CN109897099A - A kind of preparation method of Teriparatide - Google Patents
A kind of preparation method of Teriparatide Download PDFInfo
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- CN109897099A CN109897099A CN201910240604.6A CN201910240604A CN109897099A CN 109897099 A CN109897099 A CN 109897099A CN 201910240604 A CN201910240604 A CN 201910240604A CN 109897099 A CN109897099 A CN 109897099A
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- Prior art keywords
- teriparatide
- resin
- fmoc
- phe
- preparation
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Links
- 229960005460 teriparatide Drugs 0.000 title claims abstract description 98
- 108010049264 Teriparatide Proteins 0.000 title claims abstract description 97
- OGBMKVWORPGQRR-UMXFMPSGSA-N teriparatide Chemical compound C([C@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)C(C)C)[C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=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](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CNC=N1 OGBMKVWORPGQRR-UMXFMPSGSA-N 0.000 title claims abstract description 96
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 239000011347 resin Substances 0.000 claims abstract description 73
- 229920005989 resin Polymers 0.000 claims abstract description 73
- 238000005859 coupling reaction Methods 0.000 claims abstract description 36
- 230000008878 coupling Effects 0.000 claims abstract description 27
- 238000010168 coupling process Methods 0.000 claims abstract description 27
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 26
- 239000012043 crude product Substances 0.000 claims abstract description 25
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 24
- 238000006243 chemical reaction Methods 0.000 claims abstract description 22
- 150000001413 amino acids Chemical class 0.000 claims abstract description 11
- 230000000694 effects Effects 0.000 claims abstract description 11
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims abstract description 10
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000006166 lysate Substances 0.000 claims abstract description 9
- SJVFAHZPLIXNDH-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-phenylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=CC=C1 SJVFAHZPLIXNDH-QFIPXVFZSA-N 0.000 claims abstract description 7
- JFLSOKIMYBSASW-UHFFFAOYSA-N 1-chloro-2-[chloro(diphenyl)methyl]benzene Chemical compound ClC1=CC=CC=C1C(Cl)(C=1C=CC=CC=1)C1=CC=CC=C1 JFLSOKIMYBSASW-UHFFFAOYSA-N 0.000 claims abstract description 7
- 238000005406 washing Methods 0.000 claims abstract description 7
- 239000012190 activator Substances 0.000 claims abstract description 6
- 238000009833 condensation Methods 0.000 claims abstract description 5
- 230000005494 condensation Effects 0.000 claims abstract description 5
- 230000009471 action Effects 0.000 claims abstract description 3
- 230000003213 activating effect Effects 0.000 claims abstract description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 42
- 239000012071 phase Substances 0.000 claims description 37
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 36
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 25
- 238000010828 elution Methods 0.000 claims description 18
- 150000003839 salts Chemical class 0.000 claims description 18
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 14
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 12
- 239000012141 concentrate Substances 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 12
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 11
- 239000000463 material Substances 0.000 claims description 10
- 125000006239 protecting group Chemical group 0.000 claims description 10
- 239000012317 TBTU Substances 0.000 claims description 8
- CLZISMQKJZCZDN-UHFFFAOYSA-N [benzotriazol-1-yloxy(dimethylamino)methylidene]-dimethylazanium Chemical compound C1=CC=C2N(OC(N(C)C)=[N+](C)C)N=NC2=C1 CLZISMQKJZCZDN-UHFFFAOYSA-N 0.000 claims description 8
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 7
- 230000009977 dual effect Effects 0.000 claims description 7
- 238000012856 packing Methods 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 210000004899 c-terminal region Anatomy 0.000 claims description 6
- 230000008859 change Effects 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 6
- 239000007791 liquid phase Substances 0.000 claims description 6
- 238000011068 loading method Methods 0.000 claims description 6
- 238000005374 membrane filtration Methods 0.000 claims description 6
- 239000012982 microporous membrane Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 claims description 5
