CN103467574B - Purification method of desmopressin acetate - Google Patents
Purification method of desmopressin acetate Download PDFInfo
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- CN103467574B CN103467574B CN201310399955.4A CN201310399955A CN103467574B CN 103467574 B CN103467574 B CN 103467574B CN 201310399955 A CN201310399955 A CN 201310399955A CN 103467574 B CN103467574 B CN 103467574B
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- 108010000437 Deamino Arginine Vasopressin Proteins 0.000 title claims abstract description 103
- 238000000034 method Methods 0.000 title claims abstract description 29
- 238000000746 purification Methods 0.000 title abstract description 18
- FIEYHAAMDAPVCH-UHFFFAOYSA-N 2-methyl-1h-quinazolin-4-one Chemical compound C1=CC=C2NC(C)=NC(=O)C2=C1 FIEYHAAMDAPVCH-UHFFFAOYSA-N 0.000 title abstract description 7
- 229960002845 desmopressin acetate Drugs 0.000 title abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 238000011068 loading method Methods 0.000 claims abstract description 3
- 229960004281 desmopressin Drugs 0.000 claims description 121
- NFLWUMRGJYTJIN-NXBWRCJVSA-N desmopressin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSCCC(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(N)=O)=O)CCC(=O)N)C1=CC=CC=C1 NFLWUMRGJYTJIN-NXBWRCJVSA-N 0.000 claims description 94
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 36
- 239000012535 impurity Substances 0.000 claims description 33
- 239000000126 substance Substances 0.000 claims description 21
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 17
- 239000000945 filler Substances 0.000 claims description 11
- 229960000583 acetic acid Drugs 0.000 claims description 7
- 239000000523 sample Substances 0.000 claims description 7
- 238000004108 freeze drying Methods 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 2
- 239000012488 sample solution Substances 0.000 claims description 2
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims 1
- 238000005406 washing Methods 0.000 abstract description 7
- 238000010828 elution Methods 0.000 abstract description 6
- 239000000047 product Substances 0.000 abstract description 6
- 238000001035 drying Methods 0.000 abstract description 3
- 239000002253 acid Substances 0.000 abstract description 2
- 239000012043 crude product Substances 0.000 abstract description 2
- 229920000642 polymer Polymers 0.000 abstract 2
- 230000001105 regulatory effect Effects 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 69
- 108090000765 processed proteins & peptides Proteins 0.000 description 36
- 239000013558 reference substance Substances 0.000 description 31
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 25
- 238000002474 experimental method Methods 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 12
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- 238000010532 solid phase synthesis reaction Methods 0.000 description 10
- 238000003756 stirring Methods 0.000 description 10
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- 239000007864 aqueous solution Substances 0.000 description 9
- 238000004128 high performance liquid chromatography Methods 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 6
- 238000012937 correction Methods 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 3
- TZCYLJGNWDVJRA-UHFFFAOYSA-N 6-chloro-1-hydroxybenzotriazole Chemical compound C1=C(Cl)C=C2N(O)N=NC2=C1 TZCYLJGNWDVJRA-UHFFFAOYSA-N 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
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- 230000005526 G1 to G0 transition Effects 0.000 description 2
- 208000032843 Hemorrhage Diseases 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- NDKDFTQNXLHCGO-UHFFFAOYSA-N 2-(9h-fluoren-9-ylmethoxycarbonylamino)acetic acid Chemical compound C1=CC=C2C(COC(=O)NCC(=O)O)C3=CC=CC=C3C2=C1 NDKDFTQNXLHCGO-UHFFFAOYSA-N 0.000 description 1
- DKIDEFUBRARXTE-UHFFFAOYSA-N 3-mercaptopropanoic acid Chemical compound OC(=O)CCS DKIDEFUBRARXTE-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical class N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- BTWRSVTVZVCMEM-LSCKAKRGSA-N C[C@](C(N(C)C(CCSSC)=O)I)(NCC(CCCCCN[I](N)N)NC[C@H](CN)O)O Chemical compound C[C@](C(N(C)C(CCSSC)=O)I)(NCC(CCCCCN[I](N)N)NC[C@H](CN)O)O BTWRSVTVZVCMEM-LSCKAKRGSA-N 0.000 description 1
