CN111057140A - Selepressin purification method - Google Patents
Selepressin purification method Download PDFInfo
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- 238000000746 purification Methods 0.000 title claims abstract description 84
- 238000000034 method Methods 0.000 title claims abstract description 53
- JCVQBJTWWDYUFQ-MRUTUVJXSA-N selepressin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CCCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@H](N)C(=O)N1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(C)C)C(=O)NCC(N)=O)=O)[C@@H](C)CC)C1=CC=CC=C1 JCVQBJTWWDYUFQ-MRUTUVJXSA-N 0.000 title claims description 8
- 229950008103 selepressin Drugs 0.000 title claims description 8
- 238000007363 ring formation reaction Methods 0.000 claims abstract description 23
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 303
- 150000003839 salts Chemical class 0.000 claims description 72
- 239000000243 solution Substances 0.000 claims description 69
- 238000011068 loading method Methods 0.000 claims description 59
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 51
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 50
- 238000006243 chemical reaction Methods 0.000 claims description 46
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 36
- 230000005526 G1 to G0 transition Effects 0.000 claims description 32
- 238000007865 diluting Methods 0.000 claims description 31
- 238000010828 elution Methods 0.000 claims description 30
- 239000007864 aqueous solution Substances 0.000 claims description 24
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 20
- 238000002156 mixing Methods 0.000 claims description 20
- 239000000523 sample Substances 0.000 claims description 19
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 17
- 239000005695 Ammonium acetate Substances 0.000 claims description 17
- 229940043376 ammonium acetate Drugs 0.000 claims description 17
- 235000019257 ammonium acetate Nutrition 0.000 claims description 17
- 238000010829 isocratic elution Methods 0.000 claims description 16
- 238000001914 filtration Methods 0.000 claims description 15
- 238000002390 rotary evaporation Methods 0.000 claims description 15
- BAZAXWOYCMUHIX-UHFFFAOYSA-M sodium perchlorate Chemical compound [Na+].[O-]Cl(=O)(=O)=O BAZAXWOYCMUHIX-UHFFFAOYSA-M 0.000 claims description 11
- 229910001488 sodium perchlorate Inorganic materials 0.000 claims description 11
- 239000000945 filler Substances 0.000 claims description 9
- 229920000642 polymer Polymers 0.000 claims description 9
- UNXNGGMLCSMSLH-UHFFFAOYSA-N dihydrogen phosphate;triethylazanium Chemical compound OP(O)(O)=O.CCN(CC)CC UNXNGGMLCSMSLH-UHFFFAOYSA-N 0.000 claims description 7
- VLTRZXGMWDSKGL-UHFFFAOYSA-M perchlorate Inorganic materials [O-]Cl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-M 0.000 claims description 7
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims description 7
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 claims description 4
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 4
- 239000012488 sample solution Substances 0.000 claims description 4
- 239000000377 silicon dioxide Substances 0.000 claims description 4
- GDDNTTHUKVNJRA-UHFFFAOYSA-N 3-bromo-3,3-difluoroprop-1-ene Chemical compound FC(F)(Br)C=C GDDNTTHUKVNJRA-UHFFFAOYSA-N 0.000 claims description 3
- AXZAYXJCENRGIM-UHFFFAOYSA-J dipotassium;tetrabromoplatinum(2-) Chemical compound [K+].[K+].[Br-].[Br-].[Br-].[Br-].[Pt+2] AXZAYXJCENRGIM-UHFFFAOYSA-J 0.000 claims description 3
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 3
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 3
- 229910001487 potassium perchlorate Inorganic materials 0.000 claims description 3
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 3
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Natural products C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 2
- 239000011159 matrix material Substances 0.000 claims description 2
- JPYTXSWRYBJQHD-UHFFFAOYSA-N prop-2-enoic acid;prop-1-enylbenzene Chemical compound OC(=O)C=C.CC=CC1=CC=CC=C1 JPYTXSWRYBJQHD-UHFFFAOYSA-N 0.000 claims description 2
- 239000012521 purified sample Substances 0.000 claims description 2
- 125000003011 styrenyl group Chemical group [H]\C(*)=C(/[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims 2
- 235000019796 monopotassium phosphate Nutrition 0.000 claims 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims 2
- 239000012535 impurity Substances 0.000 abstract description 58
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 48
- 230000000694 effects Effects 0.000 abstract description 4
- 239000002904 solvent Substances 0.000 abstract description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 abstract description 2
- 238000004007 reversed phase HPLC Methods 0.000 abstract description 2
- 239000012071 phase Substances 0.000 description 231
- 238000005406 washing Methods 0.000 description 67
- 238000001514 detection method Methods 0.000 description 61
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 28
- 239000000741 silica gel Substances 0.000 description 28
- 229910002027 silica gel Inorganic materials 0.000 description 28
- 239000000047 product Substances 0.000 description 27
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 16
- 239000012467 final product Substances 0.000 description 15
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 14
- 239000012528 membrane Substances 0.000 description 14
- 239000000706 filtrate Substances 0.000 description 12
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 9
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- 206010040047 Sepsis Diseases 0.000 description 2
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 description 2
- 235000011009 potassium phosphates Nutrition 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 208000013223 septicemia Diseases 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 235000011008 sodium phosphates Nutrition 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 206010040070 Septic Shock Diseases 0.000 description 1
- GXBMIBRIOWHPDT-UHFFFAOYSA-N Vasopressin Natural products N1C(=O)C(CC=2C=C(O)C=CC=2)NC(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CCCN=C(N)N)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C1CC1=CC=CC=C1 GXBMIBRIOWHPDT-UHFFFAOYSA-N 0.000 description 1
- 102000002852 Vasopressins Human genes 0.000 description 1
- 108010004977 Vasopressins Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- KBZOIRJILGZLEJ-LGYYRGKSSA-N argipressin Chemical compound C([C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@@H](C(N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)=O)N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(N)=O)C1=CC=CC=C1 KBZOIRJILGZLEJ-LGYYRGKSSA-N 0.000 description 1
- 230000004872 arterial blood pressure Effects 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000000018 receptor agonist Substances 0.000 description 1
- 229940044601 receptor agonist Drugs 0.000 description 1
- 230000033458 reproduction Effects 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 230000036303 septic shock Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 229960003726 vasopressin Drugs 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
-
- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Endocrinology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention aims to provide the Selepress purification process which is simple to operate, good in impurity removal effect and high in yield. The purification method of the invention is to purify the cyclization solution by one step by an HPLC method to remove various solvents and some small polar impurities in the cyclization solution. Then, carrying out two-step purification by using a reversed-phase high performance liquid chromatography, and finally converting acetate to obtain a pure Selepress product. The purification method is simple and feasible, the purity of the prepared Selepress is high and is more than 99.5%, the maximum single impurity is less than 0.1%, and the total yield is more than 70%.
