CN114671927A - Preparation method of degarelix crude peptide - Google Patents

Abstract

The present invention relates to the field of polypeptide synthesis, in particular to a preparation method of crude degarelix peptide. In the present invention, the organic solvent in the sedimentation filtration is replaced with water, the lysate is diluted with water, and the crude peptide is obtained by stirring and filtration. The method avoids the large use of organic solvents and repeated centrifugation steps, so it is simple, efficient, environmentally friendly and cheap; at the same time, the precipitation rate of this method is 97.2% ± 1%, which is comparable to the traditional anhydrous ether sedimentation centrifugal solid precipitation rate (this patent compares the implementation of 97.8% in Example 1) is comparable.

Description

Preparation method of degarelix crude peptide

technical field

The present invention relates to the field of polypeptide synthesis, in particular to a preparation method of crude degarelix peptide.

Background technique

Degarelix acetate is a gonadotropin-releasing hormone (GnRH) receptor inhibitor drug developed by Ferring Pharmaceuticals Co., Ltd. It can reversibly inhibit the pituitary GnRH receptor to reduce the release of gonadotropins and then inhibit the release of testosterone. , and was registered and listed by the US FDA in December 2008. It is used to delay the growth and deterioration of prostate cancer and is suitable for the treatment of advanced prostate cancer. The molecular structure is as follows:

The amino acid abbreviated peptide sequence is as follows:

Ac-D-Nal-D-Cpa-D-pal-Ser-Aph(Hor)-D-Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH ₂

The synthesis process of degarelix is mainly Fmoc solid-phase synthesis, and the resin peptide of solid-phase synthesis is cleaved by trifluoroacetic acid (TFA) to obtain a crude peptide lysate. There are two methods to obtain crude peptide from crude peptide lysate. One is sedimentation and centrifugation in a solvent. In the early stage, anhydrous ether was mainly used as a solvent. The lysate was poured into pre-cooled anhydrous ether, and the crude peptide was obtained by sedimentation and centrifugation. , the patent of our company (application number: 201110292168.0), the patent of Hainan Shuangcheng Pharmaceutical Co., Ltd. (application number: 201310336446.7), the patent of Taizhou Shimeikang Peptide Drug Technology Co., Ltd. (application number: 201410427405.3), And the patents of Chengdu Shengnuo Biotechnology Co., Ltd. (application numbers: 201410427405.3 and 201610136374.5); there are also many patents using methyl tertiary butyl ether to replace anhydrous ether, and there are patents of Jinan Kanghe Pharmaceutical Technology Co., Ltd. ( Application number: 201710389088.4), the patent of Jiangsu Hansoh Pharmaceutical Group Co., Ltd. (application number: 201910614837.8), the patent of Asymchem Life Science Technology (Tianjin) Co., Ltd. (application number: 201910748640.3); another kind of crude peptide obtained The method is sedimentation filtration. First, the lysate is concentrated to remove part of trifluoroacetic acid, and then the concentrated solution is poured into pre-cooled isopropyl ether to sediment and filter to obtain crude peptides. This method is used by Chia Tai Tianqing Pharmaceutical Group Co., Ltd. Co., Ltd. patent (application number: 201711076937.7).

The original degarelix has two specifications, 80mg and 120mg lyophilized powder. According to the market's preparation requirements, industrial production is carried out. The synthesis scale of a batch of degarelix is about 4mol, and the resin substitution degree used is about 0.91mmol. /g, so the weight of the obtained peptide resin is about 11kg, then according to the mass-volume ratio of the conventional peptide resin sedimentation solvent (1:64), the required volume of the sedimentation reagent is 700L.

Based on the above background information on industrial production, the volume of the sedimentation reagent is 700L. Because the sedimented solids are small, a sedimentation centrifuge is used, which requires repeated centrifugation steps in batches, which is time-consuming and unfriendly to industrial scale production. In the early days, anhydrous ethyl ether was mainly used as the solvent. Anhydrous ethyl ether has a low flash point, is flammable and explosive, and is not conducive to storage. Sedimentation and centrifugation in the base ether, but the safety hazard problem of the large-scale use of methyl tert-butyl ether is still unavoidable.

The sedimentation filtration method is simple and efficient to operate, and is more suitable for industrial scale production. However, the existing sedimentation filtration of crude peptides uses isopropyl ether as a solvent. At the same time, the extensive use of isopropyl ether has harmful effects on the environment and operators.

SUMMARY OF THE INVENTION

In view of this, the present invention provides a degarelix crude peptide precipitation method. The organic solvent in the sedimentation filtration was replaced with water, the lysate was diluted with water, and the crude peptide was obtained by stirring and filtration. The method avoids the large use of organic solvents and repeated centrifugation steps, so it is simple, efficient, environmentally friendly and cheap; at the same time, the precipitation rate of this method is 97.2% ± 1%, which is comparable to the traditional anhydrous ether sedimentation centrifugal solid precipitation rate (this patent compares the implementation of 97.8% in Example 1) is comparable.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a preparation method of degarelix crude peptide, comprising the following steps:

Step 1: prepare and obtain degarelix peptide resin, crack to obtain degarelix crude peptide lysate;

Step 2: Take the degarelix crude peptide lysate obtained in step 1, concentrate under reduced pressure to obtain a concentrated solution, mix with water, adjust the pH value to 0-12, settle and filter to collect the filter cake, and prepare the Rick crude peptide;

No organic solvent is used in step 2.

