CN113307847A - Purifying and refining method of carbetocin - Google Patents
Purifying and refining method of carbetocin Download PDFInfo
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- CN113307847A CN113307847A CN202110634151.2A CN202110634151A CN113307847A CN 113307847 A CN113307847 A CN 113307847A CN 202110634151 A CN202110634151 A CN 202110634151A CN 113307847 A CN113307847 A CN 113307847A
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- 108700021293 carbetocin Proteins 0.000 title claims abstract description 100
- 229960001118 carbetocin Drugs 0.000 title claims abstract description 100
- NSTRIRCPWQHTIA-DTRKZRJBSA-N carbetocin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSCCCC(=O)N1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)NCC(N)=O)=O)[C@@H](C)CC)C1=CC=C(OC)C=C1 NSTRIRCPWQHTIA-DTRKZRJBSA-N 0.000 title claims abstract description 100
- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000007670 refining Methods 0.000 title claims abstract description 18
- 238000002953 preparative HPLC Methods 0.000 claims abstract description 34
- 238000010828 elution Methods 0.000 claims abstract description 31
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000012065 filter cake Substances 0.000 claims abstract description 18
- 238000004537 pulping Methods 0.000 claims abstract description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 16
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000001035 drying Methods 0.000 claims abstract description 15
- 238000000746 purification Methods 0.000 claims abstract description 14
- 238000004108 freeze drying Methods 0.000 claims abstract description 11
- 239000000047 product Substances 0.000 claims abstract description 9
- 238000000967 suction filtration Methods 0.000 claims abstract description 9
- 239000007864 aqueous solution Substances 0.000 claims abstract description 8
- 238000001914 filtration Methods 0.000 claims abstract description 3
- 239000007787 solid Substances 0.000 claims abstract description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 30
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 10
- 229960000583 acetic acid Drugs 0.000 claims description 10
- 238000001514 detection method Methods 0.000 claims description 10
- 238000010438 heat treatment Methods 0.000 claims description 10
- 238000012856 packing Methods 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 5
- 238000007710 freezing Methods 0.000 claims description 5
- 230000008014 freezing Effects 0.000 claims description 5
- 239000012362 glacial acetic acid Substances 0.000 claims description 5
- 239000001632 sodium acetate Substances 0.000 claims description 5
- 235000017281 sodium acetate Nutrition 0.000 claims description 5
- 238000010009 beating Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 2
- 239000000377 silicon dioxide Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 abstract description 6
- 239000002904 solvent Substances 0.000 abstract description 3
- 239000012071 phase Substances 0.000 description 27
- 108090000765 processed proteins & peptides Proteins 0.000 description 9
- 208000018525 Postpartum Hemorrhage Diseases 0.000 description 7
- 239000012141 concentrate Substances 0.000 description 6
- 239000012535 impurity Substances 0.000 description 6
- 238000005303 weighing Methods 0.000 description 6
- 239000003814 drug Substances 0.000 description 5
- 229920003180 amino resin Polymers 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- XNOPRXBHLZRZKH-DSZYJQQASA-N oxytocin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@H](N)C(=O)N1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)NCC(N)=O)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 XNOPRXBHLZRZKH-DSZYJQQASA-N 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 239000007790 solid phase Substances 0.000 description 4
- 238000001308 synthesis method Methods 0.000 description 4
- 101800000989 Oxytocin Proteins 0.000 description 3
- 102400000050 Oxytocin Human genes 0.000 description 3
- XNOPRXBHLZRZKH-UHFFFAOYSA-N Oxytocin Natural products N1C(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CC(C)C)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C(C(C)CC)NC(=O)C1CC1=CC=C(O)C=C1 XNOPRXBHLZRZKH-UHFFFAOYSA-N 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 229960001723 oxytocin Drugs 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 230000035935 pregnancy Effects 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000002270 exclusion chromatography Methods 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000007363 ring formation reaction Methods 0.000 description 2
- GRHQDJDRGZFIPO-UHFFFAOYSA-N 4-bromobutanoic acid Chemical compound OC(=O)CCCBr GRHQDJDRGZFIPO-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 241001183967 Isodon Species 0.000 description 1
- 206010058046 Post procedural complication Diseases 0.000 description 1
- 208000035965 Postoperative Complications Diseases 0.000 description 1