- ODKSFYDXXFIFQN-SCSAIBSYSA-N D-arginine Chemical compound OC(=O)[C@H](N)CCCNC(N)=N ODKSFYDXXFIFQN-SCSAIBSYSA-N 0.000 description 1
- 206010051055 Deep vein thrombosis Diseases 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 208000031220 Hemophilia Diseases 0.000 description 1
- 208000009292 Hemophilia A Diseases 0.000 description 1
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 description 1
- 102100026383 Vasopressin-neurophysin 2-copeptin Human genes 0.000 description 1
- 206010047249 Venous thrombosis Diseases 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 125000004202 aminomethyl group Chemical group [H]N([H])C([H])([H])* 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229940078916 carbamide peroxide Drugs 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 201000010064 diabetes insipidus Diseases 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 125000005519 fluorenylmethyloxycarbonyl group Chemical group 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 230000000025 haemostatic effect Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 239000002932 luster Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 1
- 235000011837 pasties Nutrition 0.000 description 1
- 229960003742 phenol Drugs 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- REFMEZARFCPESH-UHFFFAOYSA-M sodium;heptane-1-sulfonate Chemical compound [Na+].CCCCCCCS([O-])(=O)=O REFMEZARFCPESH-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
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- 231100000419 toxicity Toxicity 0.000 description 1
- 150000003852 triazoles Chemical class 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
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- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a purification method of desmopressin acetate. The purification method of desmopressin acetate is characterized by comprising the following steps: firstly, dissolving a desmopressin acetate crude product with water, and regulating pH to be acid; secondly, setting a gradient according to volume fraction, and washing a polymer reversed phase chromatographic column; thirdly, loading the solution obtained in the step 1 into the polymer reversed phase chromatographic column; fourthly, setting the gradient according to the volume fraction, maintaining the set gradient for 2 minutes, then increasing the ratio of the flowing phase B to 25% within 5 minutes, then increasing the ratio of the flowing phase B to 40% within 30 minutes, and partially collecting an elution fraction, wherein a flowing phase during an initial state of an elution gradient is 5%; fifthly, drying the elution fraction to obtain the purified desmopressin acetate product.
Description
Technical field:
The present invention relates to a kind of purification process of Desmopressin.
Background technology:
Desmopressin, illustrious name is: Desmopressin, and be a kind of ring type polypeptide, structural formula is as follows:
Can be abbreviated as:
Desmopressin is the analog of natural smart ammonia salt vassopressin, is carry out the change of two places to the chemical structure of natural hormone and obtain, i.e. Cys deaminize and replace 8-L-arginine with 8-D-arginine.Desmopressin acetate has good haemostatic effect and can not produce the feature of deep venous thrombosis.Be mainly used to treatment hemophilia, diabetes insipidus and therapeutic Bleeding control and operation consent prevention hemorrhage.Effective and side effect is little, be a kind of polypeptide drugs of good market prospect.
Chinese patent CN101372504 discloses a kind of purification process of Desmopressin, concrete steps are: the first step purifying: be stationary phase by thick peptide solution in order to octadecylsilane chemically bonded silica, take phosphate buffered saline buffer as A phase, trifluoroacetic acid aqueous solution is B phase, carry out gradient elution purifying.Second step purifying: be stationary phase in order to octadecylsilane chemically bonded silica after the first step purifying gained object peptide solution is concentrated, take aqueous acetic acid as A phase, trifluoroacetic acid aqueous solution is B phase, carry out gradient elution purifying.Turn salt: adopt anion exchange method that phosphoric acid salt, trifluoroacetate are changed into acetate.Purification yield more than 85%, finished product purity is more than 99%.
