阅读说明：本技术 一种利拉鲁肽裂解液的后处理方法 (Post-processing method of liraglutide lysate ) 是由 周黎 黄保胜 郑春旭 宋亮 王良友 于 2019-12-10 设计创作，主要内容包括：本发明公开了一种利拉鲁肽裂解液的后处理方法,属于多肽提取纯化技术领域。本发明提供一种利拉鲁肽裂解液的后处理方法,本发明方法利用利拉鲁肽等电点4.9,在碱性水条件下易溶,在酸性条件下易析出,以水为洗涤溶剂,通过调整pH达到析出的目的,避免了使用常规固相裂解沉降和洗涤用到的无水乙醚或甲基叔丁基醚,降低了安全风险,同时减少了采用醚类沉降利拉鲁肽粗品携带的杂质,有助于延长制备柱的使用寿命,降低了纯化的难度,有利于进行工业化生产。(The invention discloses a post-treatment method of liraglutide lysate, belonging to the technical field of polypeptide extraction and purification. The invention provides a post-treatment method of liraglutide lysate, which utilizes the isoelectric point of liraglutide of 4.9, is easy to dissolve under the condition of alkaline water and is easy to separate out under the condition of acidity, water is used as a washing solvent, and the purpose of separating out is achieved by adjusting pH, so that anhydrous ether or methyl tert-butyl ether used in conventional solid-phase cracking and settling and washing is avoided, the safety risk is reduced, impurities carried by ether-based settling liraglutide crude product are reduced, the service life of a preparation column is prolonged, the difficulty of purification is reduced, and the industrial production is facilitated.)

1. A post-processing method of liraglutide lysate is characterized by comprising the following steps:

(1) adding the liraglutide lysate into an inorganic alkali solution, adjusting the using amount of the inorganic alkali until a large amount of samples are separated out, carrying out solid-liquid separation after the samples are separated out, and collecting precipitates;

(2) dissolving the precipitate collected in the step (1) with an inorganic alkali solution, stirring and dropwise adding an acid solution after dissolving until a large amount of sample is separated out, carrying out solid-liquid separation after separating out, and collecting the precipitate;

(3) and (3) drying the precipitate collected in the step (2) to obtain the crude liraglutide.

2. The post-treatment method according to claim 1, wherein in the step (1), the volume ratio of the liraglutide lysate to the inorganic base solution is 1: 5-10.

3. The post-treatment method according to claim 1, wherein the inorganic alkali solution is dilute ammonia or sodium hydroxide solution.

4. The post-treatment method according to claim 3, wherein the dilute ammonia water has a concentration of 2 to 10%.

5. The post-treatment method according to claim 3, wherein the concentration of the sodium hydroxide is 30 to 500 mg/L.

6. The post-treatment method according to claim 1, wherein the acid solution is trifluoroacetic acid, acetic acid or phosphoric acid.

7. The post-treatment method according to claim 1, wherein the solid-liquid separation is centrifugation or filtration.

8. The post-treatment method according to claim 1, wherein the step (2) is repeated 1 to 2 times.

Technical Field

The invention relates to a post-treatment method of liraglutide lysate, belonging to the technical field of polypeptide extraction and purification.

Background

The liraglutide is developed by Danish Novonide company, is a glucagon-like peptide 1(GLP-1) analogue for long-acting treatment of type II diabetes, and belongs to a GLP-1 receptor agonist. The drug is approved by FDA to be marketed in 1 month and 25 2010, and approved by SFDA to be marketed in China in 3 months and 4 days 2011. The liraglutide serving as a new generation of hypoglycemic drugs based on incretins has long action time, fully retains multiple physiological activities of natural GLP-1, can safely and effectively reduce blood sugar, has a protection effect on various cardiovascular risk factors, brings a new choice for treating type 2 diabetes, and has encouraging clinical treatment effect and huge market potential.