Description
Technical Field
The invention relates to a method for purifying Selepress, belonging to the technical field of pharmaceutical chemistry.
Background
The amino acid sequence of Selepressin is Cyclo- (Cys-Phe-Ile-Hgn-Asn-Cys) -Pro-Orn (iPr) -Gly-NH2Molecular weight 1048.29, is a selective vasopressin V1a type receptor agonist, which increases arterial blood pressure and is useful for treating septic shock. Septicemia is acute systemic infection caused by invasion of pathogenic bacteria or conditioned pathogenic bacteria into blood circulation, growth and reproduction in blood and toxin production, and infectious shock can occur in severe cases, so tens of millions of people suffer from the septicemia every year in the global range. Therefore, the development of Selepressin is of great significance.
At present, very few reports about the purification method of Selepress at home and abroad are reported, and the purification method is only roughly introduced in patent EP1984012, wherein the purification method comprises the steps of performing one-step purification by using HPLC (high performance liquid chromatography), using TEAP pH5.2 as a buffer salt system and acetonitrile as an organic phase, then performing salt conversion, freezing and drying to obtain a finished product, wherein the purity of the finished product is 98.8%, and the purification yield is only 30%.
The existing reported purification method for Selepress is difficult to obtain a high-purity sample with the purity of more than 99.5 percent and the single impurity of less than 0.1 percent, and the purification yield is not high. Through the creative work of the invention, the impurity 1 is found to be a key impurity which affects the purity and yield of the final product, namely the maximum single impurity, and is relatively stable, and is confirmed by a primary mass spectrum after extraction (the primary mass spectrum is shown in figure 1), the impurity is an impurity of a plurality of Gly residues formed in the Selepress synthesis process, and the chemical structure of the impurity is as follows:
as Gly has small steric hindrance and polypeptide conformation self-synergistic effect, the impurity is inevitably generated in Selepress synthesis, and the structure and the property of the impurity are similar to those of Selepress, so that the impurity is difficult to purify to be within 0.1 percent, and in addition, the high yield is ensured.
In order to solve the problems of low purity, low yield, low automation degree of the purification process and the like in the prior art, the Selepress purification process is developed, and has very important significance for providing high-quality medicines for patients.
Disclosure of Invention
The invention aims to provide the Selepress purification process which is simple to operate, good in impurity removal effect and high in yield.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for purifying Selepress, comprising the following steps:
step 1: performing one-step purification, namely filtering the cyclized solution, and performing isocratic elution in the range of 25-45% of acetonitrile volume by taking a polymer filler as a stationary phase, a trifluoroacetic acid aqueous solution as an A phase and acetonitrile as a B phase to obtain a one-step purified sample for two-step purification;
step 2: performing two-step purification, namely diluting the sample solution obtained in the step 1 by using water and then loading the sample by using octadecylsilane or octaalkylsilane bonded silica as a stationary phase, performing linear gradient elution within the range of 10-30% of acetonitrile volume ratio by using a perchlorate aqueous solution or a phosphate solution as a phase A and acetonitrile as a phase B, and combining the samples for salt transfer;
and step 3: salt conversion, using octadecylsilane or octaalkylsilane bonded silica as stationary phase, diluting the sample solution obtained in step 2 with water, loading, and using ammonium acetate aqueous solution as A1Phase B, acetonitrile, changing salt at equal degree within the range of acetonitrile volume ratio of 2% -3% for 15min-20min, and then taking acetic acid water solution as A2Phase B, acetonitrile, 2-3% acetonitrile volume ratio, isocratic equilibrium for 10-20 min, and acetic acid water solution A2Phase B, acetonitrile, eluting linearly in the acetonitrile volume ratio of 4 v/v% -15 v/v%, combining samples, concentrating and freeze-drying.
In some examples, the Selepressin purity in the cyclization solution is 55% or more.
In some embodiments, the purity of selepress in the cyclization solution is preferably 60% or more.
In the step 1, the polymer filler matrix is styrene or methyl styrene acrylate.
The polymer filler in step 1 is selected from UniPS 30-100, UniPS30-300, UniPS 40-300, UniPS10-300, UniPSN 30-300, UniPSN 40-300 and UniPMM 40-500.
In some embodiments, the polymer filler in step 1 is selected from UniPS 30-100, UniPS30-300, UniPS 40-300, UniPS 10-300.
In some embodiments, the aqueous trifluoroacetic acid solution in step 1 is in a volume ratio of 0.05 v/v% to 0.5 v/v%.
The volume of the isocratic elution acetonitrile in the step 1 is preferably 30-40%.
In the step 2, the A-phase perchlorate aqueous solution is selected from sodium perchlorate, potassium perchlorate or ammonium perchlorate aqueous solution.
Preferably, the A-phase perchlorate aqueous solution used in the step 2 is sodium perchlorate aqueous solution.
In some embodiments, the phase A perchlorate aqueous solution in step 2 has a pH of 2.0 to 3.5 and a salt concentration of 20mM/L to 200 mM/L.
In the step 2, the A-phase phosphate solution is selected from triethylamine phosphate, sodium phosphate and potassium phosphate.
Further, the pH value of the triethylamine phosphate solution is 2.5-3.5, and the salt concentration is 0.1-1.2 v/v%.
Further, the pH of the sodium phosphate or potassium phosphate solution is 2.5 to 3.5, and the salt concentration is 30mM/L to 100 mM/L.
In some embodiments, step 3 is a1The concentration of the phase ammonium acetate aqueous solution is 45mM/L-100mM/L, A2The concentration of the phase acetic acid aqueous solution is 0.01 v/v% -0.5 v/v%.
The concentration in the step 3 is water bath rotary evaporation concentration, wherein the temperature of the water bath is 25-35 ℃.
The steps 1, 2 and 3 of the invention adopt HPLC method, wherein the steps 2 and 3 adopt Rp-HPLC method, and the cyclized liquid is: the Selepress which is prepared by oxidizing the linear peptide synthesized by the solid phase method in an acetic acid aqueous solution, is not refined and has purity which can not meet the requirement of medical use.
Those skilled in the art can select columns of different sizes (diameter. times. length), such as 26 mm. times.460 mm, 5 cm. times.25 cm, 15 cm. times.25 cm, 30 cm. times.25 cm, depending on the content of the Selepressin cyclization solution.
And (3) diluting with water, preferably adding 1 volume of water for dilution.
