CN113683663A - Purification method of organism-protected polypeptide crude product - Google Patents
Purification method of organism-protected polypeptide crude product Download PDFInfo
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 103
- 238000000746 purification Methods 0.000 title claims abstract description 103
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 95
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 95
- 238000000034 method Methods 0.000 title claims abstract description 57
- 239000012043 crude product Substances 0.000 title claims abstract description 54
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 99
- 238000000926 separation method Methods 0.000 claims abstract description 11
- 239000000047 product Substances 0.000 claims abstract description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 90
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 54
- 238000001514 detection method Methods 0.000 claims description 46
- 238000010828 elution Methods 0.000 claims description 37
- 239000000243 solution Substances 0.000 claims description 29
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 239000007864 aqueous solution Substances 0.000 claims description 17
- 238000004108 freeze drying Methods 0.000 claims description 10
- 230000001681 protective effect Effects 0.000 claims description 10
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 8
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 8
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 8
- 239000000945 filler Substances 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 3
- 239000012264 purified product Substances 0.000 claims description 2
- 239000012045 crude solution Substances 0.000 claims 1
- 239000012535 impurity Substances 0.000 abstract description 27
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 abstract description 12
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 abstract description 6
- 241000282414 Homo sapiens Species 0.000 abstract description 4
- DHBXNPKRAUYBTH-UHFFFAOYSA-N 1,1-ethanedithiol Chemical compound CC(S)S DHBXNPKRAUYBTH-UHFFFAOYSA-N 0.000 abstract description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 abstract description 3
- 125000006239 protecting group Chemical group 0.000 abstract description 3
- 239000013557 residual solvent Substances 0.000 abstract description 3
- 229910001415 sodium ion Inorganic materials 0.000 abstract description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 abstract description 2
- 238000010521 absorption reaction Methods 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 239000012071 phase Substances 0.000 description 61
- 238000004587 chromatography analysis Methods 0.000 description 12
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical group O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 description 10
- 239000011347 resin Substances 0.000 description 8
- 229920005989 resin Polymers 0.000 description 8
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 8
- ZPGDWQNBZYOZTI-SFHVURJKSA-N (2s)-1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZPGDWQNBZYOZTI-SFHVURJKSA-N 0.000 description 7
- 150000001413 amino acids Chemical class 0.000 description 7
- 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 6
- 238000001914 filtration Methods 0.000 description 6
- 239000008213 purified water Substances 0.000 description 6
- 238000007865 diluting Methods 0.000 description 5
- 239000012467 final product Substances 0.000 description 5
- 238000011068 loading method Methods 0.000 description 5
- 150000003839 salts Chemical group 0.000 description 5
- 238000005303 weighing Methods 0.000 description 5
- QWXZOFZKSQXPDC-NSHDSACASA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C)C(O)=O)C3=CC=CC=C3C2=C1 QWXZOFZKSQXPDC-NSHDSACASA-N 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- FODJWPHPWBKDON-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[(2-methylpropan-2-yl)oxy]-4-oxobutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 FODJWPHPWBKDON-IBGZPJMESA-N 0.000 description 2
- CBPJQFCAFFNICX-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(C)C)C(O)=O)C3=CC=CC=C3C2=C1 CBPJQFCAFFNICX-IBGZPJMESA-N 0.000 description 2
- OTKXCALUHMPIGM-FQEVSTJZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-[(2-methylpropan-2-yl)oxy]-5-oxopentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 OTKXCALUHMPIGM-FQEVSTJZSA-N 0.000 description 2
- UMRUUWFGLGNQLI-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UMRUUWFGLGNQLI-QFIPXVFZSA-N 0.000 description 2
- PMZXXNPJQYDFJX-UHFFFAOYSA-N acetonitrile;2,2,2-trifluoroacetic acid Chemical compound CC#N.OC(=O)C(F)(F)F PMZXXNPJQYDFJX-UHFFFAOYSA-N 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- XXMFJKNOJSDQBM-UHFFFAOYSA-N 2,2,2-trifluoroacetic acid;hydrate Chemical compound [OH3+].[O-]C(=O)C(F)(F)F XXMFJKNOJSDQBM-UHFFFAOYSA-N 0.000 description 1
- HEEWEZGQMLZMFE-RKGINYAYSA-N CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@H]1N(C(=O)[C@H]2N(CCC2)C(=O)[C@H]2N(CCC2)C(=O)[C@H](CCC(O)=O)NC(=O)CN)CCC1 Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@H]1N(C(=O)[C@H]2N(CCC2)C(=O)[C@H]2N(CCC2)C(=O)[C@H](CCC(O)=O)NC(=O)CN)CCC1 HEEWEZGQMLZMFE-RKGINYAYSA-N 0.000 description 1
