A purification method of recombinant human glucagon-like peptide-1 analog

A technique for glucagon and a purification method, which is applied in the field of purification of recombinant human glucagon-like peptide-1 analogs, and can solve problems such as slow flow rate, inability to carry out industrial scale-up production, and increased pressure in the chromatography process. Achieve the effect of low production cost and improve the feasibility of enlarged production

Active Publication Date: 2021-10-01
YICHANG HEC CHANGJIANG PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This purification method is relatively simple and the production cost is low. However, when GLP-1 is added with an organic solvent under acidic pH conditions, the stability of the sample becomes poor, and protein denaturation and aggregation are prone to occur, resulting in an increase in the pressure of the chromatography process and a slow flow rate. Carry out industrial scale-up production

Method used

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  • A purification method of recombinant human glucagon-like peptide-1 analog
  • A purification method of recombinant human glucagon-like peptide-1 analog
  • A purification method of recombinant human glucagon-like peptide-1 analog

Examples

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Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Recombinant expression of precursor fusion protein in Escherichia coli was pretreated and denatured, captured by anion chromatography and digested with enterokinase to remove the tagged protein to obtain GLP-1 analogs

[0041] Recombinant Expression of DsbC-DDDK-Arg in Escherichia coli by DNA Recombination Technology 34 GLP-1(7-37) fusion protein, the recombinant cells were fermented and centrifuged to collect the cells, the recombinant cells were used to extract the inclusion bodies and denatured, then use anion chromatography to capture the fusion protein, and use 200mM NaCl for linear gradient elution to collect the main peak . After capturing the fusion protein, adjust the pH to 8.0, and add recombinant enterokinase at a ratio of 10 IU / mg for enzyme digestion.

Embodiment 2

[0042] Example 2: Purification using Source 30Q media

[0043] This embodiment provides a purification method for GLP-1 analogues, specifically:

[0044] (1) Dilute hydrochloric acid is added to the feed solution after enzymatic digestion in Example 1, and the pH is adjusted to 5.8, and the isoelectric point precipitation is carried out. After standing overnight, the precipitate is collected by centrifugation, which is Arg 34 GLP-1(7-37), the purity is about 80%, the recovery rate is more than 90%;

[0045] (2) Resuspend the precipitate obtained in step (1) in 20mM Tris and adjust the pH to 7.0 with sodium hydroxide to make Arg 34 GLP-1(7-37) was redissolved, adding purified water to adjust the conductance to no more than 6ms / cm 2 , filtered and clarified;

[0046] (3) Take the feed liquid obtained in step (2) and apply it to Source 30Q filler, the loading amount is 20g / L gel. After the sample is loaded, wash 3 column volumes with pH7.0 5mMTris-HCl, and then wash with pH7.0...

Embodiment 3

[0049] Example 3: Purification using UniGel 30Q media

[0050] This embodiment provides a purification method for GLP-1 analogues, specifically:

[0051] (1) Dilute hydrochloric acid is added to the feed liquid after enzymatic digestion in Example 1, the pH is adjusted to 5.2, and the isoelectric point precipitation is carried out. After standing overnight, the precipitate is collected by centrifugation, which is Arg 34 GLP-1(7-37), the purity is about 78%, and the recovery rate is more than 91%;

[0052] (2) Resuspend the precipitate obtained in step (1) in 20mM Tris and adjust the pH to 9.0 with sodium hydroxide to make Arg 34 GLP-1(7-37) was redissolved, adding purified water to adjust the conductance to no more than 6ms / cm 2 , filtered to clarify.

[0053] (3) Take the feed liquid obtained in step (2) and apply it to UniGel 30Q filler, the loading amount is 20g / L gel. After the sample loading is completed, wash 3 column volumes with pH9.0 50mMTris-HCl, then use pH9.0, ...

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Abstract

The present invention relates to a method for purifying recombinant human glucagon-like peptide-1 analogs, the method comprising the following steps: (1) taking a product to be purified containing recombinant human glucagon-like peptide-1 analogs, Adjust its pH value to the isoelectric point of the analogue, let it stand fully, and then centrifuge to discard the supernatant, collect the precipitate, and redissolve; (2) load the solution obtained in step (1) on an anion chromatography column, rinse, wash The eluate containing the analog was collected. The method provided by the invention has high purification efficiency, high product purity and high recovery rate. The precipitation process adopted is simple and easy to operate, the anion chromatography process is easy to perform linear amplification, and the production cost is low, which greatly improves the feasibility of industrial scale-up production.

Description

technical field [0001] The invention relates to the field of production of diabetes drugs, in particular to a method for purifying recombinant human glucagon-like peptide-1 analogs. Background technique [0002] Human glucagon-like peptide-1 (glucose-dependent insulinotropic peptide-1, GLP-1) is a polypeptide hormone secreted by intestinal L cells, and its mechanism of action is through the GLP-1 of pancreatic β cells. 1 receptor specific binding, after the interaction, the insulin secretion induced by glucose is significantly enhanced, and the function of GLP-1 is dependent on the blood sugar concentration. If it is used for the treatment of diabetes, the symptoms of hypoglycemia will not occur. With the continuous deepening of the research on its structure and physiological function, GLP-1 has become a very potential therapeutic drug for diabetes. However, the biological half-life of GLP-1 active peptides in the body is relatively short, mainly due to the rapid degradatio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/605C07K1/36C07K1/30C07K1/18
CPCC07K14/605
Inventor 李志雄张晓焰王振伟高相雷莫隆兴林小鹊章琛郭林峰李晓平陈小锋李文佳
Owner YICHANG HEC CHANGJIANG PHARMA CO LTD
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