Has the advantages that: the purification method of the invention is to purify the cyclization solution by one step by an HPLC method to remove various solvents and some small polar impurities in the cyclization solution. Then, carrying out two-step purification by using a reversed-phase high performance liquid chromatography, and finally converting acetate to obtain a pure Selepress product. The purification method is simple and feasible, the purity of the prepared Selepress is high and can reach more than 99.5%, the maximum single impurity is less than 0.1%, and the total yield is higher than 70%, while the purity of the finished product obtained by the purification method of the original patent EP1984012 is 98.8%, and the yield is 30%, so that the method has considerable advantages compared with the prior art.
The invention finds that the impurity 1 is an impurity which is easy to generate in the synthesis process and has one more Gly residue formed in the Selepress synthesis process, the chemical property and the structure are similar to those of Selepress, the impurity is a key impurity which affects the product yield and the quality, the impurity is difficult to purify within 0.1% by a common method, the impurity has no great purification effect on the impurity by salt conversion, and the impurity can be purified within 0.1% by a preferred purification method in the step 2, so that the maximum single impurity after the salt conversion is reduced to within 0.1%.
The one-step purification of the step 1 of the invention adopts polymer filler, has strong acid and alkali resistance, is used for the primary purification of the cyclization solution, has the advantages of wide pH application range, large sample loading amount and good reproducibility, can quickly change the salt form and the solvent of a sample, simultaneously removes impurities with small polarity and extremely difficult elution, improves the purity to more than 90 percent after the Selepress one-step purification, and has the yield of up to 95 percent.
Drawings
FIG. 1 is a mass spectrometric detection of impurity 1;
FIG. 2 is a detection chart of the product of example 1;
FIG. 3 is a product inspection chart of example 2;
FIG. 4 is a detection chart of the product of example 3;
FIG. 5 is a detection chart of the product of example 4;
FIG. 6 is a detection chart of the product of example 5;
FIG. 7 is a detection chart of the product of comparative example 1;
FIG. 8 is a detection chart of the product of comparative example 2;
Detailed Description
The present invention is described in further detail below with reference to specific examples so that those skilled in the art can further understand the present invention. The examples should not be construed as limiting the scope of protection.
The Chinese names corresponding to the English abbreviations related to the invention are shown in Table 1:
TABLE 1 Chinese names corresponding to English abbreviations related to the present invention
| English abbreviation | Name of Chinese |
| HPLC | High performance liquid phase |
| TEAP | Aqueous solution of triethylamine phosphate |
| Gly | Glycine |
| v | Volume of |
| Rp-HPLC | Reversed phase liquid chromatography |
| TFA | Trifluoroacetic acid |
Example 1
Step 1: one-step purification of Selepress cyclization solution
HPLC conditions: a chromatographic column: UniPS 40-300, column length diameter: 26mm 46cm, phase a: 0.1% TFA, phase B: acetonitrile, flow rate 19mL/min, detection wavelength 230nm, B phase 30% isocratic elution.
One-step purification process: filtering the cyclized solution with 0.45 μm membrane, collecting filtrate, washing chromatographic column with acetonitrile of more than 60%, loading, isocratic eluting for 30min, collecting fractions with purity of more than or equal to 90%, mixing, and purifying in two steps.
Step 2: two-step purification
HPLC conditions: a chromatographic column: octadecyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 5cm by 25cm, phase a: 50mM/L sodium perchlorate, pH3.0 adjusted by phosphoric acid, phase B: acetonitrile, flow rate 80mL/min, detection wavelength 230nm, mobile phase B11% -14%, linear elution for 60 min.
And (2) a two-step purification process: diluting the qualified fraction collected in one step by 1 time volume with water, washing a chromatographic column with acetonitrile of more than 60%, loading, linearly eluting, taking a peak after 45min, collecting fractions with the purity of more than or equal to 99% and the impurity of less than or equal to 0.1%, and combining the fractions for salt conversion.
And step 3: salt conversion
HPLC conditions: a chromatographic column: octadecyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 5cm by 25cm, A1Phase (1): 50mM/L aqueous ammonium acetate solution, A2Phase (1): 0.06 v/v% aqueous acetic acid, phase B: acetonitrile, flow rate 80mL/min, detection wavelength 230 nm.
A salt conversion process:
1) loading: diluting the qualified fraction collected in the second step by 1 time of volume with water, washing a chromatographic column by using acetonitrile with the concentration of more than 60%, and then loading the chromatographic column;
2) salt exchange: mobile phase A1Washing the mobile phase B2% at equal temperature for 15min, wherein the washing time is 98%;
3) balancing: mobile phase A2Washing the mobile phase B2% for 10min at an isocratic rate of 98%;
4) and (3) elution: mobile phase A2And mobile phase B to flowAnd (4) linearly eluting the phase B by 4-7%, taking a peak after 30min, and collecting qualified fractions.
Mixing, concentrating in 30 deg.C water bath, and freeze drying to obtain pure product with total yield of 73.20%, wherein the purity of the mixed sample is 99.900%, and the maximum single impurity is less than 0.1%, and the final product detection is shown in FIG. 2.
Example 2
Step 1: one-step purification of Selepress cyclization solution
HPLC conditions: a chromatographic column: UniPS10-300, column length diameter: 26mm 46cm, phase a: 0.05% TFA, phase B: acetonitrile, flow rate 19mL/min, detection wavelength 230nm, B phase 30% isocratic elution.
One-step purification process: filtering the cyclized solution with 0.45 μm membrane, collecting filtrate, washing chromatographic column with acetonitrile of more than 60%, loading, isocratic eluting for 35min, collecting fractions with purity of more than or equal to 90%, mixing, and purifying in two steps.
Step 2: two-step purification
HPLC conditions: a chromatographic column: octadecyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 5cm by 25cm, phase a: 50mM/L sodium perchlorate, pH3.0 adjusted by phosphoric acid, phase B: acetonitrile, flow rate 80mL/min, detection wavelength 230nm, mobile phase B11% -14%, linear elution for 60 min.
And (2) a two-step purification process: diluting the qualified fraction collected in one step by 1 time volume with water, washing a chromatographic column with acetonitrile of more than 60%, loading, linearly eluting, taking a peak after 45min, collecting fractions with the purity of more than or equal to 99% and the impurity of less than or equal to 0.1%, and combining the fractions for salt conversion.
And step 3: salt conversion
HPLC conditions: a chromatographic column: octadecyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 5cm by 25cm, A1Phase (1): 60mM/L aqueous ammonium acetate solution, A2Phase (1): 0.06 v/v% aqueous acetic acid, phase B: acetonitrile, flow rate 80mL/min, detection wavelength 230 nm.
A salt conversion process:
1) loading: diluting the qualified fraction collected in the second step by 1 time of volume with water, washing a chromatographic column by using acetonitrile with the concentration of more than 60%, and then loading the chromatographic column;
2) salt exchange: mobile phase A1Washing with 98% mobile phase B2% for 20 min;
3) balancing: mobile phase A2Washing with 98% mobile phase B2% for 20 min;
4) and (3) elution: mobile phase A2And a mobile phase B, linearly eluting by 4-7% of the mobile phase B, peaking after 30min, and collecting qualified fractions.
The purity of the combined sample is 99.899%, the maximum single impurity is less than 0.1%, the pure product is obtained by water bath rotary evaporation and concentration at 30 ℃, the total yield is 75.08%, and the detection of the final product is shown in figure 3.
Example 3
Step 1: one-step purification of Selepress cyclization solution
HPLC conditions: a chromatographic column: UniPSN 30-300, column length diameter: 26mm 46cm, phase a: 0.06% TFA, phase B: acetonitrile, flow rate 19mL/min, detection wavelength 230nm, B phase 30% isocratic elution.
One-step purification process: filtering the cyclized solution with 0.45 μm membrane, collecting filtrate, washing chromatographic column with acetonitrile of more than 60%, loading, isocratic eluting for 35min, collecting fractions with purity of more than or equal to 90%, mixing, and purifying in two steps.
Step 2: two-step purification
HPLC conditions: a chromatographic column: octadecyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 5cm by 25cm, phase a: 50mM/L sodium perchlorate, pH3.0 adjusted by phosphoric acid, phase B: acetonitrile, flow rate 80mL/min, detection wavelength 230nm, mobile phase B11% -14%, linear elution for 60 min.
And (2) a two-step purification process: diluting the qualified fraction collected in one step by 1 time volume with water, washing a chromatographic column with acetonitrile of more than 60%, loading, linearly eluting, taking a peak after 45min, collecting fractions with the purity of more than or equal to 99% and the impurity of less than or equal to 0.1%, and combining the fractions for salt conversion.
And step 3: salt conversion
HPLC conditions: a chromatographic column: octadecyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 5cm by 25cm, A1Phase (1): 70mM/L aqueous ammonium acetate solution, A2Phase (1): 0.07 v/v% acetic acid solutionLiquid, phase B: acetonitrile, flow rate 80mL/min, detection wavelength 230 nm.
A salt conversion process:
1) loading: diluting the qualified fraction collected in the second step by 1 time of volume with water, washing a chromatographic column by using acetonitrile with the concentration of more than 60%, and then loading the chromatographic column;
2) salt exchange: mobile phase A197%, washing the mobile phase B for 15min at 3% isocratic;
3) balancing: mobile phase A297%, and isocratic washing of the mobile phase B3% for 20 min;
4) and (3) elution: mobile phase A2And a mobile phase B, linearly eluting by 4-7% of the mobile phase B, peaking after 30min, and collecting qualified fractions.
The purity of the combined sample is 99.788%, the maximum single impurity is less than 0.1%, the pure product is obtained by water bath rotary evaporation concentration at 30 ℃, the total yield is 73.40%, and the detection of the final product is shown in figure 4.
Example 4:
step 1: one-step purification of Selepress cyclization solution
HPLC conditions: a chromatographic column: UniPS10-300, column length diameter: 26mm 46cm, phase a: 0.07% TFA, phase B: acetonitrile, flow rate 19mL/min, detection wavelength 230nm, B phase 30% isocratic elution.
One-step purification process: filtering the cyclized solution with 0.45 μm membrane, collecting filtrate, washing chromatographic column with acetonitrile of more than 60%, loading, isocratic eluting for 35min, collecting fractions with purity of more than or equal to 90%, mixing, and purifying in two steps.
Step 2: two-step purification
HPLC conditions: a chromatographic column: octadecyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 5cm by 25cm, phase a: 0.5 v/v% phosphoric acid aqueous solution, triethylamine adjusted to pH3.0, phase B: acetonitrile, flow rate 80mL/min, detection wavelength 230nm, mobile phase B11% -14%, linear elution for 60 min.
And (2) a two-step purification process: diluting the qualified fraction collected in one step by 1 time volume with water, washing a chromatographic column with acetonitrile of more than 60%, loading, linearly eluting, taking a peak after 45min, collecting fractions with the purity of more than or equal to 99% and the impurity of less than or equal to 0.1%, and combining the fractions for salt conversion.
And step 3: salt conversion
HPLC conditions: a chromatographic column: octadecyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 5cm by 25cm, A1Phase (1): 80mM/L aqueous ammonium acetate solution, A2Phase (1): 0.08 v/v% aqueous acetic acid, phase B: acetonitrile, flow rate 80mL/min, detection wavelength 230 nm.
A salt conversion process:
1) loading: diluting the qualified fraction collected in the second step by 1 time of volume with water, washing a chromatographic column by using acetonitrile with the concentration of more than 60%, and then loading the chromatographic column;
2) salt exchange: mobile phase A197%, washing the mobile phase B for 15min at 3% isocratic;
3) balancing: mobile phase A297%, washing the mobile phase B for 15min at 3% isocratic;
4) and (3) elution: mobile phase A2And a mobile phase B, linearly eluting by 4-7% of the mobile phase B, peaking after 30min, and collecting qualified fractions.
The purity of the combined sample is 99.725%, the maximum single impurity is less than 0.1%, the pure product is obtained by water bath rotary evaporation concentration at 30 ℃, the total yield is 71.91%, and the detection of the final product is shown in figure 5.
Example 5:
step 1: one-step purification of Selepress cyclization solution
HPLC conditions: a chromatographic column: UniPS10-300, column length diameter: 26mm 46cm, phase a: 0.09% TFA, phase B: acetonitrile, flow rate 19mL/min, detection wavelength 230nm, B phase 30% isocratic elution.
One-step purification process: filtering the cyclized solution with 0.45 μm membrane, collecting filtrate, washing chromatographic column with acetonitrile of more than 60%, loading, isocratic eluting for 35min, collecting fractions with purity of more than or equal to 90%, mixing, and purifying in two steps.
Step 2: two-step purification
HPLC conditions: a chromatographic column: octadecyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 5cm by 25cm, phase a: 50mM/L potassium perchlorate, pH3.0 adjusted by phosphoric acid, phase B: acetonitrile, flow rate 80mL/min, detection wavelength 230nm, mobile phase B11% -14%, linear elution for 60 min.
And (2) a two-step purification process: diluting the qualified fraction collected in one step by 1 time volume with water, washing a chromatographic column with acetonitrile of more than 60%, loading, linearly eluting, taking a peak after 45min, collecting fractions with the purity of more than or equal to 99% and the impurity of less than or equal to 0.1%, and combining the fractions for salt conversion.
And step 3: salt conversion
HPLC conditions: a chromatographic column: octadecyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 5cm by 25cm, A1Phase (1): 80mM/L aqueous ammonium acetate solution, A2Phase (1): 0.09 v/v% aqueous acetic acid, phase B: acetonitrile, flow rate 80mL/min, detection wavelength 230 nm.
A salt conversion process:
1) loading: diluting the qualified fraction collected in the second step by 1 time of volume with water, washing a chromatographic column by using acetonitrile with the concentration of more than 60%, and then loading the chromatographic column;
2) salt exchange: mobile phase A1Washing the mobile phase B2% at equal temperature for 15min, wherein the washing time is 98%;
3) balancing: mobile phase A2Washing the mobile phase B2% for 10min at an isocratic rate of 98%;
4) and (3) elution: mobile phase A2And a mobile phase B, linearly eluting by 4-7% of the mobile phase B, peaking after 30min, and collecting qualified fractions.
The purity of the combined sample is 99.832%, the maximum single impurity is less than 0.1%, the pure product is obtained by water bath rotary evaporation concentration at 30 ℃, the total yield is 70.09%, and the detection of the final product is shown in figure 6.
Example 6
Step 1: one-step purification of Selepress cyclization solution
HPLC conditions: a chromatographic column: UniPS 40-300, column length diameter: 26mm 46cm, phase a: 0.1% TFA, phase B: acetonitrile, flow rate 19mL/min, detection wavelength 230nm, B phase 40% isocratic elution.
One-step purification process: filtering the cyclized solution with 0.45 μm membrane, collecting filtrate, washing chromatographic column with acetonitrile of more than 60%, loading, isocratic eluting for 35min, collecting fractions with purity of more than or equal to 90%, mixing, and purifying in two steps.
Step 2: two-step purification
HPLC conditions: a chromatographic column: the octaalkyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 5cm by 25cm, phase a: 50mM/L sodium dihydrogen phosphate, pH2.5 adjusted with phosphoric acid, phase B: acetonitrile, flow rate 80mL/min, detection wavelength 230nm, mobile phase B11% -14%, linear elution for 60 min.
And (2) a two-step purification process: diluting the qualified fraction collected in one step by 1 time volume with water, washing a chromatographic column with acetonitrile of more than 60%, loading, linearly eluting, taking a peak after 45min, collecting fractions with the purity of more than or equal to 99% and the impurity of less than or equal to 0.1%, and combining the fractions for salt conversion.
And step 3: salt conversion
HPLC conditions: a chromatographic column: the octaalkyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 5cm by 25cm, A1Phase (1): 100mM/L aqueous ammonium acetate solution, A2Phase (1): 0.1 v/v% aqueous acetic acid, phase B: acetonitrile, flow rate 80mL/min, detection wavelength 230 nm.
A salt conversion process:
1) loading: diluting the qualified fraction collected in the second step by 1 time of volume with water, washing a chromatographic column by using acetonitrile with the concentration of more than 60%, and then loading the chromatographic column;
2) salt exchange: mobile phase A1Washing with 98% mobile phase B2% for 20 min;
3) balancing: mobile phase A2Washing with 98% mobile phase B2% for 20 min;
4) and (3) elution: mobile phase A2And a mobile phase B, linearly eluting by 4-7% of the mobile phase B, peaking after 30min, and collecting qualified fractions.
The purity of the combined sample is 99.782%, the maximum single impurity is less than 0.1%, the pure product is obtained by water bath rotary evaporation concentration at 30 ℃, the total yield is 72.20%, and the detection of the final product is similar to the pictures of the above examples.
Example 7
Step 1: one-step purification of Selepress cyclization solution
HPLC conditions: a chromatographic column: UniPS10-300, column length diameter: 70mm x 50cm, phase a: 0.06% TFA, phase B: acetonitrile, flow rate 150mL/min, detection wavelength 230nm, B phase 30% isocratic elution.
One-step purification process: filtering the cyclized solution with 0.45 μm membrane, collecting filtrate, washing chromatographic column with acetonitrile of more than 60%, loading, isocratic eluting for 30min, collecting fractions with purity of more than or equal to 90%, mixing, and purifying in two steps.
Step 2: two-step purification
HPLC conditions: a chromatographic column: octadecyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 15cm by 25cm, phase a: 50mM/L sodium perchlorate, pH3.0 adjusted by phosphoric acid, phase B: acetonitrile, flow rate 400mL/min, detection wavelength 230nm, mobile phase B11% -14%, linear elution for 60 min.
And (2) a two-step purification process: diluting the qualified fraction collected in one step by 1 time volume with water, washing a chromatographic column with acetonitrile of more than 60%, loading, linearly eluting, taking a peak after 40min, collecting fractions with purity of more than or equal to 99% and impurity 1 of less than or equal to 0.1%, and mixing for salt conversion.
And step 3: salt conversion
HPLC conditions: a chromatographic column: octadecyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 15cm by 25cm, A1Phase (1): 70mM/L aqueous ammonium acetate solution, A2Phase (1): 0.05 v/v% aqueous acetic acid, phase B: acetonitrile, flow rate 400mL/min, detection wavelength 230 nm.
A salt conversion process:
1) loading: diluting the qualified fraction collected in the second step by 1 time of volume with water, washing a chromatographic column by using acetonitrile with the concentration of more than 60%, and then loading the chromatographic column;
2) salt exchange: mobile phase A1Washing with 98% mobile phase B2% for 20 min;
3) balancing: mobile phase A2Washing with 98% mobile phase B2% for 20 min;
4) and (3) elution: mobile phase A2And a mobile phase B, linearly eluting by 4-7% of the mobile phase B, peaking after 30min, and collecting qualified fractions.
The purity of the combined sample is 99.829%, the maximum single impurity is less than 0.1%, the pure product is obtained by water bath rotary evaporation concentration at 30 ℃, the total yield is 72.51%, and the detection of the final product is similar to the pictures of the above examples.
Example 8
Step 1: one-step purification of Selepress cyclization solution
HPLC conditions: a chromatographic column: UniPS 30-100, column length diameter: 15 × 25cm, phase a: 0.05% TFA, phase B: acetonitrile, flow rate 400mL/min, detection wavelength 230nm, B phase 30% isocratic elution.
One-step purification process: filtering the cyclized solution with 0.45 μm membrane, collecting filtrate, washing chromatographic column with acetonitrile of more than 60%, loading, isocratic eluting for 30min, collecting fractions with purity of more than or equal to 90%, mixing, and purifying in two steps.
