CN110845602A

CN110845602A - Method for separating and purifying somaglutide

Info

Publication number: CN110845602A
Application number: CN201911201916.2A
Authority: CN
Inventors: 沈杰; 王良友; 郑春旭; 陈维维; 张璐
Original assignee: SUZHOU TIANMA GROUP TIANJI BIO-PHARMACEUTICAL CO LTD
Current assignee: SUZHOU TIANMA GROUP TIANJI BIO-PHARMACEUTICAL CO LTD
Priority date: 2019-11-29
Filing date: 2019-11-29
Publication date: 2020-02-28

Abstract

The invention relates to a method for efficiently and quickly separating and purifying Somalutide, which comprises the following steps: filtering the crude soxhlet peptide solution, purifying the filtrate by a polymer packed column and a reverse phase silica gel packed column respectively, separating and purifying by combining purification conditions of different systems, collecting a target product, and finally obtaining a product with the total purity of more than 99.5 percent and the maximum single impurity of less than 0.1 percent.

Description

Method for separating and purifying somaglutide

Technical Field

The invention relates to the technical field of polypeptide purification, and particularly relates to a method for separating and purifying somaglutide.

Background

The oral somaglutide is the best medicine for reducing blood sugar and losing weight in the GLP-1 medicine at present. Currently, NovoNordisk company has obtained US FDA approval in 2019, 9/20, bringing about a great influence on polypeptides, and insulin injections are actively developing oral preparations. In the field of diabetes in the future, polypeptide oral preparations are a necessary trend in the market. Somaltulip is a long-acting GLP-1 analog developed by NovoNordisk, injected subcutaneously once a week, and structurally, it is a GLP-1(7-37) chain in which Aib at position 8 has replaced Ala, Arg at position 34 has replaced Lys, and Lys at position 26 has been linked to an octadecanoic acid aliphatic chain. Compared with the liraglutide, the fat chain of the soxhlet peptide is longer, the hydrophobicity is increased, but the hydrophilicity of the soxhlet peptide is greatly enhanced through short-chain PEG modification.

At present, many published technologies related to polypeptide purification appear, for example, CN201811601939.8 discloses a method for purifying triptorelin, CN201510004136.4 discloses a method for purifying and preparing triptorelin, and CN201811407810.3 and CN201811603717.x respectively disclose a method for separating and purifying liraglutide. However, due to the sequence difference of different polypeptides, the method is not suitable for the separation and purification of the somaglutide. There are also many methods for purifying somaglutide and related patents, but the steps are cumbersome and the yields are low.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a method for separating and purifying the somaglutide, which has the technical problems of low purity, low yield, concentration and the like of the somaglutide separated and obtained in the prior art, and is high in product purity, good in yield and easy to industrialize.

The invention relates to a method for separating and purifying somaglutide, which comprises the following steps:

(1) filtering the crude soxhlet peptide solution with the pH value of 8.0-9.0 (preferably pH value of 9.0), and purifying the obtained filtrate by HPLC gradient separation through a polymer packed column;

wherein the crude soxhlet peptide solution comprises crude soxhlet peptide and dilute ammonia water containing 5 v% of acetonitrile; in the HPLC gradient separation and purification process, an eluent A phase is 20-30mmol/L (preferably 20mmol/L) of a carbonate aqueous solution, and the pH value of the eluent A phase is adjusted to 8.0-9.0 (preferably 9.0) by ammonia water; phase B is acetonitrile; gradient purification condition is 25-40% phase A, gradient elution time is 40-50min (preferably 40min), and primary purified product with main peak purity of more than 95% is collected at 220nm wavelength;

(2) subjecting the primary purified product to further HPLC gradient separation and purification by using a reverse phase silica gel packed column, wherein an eluent A phase is 0.1 v% TFA aqueous solution, a eluent B phase is acetonitrile, gradient purification conditions are 30-45% of the eluent A phase, gradient elution time is 40-50min (preferably 40min), and a secondary purified product with the main peak purity of more than 99% and the maximum single impurity of less than 0.2% is collected at a wavelength of 220 nm;