- 230000035484 reaction time Effects 0.000 claims description 4
- GQHTUMJGOHRCHB-UHFFFAOYSA-N 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine Chemical compound C1CCCCN2CCCN=C21 GQHTUMJGOHRCHB-UHFFFAOYSA-N 0.000 claims description 3
- 238000010511 deprotection reaction Methods 0.000 claims description 3
- 239000007790 solid phase Substances 0.000 claims description 3
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical group CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 10
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 3
- 230000008569 process Effects 0.000 abstract description 3
- 238000011084 recovery Methods 0.000 abstract description 3
- 231100001261 hazardous Toxicity 0.000 abstract description 2
- 238000003756 stirring Methods 0.000 description 8
- 239000012535 impurity Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 230000009286 beneficial effect Effects 0.000 description 5
- 238000004587 chromatography analysis Methods 0.000 description 5
- BGRWYRAHAFMIBJ-UHFFFAOYSA-N diisopropylcarbodiimide Natural products CC(C)NC(=O)NC(C)C BGRWYRAHAFMIBJ-UHFFFAOYSA-N 0.000 description 5
- 239000000945 filler Substances 0.000 description 5
- 208000001132 Osteoporosis Diseases 0.000 description 4
- 238000005336 cracking Methods 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- 238000011049 filling Methods 0.000 description 4
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000000963 osteoblast Anatomy 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 208000010392 Bone Fractures Diseases 0.000 description 2
- HNDVDQJCIGZPNO-RXMQYKEDSA-N D-histidine Chemical compound OC(=O)[C@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-RXMQYKEDSA-N 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000006340 racemization Effects 0.000 description 2
- 238000007086 side reaction Methods 0.000 description 2
- 206010065687 Bone loss Diseases 0.000 description 1
- 206010010904 Convulsion Diseases 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- DXRFZHILMCWCNG-UHFFFAOYSA-N N,N-dimethyl-1,8-naphthyridin-2-amine Chemical compound C1=CC=NC2=NC(N(C)C)=CC=C21 DXRFZHILMCWCNG-UHFFFAOYSA-N 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 108090000445 Parathyroid hormone Proteins 0.000 description 1
- 102000003982 Parathyroid hormone Human genes 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102100040247 Tumor necrosis factor Human genes 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 230000036461 convulsion Effects 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- OGBMKVWORPGQRR-UHFFFAOYSA-N forteo Chemical compound C=1NC=NC=1CC(C(=O)NC(CC(C)C)C(=O)NC(CC(N)=O)C(=O)NC(CO)C(=O)NC(CCSC)C(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NC(C(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC=1C2=CC=CC=C2NC=1)C(=O)NC(CC(C)C)C(=O)NC(CCCNC(N)=N)C(=O)NC(CCCCN)C(=O)NC(CCCCN)C(=O)NC(CC(C)C)C(=O)NC(CCC(N)=O)C(=O)NC(CC(O)=O)C(=O)NC(C(C)C)C(=O)NC(CC=1N=CNC=1)C(=O)NC(CC(N)=O)C(=O)NC(CC=1C=CC=CC=1)C(O)=O)NC(=O)C(CCCCN)NC(=O)CNC(=O)C(CC(C)C)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CCSC)NC(=O)C(CC(C)C)NC(=O)C(CCC(N)=O)NC(=O)C(NC(=O)C(CCC(O)=O)NC(=O)C(CO)NC(=O)C(NC(=O)C(N)CO)C(C)C)C(C)CC)CC1=CNC=N1 OGBMKVWORPGQRR-UHFFFAOYSA-N 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 230000009245 menopause Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 210000002997 osteoclast Anatomy 0.000 description 1
- 229960001319 parathyroid hormone Drugs 0.000 description 1
- 239000000199 parathyroid hormone Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011519 second-line treatment Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- 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
Abstract
The invention discloses a kind of preparation methods of Teriparatide, comprising the following steps: under the action of activator and coupling reagent, is coupled to obtain Fmoc-Phe- resin as resin carrier and Fmoc-Phe-OH using 2-CTC resin；Fmoc deprotecting regent is added into Fmoc-Phe- resin, obtains Phe- resin after washing after reaction and with N, N-DMF, under condensation reagent and activating reagent effect, extension one by one is carried out to amino acid and is coupled, Teriparatide-resin is obtained；The lysate containing PhSMe/PhOH/EDT system is added into Teriparatide-resin, obtains Teriparatide crude product；The thick peptide of Teriparatide is isolated and purified using C18 column, is lyophilized, obtains high-purity Teriparatide.The preparation method of Teriparatide of the present invention is at low cost, simple process, and the present invention improves the total recovery of Teriparatide, and non-hazardous to any environment.It is suitble to large-scale production.