- 208000013875 Heart injury Diseases 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- UNXNGGMLCSMSLH-UHFFFAOYSA-N dihydrogen phosphate;triethylazanium Chemical compound OP(O)(O)=O.CCN(CC)CC UNXNGGMLCSMSLH-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 208000016616 gastric juice peptides Diseases 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 238000005583 trifluoroacetylation reaction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the technical field of biomolecule separation and purification, and provides a purification method of a crude product of a body-protecting polypeptide. The first high performance liquid chromatography purification of the invention can remove residual solvents (including trifluoroacetic acid, ethanedithiol, methyl tert-butyl ether), residual protecting groups and most impurities contained in the crude product; the second high performance liquid chromatography purification is capable of removing impurities and phosphate ions and sodium ions that were not removed by the first high performance liquid chromatography purification. The acetate of the organism protection polypeptide is obtained by the purification method provided by the invention, and is beneficial to the absorption of a human body; and the obtained organism protection polypeptide has high purity and high yield. The data of the examples show that: the purity of the pure product of the organism protection polypeptide is more than 99.58 percent, and the content of single impurities is less than 0.1 percent.
Description
Technical Field
The invention relates to the technical field of biomolecule separation and purification, in particular to a purification method of a crude product of body-protecting polypeptide.
Background
The body protective polypeptide (BPC-157) is a fragment of gastric juice peptide, contains 15 amino acids, has a molecular weight of 1419.5, and has a sequence of Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val. The BPC-157 can promote the regeneration of muscles and ligaments, promote the healing of wound surfaces and improve the hardness and elasticity of tendons; has important effect on enhancing muscle of body building athletes. Research shows that BPC-157 has multiple protective effects in human organs, such as pancreas, liver injury, endothelium, heart injury and joints, so that BPC-157 becomes a multifunctional peptide and can bring huge benefits to human beings. Chinese patent publication No. CN102229649a discloses that the synthesized crude product is purified, eluted and lyophilized by HPLC using 0.1% trifluoroacetic acid-water, 0.1% trifluoroacetic acid-acetonitrile system to obtain BPC-157 sample. The obtained BPC-157 sample has low purity and high single impurity content.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for purifying a crude product of an organism-protected polypeptide. The purification method provided by the invention has the advantages that the obtained organism protection polypeptide has high purity and low single impurity content.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a purification method of a crude product of body-protecting polypeptide, which comprises the following steps:
dissolving the organism protection polypeptide crude product to obtain an organism protection polypeptide crude product solution;
performing high performance liquid chromatography separation and purification on the organism protection polypeptide crude product solution to obtain an organism protection polypeptide pure product;
the high performance liquid chromatography separation and purification comprises a first high performance liquid chromatography purification and a second high performance liquid chromatography purification which are sequentially carried out;
the parameters of the first high performance liquid chromatography purification include:
a first chromatographic column: c18A column;
first mobile phase a: the concentration of the sodium dihydrogen phosphate aqueous solution is 3-30 g/L;
first mobile phase B: acetonitrile;
the first flow rate: 1-1000 mL/min;
the first gradient elution procedure was:
0~60min:n1vol%→m1vol% first mobile phase B; n is1Is 5 to 10, m130 to 60;
first detection wavelength: 210-260 nm;
the parameters of the second high performance liquid chromatography purification include:
second mobile phase a: the volume ratio of acetic acid to water in the acetic acid aqueous solution is (0.1-5): 100, respectively;
second mobile phase B: acetonitrile;
the second flow rate: 1-1000 mL/min;
the second gradient elution procedure was:
0~60min:n2vol%→m2vol% second mobile phase B; n is2Is 5 to 10, m230 to 60;
second detection wavelength: 210-260 nm.
Preferably, the solvent for dissolving the crude product of the body protection polypeptide is acetic acid water solution; the volume percentage of acetic acid in the acetic acid aqueous solution is 5-15%.
Preferably, the concentration of the organism protection peptide in the organism protection polypeptide crude product solution is 10-100 mg/mL.
Preferably, the first high performance liquid chromatography purification yields a first purified body protective polypeptide; and performing second high performance liquid chromatography purification on the first purified body-protecting polypeptide.
Preferably, the first detection wavelength and the second detection wavelength are independently 220-250 nm.
Preferably, the effective lengths of the first chromatographic column and the second chromatographic column are independently more than or equal to 150 mm; the diameters are independently 10-200 mm; the particle size of the filler of the first chromatographic column and the second chromatographic column is 10-30 μm independently.
Preferably, the parameters of the first high performance liquid chromatography purification include: the gradient elution procedure was: 0-60 min: 5 vol% → 55 vol% first flow B phase; the parameters of the second high performance liquid chromatography purification include: the gradient elution procedure was: 0-60 min: 10 vol% → 60 vol% second mobile B phase.
Preferably, the parameters of the first high performance liquid chromatography purification include: the gradient elution procedure was: 0-60 min: 10 vol% → 30 vol% first mobile phase B; the parameters of the second high performance liquid chromatography purification include: the gradient elution procedure was: 0-60 min: 10 vol% → 50 vol% of the second mobile phase B.
Preferably, the parameters of the first high performance liquid chromatography purification include: the gradient elution procedure was: 0-60 min: 5 vol% → 55 vol% first flow B phase; the parameters of the second high performance liquid chromatography purification include: the gradient elution procedure was: 0-60 min: 10 vol% → 40 vol% of the second mobile phase B.
Preferably, after the second high performance liquid chromatography purification, the method further comprises freeze-drying the obtained second high performance liquid chromatography purified fraction to obtain the purified product of the body-protecting polypeptide.
The invention provides a purification method of a crude product of body-protecting polypeptide, which comprises the following steps: dissolving the organism protection polypeptide crude product to obtain an organism protection polypeptide crude product solution; performing high performance liquid chromatography separation and purification on the organism protection polypeptide crude product solution; the high performance liquid chromatography separation and purification comprises a first high performance liquid chromatography purification and a second high performance liquid chromatography purification which are sequentially carried out; the parameters of the first high performance liquid chromatography purification include: a first chromatographic column: c18A column; first mobile phase a: the concentration of the sodium dihydrogen phosphate aqueous solution is 3-30 g/L; first mobile phase B: acetonitrile; the first flow rate: 1-1000 mL/min; the first gradient elution procedure was: 0-60 min: n is1vol%→m1vol% first mobile phase B; n is1Is 5 to 10, m130 to 60; first detection wavelength: 210-260 nm; the parameters of the second high performance liquid chromatography purification include: second mobile phase a: the volume ratio of acetic acid to water in the acetic acid aqueous solution is (0.1-5): 100, respectively; second mobile phase B: acetonitrile; the second flow rate: 1-1000 mL/min; the second gradient elution procedure was: 0-60 min: n is2vol%→m2vol% second mobile phase B; n is2Is 5 to 10, m230 to 60; second detection wavelength: 210-260 nm.
The first high performance liquid chromatography purification of the invention can remove residual solvents (including trifluoroacetic acid, ethanedithiol, methyl tert-butyl ether), residual protecting groups and most impurities contained in the crude product; the second high performance liquid chromatography purification is capable of removing impurities and phosphate ions and sodium ions that were not removed by the first high performance liquid chromatography purification. The organism protection polypeptide obtained by the purification method provided by the invention has high purity and low single impurity content; meanwhile, the obtained acetate of the polypeptide is protected by the organism and is beneficial to the absorption of the human body. The data of the examples show that: the purity of the pure product of the organism protection polypeptide is more than 99.58 percent, and the content of single impurities is less than 0.1 percent.
Detailed Description
The invention provides a purification method of a crude product of body-protecting polypeptide, which comprises the following steps:
dissolving the organism protection polypeptide crude product to obtain an organism protection polypeptide crude product solution;
and (3) carrying out high performance liquid chromatography separation and purification on the crude organism protective polypeptide solution to obtain a pure organism protective polypeptide product.
In the present invention, the starting materials used in the present invention are preferably commercially available products unless otherwise specified.
The invention dissolves the organism protection polypeptide crude product to obtain the organism protection polypeptide crude product solution.
In the invention, the purity of the organism protection polypeptide crude product is preferably more than or equal to 60%. In the invention, the impurities in the crude product of the organism protection polypeptide preferably comprise acetylation impurities, trifluoroacetylation impurities, impurities which lack single or multiple amino acids and are connected with single or multiple amino acids.
In the invention, the crude product of the body protection polypeptide is preferably synthesized by Fmoc method solid phase; the preparation method of the matrix-protected polypeptide crude product preferably comprises the following steps: Fmoc-Val-Resin is used as initial Resin, and the following protected amino acids are connected in sequence: Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Gly-OH; obtaining the peptide Resin H-Gly-Glu (OtBu) -Pro-Pro-Gly-Lys (Boc) -Pro-Ala-Asp (OtBu) -Ala-Gly-Leu-Val-Resin; trifluoroacetic acid was used: water volume ratio 95: 5, the peptide Resin H-Gly-Glu (OtBu) -Pro-Pro-Pro-Gly-Lys (Boc) -Pro-Ala-Asp (OtBu) -Ala-Gly-Leu-Val-Resin is cracked by the mixed solution, filtrate is filtered, and the crystal ether is precipitated and washed to finally obtain the crude product of the matrix protection polypeptide.
In the invention, the solvent for dissolving the crude organism protection polypeptide is preferably acetic acid water solution; the volume percentage content of acetic acid in the acetic acid aqueous solution is preferably 5% to 15%, and more preferably 10%.
In the invention, the concentration of the organism protection peptide in the organism protection polypeptide crude product solution is preferably 10-100 mg/mL.
After the organism protection polypeptide crude product is dissolved, preferably, filtering is further carried out, so that an organism protection polypeptide crude product solution is obtained.
After the organism protection polypeptide crude product solution is obtained, the invention performs high performance liquid chromatography separation and purification on the organism protection polypeptide crude product solution to obtain an organism protection polypeptide pure product.
In the invention, the high performance liquid chromatography separation and purification comprises sequentially carrying out first high performance liquid chromatography purification and second high performance liquid chromatography purification.
The parameters of the first high performance liquid chromatography purification include:
a first chromatographic column: c18A column; the effective length of the first chromatographic column is preferably not less than 150mm, and is further preferably 150-250 mm; the diameter is preferably 10-200 mm; the particle size of the filler of the first chromatographic column is preferably 10 to 30 μm, and more preferably 10 μm.
First mobile phase a: the concentration of the sodium dihydrogen phosphate aqueous solution is 3-30 g/L, preferably 10-20 g/L;
first mobile phase B: acetonitrile;
the first flow rate: 1-1000 mL/min, in the present invention, the first flow rate is preferably related to the diameter of the first chromatographic column, specifically: the first flow rate is 5mL/min when the diameter of the first chromatography column is 10 mm; the first flow rate is 10mL/min when the diameter of the first chromatography column is 20 mm; the first flow rate is 25mL/min when the diameter of the first chromatography column is 30 mm; the first flow rate is 60mL/min when the diameter of the first chromatography column is 50 mm; the first flow rate is 250mL/min when the diameter of the first chromatography column is 100 mm; the first flow rate is 1000mL/min when the diameter of the first chromatography column is 200 mm;
the first gradient elution procedure was:
0~60min:n1vol%→m1vol% first mobile phase B; n is1Is 5 to 10, m130 to 60; specifically, it is preferable that: 0-60 min: 5 vol% → 55 vol% first flow B phase; or, 0-60 min: 10 vol% → 30 vol% first mobile phase B; or, 0-60 min: 5 vol% → 55 vol% first flow B phase.
First detection wavelength: 210 to 260nm, preferably 220 to 250 nm.
After the first high performance liquid chromatography purification, the first purified body-protecting polypeptide is preferably obtained; and performing second high performance liquid chromatography purification on the first purified body-protecting polypeptide. In the invention, the fraction collection is controlled and combined according to the determined process and the intermediate control standard. In the present invention, the fraction N subjected to the second high performance liquid chromatography purification is preferably diluted before being subjected to the second high performance liquid chromatography purification; the diluted reagent is preferably purified water; the dilution factor is preferably 1 fold.
In the present invention, the first high performance liquid chromatography purification is capable of removing residual solvents (including trifluoroacetic acid, ethanedithiol, methyl tert-butyl ether), residual protecting groups and most of impurities contained in the crude product.
In the present invention, the parameters of the second high performance liquid chromatography purification include:
second chromatographic column: c18A column; the effective length of the second chromatographic column is preferably not less than 150mm, and is further preferably 150-250 mm; the diameter is preferably 10-200 mm; the particle size of the filler of the second chromatographic column is preferably 10-30 μm, and more preferably 10 μm.
A second mobile phase A: the volume ratio of acetic acid to water in the acetic acid aqueous solution is (0.1-5): 100, preferably (0.5-2): 100, respectively;
a second mobile phase B: acetonitrile;
the second flow rate: 1-1000 mL/min; 1-1000 mL/min, in the present invention, the second flow rate is preferably related to the diameter of the second chromatographic column, specifically: the second flow rate is 5mL/min when the diameter of the second chromatography column is 10 mm; the second flow rate is 10mL/min when the diameter of the second chromatography column is 20 mm; the second flow rate is 25mL/min when the diameter of the second chromatography column is 30 mm; the second flow rate is 60mL/min when the diameter of the second chromatography column is 50 mm; the second flow rate is 250mL/min when the diameter of the second chromatography column is 100 mm; the second flow rate is 1000mL/min when the diameter of the second chromatography column is 200 mm;
the second gradient elution procedure was:
0~60min:n2vol%→m2vol% second mobile phase B; n is2Is 5 to 10, m230 to 60; specifically, it is preferable that: 0-60 min: 10 vol% → 60 vol% second mobile B phase; or, 0-60 min: 10 vol% → 50 vol% second mobile B phase; or, 0-60 min: 10 vol% → 40 vol% of the second mobile phase B.
Second detection wavelength: 210 to 260nm, preferably 220 to 250 nm.
In the present invention, the second high performance liquid chromatography purification can remove impurities, phosphate ions and sodium ions, which are not removed by the first high performance liquid chromatography purification.
After the second high performance liquid chromatography purification, the invention preferably further comprises freeze-drying the obtained second high performance liquid chromatography purified fraction to obtain the pure product of the organism protection polypeptide.
In the present invention, when the volume of the second HPLC-purified fraction is < 1L, flask-hanging freeze-drying is preferably employed; when the volume of the second high performance liquid chromatography purification fraction is more than or equal to 1L, plate freeze drying is preferably adopted.
The following examples are provided to illustrate the purification method of crude body-protecting polypeptide of the present invention in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
The preparation method of the BPC-157 polypeptide crude product comprises the following steps:
Fmoc-Val-Resin is used as a starting Resin, coupling is carried out according to the size of 80mmol polypeptide, and the following protected amino acids of Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Gly-OH and amino acid raw materials are sequentially connected, wherein the amino acid raw materials are added according to the molar quantity of 2 times, namely 160 mmol; obtaining the peptide Resin H-Gly-Glu (OtBu) -Pro-Pro-Gly-Lys (Boc) -Pro-Ala-Asp (OtBu) -Ala-Gly-Leu-Val-Resin; trifluoroacetic acid was used: water volume ratio 95: 5, the peptide Resin H-Gly-Glu (OtBu) -Pro-Pro-Pro-Gly-Lys (Boc) -Pro-Ala-Asp (OtBu) -Ala-Gly-Leu-Val-Resin is cracked by the mixed solution, filtrate is filtered, and the filtrate is precipitated and washed by glacial ethyl ether, so that the crude substrate protection polypeptide (hereinafter referred to as BPC-157 polypeptide crude product) is finally obtained, wherein the purity is 72 percent, and the mass is 115.2 g.
Weighing 182mg of BPC-157 polypeptide crude product, dissolving the BPC-157 polypeptide crude product into 20mL of acetic acid-water (the volume ratio of acetic acid to water is 5: 100), and filtering to obtain the organism protection polypeptide crude product solution.
After loading, performing a first high performance liquid chromatography purification, wherein parameters of the first high performance liquid chromatography purification comprise: the first chromatographic column is 20mm x 250mm C18-20 μm; the first flow rate is 10 mL/min; 5.2g/L sodium dihydrogen phosphate aqueous solution is used as a first mobile phase A, and acetonitrile is used as a first mobile phase B; the gradient elution procedure was: 0-60 min: 5 vol% → 55 vol% first flow B phase; detecting with detection wavelength of 210nm, performing high performance liquid chromatography on each eluate, collecting No. 3-8 fraction with purity of 98.53% and maximum single impurity of 0.38%, and obtaining first purified organism protective polypeptide.
The conditions of the high performance liquid chromatography detection are as follows:
mobile phase: a: trifluoroacetic acid aqueous solution with volume concentration of 0.1 percent; b: trifluoroacetic acid acetonitrile solution with volume concentration of 0.1%;
column temperature: 40 ℃;
detection wavelength: 220 nm;
flow rate: 1.0 mL/min;
the elution procedure is shown in table 1:
TABLE 1 elution procedure
Diluting the collected first purified organism protection polypeptide by one volume with purified water, and then carrying out second high performance liquid chromatography purification and salt exchange elution: the parameters of the second high performance liquid chromatography purification include: the second column is 20mm X250 mm C18-20 μm; the second flow rate is 10 mL/min; taking an acetic acid water solution with the acetic acid volume percentage content of 0.5% as a second mobile phase A, and taking acetonitrile as a second mobile phase B; the gradient elution procedure is 0-60 min: 10 vol% → 60 vol% second mobile phase B, detection wavelength 220nm, fraction 1-7# was collected and subjected to vial-hanging lyophilization, and detection was performed according to the conditions of the high performance liquid chromatography detection described above, and results were: the chromatographic purity of the final product was 99.82% with a maximum single impurity of 0.06%; the mass was 83mg, the yield was 46%, and the acetic acid content was 5.8%.
Example 2
Weighing 10.5g of BPC-157 polypeptide crude product, dissolving the BPC-157 polypeptide crude product into 200mL of acetic acid-water (the volume ratio of acetic acid to water is 10: 100), and filtering to obtain the organism protection polypeptide crude product solution.
After loading, performing a first high performance liquid chromatography purification, wherein parameters of the first high performance liquid chromatography purification comprise: the first chromatographic column is C of 100mm multiplied by 250mm18-10 μm; the first flow rate is 250 mL/min; taking 15.6g/L sodium dihydrogen phosphate aqueous solution as a first mobile phase A and acetonitrile as a first mobile phase B, and carrying out gradient elution procedures as follows: 0-60 min, 10 vol% → 30 vol% of a first mobile phase B, a detection wavelength of 220nm, subjecting each eluate to high performance liquid chromatography detection, collecting fractions # 1-7, detecting according to the conditions of the high performance liquid chromatography detection of example 1,the results were: the purity is 98.45 percent, the maximum single impurity is 0.32 percent, and the first purified organism protection polypeptide is obtained.
Diluting the first purified organism protection polypeptide by one volume with purified water, and then carrying out second high performance liquid chromatography purification, salt exchange and elution: the parameters of the second high performance liquid chromatography purification include: the second column is 100mm X250 mm C18-10 μm; the second flow rate is 250 mL/min; taking an acetic acid aqueous solution with the acetic acid volume percentage content of 1% as a second mobile phase A, and taking acetonitrile as a second mobile phase B; the gradient elution procedure was: 0-60 min, 10 vol% → 50 vol% of a second mobile phase B; the detection wavelength is 220nm, fractions 1-7# are collected and subjected to hanging bottle freeze-drying, and detection is carried out according to the conditions of the high performance liquid chromatography detection of example 1, and the results are as follows: the chromatographic purity of the final product was 99.75% with a maximum single impurity of 0.08%; the mass was 5.04g, the yield was 48%, and the acetic acid content was 6.1%.
The crude BPC-157 polypeptide was the same as in example 1.
Example 3
Weighing 82.7g of BPC-157 polypeptide crude product, dissolving to 3.5L with acetic acid-water (the volume ratio of acetic acid to water is 15: 100), and filtering to obtain organism protection polypeptide crude product solution.
After loading, performing a first high performance liquid chromatography purification, wherein parameters of the first high performance liquid chromatography purification comprise: the first chromatographic column is C of 200mm multiplied by 150mm18-10 μm, the first flow rate being 1000 mL/min; using 20.8g/L sodium dihydrogen phosphate water solution as a first mobile phase A and acetonitrile as a second mobile phase B, and performing gradient elution by the following procedures: 0-60 min, 5 vol% → 55 vol% of the first mobile phase B, the detection wavelength is 250nm, the eluates are subjected to high performance liquid chromatography detection, 4# -8# fractions are collected and detected according to the conditions of the high performance liquid chromatography detection of the embodiment 1, and the results are as follows: the purity is 98.35 percent, the maximum single impurity is 0.41 percent, and the first purified organism protection polypeptide is obtained.
