CN112175068A - Method for purifying semaglutide - Google Patents
Method for purifying semaglutide Download PDFInfo
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- CN112175068A CN112175068A CN202011039848.7A CN202011039848A CN112175068A CN 112175068 A CN112175068 A CN 112175068A CN 202011039848 A CN202011039848 A CN 202011039848A CN 112175068 A CN112175068 A CN 112175068A
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- DLSWIYLPEUIQAV-UHFFFAOYSA-N Semaglutide Chemical compound CCC(C)C(NC(=O)C(Cc1ccccc1)NC(=O)C(CCC(O)=O)NC(=O)C(CCCCNC(=O)COCCOCCNC(=O)COCCOCCNC(=O)CCC(NC(=O)CCCCCCCCCCCCCCCCC(O)=O)C(O)=O)NC(=O)C(C)NC(=O)C(C)NC(=O)C(CCC(N)=O)NC(=O)CNC(=O)C(CCC(O)=O)NC(=O)C(CC(C)C)NC(=O)C(Cc1ccc(O)cc1)NC(=O)C(CO)NC(=O)C(CO)NC(=O)C(NC(=O)C(CC(O)=O)NC(=O)C(CO)NC(=O)C(NC(=O)C(Cc1ccccc1)NC(=O)C(NC(=O)CNC(=O)C(CCC(O)=O)NC(=O)C(C)(C)NC(=O)C(N)Cc1cnc[nH]1)C(C)O)C(C)O)C(C)C)C(=O)NC(C)C(=O)NC(Cc1c[nH]c2ccccc12)C(=O)NC(CC(C)C)C(=O)NC(C(C)C)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CCCNC(N)=N)C(=O)NCC(O)=O DLSWIYLPEUIQAV-UHFFFAOYSA-N 0.000 title claims abstract description 42
- 229950011186 semaglutide Drugs 0.000 title claims abstract description 41
- 108010060325 semaglutide Proteins 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 29
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 72
- 238000000746 purification Methods 0.000 claims abstract description 46
- 239000012071 phase Substances 0.000 claims abstract description 41
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 28
- 239000000945 filler Substances 0.000 claims abstract description 22
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000003960 organic solvent Substances 0.000 claims abstract description 19
- 238000010828 elution Methods 0.000 claims abstract description 18
- 239000000243 solution Substances 0.000 claims abstract description 14
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 10
- 238000004108 freeze drying Methods 0.000 claims abstract description 8
- 239000000706 filtrate Substances 0.000 claims abstract description 5
- 238000001914 filtration Methods 0.000 claims abstract description 5
- 239000012528 membrane Substances 0.000 claims abstract description 5
- 239000012468 concentrated sample Substances 0.000 claims abstract description 3
- 238000005336 cracking Methods 0.000 claims abstract description 3
- 125000006239 protecting group Chemical group 0.000 claims abstract description 3
- 239000011347 resin Substances 0.000 claims abstract description 3
- 229920005989 resin Polymers 0.000 claims abstract description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 claims abstract description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 24
- 239000012535 impurity Substances 0.000 claims description 23
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 239000000377 silicon dioxide Substances 0.000 claims description 12
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 5
- 239000002245 particle Substances 0.000 claims description 5
- 230000006340 racemization Effects 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 4
- 101100338242 Drosophila virilis His1.1 gene Proteins 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 2
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- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical class C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
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- 239000000337 buffer salt Substances 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- UNXNGGMLCSMSLH-UHFFFAOYSA-N dihydrogen phosphate;triethylazanium Chemical compound OP(O)(O)=O.CCN(CC)CC UNXNGGMLCSMSLH-UHFFFAOYSA-N 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- FUOOLUPWFVMBKG-UHFFFAOYSA-N 2-Aminoisobutyric acid Chemical compound CC(C)(N)C(O)=O FUOOLUPWFVMBKG-UHFFFAOYSA-N 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 102000007446 Glucagon-Like Peptide-1 Receptor Human genes 0.000 description 2
- 108010086246 Glucagon-Like Peptide-1 Receptor Proteins 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000003795 desorption Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000002390 rotary evaporation Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 101710198884 GATA-type zinc finger protein 1 Proteins 0.000 description 1
- 102000051325 Glucagon Human genes 0.000 description 1
- 108060003199 Glucagon Proteins 0.000 description 1
- 229940089838 Glucagon-like peptide 1 receptor agonist Drugs 0.000 description 1
- 101000788682 Homo sapiens GATA-type zinc finger protein 1 Proteins 0.000 description 1
- 206010021518 Impaired gastric emptying Diseases 0.000 description 1
- YSDQQAXHVYUZIW-QCIJIYAXSA-N Liraglutide Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCNC(=O)CC[C@H](NC(=O)CCCCCCCCCCCCCCC)C(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=C(O)C=C1 YSDQQAXHVYUZIW-QCIJIYAXSA-N 0.000 description 1