Description
Technical field
The invention belongs to polypeptide drugs fields, are related to a kind of preparation method of Teriparatide.
Background technique
Teriparatide, illustrious name are as follows: Teriparatide is a kind of a kind of polypeptide synthesized developed by Lilly Co., Eli.
Hormone, U.S. Food and Drug Administration have approved Teriparatide on November 26th, 2002 for treating man and menopause
The osteoporosis with height risk of bone fracture of women is also used for the primary for the treatment of man and/or since sexual hypofunction causes
The osteoporosis with high risk of bone fracture.Current conventional medicine for treating osteoporosis typically just acts on osteoclast and slows down
Or bone-loss is blocked, and the derivative of this parathyroid hormone of Teriparatide has increase osteoblast number, enhances its work
Property and prevent TNF-a Induced Apoptosis in Osteoblasts effect.Because it can increase the activity and quantity of osteoblast, so as to promote bone uptake,
Second line treatment for severe osteoporosis.Furthermore Teriparatide side effect very little is generally only nausea, dizziness and skelagia convulsion
Contraction.Therefore Teriparatide has very high medical value and vast market prospect.
The molecular formula of Teriparatide is C181H291N55O51S2, relative molecular mass 4117.75, Teriparatide is by 34
Natural amino acid composition, peptide sequence are as follows: H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-
Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-
His-Asn-Phe-OH.Li Lai company is to obtain Teriparatide, such as patent US6590081 using gene expression, however, gene
Representation has the defects of technical threshold, work is complicated, serious three wastes.
However, L-His is easy racemization into D-His in known preparation method, and then racemization impurity is generated, i.e., can given birth to
At D-His32Teriparatide.D-His32The structure of Teriparatide is as follows :-Ser-Val-Ser-Glu-Ile-Gln-Leu-
Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-
Lys-Leu-Gln-Asp-Val-D-His-Asn-Phe-OH.The presence of the impurity seriously affects the content of Teriparatide and makes
With safety.Therefore it needs to find effective method to remove it and reach 0.1% or less gold standard rank.Existing conjunction
At method, prepare Teriparatide and find that technical problem of the existing technology is: synthesis purity is relatively low, at high cost, especially miscellaneous
The problem of matter D-His 32- Teriparatide content is larger, is not suitable for large-scale production.
Summary of the invention
In view of this, the present invention provides a kind of preparation method of Teriparatide, solve defect existing for current technology and
It is insufficient.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of preparation method of Teriparatide, comprising the following steps:
(1) Fmoc-Phe- resin is prepared: under the action of activator and coupling reagent, using 2-CTC resin as resin
Carrier and Fmoc-Phe-OH are coupled to obtain Fmoc-Phe- resin；
(2) Teriparatide-resin complexes are prepared: Fmoc remove-insurance being added in the Fmoc-Phe- resin obtained to step (1)
Reagent is protected, Phe- resin is obtained after washing after reaction and with N, N-DMF, under condensation reagent and activating reagent effect, presses
According to sequence of the Teriparatide from C-terminal to N-terminal, amino acid is carried out to extend coupling one by one, is corresponded to after extending coupling every time
Peptide resin, obtain Teriparatide-resin；
(3) it prepares Teriparatide crude product: being added into Teriparatide-resin obtained in step (2) and contain PhSMe/
The lysate of PhOH/EDT system removes peptide side chain protecting group and takes off Teriparatide from resin, obtains Teriparatide
Crude product；
(4) refine Teriparatide: the thick peptide of Teriparatide is isolated and purified using C18 column, is lyophilized, and it is vertical to obtain high-purity spy
Pa peptide.
Further, above-mentioned steps (1) activator is DIEA, TEA or DBU；
Further, it is N, N- diisopropylcarbodiimide/4- that above-mentioned steps (1) coupling reagent, which is the coupling reagent,
Dimethylamino naphthyridine Dual system coupling reagent；
It is using above-mentioned further beneficial effect, the use of DIEA, TEA or DBU is activator and uses N, N- bis-
Diisopropylcarbodiimide/4-dimethylaminopyridine Dual system coupling reagent is that coupling reagent can effectively improve Fmoc-Phe-OH
Reactivity, and material purity and conversion after coupling reaction can be effectively improved using coupling reagent of the present invention
Rate.
