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A kind of purification method of glucagon

A technology for glucagon and purification methods, which is applied in the direction of preparation methods, chemical instruments and methods of glucagon and peptides, and can solve the problems that glucagon cannot achieve high production efficiency

Active Publication Date: 2021-05-14
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These phenomena have always restricted glucagon from being able to achieve high production efficiency. How to break through the technical bottleneck of the current purification process has always been the goal of continuous exploration by technicians

Method used

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  • A kind of purification method of glucagon
  • A kind of purification method of glucagon
  • A kind of purification method of glucagon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: purification method of the present invention

[0063] 1. Crude peptide pretreatment

[0064] Dissolve 5 g of crude glucagon peptide synthesized in solid phase with pure water until the sample is dissolved, then slowly add ammonia water to adjust the pH to 10, then raise the temperature of the crude peptide solution to 60 degrees Celsius, and slowly add 100 mmol / 1 of guanidine hydrochloride and 300mmol / l of sodium chloride were kept stirring during the addition process. After 20 minutes of reaction, the reverse phase detection reaction was completed, and the temperature of the crude peptide solution was immediately lowered to 37 degrees Celsius with ice water, the reaction was terminated, and the IM conversion process was over. .

[0065] 2. Gradient elution purification

[0066] Purification conditions: chromatographic column: a chromatographic column with octadecyl bonded silica gel as the stationary phase, and the diameter and length of the column are...

Embodiment 2

[0071] Embodiment 2: purification method of the present invention

[0072] 1. Crude peptide pretreatment

[0073] Dissolve 15 g of the crude glucagon peptide synthesized in the liquid phase with pure water until the sample is dissolved, then slowly add ammonia water to adjust the pH to 9.5, then raise the temperature of the crude peptide solution to 55 degrees Celsius, and slowly add 100 mmol / 1 of guanidine hydrochloride and 200mmol / l of sodium chloride, kept stirring during the addition process, after 20 minutes of reaction, the reverse phase detection reaction was completed, immediately lowered the temperature of the crude peptide solution to 37 degrees Celsius with ice water, the reaction was terminated, and the IM conversion process was over .

[0074] 2. Gradient elution purification

[0075] Purification conditions: chromatographic column: a chromatographic column with octadecyl bonded silica gel as the stationary phase, and the diameter and length of the column are: ...

Embodiment 3

[0080] Embodiment 3: purification method of the present invention

[0081] 1. Crude peptide pretreatment

[0082] Dissolve 15 g of the crude glucagon peptide in Example 2 with pure water until the sample is dissolved, then slowly add ammonia water to adjust the pH to 9.5, then raise the temperature of the crude peptide solution to 55 degrees Celsius, and slowly add 100 mmol The guanidine hydrochloride of / l and the sodium chloride of 300mmol / l, keep stirring during the addition process, after reacting for 20min, the reversed-phase detection reaction is completed, and the temperature of the crude peptide solution is immediately lowered to 37 degrees Celsius with ice water, the reaction is terminated, and the IM conversion process End.

[0083] 2. Gradient elution purification

[0084] Purification conditions: chromatographic column: a chromatographic column with octadecyl bonded silica gel as the stationary phase, and the diameter and length of the column are: 10cm×25cm. Mob...

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Abstract

The invention relates to the field of biotechnology and discloses a method for purifying glucagon. After the purification method of the present invention dissolves the crude glucagon peptide, adjust the pH of the crude peptide solution to alkaline, add protein denaturant and chloride salt to the crude peptide solution to react until the glucagon content no longer increases, Cool down and carry out RP-HPLC purification and turn salt then. The present invention discovers the key factor restricting the low purification efficiency of glucagon crude peptide, and then finds that the crude peptide is pretreated with protein denaturant and chloride salt, and then purified, thus the purification yield and total yield can be reduced. rate increased substantially. On this basis, the compound salt solution can be used as the mobile phase for further purification, which can improve the quality of glucagon. In the existing purification process of glucagon, the purification method of the present invention has obvious advantages, the yield and quality are obviously improved, and the economic benefits brought are also very considerable.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for purifying glucagon. Background technique [0002] Glucagon (glucagon) is a hormone secreted by pancreatic islet α-cells. It consists of 29 amino acids and is a linear polypeptide with a molecular weight of 3485 Daltons. The glucagon peptide sequence is as follows: [0003] NH 2 -His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp -Leu-Met-Asn-Thr-COOH [0004] Glucagon binds to receptors in the body and releases glucose stored in the liver in the form of glycogen through glycogenolysis, thereby helping to maintain the glucose level in the blood. It can raise blood sugar, and its half-life is short. Existing literature shows that glucagon plays an important role in energy metabolism or energy intake mediation in the body [Habegger et al., Nature Reviews Endocrinology 2010, 6, 689-697], so glucagon may also play a role in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/605C07K1/20
Inventor 尹传龙赵忠卫康旭宓鹏程陶安进袁建成
Owner HYBIO PHARMA