Step 2: two-step purification
HPLC conditions: a chromatographic column: octadecyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 30cm by 25cm, phase a: 50mM/L sodium perchlorate, pH3.0 adjusted by phosphoric acid, phase B: acetonitrile, flow rate 1500mL/min, detection wavelength 230nm, mobile phase B11% -14%, linear elution for 60 min.
And (2) a two-step purification process: diluting the qualified fraction collected in one step by 1 time volume with water, washing a chromatographic column with acetonitrile of more than 60%, loading, linearly eluting, taking a peak after 40min, collecting fractions with purity of more than or equal to 99% and impurity 1 of less than or equal to 0.1%, and mixing for salt conversion.
And step 3: salt conversion
HPLC conditions: a chromatographic column: octadecyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 15cm by 25cm, A1Phase (1): 50mM/L aqueous ammonium acetate solution, A2Phase (1): 0.05 v/v% aqueous acetic acid, phase B: acetonitrile, flow rate 1500mL/min, detection wavelength 230 nm.
A salt conversion process:
1) loading: diluting the qualified fraction collected in the second step by 1 time of volume with water, washing a chromatographic column by using acetonitrile with the concentration of more than 60%, and then loading the chromatographic column;
2) salt exchange: mobile phase A1Washing with 98% mobile phase B2% for 20 min;
3) balancing: mobile phase A2Washing with 98% mobile phase B2% for 20 min;
4) and (3) elution: mobile phase A2And a mobile phase B, linearly eluting by 4-7% of the mobile phase B, peaking after 30min, and collecting qualified fractions.
The purity of the combined sample is 99.790%, the maximum single impurity is less than 0.1%, the pure product is obtained by water bath rotary evaporation concentration at 30 ℃, the total yield is 71.01%, and the detection of the final product is similar to the pictures of the above examples.
Example 9
Step 1: one-step purification of Selepress cyclization solution
HPLC conditions: a chromatographic column: UniPS 40-300, column length diameter: 26mm 46cm, phase a: 0.1% TFA, phase B: acetonitrile, flow rate 19mL/min, detection wavelength 230nm, B phase 35% isocratic elution.
One-step purification process: filtering the cyclized solution with 0.45 μm membrane, collecting filtrate, washing chromatographic column with acetonitrile of more than 60%, loading, isocratic eluting for 30min, collecting fractions with purity of more than or equal to 90%, mixing, and purifying in two steps.
Step 2: two-step purification
HPLC conditions: a chromatographic column: the octaalkyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 5cm by 25cm, phase a: 50mM/L sodium perchlorate, pH2.9 adjusted by phosphoric acid, phase B: acetonitrile, flow rate 80mL/min, detection wavelength 230nm, mobile phase B11% -14%, linear elution for 60 min.
And (2) a two-step purification process: diluting the qualified fraction collected in one step by 1 time volume with water, washing a chromatographic column with acetonitrile of more than 60%, loading, linearly eluting, taking a peak after 45min, collecting fractions with the purity of more than or equal to 99% and the impurity of less than or equal to 0.1%, and combining the fractions for salt conversion.
And step 3: salt conversion
HPLC conditions: a chromatographic column: the octaalkyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 5cm by 25cm, A1Phase (1): 50mM/L aqueous ammonium acetate solution, A2Phase (1): 0.1 v/v% aqueous acetic acid, phase B: acetonitrile, flow rate 80mL/min, detection wavelength 230 nm.
A salt conversion process:
1) loading: diluting the qualified fraction collected in the second step by 1 time of volume with water, washing a chromatographic column by using acetonitrile with the concentration of more than 60%, and then loading the chromatographic column;
2) salt exchange: mobile phase A1Washing the mobile phase B2% at equal temperature for 15min, wherein the washing time is 98%;
3) balancing: mobile phase A2Washing the mobile phase B2% for 10min at an isocratic rate of 98%;
4) and (3) elution: mobile phase A2And a mobile phase B, linearly eluting by 4-7% of the mobile phase B, peaking after 30min, and collecting qualified fractions.
The purity of the combined sample is 99.834%, the maximum single impurity is less than 0.1%, the pure product is obtained by water bath rotary evaporation concentration at 32 ℃, the total yield is 70.13%, and the detection of the final product is similar to the pictures of the above examples.
Example 10
Step 1: one-step purification of Selepress cyclization solution
HPLC conditions: a chromatographic column: UniPS 40-300, column length diameter: 70mm x 50cm, phase a: 0.1% TFA, phase B: acetonitrile, flow rate 150mL/min, detection wavelength 230nm, B phase 33% isocratic elution.
One-step purification process: filtering the cyclized solution with 0.45 μm membrane, collecting filtrate, washing chromatographic column with acetonitrile of more than 60%, loading, isocratic eluting for 30min, collecting fractions with purity of more than or equal to 90%, mixing, and purifying in two steps.
Step 2: two-step purification
HPLC conditions: a chromatographic column: the octaalkyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 15cm by 25cm, phase a: 50mM/L ammonium perchlorate, pH3.2 adjusted by phosphoric acid, phase B: acetonitrile, flow rate 400mL/min, detection wavelength 230nm, mobile phase B11% -14%, linear elution for 60 min.
And (2) a two-step purification process: diluting the qualified fraction collected in one step by 1 time volume with water, washing a chromatographic column with acetonitrile of more than 60%, loading, linearly eluting, taking a peak after 43min, collecting fractions with purity of more than or equal to 99% and impurity 1 of less than or equal to 0.1%, and mixing for salt conversion.
And step 3: salt conversion
HPLC conditions: a chromatographic column: the octaalkyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 15cm by 25cm, A1Phase (1): 60mM/L acetic acidAqueous ammonium solution, A2Phase (1): 0.05 v/v% aqueous acetic acid, phase B: acetonitrile, flow rate 400mL/min, detection wavelength 230 nm.
A salt conversion process:
1) loading: diluting the qualified fraction collected in the second step by 1 time of volume with water, washing a chromatographic column by using acetonitrile with the concentration of more than 60%, and then loading the chromatographic column;
2) salt exchange: mobile phase A197%, washing the mobile phase B for 15min at 3% isocratic;
3) balancing: mobile phase A297%, and isocratic washing of the mobile phase B3% for 20 min;
4) and (3) elution: mobile phase A2And a mobile phase B, linearly eluting by 4-7% of the mobile phase B, peaking after 30min, and collecting qualified fractions.
The purity of the combined sample is 99.798%, the maximum single impurity is less than 0.1%, the pure product is obtained by water bath rotary evaporation concentration at 25 ℃, the total yield is 72.83%, and the detection of the final product is similar to the pictures of the above examples.