(3) desalting the secondary purified product by using a reverse phase silica gel packed column, wherein an eluent A phase is water, a eluent B phase is acetonitrile, a gradient elution method is adopted, the gradient elution condition is 30-60% of the A phase, the gradient elution time is 40-50min (preferably 40min), and the desalted product with the main peak purity of more than 99.5% and the maximum single impurity of less than 0.1% is collected under the wavelength of 220 nm;

(4) and (3) performing rotary evaporation concentration on the desalted product by adopting a thin film evaporator to remove acetonitrile and a large amount of water in the product, wherein the vacuum degree of the thin film evaporator is not higher than-0.09 MPa (preferably-0.097 MPa), the evaporation temperature is 40-50 ℃ (preferably 40 ℃), the rotation speed is 1400-1800r/min (preferably 1400-1500r/min), and then freeze-drying to obtain the purified somnumutide.

Further, in the step (1), filtration was performed with an organic filter having a pore size of 0.22. mu.m.

Further, in the step (1), the dissolution concentration of the crude soxhlet peptide in the crude soxhlet peptide solution is 20-40mmol/L, preferably 20-25 mmol/L. The dissolution system of the crude product of the soxhlet peptide is diluted ammonia water with 5 v% of acetonitrile, the soxhlet peptide is completely dissolved by controlling the pH value of the solution, and the polymer filler can be effectively protected by adding the 5 v% of acetonitrile.

Further, in the step (1), the filler in the polymer filler column is polymethacrylate polymer. The polymethacrylate polymer filler is adopted, so that the filling is more resistant to alkali flushing, the filler separation degree is good, and the filler service life is longer.

Further, in the step (1), the aqueous carbonate solution is an aqueous ammonium carbonate solution.

Further, in the step (1), the carbonate aqueous solution further includes phosphoric acid.

Further, in the step (2), the filler of the reversed phase silica gel filler column is an octaalkylsilane bonded silica gel filler.

Further, in the step (3), the filler of the reversed phase silica gel filler column is an octaalkylsilane bonded silica gel filler.

Further, in the step (4), the concentration rate of the evaporative concentration was 40 kg/h. The rotary evaporation concentration is carried out by adopting a film evaporator, the film evaporator is a novel efficient evaporator which is forced to form a film through a rotary film scraper and flows at a high speed, the heat transfer efficiency is high, the retention time is short, and the film evaporation is carried out under the vacuum condition, so that the solvent in the product can be evaporated quickly, the volume of the product is reduced, and the concentration effect is achieved.

Further, in the step (4), the desorption temperature of freeze-drying is 30-35 ℃.

Further, in the step (4), the bead peak purity of the purified somaglutide is more than 99.5%, and the maximum single impurity is less than 0.1%.

By the scheme, the invention at least has the following advantages:

the invention provides a method for separating and purifying Somalutide by adopting different stationary phase matrixes, different separation conditions and a desalting method and concentrating through a thin film evaporator.

According to the method, carbonate is used for primary purification, and the carbonate ions are used for separating the somaglutide, so that the removal effect of impurities with the peak emergence time later than the main peak is better, and the yield is higher.

According to the method, TFA is used for secondary purification, and trifluoroacetic acid is used for separating the somaglutide, so that the removal effect of impurities with the peak emergence time earlier than that of a main peak is good, and the yield is higher.

The invention uses water and acetonitrile as mobile phases, which are further purified in preparative separations and desalinize the somaglutide.

The invention uses the thin film evaporator for concentration, has no sample loss and quicker concentration efficiency which is more than 10 times of the concentration efficiency of rotary evaporation.

