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Method for low-cost purification of exenatide

A low-cost exenatide technology, applied in the field of peptide purification, to achieve high yield, cost saving, and environmental protection of the purification process

Active Publication Date: 2014-03-05
SHAANXI HUIKANG BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of existing exenatide purification methods, and provide a low-cost, high-purity exenatide purification method suitable for industrialization

Method used

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  • Method for low-cost purification of exenatide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1. Sample dissolution

[0028] Dissolve 0.2 g of the crude exenatide obtained by solid-phase synthesis in 5 mL of 20% acetic acid aqueous solution, adjust the pH value to 4.0 with 15% sodium hydroxide aqueous solution, and disperse it ultrasonically. After the solution is completely clarified, Filter through a 0.45 μm membrane filter and collect the filtrate.

[0029] 2. Crude and pure

[0030] The filtrate was crudely purified by ion-exchange high-performance liquid chromatography. The filler was a strong cation-exchange column of SP high-flow rate agarose microspheres (provided by GE Healthcare) with a particle size of 45-165 μm. The column packing volume was 15 mL. Phase A is 0.02mol / L acetic acid-sodium acetate aqueous solution with a pH value of 4.0, mobile phase B is 0.02mol / L acetic acid-sodium acetate aqueous solution with a pH value of 4.0 containing 1mol / L sodium chloride, and the flow rate is 4mL / min , the column temperature is 40°C, and the detection wavel...

Embodiment 2

[0036] 1. Sample dissolution

[0037] Dissolve 1.5 g of the crude exenatide obtained by solid-phase synthesis in 50 mL of 20% acetic acid aqueous solution, adjust the pH value to 4.0 with 15% sodium hydroxide aqueous solution, and disperse ultrasonically until the solution is completely clarified. Filter through a 0.45 μm membrane filter and collect the filtrate.

[0038] 2. Crude and pure

[0039]The filtrate was crudely purified by ion-exchange high-performance liquid chromatography, and the filler was a strong cation exchange column of SP high-flow rate agarose microspheres with a particle size of 50-160 μm (provided by Xi’an Jiaotong University Baosai Biotechnology Co., Ltd.). The filling volume is 150mL, the mobile phase A is 0.02mol / L acetic acid-sodium acetate aqueous solution with a pH value of 4.0, and the mobile phase B is 0.02mol / L acetic acid-sodium acetate with a pH value of 4.0 containing 1mol / L sodium chloride Aqueous solution, the flow rate is 8mL / min, the co...

Embodiment 3

[0045] In the crude purification step 2 of this embodiment, the mobile phase gradient is selected from 0 to 60 minutes A:B from 60:40 to 30:70. The other steps were the same as in Example 2 to obtain exenatide with a purity greater than 98%, and the purification yield was 50%.

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Abstract

The invention discloses a method for low-cost purification of exenatide. The method comprises the steps of firstly, performing initial purification on a large quantity of exenatide crude products by using a strong cation exchange column by adopting an efficient liquid phase chromatography so as to remove most of impurities in the exenatide crude products and provide convenience for refining purification from two aspects of quality and quantity, then performing desalination by using a reverse phase polymer column, and then performing refining purification by using a C18 reverse phase silica gel column. By adopting the method, the production cost is reduced, the exenatide with the concentration of more than 98% can be obtained, and the requirements of low cost, high yield and industrialization for purifying exenatide can be met.

Description

technical field [0001] The invention belongs to the field of polypeptide purification, and in particular relates to a method for purifying exenatide. Background technique [0002] In 1995, U.S. Patent (US5424286) disclosed a kind of polypeptide Exendin-4 (H-His-Gly-Glu-H-His-Gly-Glu- Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn- Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Pro-Ser-NH2), its structure has 48% homology with human glucagon (Glucagon), and human GLP- 1 (glucagon-like peptide-1) has 53% homology. [0003] Studies have shown that, as an analog of GLP-1, Exendin-4 can interact with GLP-1 receptors, stimulate the regeneration of pancreatic β cells, promote insulin secretion, inhibit the release of glucagon, slow down the rate of gastric emptying, and inhibit food intake. Its role in promoting insulin secretion is based on blood sugar levels, so it can reduce the incidence of hypoglycemia, and it still has a hypog...

Claims

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Application Information

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IPC IPC(8): C07K14/575C07K1/20C07K1/18
CPCC07K14/575
Inventor 冯凌云韩彬张腾韩广杨晓琳赵金礼
Owner SHAANXI HUIKANG BIO TECH CO LTD
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