In the solid phase synthesis process of existing Desmopressin, because synthesis step is more, the raw material quantity added is more, and cause impurity in products kind many, known impurity comprises: [des-Gln
4-Desmopressin, des-Asn
5-Desmopressins etc., unknown impuritie also has a lot.Des-Gln in relative substance
4-Desmopressin, structure is as follows:
This impurity is one of maximum impurity of toxicity, and extremely unstable, and easy to change, the existence of this impurity has a strong impact on the color and luster of Desmopressin, content and use safety.Therefore need to find effective means to be removed and make it reach gold standard rank less than 0.1%.The present inventor studies discovery, and the means of this impurity prior art are difficult to removing, though some method can remove part, removal effect is undesirable, is difficult to reach gold standard rank and easily causes the yield of Desmopressin own to reduce simultaneously.For this reason, how the present inventor's primary study removes impurity des-Gln
4-Desmopressin and do not affect the yield problem of Desmopressin.
The present inventor uses existing purification process, has carried out purifying to the Desmopressin prepared with solid-phase synthesis, finds impurity des-Gln
4the content of-Desmopressin all cannot reach and fix one's aim 0.1% in advance.For this reason, the purification process of the present inventor to Desmopressin is studied, thus obtains technical scheme of the present invention.
The Desmopressin that prior art obtains 40 DEG C of thermostat containers are placed, and observe outward appearance and proterties, and detect related substance, test-results is as follows
| Time, sky | Outward appearance, proterties | Related substance |
| 0 | For white powder | Have no impurity spot |
| 10 | For white powder | Have no impurity spot |
| 30 | For white powder | Have no impurity spot |
| 60 | For white powder | Have no impurity spot |
| 90 | For buff powder | Visible foreign spot |
The invention has the beneficial effects as follows: the Desmopressin purification process providing a kind of high purity (99.9%), high yield (more than 90%) and low impurity (des-Gln4-Desmopressin content is less than 0.05%).
Summary of the invention:
The object of this invention is to provide the purification process of the Desmopressin of a kind of high yield, high purity and low impurity.The technical issues that need to address of the present invention are: the purification process selecting a kind of Desmopressin, solve (1) Desmopressin finished product purity not high, (2) Desmopressin product yield is not high, the problem that (3) Desmopressin can not effectively be separated with impurity des-Gln4-Desmopressin.
In the present invention, some conventional abbreviations have following implication;
Fmoc: fluorenylmethyloxycarbonyl
Fmoc-AA-OH:N
αthe amino acid of-fluorenylmethyloxycarbonyl protection
TBu: the tertiary butyl
Cl-HOBt:6-chloro-1-hydroxybenzene a pair of horses going side by side triazole
DIC:N, N '-DIC
Mpr:3-thiohydracrylic acid
Tyr: tyrosine
Phe: phenylalanine
Gln: glutamine
Asn: asparagine
Cys: halfcystine
Pro: proline(Pro)
D-Arg:D type arginine
Gly: glycine
Trt: trityl
DMF:N, N '-dimethyl formamide
Piperidine: hexahydropyridine
Fmoc-Rink Amide Am resin: with the Rink aminomethyl resin of Fmoc protection
TFA: trifluoracetic acid
MeOH: methyl alcohol
DCM: methylene dichloride
Technical scheme of the present invention is:
Use polymkeric substance reverse phase filler post can make Desmopressin and impurity des-Gln
4-Desmopressin is effectively separated.
The invention provides a kind of purification process of Desmopressin for this reason, it is characterized in that, step is as follows:
Step 1, by Desmopressin crude product water dissolution, regulates pH to acid;
Step 2, by volume mark arranges gradient, uses mobile phase A to rinse polymkeric substance reverse-phase chromatographic column;
Step 3, is loaded into step 1 solution in polymkeric substance reverse-phase chromatographic column;
Step 4, by volume mark arranges gradient, and the original state Mobile phase B of gradient is 5%, keeps 2 minutes, then in 5 minutes, Mobile phase B ratio is increased to 25%, then in 30 minutes, Mobile phase B ratio is increased to 40%.Fraction collection eluting fraction;
Step 5, obtains sterling Desmopressin by eluting fraction through super-dry.