By adopting the method, the final total purification yield of the somaglutide reaches 65-70%, the purity is over 99.5%, and the maximum single impurity is not more than 0.1%.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

FIG. 1 is a high performance liquid chromatography chromatogram of crude Somalglutide in step 1;

FIG. 2 is a HPLC analysis chart of 96.80% purity Somalutide after separation and purification in step 2;

FIG. 3 is a HPLC analysis chart of the isolated and purified 99.82% purity somaglutide of step 3;

fig. 4 is a high performance liquid chromatography analysis spectrum of the finished product of the 99.89% purity somaglutide obtained in step 4 after separation and purification.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

The implementation provides a method for separating and purifying somaglutide, which comprises the following steps:

1. sample dissolution: 10 g of the crude product (the target peptide is the somaglutide), dissolved in diluted ammonia water with pH9 and 5 v% acetonitrile, ultrasonically treated, and filtered by a 0.22 mu m organic filter membrane to obtain a crude product solution.

2. Primary purification conditions: separating and purifying the crude product solution by polymethacrylate polymer packing, wherein the phase A is as follows: 20mmol/L sodium carbonate solution (pH adjusted to 9 with phosphoric acid), phase B is: acetonitrile, gradient elution condition of 25-40% phase A, gradient elution time of 40min, detection wavelength of 220nm, A, B phase flow rate of 80mL/min, room temperature, and sample injection of 4g crude product solution.

And (3) purification process: the column was rinsed clean with 95% acetonitrile and the sample was equilibrated to 4g target. And (3) carrying out linear gradient elution for 40min according to the primary purification conditions, collecting a target peak to obtain a component with the purity of more than 95%, wherein the component is used as a sample before the second purification, and the yield of the primary purification is about 88%.

3. And (3) secondary purification conditions: and (3) further separating and purifying the sample obtained by primary purification by virtue of an octaalkylsilane bonded silica filler, wherein the phase A is as follows: 0.1 v% TFA, aqueous B phase: acetonitrile, gradient elution condition of 30-45% of phase A, gradient elution time of 40min, detection wavelength of 220nm, A, B phase flow rate of 80mL/min, room temperature, and sample injection of 3.52g of target product.

And (3) purification process: the column was rinsed clean with 95% acetonitrile and the sample was equilibrated to 3.52g of target. And (3) carrying out linear gradient elution for 40min according to the secondary purification conditions, collecting a target peak, and obtaining a component with the purity of more than 99% and the maximum single impurity of less than 0.2%, wherein the component is used as a sample before the third purification, and the yield of the steps is about 80%.

4. And (3) three-time purification conditions: and (3) carrying out further separation and purification and desalting on the sample obtained by secondary purification by virtue of an octaalkylsilane bonded silica filler, wherein the phase A is as follows: the water phase B is as follows: acetonitrile, gradient elution condition of 30-60% phase A, gradient elution time of 30min, detection wavelength of 220nm, A, B phase flow rate of 80mL/min, room temperature, sample injection of 2.816 g of target product.

And (3) purification process: the column was rinsed clean with 95% acetonitrile and equilibrated to 2.816 g of target. And (3) carrying out linear gradient elution for 40min according to the conditions, collecting a target peak, and obtaining a component with the purity of more than 99.5 percent and the maximum single impurity of less than 0.1 percent, wherein the component is used as a sample before thin film evaporation, the yield of the steps is about 95 percent, and the total yield of the product after three times of purification is about 66.88 percent.

4. Film evaporation: and concentrating and recovering the sample after the three times of purification by using a thin film evaporator to obtain a high-concentration sample. The vacuum degree of the film evaporator is-0.097 MPa, the evaporation temperature is 40 ℃, and the rotating speed is 1400 r/min.

5. Freezing and drying: and putting the sample into a freeze-drying tray, and freeze-drying at the desorption temperature of 35 ℃ under the vacuum pressure of 0.1pa to obtain 2.67 g of the somaglutide with the maximum single impurity content of less than 0.1 percent and the purification total yield of 66.75 percent, wherein the freeze-drying desorption temperature is 35 ℃.