Further, the molar ratio of resin solid phase carrier and Fmoc-Phe-OH are 2-3.5: 1 in step (1)；
Be using above-mentioned further beneficial effect, limit the molar ratio of resin solid phase carrier and Fmoc-Phe-OH as
2-3.5: 1 can be improved the accuracy of reaction, reduces impurity and generates, to improve the purity of gained Fmoc-Phe- resin.
Further, the Fmoc deprotecting regent in above-mentioned steps (2) be volume ratio be 1: 1.5~4.5 triethylamine and
The mixture of DMF, the temperature of the Fmoc deprotection reaction are 30 DEG C~65 DEG C, and the reaction time is 5min~70min；
Be using above-mentioned further beneficial effect, the Fmoc deprotecting regent that the present invention limits be volume ratio and
Fmoc deprotecting regent type can effectively remove the Fmoc in Fmoc-Phe- resin, and be not susceptible to side reaction, prepare item
Part is simple and effective；In extending coupling, since there is protecting group at each amino acid N end, it is therefore desirable to first remove N-terminal protecting group again
Coupling, this is common knowledge for a person skilled in the art.The present invention preferably uses PIP/DMF (triethylamine/N, N-DMF) to mix
Solution removal N-terminal protecting group containing triethylamine is 10~30% (V) in mixed solution, remaining is DMF；Remove N-terminal protecting group reagent
Dosage is preferably every 0.05mol peptide resin 1000-1600mL.
Further, above-mentioned steps (2) condensing agent is TBTU/HOBT/DIEA or TBTU/Cl-HOBT/DIEA, condensing agent
In three kinds of combinations of materials amount dosage it is equal；
It is using above-mentioned further beneficial effect, the condensing agent composition and dosage configuration that the present invention limits are convenient,
It is obvious to the condensation effect of raw material using safe, it is generated without any impurity, does not generate any side reaction, it is nontoxic.
Further, the temperature of above-mentioned steps (2) condensation reaction is 30 DEG C~65 DEG C, reaction time 40-200min.
Further, above-mentioned steps (2) extend the molar ratio of the protected amino acid and corresponding peptide resin when coupling every time
It is 0.5-5: 1.
Further, above-mentioned steps (3) lysate is that volume ratio is PhSMe: PhOH: EDT: solvent=4: 2: 4: 90；
It is using above-mentioned further beneficial effect, lytic reagent of the present invention, residue Met in synthesis process can be aoxidized
By-product Met (O) is reduced to Met, improves thick peptide purity, reduces HPLC purifying difficulty.After purification through HPLC, impurity Met8
(O)-Teriparatide content is reduced to 0.1% or less.
Further, above-mentioned steps (4) purification step are as follows:
Teriparatide crude product adds 10% acetum to dissolve, and 0.45 μm of filtering with microporous membrane of solution purifies spare；
It is purified using high performance liquid chromatography, the reverse phase C18 that purifying is 10 μm with chromatograph packing material, flow phase system is
0.2%TFA/ aqueous solution -0.2%TFA/ acetonitrile solution, column flow rate 80mL/min are eluted using gradient system, circulation
Sample introduction takes crude product solution to be splined in chromatographic column, and it is pure to obtain Teriparatide after collection main peak boils off acetonitrile for starting mobile phase elution
Change intermediate concentrate；
Teriparatide is taken to purify intermediate concentrate, it is spare with 0.45 μm of filter membrane filtration；
It carries out changing salt using high performance liquid chromatography, flow phase system is 1% acetic acid/water solution-acetonitrile, and chromatography is used in purifying
Reverse phase C18, the column flow rate 80mL/min that filler is 10 μm are recycled loading, are splined on chromatographic column using gradient elution
In, starting mobile phase elution, acquire map, observes the variation of trap, collection changes salt main peak and detects purity with analysis liquid phase,
Salt main peak solution is changed in merging, is concentrated under reduced pressure, and obtains Teriparatide aqueous acetic acid, and it is pure to obtain Teriparatide for -10 DEG C of freeze-dryings
Product.