Polymkeric substance reverse phase filler post is (UniPS
tM10-100,50mm × 250mm);
Moving phase: A is 0.3% aqueous acetic acid;
Mobile phase B is acetonitrile;
Gradient program is: original state Mobile phase B is 5%, keeps 2 minutes, then in 5 minutes, Mobile phase B ratio is increased to 25%, then in 30 minutes, Mobile phase B ratio is increased to 40%.
Flow velocity is: 80mL/min,
Determined wavelength is 220nm.
Preferred operation steps is as follows:
Step 1, sample preparation:
Thick for the Desmopressin of 1g peptide is dissolved in 50ml pure water, uses vinegar acid for adjusting pH to be 4.5.
Step 2, balance
By volume mark arranges gradient, uses the mobile phase A of 100% to rinse chromatographic column 10min;
Step 3, loading
Sample solution is loaded in chromatographic column;
Step 4, wash-out
By volume mark arranges gradient, and original state Mobile phase B is 5%, keeps 2 minutes, then in 5 minutes, Mobile phase B ratio is increased to 25%, then in 30 minutes, Mobile phase B ratio is increased to 40%, fraction collection eluting fraction.
Step 5, freeze-drying
Eluting fraction is obtained sterling Desmopressin through freeze-drying.
Beneficial effect of the present invention is further illustrated below by way of experimental data:
Adopt Chinese patent CN101372504 method purifying crude Desmopressin, measure impurity des-Gln
4the content of-Desmopressin is 0.45%
Adopt method purifying crude Desmopressin of the present invention, measure impurity des-Gln
4the content of-Desmopressin is 0.01%.
Method of the present invention is through screening acquisition, and screening process is as follows:
1, the selection of polymkeric substance reverse phase filler:
UniPS
TM10-100；UniPS
TM20-300
2, the selection of moving phase:
Aqueous acetic acid/acetonitrile; The TFA aqueous solution/acetonitrile
3, the selection of gradient elution program:
Gradient program is: (1) by volume mark arranges gradient, and original state Mobile phase B is 5%, keeps 2 minutes, then in 5 minutes, Mobile phase B ratio is increased to 25%, then in 30 minutes, Mobile phase B ratio is increased to 40%.(2) by volume mark arranges gradient, and original state Mobile phase B is 5%, keeps 2 minutes, then in 5 minutes, Mobile phase B ratio is increased to 20%, then in 30 minutes, Mobile phase B ratio is increased to 40%.
Propose 6 kinds of experiment conditions for this reason:
Experiment condition 1: polymkeric substance reverse phase filler post (UniPS
tM10-100,50mm × 250mm); Moving phase: A is: the 0.3%HAc aqueous solution; B is: acetonitrile; Gradient program is: original state Mobile phase B is 5%, keeps 2 minutes, then in 5 minutes, Mobile phase B ratio is increased to 25%, then in 30 minutes, Mobile phase B ratio is increased to 40%.Flow velocity is: 80mL/min, and determined wavelength is 220nm;
Experiment condition 2: polymkeric substance reverse phase filler post (UniPS
tM20-300,50mm × 250mm); Moving phase: A is: the 0.3%HAc aqueous solution; B is: acetonitrile; Gradient program is: original state Mobile phase B is 5%, keeps 2 minutes, then in 5 minutes, Mobile phase B ratio is increased to 25%, then in 30 minutes, Mobile phase B ratio is increased to 40%.Flow velocity is: 80mL/min, and determined wavelength is 220nm;
Experiment condition 3: polymkeric substance reverse phase filler post (UniPS
tM10-100,50mm × 250mm); Moving phase: A is: the 0.1%TFA aqueous solution; B is: acetonitrile; Gradient program is: original state Mobile phase B is 5%, keeps 2 minutes, then in 5 minutes, Mobile phase B ratio is increased to 25%, then in 30 minutes, Mobile phase B ratio is increased to 40%.Flow velocity is: 80mL/min, and determined wavelength is 220nm;
Experiment condition 4: polymkeric substance reverse phase filler post (UniPS
tM20-300,50mm × 250mm); Moving phase: A is: the 0.1%TFA aqueous solution; B is: acetonitrile; Gradient program is: original state Mobile phase B is 5%, keeps 2 minutes, then in 5 minutes, Mobile phase B ratio is increased to 25%, then in 30 minutes, Mobile phase B ratio is increased to 40%.Flow velocity is: 80mL/min, and determined wavelength is 220nm;
Experiment condition 5: polymkeric substance reverse phase filler post (UniPS
tM10-100,50mm × 250mm); Moving phase: A is: the 0.3%HAc aqueous solution; B is: acetonitrile; Gradient program is: original state Mobile phase B is 5%, keeps 2 minutes, then in 5 minutes, Mobile phase B ratio is increased to 20%, then in 30 minutes, Mobile phase B ratio is increased to 40%.Flow velocity is: 80mL/min, and determined wavelength is 220nm;
Experiment condition 6: polymkeric substance reverse phase filler post (UniPS
tM20-300,50mm × 250mm); Moving phase: A is: the 0.3%HAc aqueous solution; B is: acetonitrile; Gradient program is: original state Mobile phase B is 5%, keeps 2 minutes, then in 5 minutes, Mobile phase B ratio is increased to 20%, then in 30 minutes, Mobile phase B ratio is increased to 40%.Flow velocity is: 80mL/min, and determined wavelength is 220nm;
Adopt the method purifying crude Desmopressin of experiment condition 1, measure impurity des-Gln
4the content of-Desmopressin is as follows: 0.01%
Adopt the method purifying crude Desmopressin of experiment condition 2, measure impurity des-Gln
4the content of-Desmopressin is as follows: 0.07%
Adopt the method purifying crude Desmopressin of experiment condition 3, measure impurity des-Gln
4the content of-Desmopressin is as follows: 0.03%
Adopt the method purifying crude Desmopressin of experiment condition 4, measure impurity des-Gln
4the content of-Desmopressin is as follows: 0.04%
Adopt the method purifying crude Desmopressin of experiment condition 5, measure impurity des-Gln
4the content of-Desmopressin is as follows: 0.07%
Adopt the method purifying crude Desmopressin of experiment condition 6, measure impurity des-Gln
4the content of-Desmopressin is as follows: 0.09%
Above result shows, the purification effect of experiment condition 1 is optimum.
The Desmopressin that the inventive method obtains 40 DEG C of thermostat containers are placed, and observe outward appearance and proterties, and detect related substance, test-results is as follows
| Time, sky | Outward appearance, proterties | Related substance |
| 0 | For white powder | Have no impurity spot |
| 10 | For white powder | Have no impurity spot |
| 30 | For white powder | Have no impurity spot |
| 60 | For white powder | Have no impurity spot |
| 90 | For white powder | Have no impurity spot |
Accompanying drawing illustrates:
The HPLC collection of illustrative plates of the thick peptide of Fig. 1
Fig. 2: patent CN101372504 method carries out Desmopressin HPLC after purifying
Desmopressin HPLC after Fig. 3 the inventive method purifying
Fig. 4 Desmopressin reference substance HPLC
Embodiment:
Embodiment 1:
The synthesis of step 1 Fmoc-Gly-RinkAmide-AM-Resin
Take Rink Amide AM resin (substitution degree 0.73mmol/g) (3.29g, 2.4mmol, 1.0equiv) to add in solid phase synthesis pipe, add DCM(40mL), temperature control 10-30 DEG C is stirred swelling 30min, suction filtration removing liquid.Add DMF(20mL × 2 again) washing.Measure 20%(v/v, lower same) Piperidine/DMF solution (17mL), add in solid phase synthesis pipe, temperature control 20-30 DEG C of stirring reaction 5min, suction filtration removing liquid.Again measure 20%Piperidine/DMF solution (17mL), add in solid phase synthesis pipe, temperature control 20-30 DEG C of stirring reaction 10min, suction filtration removing liquid.Measure DMF(20mL × 6) add solid phase synthesis pipe washing rear suction filtration removing liquid.Separately take Fmoc-Gly-OH(2.14g, 3.0equiv), Cl-HOBt(1.34g, 3.3equiv) add beaker.Measure DMF(10mL) add beaker, stir after dissolving completely and be cooled to 10-15 DEG C with cryosel bath, add DIC(1.1mL, 3.0equiv), stir, temperature control 10-25 DEG C of reaction 5min, then this reaction solution is joined in solid phase synthesis pipe, suction filtration removing liquid after 2h.Measure DMF(20mL × 3) add solid phase synthesis pipe washing rear suction filtration removing liquid.Triketohydrindene hydrate detects and is negative.