Diluting the first purified organism protection polypeptide by one volume with purified water, and then carrying out second high performance liquid chromatography purification, salt exchange and elution: the parameters of the second high performance liquid chromatography purification include: the second column is 200mm X250 mm C18-10 μm; the second flow rate is 1000 mL/min; taking an acetic acid water solution with the acetic acid volume percentage content of 2% as a second mobile phase A, and taking acetonitrile as a second mobile phase B; the gradient elution procedure is 0-60 min: 10 vol% → 40 vol% of a second mobile phase B, a detection wavelength of 220nm, fractions 1-8# were collected and subjected to vial-hanging lyophilization and detection according to the conditions of high performance liquid chromatography detection of example 1, and the results were: the chromatographic purity of the final product was 99.58% with a maximum single impurity of 0.09%; the mass was 41.3g, the yield was 50%, and the acetic acid content was 6.2%.
The crude BPC-157 polypeptide was the same as in example 1.
Comparative example 1
Weighing 9.8g of BPC-157 polypeptide crude product, dissolving the BPC-157 polypeptide crude product into 200mL of acetic acid-water (the volume ratio of acetic acid to water is 10: 100), and filtering to obtain the organism protection polypeptide crude product solution.
After loading, performing a first high performance liquid chromatography purification, wherein parameters of the first high performance liquid chromatography purification comprise: the first chromatographic column is C of 100mm multiplied by 250mm18-10 μm; the first flow rate is 250 mL/min; taking a 1% triethylamine phosphate solution as a first mobile phase A, taking acetonitrile as a first mobile phase B, and carrying out a gradient elution procedure: 0-60 min, 10 vol% → 30 vol% of the first mobile phase B, the detection wavelength 220nm, and high performance liquid chromatography detection of each eluent, collecting the 2# -5# fraction, and detecting according to the conditions of the high performance liquid chromatography detection of example 1, the results are: the purity is 95.6 percent, the maximum single impurity is 3.23 percent, and the first purified organism protection polypeptide is obtained.
Diluting the first purified organism protection polypeptide by one volume with purified water, and then carrying out second high performance liquid chromatography purification, salt exchange and elution: the parameters of the second high performance liquid chromatography purification include: the second column is 100mm X250 mm C18-10 μm; the second flow rate is 250 mL/min; taking an acetic acid aqueous solution with the acetic acid volume percentage content of 1% as a second mobile phase A, and taking acetonitrile as a second mobile phase B; the gradient elution procedure was: 0-60 min, 10 vol% → 50 vol% of a second mobile phase B; the detection wavelength is 220nm, fractions 1-6# are collected and subjected to hanging bottle freeze-drying, and detection is carried out according to the conditions of the high performance liquid chromatography detection of example 1, and the results are as follows: most preferablyThe chromatographic purity of the final product was 97.2% with a maximum single impurity of 0.75%; the mass was 4.6g, and the yield was 47%.
The crude BPC-157 polypeptide was the same as in example 1.
Comparative example 2
Weighing 203mg of BPC-157 polypeptide crude product, dissolving the BPC-157 polypeptide crude product into 20mL of acetic acid-water (the volume ratio of acetic acid to water is 5: 100), and filtering to obtain the organism protection polypeptide crude product solution.
After loading, performing a first high performance liquid chromatography purification, wherein parameters of the first high performance liquid chromatography purification comprise: the first chromatographic column is 20mm x 250mm C8-10 μm; the first flow rate is 10 mL/min; 5.2g/L sodium dihydrogen phosphate aqueous solution is used as a first mobile phase A, and acetonitrile is used as a first mobile phase B; the gradient elution procedure was: 0-60 min: 5 vol% → 55 vol% first flow B phase; the detection wavelength is 210nm, the high performance liquid chromatography detection is carried out on each eluent, 3# -5# fractions are collected and detected according to the conditions of the high performance liquid chromatography detection of the embodiment 1, and the result is that: the purity is 96.8 percent, the maximum single impurity is 2.12 percent, and the first purified organism protection polypeptide is obtained.
Diluting the collected first purified organism protection polypeptide by one volume with purified water, and then carrying out second high performance liquid chromatography purification and salt exchange elution: the parameters of the second high performance liquid chromatography purification include: the second column is 20mm X250 mm C8-10 μm; the second flow rate is 10 mL/min; taking an acetic acid water solution with the acetic acid volume percentage content of 0.5% as a second mobile phase A, and taking acetonitrile as a second mobile phase B; the gradient elution procedure is 0-60 min: 10 vol% → 60 vol% of a second mobile phase B, a detection wavelength of 220nm, fractions 1-7# were collected and subjected to vial-hanging lyophilization and detection according to the conditions of high performance liquid chromatography detection of example 1, and the results were: the chromatographic purity of the final product was 97.5% with a maximum single impurity of 0.96%; the mass was 63mg, and the yield was 31.03%.
Comparative example 3
The preparation and purification of the polypeptide were carried out according to example 1 of publication No. CN102229649A, and the detection was carried out according to the conditions of the high performance liquid chromatography detection of example 1, with the results that: the purity of the purified organic polypeptide was 98.02% with a maximum single impurity of 0.76%.
Comparative example 4
The organic polypeptide prepared in example 1 was purified according to the purification procedure of example 1 disclosed in publication No. CN102229649A, and the detection was performed according to the conditions of the high performance liquid chromatography detection of example 1, and the results were: the purity of the purified organic polypeptide was 97.58% with a maximum single impurity of 0.84%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A purification method of a crude product of an organism protection polypeptide comprises the following steps:
dissolving the organism protection polypeptide crude product to obtain an organism protection polypeptide crude product solution;
performing high performance liquid chromatography separation and purification on the organism protection polypeptide crude product solution to obtain an organism protection polypeptide pure product;
the high performance liquid chromatography separation and purification comprises a first high performance liquid chromatography purification and a second high performance liquid chromatography purification which are sequentially carried out;
the parameters of the first high performance liquid chromatography purification include:
a first chromatographic column: c18A column;
first mobile phase a: the concentration of the sodium dihydrogen phosphate aqueous solution is 3-30 g/L;
first mobile phase B: acetonitrile;
the first flow rate: 1-1000 mL/min;
the first gradient elution procedure was:
0~60min:n1vol%→m1vol% first mobile phase B; n is1Is 5 to 10, m130 to 60;
first detection wavelength: 210-260 nm;
the parameters of the second high performance liquid chromatography purification include:
second mobile phase a: the volume ratio of acetic acid to water in the acetic acid aqueous solution is (0.1-5): 100, respectively;
second mobile phase B: acetonitrile;
the second flow rate: 1-1000 mL/min;
the second gradient elution procedure was:
0~60min:n2vol%→m2vol% second mobile phase B; n is2Is 5 to 10, m230 to 60;
second detection wavelength: 210-260 nm.
2. The purification method according to claim 1, wherein the solvent in which the crude organism protective polypeptide is dissolved is an aqueous acetic acid solution; the volume percentage of acetic acid in the acetic acid aqueous solution is 5-15%.
3. The purification method according to claim 1 or 2, wherein the concentration of the organic protective peptide in the crude solution of the organic protective polypeptide is 10-100 mg/mL.
4. The purification method of claim 1, wherein the first high performance liquid chromatography purification yields a first purified body protective polypeptide; and performing second high performance liquid chromatography purification on the first purified body-protecting polypeptide.
5. The purification method according to claim 1, wherein the first detection wavelength and the second detection wavelength are independently 220 to 250 nm.
6. The purification method according to claim 1, wherein the effective length of the first chromatographic column and the second chromatographic column is independently not less than 150 mm; the diameters are independently 10-200 mm; the particle size of the filler of the first chromatographic column and the second chromatographic column is 10-30 μm independently.
7. The purification method according to claim 1, wherein the parameters of the first high performance liquid chromatography purification comprise: the gradient elution procedure was: 0-60 min: 5 vol% → 55 vol% first flow B phase; the parameters of the second high performance liquid chromatography purification include: the gradient elution procedure was: 0-60 min: 10 vol% → 60 vol% second mobile B phase.
8. The purification method according to claim 1, wherein the parameters of the first high performance liquid chromatography purification comprise: the gradient elution procedure was: 0-60 min: 10 vol% → 30 vol% first mobile phase B; the parameters of the second high performance liquid chromatography purification include: the gradient elution procedure was: 0-60 min: 10 vol% → 50 vol% of the second mobile phase B.
9. The purification method according to claim 1, wherein the parameters of the first high performance liquid chromatography purification comprise: the gradient elution procedure was: 0-60 min: 5 vol% → 55 vol% first flow B phase; the parameters of the second high performance liquid chromatography purification include: the gradient elution procedure was: 0-60 min: 10 vol% → 40 vol% of the second mobile phase B.
10. The purification method according to claim 1, further comprising freeze-drying the second HPLC purified fraction after the second HPLC purification to obtain the purified product of the body-protecting polypeptide.
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