The post-cracking treatment of the liraglutide solid phase synthetic peptide resin which is disclosed at present adopts anhydrous ether or methyl tert-butyl ether. For example, the synthesis of liraglutide in patent CN110291099A uses TFA, phenol, TIS, H₂The total cleavage of the peptide was performed with a mixture of O-thioanisole, and the peptide-TFA mixture was concentrated and added to ice MTBE 5-15 v. Separating the precipitated compound by centrifugation and/or filtration and drying in a VTD; in the patent CN108976296A, after cutting is finished in a synthesis method of liraglutide, filtering a solution in a reactor into glacial ethyl ether through a sand core, uniformly mixing, centrifuging, discarding a supernatant, and repeating the operation for 3 times to obtain a crude liraglutide product; in patent CN107903317A, a synthetic method of liraglutide includes preparing a cleavage reagent TFA, thioansole, ansole, EDT 90:5:3:2, adding the cleavage reagent into the fully protected peptide under an ice bath condition, adding anhydrous ether for precipitation after the reaction is finished, performing centrifugal precipitation, and drying to obtain a product, i.e., a crude liraglutide. Patent CN105111303A a method for preparing liraglutide by solid-liquid combination, liraglutide (unmodified) peptide resin is added into frozen lysate (volume ratio TFA/TIS/H)₂O95/2.5/2.5), filtering the resin, combining the filtrate and washing liquor, concentrating to 1600ml by rotary evaporation, pouring into 16L of frozen methyl tert-ether, separating out a white precipitate, standing for 30min, filtering, washing with methyl tert-ether for 6 times, and drying in vacuum to obtain the crude peptide of the N-terminal trifluoroacetylated liraglutide (unmodified). In the patent CN102875665A, 570ml of lysate TFA, EDT, PHOH and H are prepared in the method for synthesizing liraglutide₂Adding the lysate into a flask, reacting at room temperature for 2.5 hours, filtering the resin, and collecting the filtrate. The resin was washed with a small amount of TFA, the filtrates were combined, the filtrate was added to 5700ml of anhydrous ether for precipitation, centrifuged, washed with anhydrous ether, and dried in vacuo to give 14.1g of crude peptide of liraglutide.

The liraglutide has huge market demand, a large amount of anhydrous ether or methyl tert-butyl ether is consumed by a conventional cracking and settling process, the anhydrous ether and the methyl tert-butyl ether are inflammable, and great potential safety hazards are caused when the liraglutide is used in a large amount. The invention strives to avoid the use of anhydrous ethyl ether or methyl tert-butyl ether from the viewpoint of the solubility properties of liraglutide itself.

Disclosure of Invention

In order to solve the technical problems, the invention provides a post-treatment method of liraglutide lysate, which utilizes the isoelectric point of liraglutide to be 4.9, is easy to dissolve under the condition of alkaline water and separate out under the condition of acidity, takes water as a washing solvent, achieves the purpose of separating out by adjusting pH, avoids using anhydrous ether or methyl tert-butyl ether used for conventional solid-phase cracking and settling and washing, reduces the safety risk, reduces the impurities carried by ether settling liraglutide crude product, is beneficial to prolonging the service life of a preparation column, reduces the difficulty of purification, and is beneficial to industrial production.

The first purpose of the invention is to provide a method for post-treating liraglutide lysate, which comprises the following steps:

(1) adding the liraglutide lysate into an inorganic alkali solution, adjusting the using amount of the inorganic alkali until a large amount of samples are separated out, carrying out solid-liquid separation after the samples are separated out, and collecting precipitates;

(2) dissolving the precipitate collected in the step (1) with an inorganic alkali solution, stirring and dropwise adding an acid solution after dissolving until a large amount of sample is separated out, carrying out solid-liquid separation after separating out, and collecting the precipitate;

(3) and (3) drying the precipitate collected in the step (2) to obtain the crude liraglutide.

Further, the volume ratio of the liraglutide lysate to the alkali liquor is 1: 5-10.

Further, the inorganic alkali solution is dilute ammonia water or sodium hydroxide solution.

Further, the concentration of the dilute ammonia water is 2-10%.

Further, the concentration of the sodium hydroxide is 30-500 mg/L.

Further, the acid solution is trifluoroacetic acid, acetic acid or phosphoric acid.

Further, the solid-liquid separation is centrifugation or filtration.

Further, in the post-treatment method, the step (2) is repeated for 1 to 2 times.

The principle of the invention is as follows: based on the isoelectric point 4.9 of liraglutide, the liraglutide is easy to dissolve under the condition of an alkaline (more than pH8.0) aqueous solution and is easy to separate out under the acidic condition (the pH is about 4.0-6.0), liraglutide lysate is added into an inorganic alkaline solution in the first step, because the lysate contains a large amount of trifluoroacetic acid, a sample is dissolved at the moment, the alkaline solution is required to be added to improve the pH until the sample is separated out in a large amount, solid-liquid separation is carried out after separation, and precipitates are collected; and secondly, dissolving the collected precipitate with an inorganic alkali solution, stirring and dropwise adding an acid solution after dissolving until a large amount of samples are separated out, carrying out solid-liquid separation after separating out, and collecting the precipitate to achieve the purpose of washing.

The invention has the beneficial effects that:

the method avoids using anhydrous ether or methyl tert-butyl ether used in conventional solid-phase cracking sedimentation and washing, reduces safety risks, reduces impurities carried by ether sedimentation of the crude liraglutide, is beneficial to prolonging the service life of the preparation column, reduces the difficulty of purification, and is beneficial to industrial production.

Drawings

FIG. 1 is an HPLC chart of crude liraglutide obtained by ammonia precipitation in example 4.

FIG. 2 is a HPLC chart of crude liraglutide obtained by settling with methyl tert-butyl ether in example 4.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.