- 208000010238 Uterine Inertia Diseases 0.000 description 1
- 206010046763 Uterine atony Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000001595 contractor effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000000902 placebo Substances 0.000 description 1
- 229940068196 placebo Drugs 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000004291 uterus Anatomy 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
- C07K7/16—Oxytocins; Vasopressins; Related peptides
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)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention discloses a purifying and refining method of carbetocin, which comprises the following steps: sequentially pulping and filtering the crude carbetocin by using methyl tert-butyl ether, and drying a filter cake; drying the solid after pulping, pulping by using ethanol, performing suction filtration, and drying a filter cake; dissolving the obtained filter cake with acetic acid aqueous solution, purifying by reversed phase preparative high performance liquid chromatography, and collecting elution fractions containing carbetocin; concentrating the obtained elution fraction containing carbetocin, purifying again by adopting a reversed-phase preparative high performance liquid chromatography, and collecting the elution fraction containing carbetocin; and concentrating the obtained elution fraction containing carbetocin, and putting the elution fraction into a freeze dryer for freeze drying to obtain a pure carbetocin product. The preparation method obtains the high-purity carbetocin by pulping with a specific solvent, performing two-time purification preparation by a reversed-phase preparative high performance liquid chromatography and the like.
Description
Technical Field
The invention relates to the field of medicine purification and refining, in particular to a method for purifying and refining carbetocin.
Background
The puerpera is affected by various factors in the process of delivery, particularly, the phenomenon of postpartum hemorrhage easily causes the death of the puerpera, and the life and health of the puerpera are threatened. In recent years, the medical industry in China is rapidly developed, cesarean section delivery modes are more and more, under the action of abnormal pregnancy and high-risk factors of pregnancy, the postpartum hemorrhage rate of pregnant and lying-in women is further increased, and the successful delivery of the lying-in women is influenced to a certain extent. In order to ensure the healthy delivery of the puerpera, various technical means are needed to improve the post-pregnancy hemorrhage condition of the puerpera so as to reduce the postpartum hemorrhage rate and improve the pregnancy safety of the puerpera, but the morbidity and mortality of the postpartum hemorrhage are still high. Therefore, compared with treatment, taking more active preventive measures to reduce the occurrence of postpartum uterine atony is the key to controlling and reducing the morbidity and mortality of postpartum hemorrhage and improving the quality of obstetrical medical care.
Carbetocin is a synthetic long-acting oxytocin 9 peptide analog with agonist properties developed by rabdosia pharmaceutical and has clinical and pharmacological properties similar to naturally occurring oxytocin. The carbetocin is similar to the natural oxytocin in drug type, only plays a certain contraction effect on the uterus of a pregnant woman, has a lasting effect, and is relatively mild in drug effect. Compare with traditional oxytocin class medicine, use carbetocin can also effectively reduce postoperative complication's emergence probability on the basis of guarantee delivery quality and delivery security, improve the delivery security of lying-in woman.
Research shows that when the carbetocin is intravenously administered with a single dose of 100 mu g immediately after the cesarean section under epidural or lumbar anesthesia, the carbetocin is obviously superior to placebo in preventing insufficient uterine tension and reducing postpartum hemorrhage. Administration of carbetocin early after delivery may also promote uterine recovery.
Carbetocin has the following structure:
carbetocin can be synthesized into a Carbetocin raw material medicament with excellent performance, and the clinical requirement of preventing and treating postpartum hemorrhage is met, so that the Carbetocin raw material medicament has great significance.
The preparation of carbetocin has been reported at home and abroad, and European patent ES2115543 discloses a synthesis method of carbetocin, which comprises the following steps: firstly, by the conventional solid phase polypeptide synthesis method,obtaining 4-Cl-Butyl-Tyr (Me) -Ile-Gln-Asn-Cys (Trt) -Pro-Leu-GIy-Rink amino resin, and obtaining linear 4-Cl-Butyl-Tyr (Me) -Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2And cyclizing the linear peptide to obtain carbetocin.
