阅读说明：本技术 纯化索玛鲁肽的方法 (Method for purifying somaglutide ) 是由 何平 潘俊锋 于铁妹 刘建 于 2020-08-17 设计创作，主要内容包括：本发明涉及多肽药物的纯化领域,具体涉及一种固相片段法合成索玛鲁肽的纯化制备方法,采用柱层析和膜分离技术相结合,获得了较高产品纯度及收率,解决了目前规模化制备技术难题。其步骤如下：将固相片段法合成的索玛鲁肽粗肽溶解于醋酸水溶液,用陶瓷膜过滤,然后用反相C8填料进行分离纯化,收集索玛鲁肽馏分,低温减压回收有机溶剂,得到索玛鲁肽的纯化溶液,经过膜系统脱除盐分及多余的酸根离子,进一步浓缩到适合冻干的浓度,冷冻干燥、包装得到高质量的索玛鲁肽原料。(The invention relates to the field of purification of polypeptide drugs, in particular to a purification preparation method for synthesizing somaltulin by a solid phase fragment method. The method comprises the following steps: dissolving the crude soxhalutatide synthesized by a solid phase fragment method in an acetic acid aqueous solution, filtering by using a ceramic membrane, then separating and purifying by using a reversed phase C8 filler, collecting soxhalutatide fraction, recovering an organic solvent at low temperature under reduced pressure to obtain a purified solution of the soxhalutatide, removing salt and redundant acid radical ions by using a membrane system, further concentrating to a concentration suitable for freeze drying, and carrying out freeze drying and packaging to obtain the high-quality raw soxhalutatide.)

1. A method for purifying somaglutide, comprising the steps of:

step 1: dissolving the crude soxhlet peptide into an acetic acid water solution to obtain a crude product solution;

step 2: filtering the crude product solution by a ceramic membrane, and collecting filtrate;

and step 3: taking the filtrate, taking reversed phase C8 filler as a stationary phase, taking triethylamine phosphate solution as an A mobile phase, taking acetonitrile as a B mobile phase, carrying out linear gradient elution with the gradient of 40-60 percent and the monitoring wavelength of 230nm, and collecting a fraction solution;

and 4, step 4: adjusting the pH of the distillate solution to 6-7 by using acetic acid, and recovering acetonitrile at low temperature under reduced pressure to obtain a purified sample solution;

and 5: taking the purified sample solution, taking reversed phase C8 as a stationary phase, taking an acetic acid solution as an A mobile phase, taking acetonitrile as a B mobile phase, carrying out linear gradient elution with the gradient of 43-50 percent and the monitoring wavelength of 230nm, and collecting a fraction solution;

step 6: recovering acetonitrile at low temperature under reduced pressure to obtain purified sample solution, desalting, concentrating, and freeze drying.

2. The method of claim 1, wherein the aqueous acetic acid solution in step 1 has a mass concentration of 10%.

3. The method according to claim 1 or 2, wherein the triethylamine phosphate in step 3 has a molar concentration of 80mMol/L and a pH of 7.2.

4. The method of any one of claims 1 to 3, wherein the linear gradient elution in step 3 is for a period of 40 min.

5. The method according to any one of claims 1 to 4, wherein the temperature of the low-temperature decompression in step 4 or step 6 is 30 to 45 ℃ and the degree of vacuum is 10 to 100 mbar.

6. The method of any one of claims 1 to 5, wherein the linear gradient elution in step 5 is for a period of 30 min.

7. The method according to any one of claims 1 to 6, wherein the molar concentration of the acetic acid solution in step 5 is 50 mMol/L.

8. The method according to any one of claims 1 to 7, wherein the desalting and concentrating in the step 6 adopt a cross-flow membrane filtration system, and the concentration after the concentrating is 100-150 g/L.

Technical Field

The invention relates to the field of purification of polypeptide drugs, in particular to a method for purifying somaglutide.

Background

Diabetes is a global high-occurrence metabolic disease, more than 4 hundred million type 2 diabetes patients are globally treated, more than 1 hundred million type 2 diabetes patients in China are treated, and the proportion of the diabetes patients in chronic diseases is very high. Diabetes can cause many complications, seriously affect the labor capacity of human beings and greatly endanger the health of human bodies.

The name of the sumaglutide in English is Semaglutide, and the molecular formula is C₁₈₇H₂₉₁N₄₅O₅₉Molecular weight 4113.64. The peptide sequence structure is as follows:

H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic Acid)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH。

the somaglutide is a new generation of long-acting GLP-1 analogue developed by Danish Nonoh and Node company, structurally, the 7 th position is changed into an unnatural amino acid aminoisobutyric acid, the amino group of the Lys side chain at the 26 th position is connected with AEEA, glutamic acid and octadecanoic acid fatty chain, after the AEEA is modified, the somaglutide can be tightly combined with albumin, the DPP-4 enzyme hydrolysis site is covered, renal excretion can be reduced, the biological half-life period can be prolonged, and the long-acting glucose-reducing effect can be achieved. Clinical experiment results show that the somaglutide injection is administrated once a week, the oral preparation is administrated once a day, the effect is more stable and lasting than other hypoglycemic drugs, the somaglutide injection has the effect of reducing the blood sugar and the weight, and the application prospect of the somaglutide is very optimistic.

At present, some methods and patents aiming at purification of the somaglutide exist, but the method has the disadvantages of complicated steps, low yield, related purification to various chromatographic fillers, short service life, high cost and difficulty in realizing commercial large-scale production.