Step 2
The synthesis of Mpr (Trt)-Tyr (tBu)-Phe-Gln (Trt)-Asn (Trt)-Cys (Trt)-Pro-D-Arg (pbf)-Gly-RinkAmide-AM-Resin
Step 1 product amino acid resin is sloughed Fmoc by the same way.Take Fmoc-AA-OH(3.0equiv), Cl-HOBt(3.3equiv) add beaker.Measure DMF/DCM=1/1 solvent (10mL) and add beaker, stir after dissolving completely and be cooled to 5-10 DEG C with cryosel bath, add DIC(3.0equiv), stir, temperature control 5-25 DEG C of reaction 5min, then joins in solid phase synthesis pipe by this reaction solution, suction filtration removing liquid after 2h.Measure DMF(25mL × 3) add solid phase synthesis pipe washing rear suction filtration removing liquid.Triketohydrindene hydrate detects, and if result is positive, then repeats coupling once.After all amino acid couplings terminate, wash 6 times with DMF.
Step 3
The synthesis of Mpr-Tyr (tBu)-Phe-Gln (Trt)-Asn (Trt)-Cys-Pro-D-Arg (pbf)-Gly-RinkAmide-AM-Resin
The DCM solution (30mL) containing 1%TFA is added, stirring reaction 15min, suction filtration removing liquid in step 2 gained peptide resin.Repeat 3 times again, carry out altogether 4 times and go protection to operate.6 times are washed again with DMF.
Step 4
synthesis
Under lucifuge, in step 3 gained peptide resin, add the DMF solution (15mL) containing carbamide peroxide (5eq), control temperature 20-30 DEG C of reaction 30min.Suction filtration removing liquid, then use DMF(30mL × 3 successively), MeOH(25mL × 2), DCM(30mL × 2), MeOH(25mL × 2) washing resin.Take out Desmopressin peptide resin, after drying, obtain 9.96g.
Step 5
synthesis
Measure TFA(95mL), PhSMe(4.0mL) PhOMe(1.0mL) join in 250mL reaction flask successively, stirring and evenly mixing, controls lysate temperature at 15-20 DEG C.Step 4 gained peptide resin is all added reaction flask.Control temperature of reaction 25-30 DEG C, reaction 2h.Filter, and with TFA(10mL × 2) wash.Merging filtrate, puts under revolving steaming evaporimeter and is concentrated to about 25% of original volume.Gained concentrated solution is joined sedimentation in the methyl tertiary butyl ether of-10 ~-5 DEG C.Centrifugal, washing 6 times after the thick peptide of pasty state, obtain thick peptide 2.62g through drying under reduced pressure.
Step 6
purifying
(1) take the thick peptide 1.31g of step 5 gained, dissolve with pure water 65mL, regulate pH to 4.45, after fully stirring, filter, obtain thick peptide solution.Carry out HPLC quantified by external standard method to thick peptide solution and analyze to obtain Desmopressin content in thick peptide: 64.48%, namely containing object peptide 0.84g, synthesis yield 65.5%, HPLC is analyzed collection of illustrative plates and is seen Fig. 1.Adopt the purification process of certain patent report domestic to carry out purifying to this thick peptide solution, after purifying, HPLC analysis collection of illustrative plates is shown in Fig. 2, impurity des-Gln
4-Desmopressin content is 0.51%, Desmopressin content: 99.40%, obtains Desmopressin 0.74g, purification yield: 88.1% after freeze-drying.
(2) thicker for step 5 gained peptide residue 1.31g pure water 65mL is dissolved, regulate pH to 4.46, after fully stirring, filter, obtain thick peptide solution.Take inventive method to carry out purification, regulate as follows: chromatographic column: polymkeric substance reverse phase filler post (UniPS
tM10-100,50mm × 250mm); Mobile phase A: 0.3% aqueous acetic acid; Mobile phase B: acetonitrile; Gradient program: original state Mobile phase B is 5%, keeps 2 minutes, then in 5 minutes, Mobile phase B ratio is increased to 25%, then in 30 minutes, Mobile phase B ratio is increased to 40%; Flow velocity is: 80mL/min; Determined wavelength is 220nm.After purifying, HPLC analysis collection of illustrative plates is shown in Fig. 3, impurity des-Gln
4-Desmopressin content is 0.01%, Desmopressin content: 99.95%, obtains Desmopressin 0.77g, purification yield: 91.7% after freeze-drying.
The HPLC detection method of Fig. 1 to Fig. 4 is:
Chromatographic column: Kromasil 100-5 C18 (250mm × 4.6mm) chromatographic column or quite post;
Mobile phase A: damping fluid (3.4g potassium primary phosphate and 2.0g sodium heptanesulfonate are dissolved in 1L water and adjust pH to 4.50 ± 0.05 with phosphoric acid or sodium hydroxide);
Mobile phase B: acetonitrile;
Elution flow rate: 1ml/min;
Determined wavelength: 220nm;
Gradient:
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 78 | 22 |
| 20 | 78 | 22 |
| 35 | 66 | 34 |
| 35.1 | 78 | 22 |
| 40 | 78 | 22 |
The content assaying method of Desmopressin:
Desmopressin content=Desmopressin reference substance content × Desmopressin thick peptide main peak area/Desmopressin reference substance peak area × 100%=86.63% × 138.141 × 0.499/123.158 × 0.752=64.48% in thick peptide
Note: Desmopressin reference substance is consistent with sample size with the testing conditions of the thick peptide of Desmopressin, is all: 25 μ l.
Desmopressin reference substance concentration: 0.499mg/ml
Desmopressin reference substance peak area: 123.158mAU*min(Fig. 4)
Desmopressin reference substance content: 86.63%
The thick peptide compound concentration of Desmopressin: 0.752mg/ml
The thick peptide main peak area of Desmopressin: 138.141mAU*min(Fig. 1)
Content=Desmopressin reference substance content × Desmopressin essence peptide peak area/Desmopressin reference substance peak area × 100%=86.63% × 231.094 × 0.499/123.158 × the 0.816=99.40% of Desmopressin after patent CN101372504 method purifying
Note: Desmopressin reference substance is consistent with sample size with the testing conditions of Desmopressin essence peptide, is all: 25 μ l.
Desmopressin reference substance concentration: 0.499mg/ml
Desmopressin reference substance peak area: 123.158mAU*min(Fig. 4)
Desmopressin reference substance content: 86.63%
Desmopressin essence peptide compound concentration: 0.816mg/ml
Desmopressin essence peptide peak area: 231.094mAU*min(Fig. 2)
Content=Desmopressin reference substance content × Desmopressin essence peptide peak area/Desmopressin reference substance peak area × 100%=86.63% × 189.926 × 0.499/123.158 × the 0.667=99.95% of Desmopressin after the inventive method purifying
Note: Desmopressin reference substance is consistent with sample size with the testing conditions of Desmopressin essence peptide, is all: 25 μ l.
Desmopressin reference substance concentration: 0.499mg/ml
Desmopressin reference substance peak area: 123.158mAU*min(Fig. 4)
Desmopressin reference substance content: 86.63%
Desmopressin essence peptide compound concentration: 0.667mg/ml
Desmopressin essence peptide peak area: 189.926mAU*min(Fig. 3)
Impurity Des-Gln
4the content assaying method of-Desmopressin:
Des-Gln after patent CN101372504 method purifying
4content=Desmopressin reference substance content × the Des-Gln of-Desmopressin
4-Desmopressin peak area × correction factor/Desmopressin reference substance peak area × 100%=86.63% × 1.206 × 0.98 × 0.499/123.158 × 0.816=0.51%
Note: Desmopressin reference substance is consistent with sample size with the testing conditions of Desmopressin essence peptide, is all: 25 μ l.Correction factor Des-Gln
4correction factor=0.98 of-Desmopressin.
Desmopressin reference substance concentration: 0.499mg/ml
Desmopressin reference substance peak area: 123.158mAU*min(Fig. 4)
Desmopressin reference substance content: 86.63%
Desmopressin essence peptide compound concentration: 0.816mg/ml
Des-Gln
4-Desmopressin peak area: 1.206mAU*min(Fig. 2)
Des-Gln after the inventive method purifying
4content=Desmopressin reference substance content × the Des-Gln of-Desmopressin
4-Desmopressin peak area × correction factor/Desmopressin reference substance peak area × 100%=86.63% × 0.019 × 0.98 × 0.499/123.158 × 0.667=0.01%
Note: Desmopressin reference substance is consistent with sample size with the testing conditions of Desmopressin essence peptide, is all: 25 μ l.Correction factor Des-Gln
4correction factor=1.04 of-Desmopressin.
Desmopressin reference substance concentration: 0.499mg/ml
Desmopressin reference substance peak area: 123.158mAU*min(Fig. 4)
Desmopressin reference substance content: 86.63%
Desmopressin essence peptide compound concentration: 0.667mg/ml
Des-Gln
4-Desmopressin peak area: 0.019mAU*min(Fig. 3)
Claims (1)
1. a method for purifying desmopressin, the method adopts polymkeric substance reverse-phase chromatography, can make the impurity des-Gln in finished product Desmopressin
4-Desmopressin content is reduced to less than 0.1%, it is characterized in that, said method comprising the steps of:
Step 1, sample preparation
Thick for 1g Desmopressin peptide is dissolved in 50ml pure water, uses vinegar acid for adjusting pH to be 4.5;
Step 2, balance
By volume mark arranges gradient, uses mobile phase A to rinse polymkeric substance reverse-phase chromatographic column;
Step 3, loading
Sample solution is loaded in chromatographic column;
Step 4, wash-out
By volume mark arranges gradient, and the original state Mobile phase B of gradient is 5%, keeps 2 minutes, then in 5 minutes, Mobile phase B ratio is increased to 25%, then in 30 minutes, Mobile phase B ratio is increased to 40%, fraction collection eluting fraction;
Flow velocity is: 80mL/min,
Determined wavelength is 220 nm,
Step 5, freeze-drying
Eluting fraction is obtained sterling Desmopressin through freeze-drying;
Wherein,
Polymkeric substance reverse phase filler post is UniPS
tM10-100,50mm × 250 mm;
Moving phase: A is 0.3% aqueous acetic acid;
Moving phase: B is acetonitrile.
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| CN103102395A (en) * | 2012-12-18 | 2013-05-15 | 深圳翰宇药业股份有限公司 | Preparation method of desmopressin acetate |
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| CN103102395A (en) * | 2012-12-18 | 2013-05-15 | 深圳翰宇药业股份有限公司 | Preparation method of desmopressin acetate |
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