Chinese patent CN101555272A discloses a synthesis method of carbetocin: firstly, obtaining 4-Br-Butyl-Tyr (Me) -Ile-Gln-Asn-Cys (Alloc) -Pro-Leu-Gly-Rink amino resin by a conventional solid-phase polypeptide synthesis method, obtaining 4-Br-Butyl-Tyr (Me) -Ile-Gln-Asn-Cys-Pro-Leu-Gly-Rink amino resin by Alloc protection under the catalysis of tetratriphenylphosphine palladium, carrying out solid-phase cyclization under the catalysis of lithium chloride, and obtaining carbetocin after acid hydrolysis.
The synthesis technical scheme of carbetocin is approximately the same by integrating the patents of various countries, and the process is as follows: 1) taking amino resin as a carrier, and performing deprotection; 2) protecting carboxyl on amino acid to couple with resin amino; 3) then sequentially coupling the residual protected amino acid and 4-bromobutyric acid; 4) removing a side chain protecting group of cysteine; 5) solid phase cyclization; 6) cracking to obtain crude peptide; 9) and refining to obtain pure carbetocin.
The common purification method of chemically synthesized polypeptide is mainly reversed phase preparative high performance liquid chromatography, and purification can be carried out by combining methods such as ion exchange chromatography, molecular exclusion chromatography and the like according to the characteristics of peptide structures. The peptide sequence structure of the product consists of 9 amino acid building units, belongs to short peptides, and is not suitable for purification by adopting methods of ion exchange chromatography and molecular exclusion chromatography, so that the product is purified and researched by adopting a reversed-phase preparative high performance liquid chromatography. At present, few research reports on the separation and purification aspects of the carbetocin are reported, the highest level of the purity of the carbetocin does not exceed 99.5%, the lowest content of a single impurity is only 0.2%, the quality of the carbetocin product in China is limited by the reasons of process level and is difficult to obviously improve, and the purification and refining process needs to be improved urgently at present so as to improve the overall quality of the product.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the defects of low purification purity and high single impurity content of carbetocin in the prior art, so that a purification and refining method of carbetocin is provided, and high-purity carbetocin is obtained by pulping with a specific solvent, performing two-time purification preparation by reversed-phase preparative high performance liquid chromatography and the like.
Therefore, the invention provides a method for purifying and refining carbetocin, which comprises the following steps:
(1) sequentially pulping and filtering the crude carbetocin by using methyl tert-butyl ether, and drying a filter cake;
(2) drying the solid after pulping, pulping by using ethanol, performing suction filtration, and drying a filter cake;
(3) dissolving the filter cake obtained in the step (2) by using an acetic acid aqueous solution, purifying by adopting a reversed-phase preparative high performance liquid chromatography, and collecting elution fractions containing carbetocin;
(4) concentrating the elution fraction containing carbetocin obtained in the step (3), purifying again by adopting a reversed-phase preparative high performance liquid chromatography, and collecting the elution fraction containing carbetocin;
(5) and (4) concentrating the elution fraction containing carbetocin obtained in the step (4), and then putting the elution fraction into a freeze dryer for freeze drying to obtain a pure carbetocin product.
Preferably, the beating temperature is room temperature.
Preferably, the adding volume of the methyl tert-butyl ether is 5-10 times of the mass of the crude carbetocin, and the adding volume of the ethanol is 5-10 times of the mass of the crude carbetocin.
Preferably, in the step (3), the specific chromatographic conditions of the reversed-phase preparative high performance liquid chromatography are as follows:
mobile phase: a: 0.2% aqueous sodium acetate solution B: acetonitrile;
detection wavelength: 200-210 nm;
flow rate: 110-120 ml/min.
Preferably, the mobile phase elution gradient of the reversed-phase preparative high performance liquid chromatography is as follows:
preferably, in the step (4), the specific chromatographic conditions of the reversed-phase preparative high performance liquid chromatography are as follows:
mobile phase: a: 0.1% glacial acetic acid aqueous solution B: acetonitrile;
detection wavelength: 200-210 nm;
flow rate: 110-120 ml/min.
Preferably, the mobile phase elution gradient of the reversed-phase preparative high performance liquid chromatography is as follows:
preferably, the column of the reversed-phase preparative high performance liquid chromatography is preparative HPLC, the diameter and length of the column are 60mm × 300mm, and the packing material is C18 (octadecylsilane chemically bonded silica).