- 108010019598 Liraglutide Proteins 0.000 description 1
- 102100040918 Pro-glucagon Human genes 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 238000011978 dissolution method Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 1
- 229960004666 glucagon Drugs 0.000 description 1
- 239000003877 glucagon like peptide 1 receptor agonist Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000003914 insulin secretion Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 229960002701 liraglutide Drugs 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
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- 235000012054 meals Nutrition 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
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- 239000006186 oral dosage form Substances 0.000 description 1
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- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- 238000010200 validation analysis Methods 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
- C07K14/605—Glucagons
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a method for purifying semaglutide, which comprises the following steps: 1) obtaining a cracking resin prepared by a solid-phase synthesis method and the crude semaglutide with a protecting group removed, and dissolving the crude semaglutide with a phosphate buffer solution containing an organic solvent; 2) filtering the crude peptide solution of the Semetreuptatide by using a 0.22-micron organic filter membrane, and collecting the filtrate for later use; 3) the first step of purification is to perform gradient elution by taking reversed-phase filler as a stationary phase, phosphate buffer solution containing an organic solvent as a phase A and acetonitrile as a phase B; 4) the second step of purification is carried out by taking reversed phase filler as a stationary phase, dilute acetic acid solution as a phase A and acetonitrile as a phase B, and carrying out gradient elution; 5) and (4) freeze-drying the concentrated sample to obtain the semaglutide. According to the method, the crude peptide is dissolved by the phosphate buffer solution containing the organic solvent, the pH of the dissolved crude peptide solution is more stable and is not easy to separate out, so that the blockage of a chromatographic column and equipment is avoided, and the separation effect is improved.
Description
Technical Field
The invention relates to the technical field of purification of polypeptide drugs, in particular to a separation and purification method of semaglutide, belonging to the field of pharmaceutical chemistry.
Background
The name of Chinese: semetreutide
The name of English: semaglutide
CAS number 910463-68-2
Molecular formula C187H291N45O59
Molecular weight 4113.64
The structure is as follows:
the semaglutide is a new generation of long-acting GLP-1 analogue developed and developed by Novonide company, is used for treating type II diabetes, has the effects of reducing weight and protecting cardiovascular besides reducing blood sugar, and is a medicament with the best effects of reducing blood sugar and reducing weight in GLP-1 medicaments at present. The FDA approved the injectable dosage form to market in 2017, and approved the oral dosage form to market in No. 9/20 in 2019.
Semetreuptade is a GLP-1 analog, and has 94% homology with human GLP-1. The long-acting mechanism of semaglutide is based on the modification of the structure, and semaglutide is modified to alpha-aminoisobutyric acid at the 8 th position for increasing the stabilization effect and avoiding the degradation by DPP-4 enzyme. In addition, through structural modification, the position of the 26 th lysine of the peptide chain is connected with the 18-carbon aliphatic diacid side chain, compared with the liraglutide of C16, the affinity of the increased carbon chain to albumin is enhanced by 5-6 times, and the increased carbon chain is combined with albumin, so that the molecular weight of the product is increased, the phenomenon that the product is rapidly cleared by the kidney and prevented from metabolic degradation is avoided, and the half-life period in vivo is prolonged. And lysine at position 34 was replaced with arginine to ensure stability of the C18 side chain at position 26. Therefore, the product has a half-life of up to 1 week, and the semaglutide is a GLP-1 receptor agonist, binds and activates a GLP-1 receptor, stimulates insulin secretion in a glucose-dependent manner by mediating the GLP-1 receptor, and reduces glucagon secretion. In addition, a decrease in blood glucose is also associated with delayed gastric emptying during the initial phase of a meal.
More published technologies on polypeptide purification appear at present, for example, CN201711474148 discloses a method for purifying semaglutide, CN201811482820 discloses a filler special for semaglutide purification and a purification method thereof, CN201911201916 discloses a separation and purification method of semaglutide, and CN201810663478 discloses a method for purifying semaglutide. The existing purification method has the problems of unstable dissolution method of a crude peptide sample, unobvious removal of specific impurities, low purification yield and the like.
Disclosure of Invention
The invention provides a purification process of semaglutide, which has strong operability, simple and stable process and high yield aiming at the physicochemical characteristics of the semaglutide.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for purifying semaglutide comprising the steps of:
1) obtaining a solid-phase synthesis method cracking resin and the simetrexed crude peptide with a protective group removed, and dissolving the simetrexed crude peptide by using a phosphate buffer solution containing an organic solvent;
2) filtering the crude peptide solution of the Semetreuptatide by using a 0.22-micron organic filter membrane, and collecting the filtrate for later use;
3) the first step of purification is to use reversed phase filler as a fixed phase, use a buffer salt solution containing an organic solvent as an A phase and acetonitrile as a B phase, perform gradient elution, collect components with the purity of more than 95 percent and the single impurity of less than 0.8 percent and concentrate the components;
4) the second step of purification is to take reversed phase filler as a fixed phase, dilute acetic acid solution as an A phase and acetonitrile as a B phase, perform gradient elution and collect components with the purity of more than 99.5 percent and the single impurity of less than 0.1 percent;
5) and (4) freeze-drying the concentrated sample to obtain the semaglutide. Putting the mixture into a freeze-drying tray for freeze-drying, wherein the desorption temperature of freeze-drying is 35 ℃, and the vacuum pressure is 0.1 pa.
In an embodiment of the present invention, the organic solvent used for dissolving the sample in step 1) is a polar organic reagent, preferably at least one of methanol, acetonitrile, ethanol and isopropanol, and more preferably acetonitrile.
In an embodiment of the present invention, the organic solvent used for dissolving the sample in step 3) is a polar organic reagent, preferably at least one of methanol, acetonitrile, ethanol and isopropanol, and more preferably acetonitrile.
In an embodiment of the invention, the volume percentage of organic solvent is 10% to 30%, preferably 15% to 25%; and the volume percentage difference of the organic solvent in the step 1) and the step 3) is not more than 10 percent.
In the embodiment of the present invention, the phosphate buffer solution in step 1) and step 3) includes a buffer salt system of triethylamine phosphate, disodium hydrogen phosphate and dipotassium hydrogen phosphate, wherein triethylamine phosphate is preferred, the volume percentage of triethylamine phosphate is 0.05% -0.8%, step 1) is preferred to be 0.2%, and step 2) is preferred to be 0.2%.
In the examples of the present invention, the phosphate buffer solution described in step 1) and step 3) has a pH of 6.5 to 9, preferably a pH of 7 to 8. The pH is preferably adjusted with triethylamine.
In the embodiment of the invention, the reversed-phase filler in the step 3) comprises tetraalkylsilane bonded silica filler and octaalkylsilane bonded silica filler, and the particle size of the reversed-phase filler is between 5 and 100 micrometers; octaalkylsilane bonded silica fillers, 10 μm, are preferred.
In an embodiment of the present invention, the reversed phase filler in step 4) is an octadecylsilane bonded silica filler having a particle size of between 5 and 100 μm, preferably 10 μm.
In the embodiment of the present invention, the concentration of the dilute acetic acid in the step 4) is 0.01-1.0% (volume ratio), preferably 0.2% (volume ratio).
In the examples of the present invention, the method of gradient elution in step 3) is a%: 80% -60%, B%: 20-40%, linear gradient elution for 60 min.
In the examples of the present invention, the method of gradient elution in step 4) is a%: 70% -50%, B%: 30% -50%, linear gradient elution for 60 min.
In an embodiment of the invention, the purification method is a method for removing racemic impurities of semaglutide, preferably, the racemic impurities of semaglutide are H-D-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (AEEA-AEEA-Gamma-Glu-OCDA) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH [ His ] (His-Glu-Leu-Val-Arg-Gly-Asp-Ala-Ala-Lys1Racemization and H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-D-Ala-Ala-Lys (AEEA-AEEA-Gamma-Glu-OCDA) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH [ Ala18Racemization ].
In the examples of the present invention, H-D-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (AEEA-AEEA- γ -Glu-OCDA) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH [ His1 rac ] indicates racemization of His at position 1 in semaglutide.
In the examples of the present invention, H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-D-Ala-Ala-Lys (AEEA-AEEA-Gamma-Glu-OCDA) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH [ Ala18Racemization ] refers to racemization of Ala at position 18 in semaglutide.
Compared with the prior art, the invention has the following advantages:
1. the crude peptide of the semaglutide contains a large amount of trifluoroacetic acid, the pH value of a crude peptide solution is difficult to control by dissolving the crude peptide with dilute ammonia water or acetonitrile in the prior art, the pH value of the dissolved crude peptide solution is more stable and difficult to separate out by dissolving the crude peptide with phosphate buffer solution containing organic solvent, thereby avoiding the blockage of a chromatographic column and equipment and improving the separation effect. Meanwhile, the dissolving capacity of the crude peptide is increased, and the concentration of the crude peptide reaches 50 g/L.
2. The solubility of the semaglutide and impurities thereof is particularly poor, and the A mobile phase and the crude peptide dissolving solution in the step 3) are basically the same, so that the separation of the sample caused by overlarge difference of a mobile phase system when the sample enters a chromatographic column is avoided, and the diffusion effect of the sample on a column head is reduced.
3. The first step of purification adopts octaalkylsilane bonded silica filler, organic solvent-containing buffer salt as phase A and acetonitrile as phase B, and performs gradient elution to remove most impurities and fragment impurities of semaglutide, especially partial defective peptide.
Such as: H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH [ Des-Lys (AEEA-AEEA-gamma-Glu-OCDA)20】、H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OCDA)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH【Des-Ser11】、H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Lys(AEEA-AEEA-γ-Glu-OCDA)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH【Des-Ala18】
4. And the second step of purification adopts octadecylsilane chemically bonded silica filler, dilute acetic acid solution as phase A and acetonitrile as phase B, and performs gradient elution to remove racemized impurities which are difficult to remove in the semaglutide. Such as: [ His ]1Rac and [ Ala ]18Racemization ].
4.1 Synthesis of impurities H-D-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (AEEA-AEEA-Gamma-Glu-OCDA) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH [ His ]1Racemization it was confirmed that the racemic impurity remained for 9.851min in the first purification step (FIG. 3), which was 0.36% after the first purification step, and was completely removed after the second purification step of our invention.
4.2 Synthesis of the impurity H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Tyr-Leu-Glu-Gly-Gln-D-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OCDA)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH【Ala18Racemization it was confirmed that the racemic impurity remained for 10.256min in the first purification control process (FIG. 3), which was 0.73% after the first purification step, and was completely removed after the second purification step of our invention.
5. In the prior art, ammonia water is used for adjusting the pH value, and triethylamine is used for adjusting the pH value, so that the triethylamine can inhibit the tailing of a preparation peak more than the ammonia water; further, the purity of the fraction collected in the first step of purification is more than 95%, which is greatly improved compared with the prior art.
Drawings
FIG. 1: the crude semaglutide prepared in example 1 has a color spectrum.
FIG. 2: the first step purification chromatogram of semaglutide prepared in example 2.