Disclosure of Invention

In view of the above, the present invention provides a method for purifying somaglutide. The method improves chromatographic use conditions, improves separation effect, improves product yield, prolongs the service life of chromatographic packing, and reduces production cost.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for purifying somaglutide, which comprises the following steps:

step 1: dissolving the crude soxhlet peptide into an acetic acid water solution to obtain a crude product solution;

step 2: filtering the crude product solution by a ceramic membrane, and collecting filtrate;

and step 3: taking the filtrate, taking reversed phase C8 filler as a stationary phase, taking triethylamine phosphate solution as an A mobile phase, taking acetonitrile as a B mobile phase, carrying out linear gradient elution with the gradient of 40-60 percent and the monitoring wavelength of 230nm, and collecting a fraction solution;

and 4, step 4: adjusting the pH of the distillate solution to 6-7 by using acetic acid, and recovering acetonitrile at low temperature under reduced pressure to obtain a purified sample solution;

and 5: taking the purified sample solution, taking reversed phase C8 as a stationary phase, taking an acetic acid solution as an A mobile phase, taking acetonitrile as a B mobile phase, carrying out linear gradient elution with the gradient of 43-50 percent and the monitoring wavelength of 230nm, and collecting a fraction solution;

step 6: recovering acetonitrile at low temperature under reduced pressure to obtain purified sample solution, desalting, concentrating, and freeze drying.

In some embodiments of the present invention, the concentration of the aqueous acetic acid solution in step 1 is 10% by mass.

In some embodiments of the invention, the triethylamine phosphate in step 3 has a molarity of 80mMol/L and a pH of 7.2.

In some embodiments of the invention, the time for the linear gradient elution in step 3 is 40 min.

In some embodiments of the present invention, the temperature of the low-temperature decompression in step 4 or step 6 is 30 to 45 ℃ and the vacuum degree is 10 to 100 mbar.

In some embodiments of the invention, the time for the linear gradient elution in step 5 is 30 min.

In some embodiments of the invention, the molar concentration of the acetic acid solution in step 5 is 50 mMol/L.

In some embodiments of the present invention, the desalting and concentrating in step 6 are performed by a cross-flow membrane filtration system, and the concentration after the concentration is 100-150 g/L.

The invention provides a novel method for purifying the somagulipide in a large scale, which reduces the types of chromatographic packing, improves the chromatographic use condition, improves the separation effect, improves the product yield, prolongs the service life of the chromatographic packing and reduces the production cost. The purification and separation conditions adopted by the method are mild, the bioactivity of the somaglutide is maintained to the maximum extent, and denaturation is prevented. The membrane separation technology is combined, the dependence on a chromatographic system is reduced, the working efficiency of pretreatment and aftertreatment in a purification process is improved, and the commercial mass production is realized.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows an HPLC chromatogram of Somalutide prepared in example 1;

FIG. 2 shows an HPLC chromatogram of the Somalutide prepared in example 2;

FIG. 3 shows an HPLC chromatogram of the Somalutide prepared in example 3;

figure 4 shows an HPLC chromatogram of the somalutide prepared in the comparative example.

Detailed Description

The invention discloses a method for purifying the somaglutide, which can be realized by appropriately improving the process parameters by the technical personnel in the field by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The technical scheme of the invention is as follows: a method for purifying the somaglutenin in a large scale comprises the following steps:

1) dissolving the crude soxhlet peptide synthesized by the solid-phase fragment in 10% acetic acid aqueous solution to obtain a crude product solution;

2) the crude product solution is precisely clarified and filtered by a ceramic membrane system;

3) taking filtrate, taking reversed phase C8 as a stationary phase, triethylamine phosphate as an A mobile phase, acetonitrile as a B mobile phase, carrying out linear gradient elution with the gradient of 40-60% and the monitoring wavelength of 230nm, and collecting the first-step purified fraction solution;

4) adjusting the pH of the first-step purified fraction solution to weak acidity by using acetic acid, and recovering acetonitrile at low temperature under reduced pressure to obtain a first-step purified sample solution;

5) taking the purified sample solution in the first step, taking reversed phase C8 as a stationary phase, acetic acid as an A mobile phase, acetonitrile as a B mobile phase, carrying out linear gradient elution with the gradient of 43-50% and the monitoring wavelength of 230nm, and collecting the purified fraction solution in the second step;

6) and recovering acetonitrile at low temperature under reduced pressure to obtain a second-step purified fraction solution, desalting by a membrane concentration system, concentrating to freeze-dried concentration, and performing freeze-drying, subpackaging and packaging to obtain the high-quality somaglutide raw material.

Compared with the prior art, the method has the following advantages that:

the cross-flow ceramic membrane filtration technology is selected to process a crude peptide sample, so that impurities can be well removed, chromatographic separation filler is protected to the maximum extent, and the reverse-phase C8 filler is selected, so that the separation efficiency is high, the mechanical strength and the chemical stability are good, the sustainable use time is long, and the benefits on the purification efficiency and the yield are high. The solid-phase synthesized polypeptide has a large amount of racemization, deletion, oxidation and other impurities, and mainly depends on reversed-phase C8 chromatographic separation to achieve high-purity medicinal grade, the method adopts two times of chromatographic separation of C8 with different mobile phase systems, so that a high-purity product is obtained, and the purification yield is greatly improved. The chromatographic column chromatography is combined with a membrane separation technology, so that the steps of salt conversion and desalination processes of the traditional method are reduced, the dependence on a chromatographic system is reduced, the production cost is reduced, the production efficiency is improved, and the large-scale production is easier to realize.

The raw materials and reagents used in the method for purifying the somaglutide provided by the invention can be purchased from the market.

The invention is further illustrated by the following examples: