CN118271392A - Purification method of basic peptide - Google Patents
Purification method of basic peptide Download PDFInfo
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- CN118271392A CN118271392A CN202211725863.6A CN202211725863A CN118271392A CN 118271392 A CN118271392 A CN 118271392A CN 202211725863 A CN202211725863 A CN 202211725863A CN 118271392 A CN118271392 A CN 118271392A
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 105
- 238000000034 method Methods 0.000 title claims abstract description 35
- 238000000746 purification Methods 0.000 title abstract description 26
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims abstract description 42
- 239000012071 phase Substances 0.000 claims abstract description 35
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 19
- 150000001413 amino acids Chemical class 0.000 claims abstract description 15
- 239000000337 buffer salt Substances 0.000 claims abstract description 13
- 229920001184 polypeptide Polymers 0.000 claims abstract description 11
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims abstract description 8
- 239000003480 eluent Substances 0.000 claims abstract description 8
- 239000008346 aqueous phase Substances 0.000 claims abstract description 7
- 239000000203 mixture Substances 0.000 claims abstract description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 64
- 238000002360 preparation method Methods 0.000 claims description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 claims description 18
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 claims description 18
- 235000019837 monoammonium phosphate Nutrition 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 16
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 10
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 10
- 230000005526 G1 to G0 transition Effects 0.000 claims description 9
- 238000001514 detection method Methods 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 8
- 239000006012 monoammonium phosphate Substances 0.000 claims description 8
- 239000011148 porous material Substances 0.000 claims description 8
- 239000002245 particle Substances 0.000 claims description 7
- 150000003863 ammonium salts Chemical class 0.000 claims description 6
- 238000005303 weighing Methods 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 3
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 2
- 239000005695 Ammonium acetate Substances 0.000 claims description 2
- 230000002378 acidificating effect Effects 0.000 claims description 2
- 229940043376 ammonium acetate Drugs 0.000 claims description 2
- 235000019257 ammonium acetate Nutrition 0.000 claims description 2
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 2
- 230000003139 buffering effect Effects 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 238000004007 reversed phase HPLC Methods 0.000 claims description 2
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 claims description 2
- AVBGNFCMKJOFIN-UHFFFAOYSA-N triethylammonium acetate Chemical compound CC(O)=O.CCN(CC)CC AVBGNFCMKJOFIN-UHFFFAOYSA-N 0.000 claims description 2
- PTMFUWGXPRYYMC-UHFFFAOYSA-N triethylazanium;formate Chemical compound OC=O.CCN(CC)CC PTMFUWGXPRYYMC-UHFFFAOYSA-N 0.000 claims description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims 1
- 235000011130 ammonium sulphate Nutrition 0.000 claims 1
- 239000002131 composite material Substances 0.000 abstract description 7
- 239000000945 filler Substances 0.000 abstract description 7
- 230000003321 amplification Effects 0.000 abstract description 6
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 6
- 150000001875 compounds Chemical class 0.000 abstract description 3
- 238000010828 elution Methods 0.000 abstract description 3
- 229910052710 silicon Inorganic materials 0.000 abstract description 3
- 239000010703 silicon Substances 0.000 abstract description 3
- 238000012546 transfer Methods 0.000 abstract description 3
- 238000001742 protein purification Methods 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 14
- 239000000706 filtrate Substances 0.000 description 14
- 238000011068 loading method Methods 0.000 description 12
- 239000008213 purified water Substances 0.000 description 11
- 125000003275 alpha amino acid group Chemical group 0.000 description 7
- 239000012535 impurity Substances 0.000 description 7
- 239000002904 solvent Substances 0.000 description 7
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 7
- 239000003814 drug Substances 0.000 description 6
- 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 5
- 229940079593 drug Drugs 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000011049 filling Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000002253 acid Substances 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- -1 sulfuric acid (ammonia) -acetonitrile Chemical compound 0.000 description 2
- 208000030507 AIDS Diseases 0.000 description 1
- 101800000112 Acidic peptide Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- 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
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Engineering & Computer Science (AREA)
- Water Supply & Treatment (AREA)
- Peptides Or Proteins (AREA)
Abstract
A purification method of alkaline peptide belongs to the technical field of protein purification. The aqueous phase eluent in the purification method of the invention contains sulfuric acid composite ammonium buffer salt. The basic peptide is polypeptide with PI value between 9 and 10. The basic peptide has a composition in which the number of amino acids is less than 30. The polar amino acid in the basic peptide accounts for more than 50% of the total peptide sequence length of the basic peptide. The invention can well ensure a mobile phase system by the sulfuric acid composite ammonium buffer salt, always maintains a relatively stable pH range in the elution process, improves the tolerance of the method, reduces the risk of transfer amplification of the purification process, and can well improve the phenomenon of tailing of alkaline compounds due to free silicon hydroxyl in the reversed-phase filler.