FIG. 3: the first-step purification of the semaglutide prepared in example 2 was performed by chromatography.
FIG. 4: example 3 second step purification of Semetglutide chromatogram
FIG. 5: chromatogram of semaglutide protien peptide prepared in example 6.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below, but the present invention is not to be construed as being limited to the implementable range thereof.
EXAMPLE 1 crude peptide pretreatment
Preparing a dissolving buffer solution: 2mL of phosphoric acid was measured and poured into 750mL of water, the pH was adjusted to 8.0 with triethylamine, 200mL of acetonitrile was added, and the volume was adjusted to 1L with water. Taking 1L of prepared dissolving buffer solution, adding 30g of crude peptide, carrying out ultrasonic stirring to completely dissolve the sample, filtering by using a 0.22 mu m organic filter membrane, and collecting filtrate.
EXAMPLE 2 first step purification
And (3) purification conditions: a chromatographic column: the chromatographic column using the octaalkylsilane bonded silica as a stationary phase has the following diameters and lengths: 5cm × 25 cm. Mobile phase: phase A: 0.2% phosphoric acid (v/v) in 20% acetonitrile (v/v) water, adjusted to pH8.0 with triethylamine; phase B: acetonitrile, flow rate: 80mL/min, gradient: b%: 20-40%, and linear gradient elution for 60 min; detection wavelength: 230 nm. The amount of the sample was 5.0 g. Collecting the components with the purity of more than 95 percent and the single impurity of less than 0.8 percent to be steamed for standby.
Wherein, the preparation method of the phase A comprises the steps of measuring 2mL of phosphoric acid, pouring 750mL of water, adjusting the pH value to 8.0 by triethylamine, adding 200mL of acetonitrile, and fixing the volume to 1L by water.
EXAMPLE 3 first step concentration of purified fraction
Mixing the collected qualified fractions of the first-step purification of the semaglutide, and performing reduced pressure rotary evaporation and concentration at the water temperature of 30 ℃ and the vacuum degree of-0.09 Mbar to remove most of organic solvent for later use.
EXAMPLE 4 second step purification
And (3) purification conditions: a chromatographic column: the chromatographic column using octadecylsilane chemically bonded silica as a stationary phase has the following diameter and length: 5cm × 25 cm. Mobile phase: phase A: 0.2% (volume ratio) dilute acetic acid solution; phase B: acetonitrile, flow rate: 80mL/min, gradient: b%: 30-50%, linear gradient elution for 60 min; detection wavelength: 230 nm. The amount of sample was 4.11 g. Collecting the components with the purity of more than 99.5 percent and the single impurity of less than 0.1 percent to be rotary evaporated for standby.
EXAMPLE 5 concentration of the second purified fraction
Mixing the collected qualified fractions of the second step of purification of the semaglutide, and performing reduced pressure rotary evaporation and concentration to 20mg/mL at the water temperature of 30 ℃ and the vacuum degree of-0.09 Mbar.
EXAMPLE 6 freezing and drying
And (3) putting the sample into a freeze-drying tray, and freeze-drying at the desorption temperature of 35 ℃ under the vacuum pressure of 0.1Pa to obtain 3.693 g of the semaglutide with the maximum single impurity content of less than 0.1 percent, wherein the total purification yield is 73.86%.
EXAMPLE 7 validation of racemic product
The racemic impurity position was verified by synthesizing the racemic impurity. The racemic impurities include H-D-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (AEEA-AEEA-Gamma-Glu-OCDA) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH [ His1 racemization ] and H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Tyr-Leu-Glu-Gly-Gln-D-Ala-Ala-Lys (AEEA-AEEA-Gamma-Glu-OCDA) -Glu-Phe-Ile-Ala-Trp -Leu-Val-Arg-Gly-OH [ Ala18 rac ], the retention times of the two racemates were 9.851min and 10.256min, respectively, as verified by the conditions of example 2, and it can be seen from the results of example 2 that the impurities after the first purification were 0.36% and 0.73%, whereas the two racemates were removed after the second purification.
