following diameters and lengths: 50mm by 270 mm. 10% acetic acid is used as a mobile phase A1, acetonitrile is used as a mobile phase B, the detection wavelength is 260nm, isocratic elution is firstly carried out, then 0.1% TFA aqueous solution is used as a mobile phase A2, acetonitrile is used as a mobile phase B, the detection wavelength is 225nm, gradient elution is carried out, and the maximum sample loading amount of a single needle is 2.2 g.
The specific elution gradients are shown in the following table:
collecting the abapatulin sample fraction with the purity of more than 99.0 percent and the single impurity of less than 0.15 percent.
And a second step of desalting:
the method is characterized in that a tetraalkylsilane bonded silica gel chromatographic column is used as a stationary phase column, and the diameter and the length are as follows: 50 mm. times.260 mm, pure water as mobile phase A3, acetonitrile as mobile phase B, detection wavelength of 225nm, isocratic elution, complete the desalination treatment. Wherein the volume fraction ratio of pure water to acetonitrile is 40% to 60%, the isocratic elution time is 30min, and the maximum sample loading amount of a single needle is 4.0 g.
The specific elution gradients are shown in the following table:
all desalted samples were collected. Concentrating the collected target peak fraction by rotary evaporation at water temperature of 30 deg.C under reduced pressure (-below 0.008 MP), and freeze drying to obtain abapatatin with purity of 99.1% and purification yield of 73.0%.
Example 5: purification experiment 4 of crude Abapatide
The abapa peptide was synthesized on a solid phase with a crude peptide purity of 71%.
Sample treatment: 35.0g of solid crude peptide was dissolved in acetonitrile water, and after stirring to completely dissolve the sample, the sample was filtered through a 0.45u filter, and the filtrate was collected for use.
First-step purification:
chromatographic conditions are as follows: the chromatographic column using the octaalkylsilane bonded silica as a stationary phase has the following diameters and lengths: 100 mm. times.260 mm. 10% acetic acid is used as a mobile phase A1, acetonitrile is used as a mobile phase B, the detection wavelength is 260nm, isocratic elution is firstly carried out, then 0.1% TFA aqueous solution is used as a mobile phase A2, acetonitrile is used as a mobile phase B, the detection wavelength is 225nm, gradient elution is carried out, and the maximum sample loading amount of a single needle is 8.8 g.
The specific elution gradients are shown in the following table:
collecting the abapatulin sample fraction with the purity of more than 99.0 percent and the single impurity of less than 0.15 percent.
And a second step of desalting:
the method is characterized in that a tetraalkylsilane bonded silica gel chromatographic column is used as a stationary phase column, and the diameter and the length are as follows: 50 mm. times.260 mm, pure water as mobile phase A3, acetonitrile as mobile phase B, detection wavelength of 225nm, isocratic elution, complete the desalination treatment. Wherein the volume fraction ratio of pure water to acetonitrile is 40% to 60%, the isocratic elution time is 30min, and the maximum sample loading amount of a single needle is 4.0 g.
The specific elution gradients are shown in the following table:
all desalted samples were collected. Concentrating the collected target peak fraction by rotary evaporation at water temperature of 30 deg.C under reduced pressure (-below 0.008 MP), and freeze drying to obtain abapatatin with purity of 99.0% and purification yield of 73.8%
4 experiments show that the yield of the abamectin obtained by purifying the abamectin by the method is close to 70%, the purity of the obtained abamectin is more than 99.0%, and the method has high practical significance in production.
The above are only the main features and preferred embodiments of the present invention, and it should be noted that the above preferred embodiments should not be considered as limiting the present invention, and the scope of the present invention should be subject to the scope defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (7)
1. A purification method of abamectin is characterized by comprising the following steps:
the method comprises the following steps: taking the abapa peptide crude peptide for pretreatment, and dissolving the abapa peptide crude peptide into acetonitrile aqueous solution to obtain crude peptide solution;
step two: taking the crude peptide solution, carrying out isocratic and gradient elution on a mixed solution of the A1 solution, the A2 solution and the B solution, and collecting elution components to obtain a first purified substance;
step three: taking the first purified product, carrying out salt conversion isocratic elution on a mixed solution of A3 and B solution, and collecting elution components to obtain a final purified product;
step four: carrying out reduced pressure rotary steaming, concentration and freeze drying on the final purified product to obtain an abapatulin finished product;
the A1 solution is 10% acetic acid aqueous solution;
the a2 solution was 0.1% TFA in water;
the A3 solution is pure water;
the solution B is 100% acetonitrile.
2. The method for purifying abapatulin according to claim 1, wherein: in the second step, the volume fraction ratio of the A1 solution to the B solution is 85 percent to 15 percent, and the isocratic elution time is 20 min; the volume fraction ratio of the A2 solution to the B solution is 69%: 31% → 45%: 55%, and the gradient elution time is 50-60 min.
3. The method for purifying abapatulin according to claim 1, wherein: in the third step, the volume fraction ratio of the pure water to the solution B is 40 to 60 percent, and the isocratic elution time is 30 to 40 min;
4. the method for purifying abapatulin according to claim 1, wherein: in step one, abapa peptide is dissolved in an aqueous acetonitrile solution, insoluble particles are removed with a filter, and the filtrate is collected.
5. The method for purifying abapatulin according to claim 1, wherein: and in the second step, an octaalkylsilane bonded silica chromatographic column is used as a stationary phase, 10% acetic acid is used as a mobile phase A1, acetonitrile is used as a mobile phase B, the detection wavelength is 260nm, isocratic elution is firstly carried out, then a 0.1% TFA aqueous solution is used as a mobile phase A2, the acetonitrile is used as a mobile phase B, the detection wavelength is 225nm, gradient elution is carried out, and fractions containing the abamectin are collected.
6. The method for purifying abapatulin according to claim 1, wherein: and in the third step, the tetraalkylsilane bonded silica gel chromatographic column is used as a stationary phase, a pure water mobile phase A3 and acetonitrile are used as a mobile phase B, the detection wavelength is 225nm, isocratic elution is carried out, and the desalting treatment is completed.
7. The method for purifying abapatulin according to claim 1, wherein: in the fourth step, the water bath temperature of the rotary evaporator is 30 ℃, and the vacuum degree is-0.008 MP.
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| CN114478749A (en) * | 2021-12-28 | 2022-05-13 | 深圳翰宇药业股份有限公司 | Purification method of abamectin |
| WO2024032454A1 (en) * | 2022-08-10 | 2024-02-15 | 成都奥达生物科技有限公司 | Long-acting abaloparatide compound |
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