Description
Technical Field
The invention belongs to the technical field of protein purification, and particularly relates to a purification method of alkaline peptide.
Background
The role of peptides in humans has attracted a great deal of attention in the scientific community for decades. Numerous studies have shown that proteins are not completely hydrolyzed to amino acids after ingestion, but are largely absorbed in the form of peptides, and that polypeptides are absorbed more rapidly than the amino acids of the same composition.
The polypeptide is generally formed by dehydrating and condensing 10-100 amino acid molecules, and the molecular weight is lower than 10000Da. Among the numerous polypeptide molecules, acidic peptides, neutral peptides and basic peptides are classified according to their isoelectric points (hereinafter abbreviated as PI values). In recent years, polypeptide drugs are gradually rising in the drug market, and polypeptide drugs synthesized by utilizing modern biotechnology become one of hot spots for drug development, and have wide application, high safety and remarkable curative effect because of wide adaptation, and are widely applied to prevention, diagnosis and treatment of diseases such as tumors, hepatitis, diabetes, AIDS and the like at present, so that the polypeptide drugs have wide development prospect.
At present, chemical synthesis and biological fermentation recombination are mostly adopted for preparing the peptide of interest, and then a fine product is obtained through purification. Because the polarity of most impurities in polypeptide drugs is similar to that of target components, the chromatographic packing which is selectable for polypeptide purification is limited, and high-resolution reverse high-pressure packing is mostly selected for purification in the industry at present. The reversed phase filler has free silicon hydroxyl groups, so that the tailing phenomenon of the peak type of the alkaline compound is easy to occur. The peak type with serious tailing not only affects the separation degree of impurities and main components, but also can lose part of samples, increase solvent consumption, affect purification efficiency and the like. In addition, in order to achieve a better separation effect, products meeting quality standards are produced, and meanwhile, various acid or buffer salt systems are needed to amplify the difference between impurities and target components, and in the purification of basic drug peptides, a mobile phase system is usually an acid-organic phase system, and purification systems such as exendin acetate, tikken acetate, alpidil acetate and the like are disclosed in patent application publication No. CN 105223296A. Because the acid is singly used as the water phase, the mobile phase is difficult to maintain in a relatively stable pH range all the time to ensure enough ionic strength for component elution because of no buffer action, so that the mobile phase system has poor tolerance and poor method reproducibility, and the risk of transfer amplification of the purification process is increased.
Disclosure of Invention
In view of the above problems in the prior art, an object of the present invention is to design a method for purifying an alkaline peptide. The invention uses sulfuric acid composite buffer salt as water phase eluent in the purification process, and provides a purification method of alkaline peptide with PI value of 9-10 and polar amino acid accounting for more than 50% of the total length of peptide sequence. The purification method of the invention has the advantages of reduced organic solvent consumption, good separation effect and small difference before and after process amplification.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
The invention provides a purification method of alkaline peptide, wherein an aqueous phase eluent contains sulfuric acid composite ammonium buffer salt.
In the method for purifying the alkaline peptide by utilizing the reversed-phase high-performance liquid chromatography, the aqueous phase eluent contains sulfuric acid composite ammonium buffer salt.
The alkaline peptide is polypeptide with PI value between 9 and 10.
The purification method of the basic peptide comprises the step of forming the basic peptide with the number of amino acids smaller than 30.
According to the purification method of the basic peptide, the polar amino acid in the basic peptide accounts for more than 50% of the total peptide sequence length of the basic peptide. The content percentage of polar amino acids in the invention is equal to the number of polar amino acids to the total number of amino acids of the peptide.
The purification method of the basic peptide comprises Thr, ser, cys, asn, gln, tyr, lys, arg, his, asp, glu of the polar amino acid.
In the purification method of the alkaline peptide, the volume fraction of the sulfuric acid is 0.1%.
The ammonium salt buffer salt is ammonium salt with buffering function under acidic condition, preferably ammonium salt buffer salt comprises monoammonium phosphate, ammonium citrate, ammonium formate, ammonium acetate, triethylamine formate and triethylamine acetate.
The purification method of the alkaline peptide specifically comprises the following steps:
(1) Weighing crude peptide, dissolving with a mixed solution of pure water and acetonitrile, and filtering with a filter membrane;
(2) Adding the sample into a preparation instrument, setting a stationary phase as nano-micro C18, setting a purified phase A in an aqueous phase eluent as ammonium buffer salt and mixing with sulfuric acid, regulating the pH value to 2.50 by using ammonia water, and purifying a phase B as preparation-grade pure acetonitrile;
(3) And (3) running an AB two-phase gradient elution program on a preparation instrument, setting detection wavelength, and purifying and collecting samples.
According to the purification method of the basic peptide, the volume ratio of the pure water to the acetonitrile is 9:1; the particle diameter of the nano-micro C18 is 8 mu m, and the pore diameter is the same as that of the nano-micro C18Column gauge 20 x 250mm; the concentration of the ammonium dihydrogen phosphate solution is 50mM, and the volume fraction of sulfuric acid is 0.1%.
Compared with the prior art, the invention has the following beneficial effects:
The invention can well ensure a mobile phase system by the sulfuric acid composite ammonium buffer salt, always maintains a relatively stable pH range in the elution process, improves the tolerance of the method, reduces the risk of transfer amplification of the purification process, and is mainly reflected in the retention time of main peaks before and after amplification and the removal effect of impurities. The composite system can well improve the phenomenon of tailing of alkaline compound peaks caused by free silicon hydroxyl groups in the reversed-phase filler.
Drawings
FIG. 1 is a diagram of the preparation of example 1, chromatographic column format 20X 250mM, system 50mM monoammonium phosphate, pH adjusted to 2.50 with 0.1% sulfuric acid (ammonia water) -acetonitrile;
FIG. 2 is a preparation of example 2, chromatographic column format 50X 250mM, system 50mM monoammonium phosphate, pH adjusted to 2.50 with 0.1% sulfuric acid (ammonia water) -acetonitrile;
FIG. 3 is a diagram of the preparation of example 3, column size 50X 250mM, system 50mM monoammonium phosphate, pH adjusted to 2.50 with 0.1% sulfuric acid (ammonia water) -acetonitrile;
FIG. 4 is a diagram of the preparation of example 4, column size 100X 250mM, 50mM ammonium dihydrogen phosphate, pH adjusted to 2.50 with 0.1% sulfuric acid (ammonia water) -acetonitrile;
FIG. 5 is a graph of comparative example 1, column size 20X 250mm, system 0.1% TFA-acetonitrile;
FIG. 6 is a graph of comparative example 1 column format 50X 250mm,0.1% TFA-acetonitrile system (red) preparation versus example 150 mM ammonium dihydrogen phosphate, pH adjusted to 2.50 with 0.1% sulfuric acid (ammonia) -acetonitrile system (black);
FIG. 7 is a graph of comparative example 2, column size 50X 250mm, system 0.1% TFA-acetonitrile;
FIG. 8 is a graph of comparative example 3, column size 50X 250mm, system 0.1% TFA-acetonitrile.
Detailed Description
The invention will be further explained below with reference to the drawings and examples, but the embodiments do not limit the invention in any way.
Example 1: crude peptide: peptide sequence CTQDQKRKQLDHLQLKNGDRNVIQVHNG, PI with a value of 9.76, length 28
250Mg of the crude title peptide (PI 9.76, SEQ ID NO.1, CTQDQKRKQLDHLQLKNGDRNVIQVHNG) with a purity of about 47.74% and an amino acid sequence of 50ml of 90:10=purified water were weighed out: after acetonitrile was dissolved, the solution was filtered through a 0.45 μm filter, and the filtrate was purified by filtration. Stationary phase: nano-micro C18, particle size 8 μm, pore diameterChromatographic column format 20 x 250mm, eluting solvent: preparing a purified phase A, namely weighing 57.5g of ammonium dihydrogen phosphate solid, adding 10L of purified water to prepare 50mM ammonium dihydrogen phosphate solution, adding sulfuric acid with the volume fraction of 0.1%, adjusting the pH value to 2.50 by using ammonia water, preparing phase B as pure acetonitrile, adding the dissolved and filtered crude peptide filtrate of the embodiment 1 into an instrument for preparation, wherein the flow rate of the instrument is 20mL/min, the detection wavelength is 220nm, and preparing the gradient 0-2-4-94min and 5-5-15-45% of phase B. The crude peptide loading of the needle is 250mg, about 0.5% of the filling mass is loaded, the target peak is collected in a segmented mode, the peak outlet time of the target component is about 30min, and the peak width is about 4min. As shown in FIG. 1, the tailing tendency is significantly improved compared with 0.1% TFA, the target components are eluted together more intensively, the concentration of the sample fraction is higher, and the purity of the fraction obtained by the peptides of the examples can reach 96.80%.
Example 2: crude peptide: peptide sequence CTQDQKRKQLDHLQLKNGDRNVIQVHNG, PI with a value of 9.76, length 28
1.5G of the subject crude peptide (PI 9.76, SEQ ID NO.1 (i.e., peptide sequence CTQDQKRKQLDHLQLKNGDRNVIQVHNG) having a purity of about 47.74% and an amino acid sequence of SEQ ID NO.1 was weighed out and 50mL of 90:10=purified water: after acetonitrile was dissolved, the solution was filtered through a 0.45 μm filter, the filtrate was purified by loading, stationary phase: nano-micro C18, particle size 8 μm, pore diameterChromatographic column format 50 x 250mm, eluting solvent: preparing a purified phase A, namely weighing 57.5g of ammonium dihydrogen phosphate solid, adding 10L of purified water to prepare 50mM ammonium dihydrogen phosphate solution, adding sulfuric acid with the volume fraction of 0.1%, adjusting the pH value to 2.50 by using ammonia water, preparing phase B as pure acetonitrile, adding the dissolved and filtered crude peptide filtrate of the embodiment 2 into an instrument for preparation, wherein the flow rate of the instrument is 70mL/min, the detection wavelength is 220nm, and preparing the gradient 0-2-4-94min and 5-5-15-45% phase B. As shown in FIG. 2, the crude peptide loading of the needle is 1.5g, about the filling mass is 0.5%, the target peak is collected in a segmented manner, the peak outlet time of the target component is about 33min, the peak width is about 4min, the peak outlet time and the peak width of the amplified target component are basically consistent with those of a chromatographic column with an inner diameter of 50mm, the impurity removing capacity is similar, and the purity of the fraction obtained by the amplified example peptide is about 98.9%.
Example 3: crude peptide: peptide sequence VGYLQPRTFLLKYNENGT, PI with a value of 9.52, sequence length 18
3G of the crude peptide of the title having a purity of about 66.26% (PI value 9.52, sequence length 18) and an amino acid sequence shown in SEQ ID No.2 (i.e. peptide sequence VGYLQPRTFLLKYNENGT) was weighed out and purified water was used 150ml 90:10=: after acetonitrile was dissolved, the solution was filtered through a 0.45 μm filter, the filtrate was purified by loading, stationary phase: nano-micro C18, particle size 8 μm, pore diameterChromatographic column format 50 x 250mm, eluting solvent: purification A is matched with: weighing 57.5g of monoammonium phosphate solid, adding 10L of purified water to prepare a monoammonium phosphate solution with the concentration of 50mM, adding sulfuric acid with the volume fraction of 0.1%, adjusting the pH to 2.50 by using ammonia water, taking phase B as preparation grade pure acetonitrile, adding the dissolved and filtered crude peptide filtrate of the example 3 into an instrument to prepare, wherein the flow rate of the instrument is 70mL/min, the detection wavelength is 220/280nm, and the gradient is 0-2-4-94min, and the gradient is 5-5-19-49% of phase B. As shown in FIG. 3, the crude peptide loading was 3g, about 1% of the filler mass loading, the target peak was collected in stages, and the peak time of the target component was about 28min and the peak width was about 6min. The peptides of the examples were prepared to give fractions with a maximum purity of about 97.51% with a significant improvement in tailing tendency compared to 0.1% TFA.
Example 4: crude peptide: peptide sequence VGYLQPRTFLLKYNENGT, PI with a value of 9.52, sequence length 18
12G of the crude peptide of the title having a purity of about 66.26% (PI value 9.52, sequence length 18) and an amino acid sequence shown in SEQ ID No.2 (i.e. peptide sequence VGYLQPRTFLLKYNENGT) were weighed out and 600ml of 90:10=purified water: after acetonitrile was dissolved, the solution was filtered through a 0.45 μm filter, the filtrate was purified by loading, stationary phase: nano-micro C18, particle size 8 μm, pore diameterChromatographic column specification 100 x 250mm, eluting solvent: purification A is matched with: weighing 115g of ammonium dihydrogen phosphate solid, adding 20L of purified water to prepare 50mM ammonium dihydrogen phosphate solution, adding sulfuric acid with the volume fraction of 0.1%, adjusting the pH to 2.50 by using ammonia water, taking phase B as preparation grade pure acetonitrile, adding the dissolved and filtered crude peptide filtrate of the example 4 into an instrument to prepare, wherein the flow rate of the instrument is 200mL/min, the detection wavelength is 280nm, and the gradient is 0-2-4-94min, and the gradient is 5-5-19-49% of phase B. As shown in FIG. 4, the loading amount of the crude peptide is 12g, about 1% of the loading amount of the filler by mass, the target peak is collected in a segmented manner, the peak time of the target component is about 27min, the peak width is about 7min, the peak time and the peak width of the amplified target component are basically consistent with those of a chromatographic column with an inner diameter of 50mm, the impurity removal capacity is similar, and the highest purity of the fraction prepared by the peptides of the examples is about 97%.
Comparative example 1: peptide sequence CTQDQKRKQLDHLQLKNGDRNVIQVHNG, PI with a value of 9.76, length 28
250Mg of the subject crude peptide (PI 9.76, SEQ ID NO.1 (i.e., CTQDQKRKQLDHLQLKNGDRNVIQVHNG) having the amino acid sequence of about 47.74% pure was weighed out and 50ml 90:10=purified water: after acetonitrile was dissolved, the solution was filtered through a 0.45 μm filter, and the filtrate was purified by filtration. The stationary phase is nano-micro C18, the grain diameter is 8 mu m, and the aperture is the same as that of the nano-micro C18Chromatographic column format 20 x 250mm, eluting solvent: preparing a pure water solution of trifluoroacetic acid (hereinafter referred to as TFA) with a volume fraction of 0.1% as phase A, and using preparation grade acetonitrile as phase B; dissolving and filtering the crude peptide filtrate of comparative example 1, adding into an instrument for preparation, wherein the flow rate of the preparation instrument is 20mL/min, the detection wavelength is 220nm, and the gradient is 0-2-4-94min, and the phase B is 5-5-15-45%. As shown in FIG. 5, the crude peptide loading of the needle is 250mg, about 0.5% of the filler mass is loaded, the target peak is collected in a segmented mode, the peak outlet time of the target component is about 30min, the peak width is about 6min, the tailing is serious, and the highest purity of the fraction obtained by the peptide of the comparative example under the system is about 98.9%. FIG. 6 shows a comparison of the preparation of comparative example 1 column format 50X 250mm,0.1% TFA-acetonitrile system (red) with the preparation of example 150 mM monoammonium phosphate, pH adjusted to 2.50% with 0.1% sulfuric acid (ammonia) -acetonitrile system (black).
Comparative example 2: peptide sequence CTQDQKRKQLDHLQLKNGDRNVIQVHNG, PI with a value of 9.76, length 28
1.5G of crude peptide (PI 9.76, SEQ ID NO.1 (i.e., CTQDQKRKQLDHLQLKNGDRNVIQVHNG) having the amino acid sequence shown in SEQ ID NO.1, was weighed out to give a crude peptide of approximately 47.74% purity, 300mL 90:10=purified water: after acetonitrile was dissolved, the solution was filtered through a 0.45 μm filter, and the filtrate was purified by filtration. Stationary phase: nano-micro C18, particle size 8 μm, pore diameterChromatographic column format 50 x 250mm, eluting solvent: preparing a TFA pure water solution with the volume fraction of 0.1% as a phase A, and using preparation grade acetonitrile as a phase B; dissolving and filtering the crude peptide filtrate of comparative example 2, adding into an instrument for preparation, wherein the flow rate of the preparation instrument is 70mL/min, the detection wavelength is 220nm, and the gradient is 0-2-4-94min, and the phase B is 5-5-15-45%. As shown in FIG. 7, the crude peptide loading of the needle is 1.5g, about the filling mass is 0.5%, the target peak is collected in a segmented manner, the peak time of the target component is about 34min, the peak width is about 8min, the tailing is more serious after amplification, the impurity removing capability is poor after the peak time of the chromatographic peak is delayed, and the purity of the fraction obtained by the peptide of the comparative example can reach about 95.43%.
Comparative example 3: peptide sequence VGYLQPRTFLLKYNENGT, PI with a value of 9.52, sequence length 18
3G of the crude peptide of the title having a purity of about 66.26% (PI value 9.52, sequence length 18) and an amino acid sequence shown in SEQ ID No.2 (i.e. peptide sequence VGYLQPRTFLLKYNENGT) was weighed out and purified water was used 150ml 90:10=: after acetonitrile is dissolved, the solution is filtered by a filter membrane with the diameter of 0.45 mu m, and the filtrate is loaded and purified, and the stationary phase nano micro C18, the grain diameter of 8 mu m and the pore diameter are adoptedPreparing TFA pure water solution with volume fraction of 0.1% as phase A, and preparing acetonitrile with size of preparation grade as phase B; dissolving and filtering the crude peptide filtrate of comparative example 3, adding into an instrument for preparation, wherein the flow rate of the preparation instrument is 70mL/min, the detection wavelength is 220/280nm, and the gradient is 0-2-4-94min, and the phase B is 5-5-19-49%. As shown in FIG. 8, the crude peptide loading of the needle is 3g, about 1% of the filler mass is loaded, the target peak is collected in a segmented mode, the peak outlet time of the target component is about 22.3min, the peak width is 10min, the tailing is serious, and the highest purity of the fraction obtained by preparing the comparative peptide is about 96%.
Claims (10)
1. A method for purifying an alkaline peptide, wherein an aqueous phase eluent in the method contains sulfuric acid complex ammonium buffer salt.
2. The method for purifying an alkaline peptide according to claim 1, wherein the aqueous phase eluent contains a complex ammonium sulfate buffer salt in the method for purifying an alkaline peptide by reverse phase high performance liquid chromatography.
3. The method for purifying a basic peptide according to claim 1 or 2, wherein the basic peptide is a polypeptide having a PI value of 9 to 10.
4. The method for purifying a basic peptide according to claim 1 or 2, wherein the number of amino acids in the composition of the basic peptide is less than 30.
5. The method for purifying a basic peptide according to claim 1 or 2, wherein the polar amino acid in the basic peptide is 50% or more of the total peptide sequence length of the basic peptide.
6. The method of purifying a basic peptide according to claim 4, wherein the polar amino acid comprises Thr, ser, cys, asn, gln, tyr, lys, arg, his, asp, glu.
7. The method for purifying an alkaline peptide according to claim 1, wherein the volume fraction of sulfuric acid is 0.1%.
8. The method for purifying a basic peptide according to claim 1, wherein the ammonium salt is an ammonium salt having a buffering effect under acidic conditions, preferably the ammonium salt includes monoammonium phosphate, ammonium citrate, ammonium formate, ammonium acetate, triethylamine formate, triethylamine acetate.
9. The method for purifying a basic peptide according to claim 1, wherein the method for purifying specifically comprises the steps of:
(1) Weighing crude peptide, dissolving with a mixed solution of pure water and acetonitrile, and filtering with a filter membrane;
(2) Adding the sample into a preparation instrument, setting a stationary phase as nano-micro C18, setting a purified phase A in an aqueous phase eluent as ammonium buffer salt and mixing with sulfuric acid, regulating the pH value to 2.50 by using ammonia water, and purifying a phase B as preparation-grade pure acetonitrile;
(3) And (3) running an AB two-phase gradient elution program on a preparation instrument, setting detection wavelength, and purifying and collecting samples.
10. The method for purifying a basic peptide according to claim 9, wherein the volume ratio of pure water to acetonitrile is 9:1; the particle diameter of the nano-micro C18 is 8 mu m, and the pore diameter is the same as that of the nano-micro C18Column gauge 20 x 250mm; the concentration of the ammonium dihydrogen phosphate solution is 50mM, and the volume fraction of sulfuric acid is 0.1%.
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