Preferably, the concentration condition is that the concentration temperature is set to be 35 ℃ and the vacuum degree is at least 0.075 Mpa.
Preferably, the freeze-drying condition in the step (5) is pre-freezing to-60 ℃ to-40 ℃, then preserving heat for 1-2 h, continuously heating to-30 ℃ to-20 ℃, preserving heat for 8-12 h, then heating to 20 ℃ to 30 ℃, preserving heat for 8-12 h, and the vacuum degree is at least 0.1 mbar.
The technical scheme of the invention has the following advantages:
the purification and refining method of carbetocin provided by the invention obtains high-purity carbetocin by specific solvent pulping, reversed-phase preparation and high performance liquid chromatography two-time purification preparation and the like. The purity of carbetocin reaches 99.4%, and the maximum content of single impurities is reduced to 0.3%.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Example 1
(1) Weighing 10.0g of crude carbetocin, adding the crude carbetocin into 50ml of methyl tert-butyl ether, pulping at normal temperature for 2h, and performing suction filtration by using a sand core funnel to obtain a filter cake, and drying the filter cake at 20-30 ℃ for 2h to obtain crude carbetocin I.
(2) And adding 50ml of 95% ethanol into the crude carbetocin I, pulping for 2 hours at normal temperature, and performing suction filtration by using a sand core funnel to obtain a filter cake, and drying for 2 hours at 20-30 ℃ to obtain a crude carbetocin II.
(3) Weighing 9.0g of crude carbetocin II, adding 270ml of 10% acetic acid solution, stirring for dissolving, purifying by adopting a reversed-phase preparative high performance liquid chromatography, and collecting elution fractions containing carbetocin within 25-35 min, wherein the specific chromatographic conditions are as follows:
equipment: preparative HPLC with a column diameter and length of 60mm X300 mm, packing C18
Mobile phase: a: 0.2% aqueous sodium acetate solution B: acetonitrile
Detection wavelength: 210nm
Flow rate: 120ml/min
Elution gradient:
concentrating the eluate containing carbetocin to 35%, wherein the concentration temperature is 35 deg.C and the vacuum degree is 0.075 Mpa.
After the concentration is finished, purifying again by adopting a reversed-phase preparative high performance liquid chromatography, and collecting elution fractions containing carbetocin for 30-35 min, wherein the specific chromatographic conditions are as follows:
a chromatographic column: preparative HPLC with a column diameter and length of 60mm X300 mm, packing C18
Mobile phase: a: 0.1% glacial acetic acid aqueous solution B: acetonitrile
Detection wavelength: 210nm
Flow rate: 120ml/min
Elution gradient:
concentrating the eluate containing carbetocin to 80%, wherein the concentration temperature is 35 deg.C and the vacuum degree is 0.075 Mpa.
And after the concentration is finished, pouring the concentrate into a freeze-drying tray, putting the freeze-drying tray into a freeze dryer, pre-freezing the concentrate to the temperature of minus 50 ℃, keeping the temperature for 2h, continuously heating the mixture to the temperature of minus 25 ℃, keeping the temperature for 10h, then heating the mixture to the temperature of 20 ℃, keeping the temperature for 10h, and obtaining the carbetocin with the vacuum degree of 0.1 mbar. The purity of carbetocin is 99.4%, and the maximum content of single impurities is 0.3%.
Example 2
(1) Weighing 10.0g of crude carbetocin, adding the crude carbetocin into 50ml of methyl tert-butyl ether, pulping at normal temperature for 4 hours, and performing suction filtration by using a sand core funnel to obtain a filter cake, and drying the filter cake at 20-30 ℃ for 2 hours to obtain crude carbetocin I.
(2) And adding 50ml of 95% ethanol into the crude carbetocin I, pulping at normal temperature for 4 hours, and performing suction filtration by using a sand core funnel to obtain a filter cake, and drying at 20-30 ℃ for 2 hours to obtain a crude carbetocin II.