Example 11
Step 1: one-step purification of Selepress cyclization solution
HPLC conditions: a chromatographic column: UniPS30-300, column length diameter: 70mm x 50cm, phase a: 0.05% TFA, phase B: acetonitrile, flow rate 150mL/min, detection wavelength 230nm, B phase 37% isocratic elution.
One-step purification process: filtering the cyclized solution with 0.45 μm membrane, collecting filtrate, washing chromatographic column with acetonitrile of more than 60%, loading, isocratic eluting for 30min, collecting fractions with purity of more than or equal to 90%, mixing, and purifying in two steps.
Step 2: two-step purification
HPLC conditions: a chromatographic column: the octaalkyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 15cm by 25cm, phase a: 50mM/L sodium perchlorate, pH3.0 adjusted by phosphoric acid, phase B: acetonitrile, flow rate 400mL/min, detection wavelength 230nm, mobile phase B11% -14%, linear elution for 60 min.
And (2) a two-step purification process: diluting the qualified fraction collected in one step by 1 time volume with water, washing a chromatographic column with acetonitrile of more than 60%, loading, linearly eluting, taking a peak after 45min, collecting fractions with the purity of more than or equal to 99% and the impurity of less than or equal to 0.1%, and combining the fractions for salt conversion.
And step 3: salt conversion
HPLC conditions: a chromatographic column: the octaalkyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 15cm by 25cm, A1Phase (1): 50mM/L aqueous ammonium acetate solution, A2Phase (1): 0.06 v/v% aqueous acetic acid, phase B: acetonitrile, flow rate 400mL/min, detection wavelength 230 nm.
A salt conversion process:
1) loading: diluting the qualified fraction collected in the second step by 1 time of volume with water, washing a chromatographic column by using acetonitrile with the concentration of more than 60%, and then loading the chromatographic column;
2) salt exchange: mobile phase A197%, and isocratic washing of the mobile phase B3% for 20 min;
3) balancing: mobile phase A297%, and isocratic washing of the mobile phase B3% for 20 min;
4) and (3) elution: mobile phase A2And a mobile phase B, linearly eluting by 4-7% of the mobile phase B, peaking after 30min, and collecting qualified fractions.
The purity of the combined sample is 99.868 percent, the maximum single impurity is less than 0.1 percent, the pure product is obtained by water bath rotary evaporation concentration at 35 ℃, the total yield is 70.10 percent, and the detection of the final product is similar to the pictures of the above examples.
Example 12
Step 1: one-step purification of Selepress cyclization solution
HPLC conditions: a chromatographic column: UniPS10-300, column length diameter: 15 × 25cm, phase a: 0.1% TFA, phase B: acetonitrile, flow rate 400mL/min, detection wavelength 230nm, B phase 40% isocratic elution.
One-step purification process: filtering the cyclized solution with 0.45 μm membrane, collecting filtrate, washing chromatographic column with acetonitrile of more than 60%, loading, isocratic eluting for 35min, collecting fractions with purity of more than or equal to 90%, mixing, and purifying in two steps.
Step 2: two-step purification
HPLC conditions: a chromatographic column: the octaalkyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 30cm by 25cm, phase a: 100mM/L sodium perchlorate, pH3.0 adjusted by phosphoric acid, phase B: acetonitrile, flow rate 1500mL/min, detection wavelength 230nm, mobile phase B11% -14%, linear elution for 60 min.
And (2) a two-step purification process: diluting the qualified fraction collected in one step by 1 time volume with water, washing a chromatographic column with acetonitrile of more than 60%, loading, linearly eluting, taking a peak after 40min, collecting fractions with purity of more than or equal to 99% and impurity 1 of less than or equal to 0.1%, and mixing for salt conversion.
And step 3: salt conversion
HPLC conditions: a chromatographic column: the octaalkyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 30cm by 25cm, A1Phase (1): 50mM/L aqueous ammonium acetate solution, A2Phase (1): 0.05 v/v% aqueous acetic acid, phase B: acetonitrile, flow rate 1500mL/min, detection wavelength 230 nm.
A salt conversion process:
1) loading: diluting the qualified fraction collected in the second step by 1 time of volume with water, washing a chromatographic column by using acetonitrile with the concentration of more than 60%, and then loading the chromatographic column;
2) salt exchange: mobile phase A1Washing with 98% mobile phase B2% for 20 min;
3) balancing: mobile phase A2Washing with 98% mobile phase B2% for 20 min;
4) and (3) elution: mobile phase A2And a mobile phase B, linearly eluting by 4-7% of the mobile phase B, peaking after 30min, and collecting qualified fractions.
After combination, the purity of the sample is 99.873%, the maximum single impurity is less than 0.1%, the impurity 1 is less than 0.1%, the pure product is obtained by water bath rotary evaporation and concentration at 33 ℃, the total yield is 70.56%, and the detection of the final product is similar to the pictures of the above examples.
Comparative example 1
Step 1: pretreatment of Selepress cyclization solution
Collecting cyclized solution, and filtering with 0.45 μm membrane
Step 2: one-step purification
HPLC conditions: a chromatographic column: octadecyl bonded silica gel is a fixed chromatographic column, and the length and diameter of the column are as follows: 5cm 25 cm. Phase A: 1 percent of phosphoric acid aqueous solution and triethylamine are used for adjusting the pH value to 5.2; phase B: and (3) acetonitrile. Flow rate: 80 ml/min. Detection wavelength: 230 nm. Elution was linear for 120min with mobile phase B8% -28%.
One-step purification process: washing chromatographic column with acetonitrile water of more than 60%, loading, linearly eluting, peaking after 55min, collecting fractions with purity of more than or equal to 90%, mixing, and transferring salt
And step 3: salt conversion
HPLC conditions: a chromatographic column: octadecyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 5cm by 25cm, A1Phase (1): 50mM/L aqueous ammonium acetate solution, A2Phase (1): 0.1 v/v% aqueous acetic acid, phase B: acetonitrile, flow rate 80mL/min, detection wavelength 230 nm.
A salt conversion process:
1) loading: diluting the qualified fraction collected in the one step by 1 time of volume with water, washing a chromatographic column with more than 60% acetonitrile, and then loading the chromatographic column;
2) salt exchange: mobile phase A194 percent, and the mobile phase B is washed for 20min with 6 percent isocratic;
3) balancing: mobile phase A294 percent, and the mobile phase B is washed for 12min with 6 percent isocratic;
4) and (3) elution: mobile phase A2And a mobile phase B, linearly eluting by 2-10% of the mobile phase B, taking a peak after 40min, and collecting fractions with higher purity.
