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Polypeptide purification method

A purification method and technology of trifluoroethanol, which can be used in measuring devices, instruments, scientific instruments, etc., can solve the problems of consuming mobile phases and increasing costs, and achieve the effects of reduced dosage, cost saving and high purity

Active Publication Date: 2016-01-06
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 prepared as the A phase, and the volume ratio of pure chromatography-grade acetonitrile and chromatography-grade trifluoroethanol was 7:3 as the B phase; The 10mg / ml thymus α1 after dissolving and filtering, the crude product purity is 20%, is added to the instrument for preparation, the peak time of the sample is 15min, the collected solution is dried and then weighed and analyzed, the degree of separation is 2.27, the number of theoretical plates is 73157, The signal-to-noise ratio was 419.1, the recovery was 31%, and the purity was 90%.

[0028] Under the same preparation equipment and conditions, the thymus α1 sample of the same concentration and purity was used for preparation, and the mobile phase was changed to 0.1% TFA aqueous solution for phase A, and pure acetonitrile for phase B...

Embodiment 2

[0031] Weigh 200 mg of thymosin α1 crude product, with a crude product purity of 16%, add 10 ml of acetonitrile and stir until it becomes turbid, then add 30 ml of pure water, ultrasonically stir until it is clear and transparent, and filter with a 0.45u microporous membrane. In the liquid chromatography, other conditions remain unchanged, the mobile phase is changed to A: 0.1% TFA water, B: a mixed solution of acetonitrile and tetrahydrofuran with a volume ratio of 7:3, and the preparation gradient is changed to 0-5-45min, 5-25- 35%. The peak time is 16min, and the purity of the obtained sample is 99%. After lyophilization, it is weighed as 14mg 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 time of the sample was 28 min, the purity was 98%, and the weight after lyophilization was 9 mg, and the yield was 28.1%.

Embodiment 3

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

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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 method for purifying a class of polypeptides, belonging to the technical field of protein and polypeptide purification. Background technique [0002] Polypeptide is a kind of substance between amino acids and proteins, which is composed of one or more amino acids combined by peptide bonds in a certain order. There are tens of thousands of polypeptides found in living organisms. Polypeptide is a structural fragment of protein, which can play the role of active group of protein and is an important substance for human metabolism and regulation activities. With the development of biotechnology and the growing maturity of peptide synthesis technology, there are still low-purity peptides and complex impurities, especially the physicochemical properties of impurities are very similar to the target peptide, which brings great difficulties to separation and purification. Traditional separation and purification technologies, such as s...

Claims

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Application Information

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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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