The spy synthesized by the method for the invention is vertical to be afraid of peptide through detecting, and purity highest is greater than 99.5%, maximum single contaminant
Less than 0.1%, total recovery is higher than 50%%, much higher than conversion ratio disclosed in the prior art.
From the above technical scheme, the present invention selects suitable synthetic schemes, and optimizes entire synthesis technology, obtains
High purity product, improves the total recovery of Teriparatide, and non-hazardous to any environment, and preparation process of the invention is suitble to
The Teriparatide of large-scale production, this technique can be effectively controlled process impurity
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1
(1) Fmoc-Phe- resin is prepared:
0.1molDIEA and 20gN are taken, N- diisopropylcarbodiimide/4-dimethylaminopyridine Dual system coupling reagent adds
Enter into the reaction vessel of 2-CTC resin for filling 0.6mol, 0.3molFmoc-Phe-OH progress coupling reaction is then added and obtains
To Fmoc-Phe- resin；
(2) Teriparatide-resin complexes are prepared:
The mixture of triethylamine and DMF is added in Fmoc-Phe- resin with 1: 2 ratio, stirring makes its reaction
30min extracts liquid later, with DMF washed product 6 times, obtains Phe- resin after washing after reaction and with N, N-DMF,
It is added under 1: 1: the 1 TBTU/HOBT/DIEA mixture effect that 50ml is prepared later, according to Teriparatide from C-terminal to N-terminal
Sequentially, amino acid extend one by one and be coupled, obtain corresponding peptide resin after extending coupling every time, coupling reaction 120 is divided
Zhong Hou obtains Teriparatide-resin；
(3) Teriparatide crude product is prepared:
It is configured containing PhSMe/PhOH/EDT body that addition 200ml is slowly added into obtained Teriparatide-resin
The lysate of system stirs while being added, and can remove peptide side chain protecting group and by Teriparatide from tree after cracking 30min
It is taken off on rouge, obtains Teriparatide crude product；
(4) Teriparatide is refined:
Teriparatide crude product adds 10% acetum to dissolve, and 0.45 μm of filtering with microporous membrane of solution purifies spare；
It is purified using high performance liquid chromatography, the reverse phase C18 that purifying is 10 μm with chromatograph packing material, flow phase system is
0.2%TFA/ aqueous solution -0.2%TFA/ acetonitrile solution, column flow rate 80mL/min are eluted using gradient system, circulation
Sample introduction takes crude product solution to be splined in chromatographic column, and it is pure to obtain Teriparatide after collection main peak boils off acetonitrile for starting mobile phase elution
Change intermediate concentrate；
Teriparatide is taken to purify intermediate concentrate, it is spare with 0.45 μm of filter membrane filtration；
It carries out changing salt using high performance liquid chromatography, flow phase system is 1% acetic acid/water solution-acetonitrile, and chromatography is used in purifying
Reverse phase C18, the column flow rate 80mL/min that filler is 10 μm are recycled loading, are splined on chromatographic column using gradient elution
In, starting mobile phase elution, acquire map, observes the variation of trap, collection changes salt main peak and detects purity with analysis liquid phase,
Salt main peak solution is changed in merging, is concentrated under reduced pressure, and obtains Teriparatide aqueous acetic acid, -10 DEG C of freeze-dryings, obtaining purity is
99.6% Teriparatide sterling, conversion ratio 51.23%.
Embodiment 2
(1) Fmoc-Phe- resin is prepared:
0.15molTEA and 25gN are taken, N- diisopropylcarbodiimide/4-dimethylaminopyridine Dual system coupling reagent adds
Enter into the reaction vessel of 2-CTC resin for filling 0.75mol, 0.5molFmoc-Phe-OH is then added and carries out coupling reaction
Obtain Fmoc-Phe- resin；
(2) Teriparatide-resin complexes are prepared:
The mixture of triethylamine and DMF is added in Fmoc-Phe- resin with 1: 3.5 ratio, stirring makes its reaction
40min extracts liquid later, with DMF washed product 6 times, obtains Phe- resin after washing after reaction and with N, N-DMF,
It is added under 1: 1: the 1 TBTU/Cl-HOBT/DIEA mixture effect that 60ml is prepared later, according to Teriparatide from C-terminal to N-terminal
Sequence, to amino acid carry out one by one extend be coupled, every time extend coupling after obtain corresponding peptide resin, coupling reaction 120
After minute, Teriparatide-resin is obtained；
(3) Teriparatide crude product is prepared:
It is configured containing PhSMe/PhOH/EDT body that addition 250ml is slowly added into obtained Teriparatide-resin
The lysate of system stirs while being added, and can remove peptide side chain protecting group and by Teriparatide from tree after cracking 30min
It is taken off on rouge, obtains Teriparatide crude product；
(4) Teriparatide is refined:
Teriparatide crude product adds 10% acetum to dissolve, and 0.45 μm of filtering with microporous membrane of solution purifies spare；
It is purified using high performance liquid chromatography, the reverse phase C18 that purifying is 10 μm with chromatograph packing material, flow phase system is
0.2%TFA/ aqueous solution -0.2%TFA/ acetonitrile solution, column flow rate 80mL/min are eluted using gradient system, circulation
Sample introduction takes crude product solution to be splined in chromatographic column, and it is pure to obtain Teriparatide after collection main peak boils off acetonitrile for starting mobile phase elution
Change intermediate concentrate；
Teriparatide is taken to purify intermediate concentrate, it is spare with 0.45 μm of filter membrane filtration；
It carries out changing salt using high performance liquid chromatography, flow phase system is 1% acetic acid/water solution-acetonitrile, and chromatography is used in purifying
Reverse phase C18, the column flow rate 80mL/min that filler is 10 μm are recycled loading, are splined on chromatographic column using gradient elution
In, starting mobile phase elution, acquire map, observes the variation of trap, collection changes salt main peak and detects purity with analysis liquid phase,
Salt main peak solution is changed in merging, is concentrated under reduced pressure, and obtains Teriparatide aqueous acetic acid, -10 DEG C of freeze-dryings, obtaining purity is
99.4% Teriparatide sterling, conversion ratio 49.55%.
Embodiment 3
(1) Fmoc-Phe- resin is prepared:
0.2molDBU and 15gN are taken, N- diisopropylcarbodiimide/4-dimethylaminopyridine Dual system coupling reagent adds
Enter into the reaction vessel of 2-CTC resin for filling 0.55mol, 0.3molFmoc-Phe-OH is then added and carries out coupling reaction
Obtain Fmoc-Phe- resin；
(2) Teriparatide-resin complexes are prepared:
The mixture of triethylamine and DMF is added in Fmoc-Phe- resin with 1: 4.5 ratio, stirring makes its reaction
30min extracts liquid later, with DMF washed product 6 times, obtains Phe- resin after washing after reaction and with N, N-DMF,
It is added under 1: 1: the 1 TBTU/HOBT/DIEA mixture effect that 50ml is prepared later, according to Teriparatide from C-terminal to N-terminal
Sequentially, amino acid extend one by one and be coupled, obtain corresponding peptide resin after extending coupling every time, coupling reaction 120 is divided
Zhong Hou obtains Teriparatide-resin；
(3) Teriparatide crude product is prepared:
It is configured containing PhSMe/PhOH/EDT body that addition 200ml is slowly added into obtained Teriparatide-resin
The lysate of system stirs while being added, and can remove peptide side chain protecting group and by Teriparatide from tree after cracking 30min
It is taken off on rouge, obtains Teriparatide crude product；
(4) Teriparatide is refined:
Teriparatide crude product adds 10% acetum to dissolve, and 0.45 μm of filtering with microporous membrane of solution purifies spare；
It is purified using high performance liquid chromatography, the reverse phase C18 that purifying is 10 μm with chromatograph packing material, flow phase system is
0.2%TFA/ aqueous solution -0.2%TFA/ acetonitrile solution, column flow rate 80mL/min are eluted using gradient system, circulation
Sample introduction takes crude product solution to be splined in chromatographic column, and it is pure to obtain Teriparatide after collection main peak boils off acetonitrile for starting mobile phase elution
Change intermediate concentrate；
Teriparatide is taken to purify intermediate concentrate, it is spare with 0.45 μm of filter membrane filtration；
It carries out changing salt using high performance liquid chromatography, flow phase system is 1% acetic acid/water solution-acetonitrile, and chromatography is used in purifying
Reverse phase C18, the column flow rate 80mL/min that filler is 10 μm are recycled loading, are splined on chromatographic column using gradient elution
In, starting mobile phase elution, acquire map, observes the variation of trap, collection changes salt main peak and detects purity with analysis liquid phase,
Salt main peak solution is changed in merging, is concentrated under reduced pressure, and obtains Teriparatide aqueous acetic acid, 10 DEG C of freeze-dryings, obtaining purity is
99.5% Teriparatide sterling, conversion ratio 51.75%.
Embodiment 4
(1) Fmoc-Phe- resin is prepared:
0.3molDIEA and 45gN are taken, N- diisopropylcarbodiimide/4-dimethylaminopyridine Dual system coupling reagent adds
Enter into the reaction vessel of 2-CTC resin for filling 0.8mol, 0.3molFmoc-Phe-OH progress coupling reaction is then added and obtains
To Fmoc-Phe- resin；
(2) Teriparatide-resin complexes are prepared:
The mixture of triethylamine and DMF is added in Fmoc-Phe- resin with 1: 1.5 ratio, stirring makes its reaction
30min extracts liquid later, with DMF washed product 6 times, obtains Phe- resin after washing after reaction and with N, N-DMF,
It is added under 1: 1: the 1 TBTU/HOBT/DIEA mixture effect that 70ml is prepared later, according to Teriparatide from C-terminal to N-terminal
Sequentially, amino acid extend one by one and be coupled, obtain corresponding peptide resin after extending coupling every time, coupling reaction 120 is divided
Zhong Hou obtains Teriparatide-resin；
(3) Teriparatide crude product is prepared:
It is configured containing PhSMe/PhOH/EDT body that addition 200ml is slowly added into obtained Teriparatide-resin
The lysate of system stirs while being added, and can remove peptide side chain protecting group and by Teriparatide from tree after cracking 30min
It is taken off on rouge, obtains Teriparatide crude product；
(4) Teriparatide is refined:
Teriparatide crude product adds 10% acetum to dissolve, and 0.45 μm of filtering with microporous membrane of solution purifies spare；
It is purified using high performance liquid chromatography, the reverse phase C18 that purifying is 10 μm with chromatograph packing material, flow phase system is
0.2%TFA/ aqueous solution -0.2%TFA/ acetonitrile solution, column flow rate 80mL/min are eluted using gradient system, circulation
Sample introduction takes crude product solution to be splined in chromatographic column, and it is pure to obtain Teriparatide after collection main peak boils off acetonitrile for starting mobile phase elution
Change intermediate concentrate；
Teriparatide is taken to purify intermediate concentrate, it is spare with 0.45 μm of filter membrane filtration；
It carries out changing salt using high performance liquid chromatography, flow phase system is 1% acetic acid/water solution-acetonitrile, and chromatography is used in purifying
Reverse phase C18, the column flow rate 80mL/min that filler is 10 μm are recycled loading, are splined on chromatographic column using gradient elution
In, starting mobile phase elution, acquire map, observes the variation of trap, collection changes salt main peak and detects purity with analysis liquid phase,
Salt main peak solution is changed in merging, is concentrated under reduced pressure, and obtains Teriparatide aqueous acetic acid, -10 DEG C of freeze-dryings, obtaining purity is
99.6% Teriparatide sterling, conversion ratio 50.22%.
Each embodiment in this specification is described in a progressive manner, the highlights of each of the examples are with other
The difference of embodiment, the same or similar parts in each embodiment may refer to each other.For device disclosed in embodiment
For, since it is corresponded to the methods disclosed in the examples, so being described relatively simple, related place is said referring to method part
It is bright.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Claims (10)
1. a kind of preparation method of Teriparatide, which comprises the following steps:
(1) Fmoc-Phe- resin is prepared: under the action of activator and coupling reagent, using 2-CTC resin as resin carrier
It is coupled to obtain Fmoc-Phe- resin with Fmoc-Phe-OH；
(2) Teriparatide-resin complexes are prepared: Fmoc deprotection examination being added in the Fmoc-Phe- resin obtained to step (1)
Agent obtains Phe- resin after washing after reaction and with N, N-DMF, under condensation reagent and activating reagent effect, according to spy
Vertical sequence of the pa peptide from C-terminal to N-terminal carries out amino acid to extend coupling one by one, obtains corresponding peptide after extending coupling every time
Resin obtains Teriparatide-resin；
(3) it prepares Teriparatide crude product: being added into Teriparatide-resin obtained in step (2) and contain PhSMe/PhOH/
The lysate of EDT system removes peptide side chain protecting group and takes off Teriparatide from resin, obtains Teriparatide crude product；
(4) refine Teriparatide: the thick peptide of Teriparatide is isolated and purified using C18 column, is lyophilized, is obtained high-purity Te Lipa
Peptide.
2. a kind of preparation method of Teriparatide according to claim 1, which is characterized in that step (1) described activator
For DIEA, TEA or DBU.
3. a kind of preparation method of Teriparatide according to claim 1, which is characterized in that step (1) the coupling examination
Agent is that the coupling reagent is N, N- diisopropylcarbodiimide/4-dimethylaminopyridine Dual system coupling reagent.
4. a kind of preparation method of Teriparatide according to claim 1, which is characterized in that resin solid phase in step (1)
The molar ratio of carrier and Fmoc-Phe-OH are 2-3.5: 1.
5. a kind of preparation method of Teriparatide according to claim 1, which is characterized in that described in step (2)
Fmoc deprotecting regent be volume ratio be 1: 1.5~4.5 triethylamine and DMF mixture, the Fmoc deprotection reaction
Temperature is 30 DEG C~65 DEG C, and the reaction time is 5min~70min.
6. a kind of preparation method of Teriparatide according to claim 1, which is characterized in that step (2), the condensing agent
For TBTU/HOBT/DIEA or TBTU/Cl-HOBT/DIEA, the amount dosage of three kinds of combinations of materials is equal in condensing agent.
7. a kind of preparation method of Teriparatide according to claim 1, which is characterized in that step (2), the condensation are anti-
The temperature answered is 30 DEG C~65 DEG C, reaction time 40-200min.
8. a kind of preparation method of Teriparatide according to claim 1, which is characterized in that step (2) extends even every time
The molar ratio of the protected amino acid and corresponding peptide resin is 0.5-5: 1 when connection.
9. a kind of preparation method of Teriparatide according to claim 1, which is characterized in that step (3), the lysate
It is PhSMe: PhOH: EDT for volume ratio: solvent=4: 2: 4: 90.
10. a kind of preparation method of Teriparatide according to claim 1, which is characterized in that step (4), the purifying
Step are as follows:
Teriparatide crude product adds 10% acetum to dissolve, and 0.45 μm of filtering with microporous membrane of solution purifies spare；
It is purified using high performance liquid chromatography, the reverse phase C18 that purifying is 10 μm with chromatograph packing material, flow phase system is
0.2%TFA/ aqueous solution -0.2%TFA/ acetonitrile solution, column flow rate 80mL/min are eluted using gradient system, circulation
Sample introduction takes crude product solution to be splined in chromatographic column, and it is pure to obtain Teriparatide after collection main peak boils off acetonitrile for starting mobile phase elution
Change intermediate concentrate；
Teriparatide is taken to purify intermediate concentrate, it is spare with 0.45 μm of filter membrane filtration；
It carries out changing salt using high performance liquid chromatography, flow phase system is 1% acetic acid/water solution-acetonitrile, and chromatograph packing material is used in purifying
Loading is recycled, is splined in chromatographic column, opens using gradient elution for 10 μm of reverse phase C18, column flow rate 80mL/min
Dynamic mobile phase elution, acquires map, observes the variation of trap, and collection changes salt main peak and with liquid phase detection purity is analyzed, merges
Salt main peak solution is changed, is concentrated under reduced pressure, Teriparatide aqueous acetic acid is obtained, -10 DEG C of freeze-dryings obtain Teriparatide sterling.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114478750A (en) * | 2021-12-28 | 2022-05-13 | 深圳翰宇药业股份有限公司 | Purification method of teriparatide |
| CN115656391A (en) * | 2022-12-12 | 2023-01-31 | 哈尔滨吉象隆生物技术有限公司 | Method for detecting impurities contained in teriparatide |
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| CN115656391A (en) * | 2022-12-12 | 2023-01-31 | 哈尔滨吉象隆生物技术有限公司 | Method for detecting impurities contained in teriparatide |
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Application publication date: 20190618 |