(3) Weighing 9.0g of crude carbetocin II, adding 270ml of 10% acetic acid solution, stirring for dissolving, purifying by adopting a reversed-phase preparative high performance liquid chromatography, and collecting elution fractions containing carbetocin within 25-35 min, wherein the specific chromatographic conditions are as follows:
equipment: preparative HPLC with a column diameter and length of 60mm X300 mm, packing C18
Mobile phase: a: 0.2% aqueous sodium acetate solution B: acetonitrile
Detection wavelength: 200nm
Flow rate: 110ml/min
Elution gradient:
concentrating the eluate containing carbetocin to 35%, setting the concentration temperature at 35 deg.C and vacuum degree at 0.100 Mpa.
After the concentration is finished, purifying again by adopting a reversed-phase preparative high performance liquid chromatography, and collecting elution fractions containing carbetocin for 30-35 min, wherein the specific chromatographic conditions are as follows:
a chromatographic column: preparative HPLC with a column diameter and length of 60mm X300 mm, packing C18
Mobile phase: a: 0.1% glacial acetic acid aqueous solution B: acetonitrile
Detection wavelength: 200nm
Flow rate: 110ml/min
Elution gradient:
concentrating the eluate containing carbetocin to 80%, setting the concentration temperature at 35 deg.C and vacuum degree at 0.100 Mpa.
And after the concentration is finished, pouring the concentrate into a freeze-drying tray, putting the freeze-drying tray into a freeze dryer, pre-freezing the concentrate to-60 ℃, keeping the temperature for 1h, continuously heating the mixture to-30 ℃, keeping the temperature for 12h, then heating the mixture to 30 ℃, keeping the temperature for 8h, and obtaining the carbetocin with the vacuum degree of 0.125 mbar. The purity of carbetocin is 99.3%, and the maximum content of single impurities is 0.4%.
Example 3
(1) Weighing 10.0g of crude carbetocin, adding the crude carbetocin into 50ml of methyl tert-butyl ether, pulping at normal temperature for 4 hours, and performing suction filtration by using a sand core funnel to obtain a filter cake, and drying the filter cake at 20-30 ℃ for 2 hours to obtain crude carbetocin I.
(2) And adding 50ml of 95% ethanol into the crude carbetocin I, pulping at normal temperature for 4 hours, and performing suction filtration by using a sand core funnel to obtain a filter cake, and drying at 20-30 ℃ for 2 hours to obtain a crude carbetocin II.
(3) Weighing 9.0g of crude carbetocin II, adding 270ml of 10% acetic acid solution, stirring for dissolving, purifying by adopting a reversed-phase preparative high performance liquid chromatography, and collecting elution fractions containing carbetocin within 25-35 min, wherein the specific chromatographic conditions are as follows:
equipment: preparative HPLC with a column diameter and length of 60mm X300 mm, packing C18
Mobile phase: a: 0.2% aqueous sodium acetate solution B: acetonitrile
Detection wavelength: 205nm
Flow rate: 115ml/min
Elution gradient:
the eluent containing carbetocin is concentrated by 35 percent, the concentration temperature is set to be 35 ℃, and the vacuum degree is 0.090 Mpa.
After the concentration is finished, purifying again by adopting a reversed-phase preparative high performance liquid chromatography, and collecting elution fractions containing carbetocin for 30-35 min, wherein the specific chromatographic conditions are as follows:
a chromatographic column: preparative HPLC with a column diameter and length of 60mm X300 mm, packing C18
Mobile phase: a: 0.1% glacial acetic acid aqueous solution B: acetonitrile
Detection wavelength: 205nm
Flow rate: 115ml/min
Elution gradient:
the eluent containing carbetocin is concentrated by 80 percent, the concentration temperature is set to be 35 ℃, and the vacuum degree is 0.090 Mpa.
And after the concentration is finished, pouring the concentrate into a freeze-drying tray, putting the freeze-drying tray into a freeze dryer, pre-freezing the concentrate to-40 ℃, preserving heat for 1h, continuously heating the temperature to-20 ℃, preserving heat for 8h, then heating the temperature to 25 ℃, preserving heat for 10h, and obtaining the carbetocin with the vacuum degree of 0.110 mbar. The purity of carbetocin is 99.1%, and the maximum content of single impurities is 0.5%.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A purifying and refining method of carbetocin is characterized by comprising the following steps:
(1) sequentially pulping and filtering the crude carbetocin by using methyl tert-butyl ether, and drying a filter cake;
(2) drying the solid after pulping, pulping by using ethanol, performing suction filtration, and drying a filter cake;
(3) dissolving the filter cake obtained in the step (2) by using an acetic acid aqueous solution, purifying by adopting a reversed-phase preparative high performance liquid chromatography, and collecting elution fractions containing carbetocin;
(4) concentrating the elution fraction containing carbetocin obtained in the step (3), purifying again by adopting a reversed-phase preparative high performance liquid chromatography, and collecting the elution fraction containing carbetocin;
(5) and (4) concentrating the elution fraction containing carbetocin obtained in the step (4), and then putting the elution fraction into a freeze dryer for freeze drying to obtain a pure carbetocin product.
2. The method for purifying and refining carbetocin according to claim 1, wherein the beating temperature is room temperature.
3. The method for purifying and refining carbetocin according to claim 1, wherein the volume of methyl tert-butyl ether added is 5-10 times of the mass of the crude carbetocin, and the volume of ethanol added is 5-10 times of the mass of the crude carbetocin.
4. The method for purifying and refining carbetocin according to claim 1, wherein in step (3), the specific chromatographic conditions of the reversed-phase preparative high performance liquid chromatography are as follows:
mobile phase: a: 0.2% aqueous sodium acetate solution B: acetonitrile;
detection wavelength: 200-210 nm;
flow rate: 110-120 ml/min.
5. The method for purifying and refining carbetocin according to claim 4, wherein the mobile phase elution gradient of the reversed-phase preparative high performance liquid chromatography is as follows:
6. the method for purifying and refining carbetocin according to claim 1 or 5, wherein in step (4), the specific chromatographic conditions of the reversed-phase preparative high performance liquid chromatography are as follows:
mobile phase: a: 0.1% glacial acetic acid aqueous solution B: acetonitrile;
detection wavelength: 200-210 nm;
flow rate: 110-120 ml/min.
7. The purification and purification method of carbetocin according to claim 6, wherein the mobile phase elution gradient of the reversed-phase preparative high performance liquid chromatography is as follows:
8. the method for purifying and refining carbetocin according to claims 4-6, wherein the reversed-phase preparative high performance liquid chromatography column is preparative HPLC, the diameter and length of the column are 60mm x 300mm, and the packing material is octadecylsilane chemically bonded silica.
9. The method for purifying carbetocin according to claim 1, wherein the concentration conditions are 35 ℃ at the concentration temperature and 0.075MPa at the vacuum degree.
10. The method for purifying and refining carbetocin according to claim 9, wherein the freeze-drying conditions in step (5) are pre-freezing to-60 ℃ to-40 ℃, then preserving heat for 1-2 h, continuously heating to-30 ℃ to-20 ℃ and preserving heat for 8-12 h, then heating to 20 ℃ to 30 ℃ and preserving heat for 8-12 h, and the vacuum degree is at least 0.1 mbar.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103435687A (en) * | 2013-09-05 | 2013-12-11 | 杭州诺泰制药技术有限公司 | Method for purifying carbetocin |
| WO2017175233A1 (en) * | 2016-04-04 | 2017-10-12 | Davuluri Ramamohan Rao | Process for large scale liquid phase synthesis of carbetocin and its novel intermediates |
| CN107880111A (en) * | 2017-11-14 | 2018-04-06 | 杭州湃肽生化科技有限公司 | A kind of method for preparing Liraglutide |
| CN112409458A (en) * | 2019-08-21 | 2021-02-26 | 深圳翰宇药业股份有限公司 | Preparation method of carbetocin |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103435687A (en) * | 2013-09-05 | 2013-12-11 | 杭州诺泰制药技术有限公司 | Method for purifying carbetocin |
| WO2017175233A1 (en) * | 2016-04-04 | 2017-10-12 | Davuluri Ramamohan Rao | Process for large scale liquid phase synthesis of carbetocin and its novel intermediates |
| CN107880111A (en) * | 2017-11-14 | 2018-04-06 | 杭州湃肽生化科技有限公司 | A kind of method for preparing Liraglutide |
| CN112409458A (en) * | 2019-08-21 | 2021-02-26 | 深圳翰宇药业股份有限公司 | Preparation method of carbetocin |
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