Comparative example 1: pretreatment of crude peptide
The crude peptide was solubilized using the solubilization system shown in the table below. Adding crude peptide into the prepared dissolving buffer solution, performing ultrasonic stirring to completely dissolve the sample, filtering with a 0.22 μm organic filter membrane, and collecting the filtrate.
Because the crude semaglutide is not easy to dissolve, the invention adopts the phosphoric acid aqueous solution with 20 percent of acetonitrile and pH of 8, which can greatly improve the solvent of the crude peptide, can be more efficiently used for column chromatography separation, and overcomes the problem that the prior art can only reduce the sample loading amount and the sample loading concentration. Meanwhile, the solution prepared by the method is very stable, does not precipitate, and can avoid column blockage.
The invention uses a column of 5cm, can load 5g of crude peptide, and greatly improves the separation efficiency and the sample loading amount compared with the prior art.
Claims (10)
1. A method for purifying semaglutide comprising the steps of:
1) obtaining a cracking resin prepared by a solid-phase synthesis method and the crude semaglutide with a protecting group removed, and dissolving the crude semaglutide with a phosphate buffer solution containing an organic solvent;
2) filtering the crude peptide solution of the Semetreuptatide by using a 0.22-micron organic filter membrane, and collecting the filtrate for later use;
3) the first step of purification is to use reversed phase filler as a fixed phase, phosphate buffer solution containing organic solvent as an A phase and acetonitrile as a B phase, perform gradient elution, collect components with purity of more than 95 percent and single impurity of less than 0.8 percent and concentrate the components;
4) the second step of purification is to take reversed phase filler as a fixed phase, dilute acetic acid solution as an A phase and acetonitrile as a B phase, perform gradient elution and collect components with the purity of more than 99.5 percent and the single impurity of less than 0.1 percent;
5) and (4) freeze-drying the concentrated sample to obtain the semaglutide.
2. The purification process according to claim 1, wherein the organic solvent in step 1) is a polar organic reagent, preferably at least one of methanol, acetonitrile, ethanol, isopropanol, more preferably acetonitrile.
3. The purification process according to claim 1, wherein in step 3) the organic solvent is a polar organic reagent, preferably at least one of methanol, acetonitrile, ethanol, isopropanol, more preferably acetonitrile.
4. The purification process according to claim 1, wherein the volume percentage of organic solvent in step 1) and step 3) is 10% to 30%, preferably 15% to 25%; and the volume percentage difference of the organic solvent in the step 1) and the step 3) is not more than 10 percent.
5. The purification method as claimed in claim 1, wherein in step 1) and step 3), each 1L of the phosphate buffer solution containing the organic solvent is prepared by measuring 2mL of phosphoric acid, pouring 700-750mL of water, adjusting pH to 6.5-9 with triethylamine, adding 150-250mL of acetonitrile, and diluting to 1L with water.
6. The purification method according to claim 1, wherein the gradient elution method in step 3) is B%: 20-40%, and linear gradient elution for 60 min;
the gradient elution method in the step 4) is B%: 30% -50%, linear gradient elution for 60 min.
7. The purification process according to claim 1, wherein the reverse phase filler in step 3) is selected from tetraalkylsilane-bonded silica fillers, octaalkylsilane-bonded silica fillers having a particle size of between 5 and 100 μm; octaalkylsilane bonded silica fillers having a particle size of 10 μm are preferred.
8. The purification method according to claim 1, wherein the reverse phase filler in step 4) is an octadecylsilane bonded silica filler having a particle size of 5-100 μm, preferably 10 μm.
9. The purification method according to claim 1, wherein the concentration of the dilute acetic acid in the step 4) is 0.01-1.0% by volume.
10. The purification process according to claim 1, which is a process for removing racemization impurities of semaglutide, preferably, H-D-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (AEEA-AEEA- γ -Glu-OCDA) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH [ His1 racemization ] and H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Tyr-Leu-Glu-Gly-Gln-D -Ala-Ala-Lys (AEEA-AEEA- γ -Glu-OCDA) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH [ Ala18 rac ].
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