After combination, the purity of the sample is 98.103%, the maximum single impurity is 0.550% (namely impurity 1), the pure product is obtained by water bath rotary evaporation and concentration at 30 ℃, the total yield is 31.52%, and the detection of the final product is shown in figure 7.
Comparative example 2
Step 1: pretreatment of Selepress cyclization solution
The cyclized solution was collected and filtered through a 0.45 μm membrane.
Step 2: one-step purification
HPLC conditions: a chromatographic column: the octaalkyl bonded silica gel is a fixed chromatographic column, and the length and diameter of the column are as follows: 10cm 25 cm. Phase A: 1 percent of phosphoric acid aqueous solution and triethylamine are used for adjusting the pH value to 5.0; phase B: and (3) acetonitrile. Flow rate: 220 ml/min. Detection wavelength: 230 nm. Linearly eluting with mobile phase B8% -28% for 120min
One-step purification process: washing the chromatographic column with acetonitrile water of more than 60%, loading, linearly eluting, taking out peak after 55min, collecting fractions with purity of more than or equal to 90%, and mixing for salt conversion.
And step 3: salt conversion
HPLC conditions: a chromatographic column: the octaalkyl bonded silica gel is a stationary phase chromatographic column, and the length and diameter of the column are as follows: 10cm by 25cm, A1Phase (1): 50mM/L aqueous ammonium acetate solution, A2Phase (1): 0.1 v/v% aqueous acetic acid, phase B: and (3) acetonitrile. The flow rate was 220mL/min, and the detection wavelength was 230 nm.
A salt conversion process:
1) loading: diluting the qualified fraction collected in the one step by 1 time of volume with water, washing a chromatographic column with more than 60% acetonitrile, and then loading the chromatographic column;
2) salt exchange: mobile phase A194 percent, and the mobile phase B is washed for 20min with 6 percent isocratic;
3) balancing: mobile phase A294 percent, and the mobile phase B is washed for 12min with 6 percent isocratic;
4) and (3) elution: mobile phase A2And a mobile phase B, linearly eluting by 2-10% of the mobile phase B, taking a peak after 40min, and collecting fractions with higher purity.
After combination, the purity of the sample is 98.321%, the maximum single impurity is 0.525% (namely impurity 1), the pure product is obtained by water bath rotary evaporation concentration at 30 ℃, the total yield is 35.12%, and the detection of the final product is shown in figure 8.
In conclusion, the invention has simple operation, large upper amount and batch production, the purity of the finished product Selepress is more than 99.5 percent, the maximum single impurity is less than 0.1 percent, the difficult-to-remove impurity 1 is less than 0.1 percent, the high quality meets the medicinal standard, the yield is more than 70 percent, while the original patent EP1984012 has the purity of 98.8 percent after purification and the total yield of only 30 percent, and has great breakthrough compared with the prior art.
The above description of the embodiments is only for the purpose of assisting understanding of the method of the present invention and the core idea thereof, and should not be construed as limiting the present invention.
Claims (15)
1. A method for purifying Selepress, which is characterized by comprising the following steps:
step 1: performing one-step purification, namely filtering the cyclized solution, and performing isocratic elution in the range of 25-45% of acetonitrile volume by taking a polymer filler as a stationary phase, a trifluoroacetic acid aqueous solution as an A phase and acetonitrile as a B phase to obtain a one-step purified sample for two-step purification;
step 2: performing two-step purification, namely diluting the sample solution obtained in the step 1 by using water and then loading the sample by using octadecylsilane or octaalkylsilane bonded silica as a stationary phase, performing linear gradient elution within the range of 10-30% of acetonitrile volume ratio by using a perchlorate aqueous solution or a phosphate solution as a phase A and acetonitrile as a phase B, and combining the samples for salt transfer;
and step 3: salt conversion, using octadecylsilane or octaalkylsilane bonded silica as stationary phase, diluting the sample solution obtained in step 2 with water, loading, and using ammonium acetate aqueous solution as A1Phase B, acetonitrile, changing salt at equal degree within the range of acetonitrile volume ratio of 2% -3% for 15min-20min, and then taking acetic acid water solution as A2Phase B, acetonitrile, 2-3% acetonitrile volume ratio, isocratic equilibrium for 10-20 min, and acetic acid water solution A2Phase B, acetonitrile, eluting linearly in the acetonitrile volume ratio of 4 v/v% -15 v/v%, mixing the samples, concentrating, and lyophilizing.
2. The method of claim 1, wherein: the purity of Selepressin in the cyclization solution is more than 55%.
3. The method of claim 1, wherein: the purity of Selepressin in the cyclized solution is preferably 60% or more.
4. The method of claim 1, wherein: in the step 1, the polymer filler matrix is styrene or methyl styrene acrylate.
5. The method of claim 1, wherein: the polymer filler in the step 1 is selected from UniPS 30-100, UniPS30-300, UniPS 40-300, UniPS10-300, UniPSN 30-300, UniPSN 40-300 and UniPMM 40-500.
6. The method of claim 1, wherein: the polymer filler in step 1 is selected from UniPS 30-100, UniPS30-300, UniPS 40-300 and UniPS 10-300.
7. The method of claim 1, wherein: the volume ratio of the trifluoroacetic acid aqueous solution in the step 1 is 0.05 v/v% -0.5 v/v%.
8. The method of claim 1, wherein: the volume of the isocratic elution acetonitrile in the step 1 is preferably 30-40%.
9. The method of claim 1, wherein: in the step 2, the A-phase perchlorate aqueous solution is selected from sodium perchlorate, potassium perchlorate or ammonium perchlorate aqueous solution.
10. The method of claim 9, wherein: the pH value of the A-phase perchlorate aqueous solution is 2.0-3.5, and the salt concentration is 20mM/L-200 mM/L.
11. The method of claim 1, wherein: in the step 2, the A-phase phosphate solution is selected from a triethylamine phosphate solution, a sodium dihydrogen phosphate solution and a potassium dihydrogen phosphate solution.
12. The method of claim 11, wherein: the pH value of the triethylamine phosphate solution is 2.5-3.5, and the salt concentration is 0.1 v/v% -1.2 v/v%.
13. The method of claim 11, wherein: the pH of the sodium dihydrogen phosphate solution and the potassium dihydrogen phosphate solution is 2.5-3.5, and the salt concentration is 30mM/L-100 mM/L.
14. The method of claim 1, wherein: a in step 31The concentration of the phase ammonium acetate aqueous solution is 45mM/L-100mM/L, A2The concentration of the phase acetic acid aqueous solution is 0.01 v/v% -0.5 v/v%.
15. The method of claim 1, wherein: the concentration in the step 3 is water bath rotary evaporation concentration, wherein the temperature of the water bath is 25-35 ℃.
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