The statistical results of the contents of the components in fig. 1-4 are shown in tables 1-4 in sequence, and the numerical numbers in the tables correspond to the numbers of the peaks in the figures. As can be seen from the table, the content of the somaglutide in the crude product was 85.76%, while the content of the somaglutide in the purified product was 99.89%, and the purity was significantly improved.

TABLE 1 statistics of the content of the components in FIG. 1

TABLE 2 statistics of the contents of the components in FIG. 2

TABLE 3 statistics of the contents of the components in FIG. 3

Table 4 statistics of the contents of the components in fig. 4

Therefore, the invention provides a method suitable for purifying the somaglutide, which is high-efficient, rapid and simple in separation and purification, and finally obtains a sample with the purity of more than 99.5 percent and the maximum single impurity of less than 0.1 percent, the yield of the sample is up to 66.75 percent, the production process is simple and stable, and the method can be used for batch production and reduces the cost.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Claims

1. A method for separating and purifying somaglutide, which is characterized by comprising the following steps:

(1) filtering the crude soxhlet peptide solution with the pH value of 8.0-9.0, and performing HPLC gradient separation and purification on the obtained filtrate through a polymer packed column;

the crude soxhlet peptide solution comprises a crude soxhlet peptide product and dilute ammonia water containing acetonitrile; in the HPLC gradient separation and purification process, an eluent A phase is 20-30mmol/L of a carbonate aqueous solution, and the pH value of the eluent A phase is adjusted to 8.0-9.0 by ammonia water; phase B is acetonitrile; gradient purification condition is 25-40% phase A, gradient elution time is 40-50min, and primary purified product with main peak purity of more than 95% is collected under 220nm wavelength;

(2) subjecting the primary purified product to further HPLC gradient separation and purification by using a reverse phase silica gel packed column, wherein an eluent A phase is 0.1 v% TFA aqueous solution, a eluent B phase is acetonitrile, gradient purification conditions are 30-45% of the eluent A phase, the gradient elution time is 40-50min, and a secondary purified product with the main peak purity of more than 99% and the maximum single impurity of less than 0.2% is collected at a wavelength of 220 nm;

(3) desalting the secondary purified product by using a reverse phase silica gel packed column, wherein an eluent A phase is water, a eluent B phase is acetonitrile, a gradient elution method is adopted, the gradient elution condition is 30-60% of the A phase, the gradient elution time is 40-50min, and the desalted product with the main peak purity of more than 99.5% and the maximum single impurity content of less than 0.1% is collected under the wavelength of 220 nm;

(4) and (3) evaporating and concentrating the desalted product by adopting a film evaporator to remove the solvent in the product, wherein the vacuum degree of the film evaporator is not higher than-0.09 MPa, the evaporation temperature is 40-50 ℃, the rotation speed is 1400-1800r/min, and then freeze-drying is carried out to obtain the purified soxhlet peptide.

2. The method of claim 1, wherein: in the step (1), the concentration of the crude soxhlet peptide in the crude soxhlet peptide solution is 20-40 mmol/L.

3. The method of claim 1, wherein: in the step (1), the filler in the polymer filler column is polymethacrylate polymer.

4. The method of claim 1, wherein: in the step (1), the aqueous carbonate solution is an aqueous ammonium carbonate solution.

5. The method of claim 1, wherein: in the step (1), the aqueous carbonate solution further includes phosphoric acid.

6. The method of claim 1, wherein: in the step (2), the filler of the reversed phase silica gel filler column is an octaalkylsilane bonded silica gel filler.

7. The method of claim 1, wherein: in the step (3), the filler of the reversed phase silica gel filler column is an octaalkylsilane bonded silica gel filler.

8. The method of claim 1, wherein: in the step (4), the concentration rate of the evaporation concentration was 40 kg/h.

9. The method of claim 1, wherein: in the step (4), the desorption temperature of freeze drying is 30-35 ℃.

10. The method of claim 1, wherein: in the step (4), the bead peak purity of the purified somaglutide is more than 99.5%, and the maximum single impurity is less than 0.1%.