Purification method of a kind of polypeptide

A purification method and polypeptide technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of cost increase, consumption flow, etc., and achieve the effects of cost saving, consumption reduction, and high purity

Active Publication Date: 2017-12-05
JIANGSU SKYRUN PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that when purifying highly hydrophobic peptides with many hydrophobic amino acids like soybean polypeptide, it takes a lot of time to walk the gradient, consumes a lot of mobile phase, and greatly increases the cost

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Under the above-mentioned peptide preparation equipment and conditions, a pure aqueous solution with a volume fraction of 0.1% trifluoroacetic acid (hereinafter referred to as TFA) was configured as phase A, and pure chromatography-grade acetonitrile and chromatography-grade trifluoroethanol were used as phase B in a volume ratio of 7:3; 10mg / ml Thymus α1 after dissolution and filtration, the crude product purity is 20% is added to the instrument for preparation, the peak eluting time of the sample is 15min, the collected solution is dried and then weighed and analyzed, the resolution is 2.27, and the number of theoretical plates is 73157, The signal-to-noise ratio was 419.1, the recovery rate was 31%, and the purity was 90%.

[0028] Under the same preparation equipment and conditions, prepare thymus α1 samples with the same concentration and purity, change the mobile phase to phase A with 0.1% TFA aqueous solution, and phase B to pure acetonitrile, and the peak eluting...

Embodiment 2

[0031] Weigh 200mg of thymosin α1 crude product, crude product purity 16%, add 10ml of acetonitrile and stir until cloudy, then add 30 ml of pure water, ultrasonically stir until clear and transparent, filter with 0.45u microporous membrane, pour all the samples into LC6000 type In high performance liquid chromatography, other conditions remain unchanged, the mobile phase is changed to A: 0.1% TFA water, B: acetonitrile and tetrahydrofuran mixed solution with a volume ratio of 7:3, and the preparation gradient becomes 0-5-45min, 5-25min -35%. The peak elution time is 16 minutes, and the purity of the obtained sample is 99%. After freeze-drying, it weighs 14 mg of pure product, and the yield can reach more than 40%.

[0032] Other conditions remained unchanged, the mobile phase was changed to A: 0.1% TFA water, B: pure acetonitrile, the peak eluting time of the sample was 28 minutes, the purity was 98%, and the weight was 9 mg after lyophilization, and the yield was 28.1%.

Embodiment 3

[0034] Weigh 200mg of thymosin α1 crude product, crude product purity 20%, add 10ml of acetonitrile and stir until cloudy, then add 30 ml of pure water, ultrasonically stir until clear and transparent, filter with 0.45u microporous membrane, pour all the samples into LC6000 type In high performance liquid chromatography, other conditions remain unchanged, the mobile phase is changed to A: 0.1% TFA water, B: acetonitrile and trifluoroethanol mixed solution with a volume ratio of 7:3, and the preparation gradient is 0-5-45min, 5- 25-35%. The purity of the collected sample is 99%. After freeze-drying, it weighs 15 mg of pure product, and the yield can reach 37.5%. Replace mobile phase A with acetate according to customer requirements, mobile phase A is: 5% acetic acid water, mobile phase B: mixed solution of acetonitrile and trifluoroethanol with a volume ratio of 7:3, and the preparation gradient is 0-5-45min , 5-25-35%. Other conditions remain unchanged, the purity of the sampl...

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Abstract

The invention discloses a polypeptide purification method and belongs to the technical field of protein and polypeptide purification. A preparative HPLC (high performance liquid chromatograph) is used for purifying polypeptides, 2,2,2-trifluoroethanol or tetrahydrofuran is added to an organic phase of a mobile phase of the preparative HPLC, the elution gradient, flow velocity and detection wavelength are set, and high-purity peptides are obtained. The polypeptide purification method has the advantages that the elution time is shortened, the use quantity of an organic solvent is reduced, the purity is high, the separation effect is good, and the cost is saved.

Description

technical field [0001] The invention discloses a purification method of a kind of polypeptide, which belongs to the technical field of protein and polypeptide purification. Background technique [0002] Polypeptide is a kind of substance between amino acid and protein, which is composed of one or more amino acids bound by peptide bonds in a certain order. Tens of thousands of polypeptides have been found to exist in living organisms. Polypeptide is a structural fragment of a protein, which can act as an active group of a protein and is an important substance for human metabolism and regulation activities. With the development of biotechnology, the peptide synthesis technology is becoming more and more mature, but there are still low purity of the initial peptide product and complex impurities, especially the physical and chemical properties of the impurities are very similar to the target peptide, which brings great difficulties to the separation and purification. Traditio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/06G01N30/02
Inventor 燕立波夏树君李佼佼金永华王军花杨振伟
Owner JIANGSU SKYRUN PHARMA CO LTD
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