In some specific embodiments of the present invention, the pH value in step 2 is 1-11.

In some specific embodiments of the present invention, the pH value in step 2 is 3-8.

In some specific embodiments of the present invention, the temperature for adjusting the pH value in step 2 is -10°C to 20°C.

In some specific embodiments of the present invention, the temperature for adjusting the pH value in step 2 is -10°C to 20°C.

In some specific embodiments of the present invention, the temperature for adjusting the pH value in step 2 is 0°C to 15°C.

In some specific embodiments of the present invention, the temperature for adjusting the pH value in step 2 is 0°C to 5°C, 5°C to 10°C, 10°C to 20°C, -10°C to 5°C, and 0°C to 10°C. ℃ or -5℃～5℃.

In some specific embodiments of the present invention, the pH adjusting agent for adjusting the pH value in step 2 includes sodium hydroxide, potassium hydroxide, ammonia water, pyridine, triethylamine, sodium citrate, sodium bicarbonate, calcium bicarbonate , one or more mixtures of sodium hydrosulfide, sodium carbonate, potassium bicarbonate and disodium hydrogen phosphate.

In some specific embodiments of the present invention, the volume ratio of water to the concentrated solution in step 2 is (2-10):1.

In some specific embodiments of the present invention, the volume ratio of water to the concentrated solution in step 2 is (3-6):1.

In some specific embodiments of the present invention, the pressure of concentration under reduced pressure in step 2 is 100 hpa, and the temperature is 15°C to 45°C.

In some specific embodiments of the present invention, the temperature of the concentration under reduced pressure is 20°C to 30°C.

In some specific embodiments of the present invention, the temperature of the concentration under reduced pressure is 15°C to 20°C, 20°C to 25°C, 25°C to 35°C, 35°C to 45°C, 20°C to 25°C or 15°C ~20℃.

In some specific embodiments of the present invention, the volume of the concentrated solution in step 2 is 10% to 100% of the volume of the degarelix crude peptide lysate before concentration.

In some specific embodiments of the present invention, the volume of the concentrated solution after concentration is 30% to 50% of the volume of the degarelix crude peptide lysate before concentration.

In some specific embodiments of the present invention, after collecting the filter cake in step 2, a step of vacuuming for 15 min is further included.

In some specific embodiments of the present invention, the degarelix peptide resin described in step 1 is obtained by the solid-phase synthesis method described in the applicant's patent (CN 102329373A).

In some specific embodiments of the present invention, the lysis solution used in the lysis in step 1 is TFA:H ₂ O=95:5.

In some specific embodiments of the present invention, in g/mL, the mass-volume ratio of the degarelix peptide resin to the lysing solution in step 1 is 1:8.

The invention provides a degarelix crude peptide precipitation method, which comprises the steps of concentrating a degarelix lysate to varying degrees within a suitable temperature range, and then adding a certain proportion of water to the concentrated lysate for dilution, Then, the pH value is adjusted with alkali under the condition of controlling the temperature, and finally the filter cake is obtained by standing and filtration, and the crude degarelix peptide is obtained by vacuum pumping for 15 minutes.

Compared with the existing precipitation method, the beneficial effects of the present invention include but are not limited to:

This technology avoids sedimentation centrifugation, thereby avoiding the repeated operation of the centrifugation step in industrial scale production, resulting in a reduction in the time used for crude peptides and an increase in efficiency (a large amount of solvent is required during industrial production in the prior art, so it is necessary to repeat the operation of the centrifugation step, which takes a long time, Instead, it is changed to add water to dilute and filter, which is simple to operate and has high efficiency.); meanwhile, the large-scale use of anhydrous ethyl ether and methyl tert-butyl ether is avoided, so that the consumption of organic solvents is reduced, and the safety of the production environment is greatly improved; the present invention The method of diluting the lysate with water and then sedimentation filtration to obtain a solid is environmentally friendly, simple and efficient, avoiding the use of isopropyl ether as a solvent in the second sedimentation filtration described in the background, reducing waste liquid discharge, and improving the quality of the production and operation environment.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required in the description of the embodiments or the prior art.

Fig. 1 shows the liquid phase spectrum of embodiment 2;

Fig. 2 shows the liquid phase spectrum of embodiment 3;

Fig. 3 shows the liquid phase spectrum of embodiment 4;

Fig. 4 shows the liquid phase spectrum of embodiment 5;

Fig. 5 shows the liquid phase spectrum of embodiment 6;

Fig. 6 shows the liquid phase spectrum of embodiment 7;

Fig. 7 shows the liquid phase spectrum of embodiment 8;

Fig. 8 shows the liquid phase spectrum of Comparative Example 1;

Fig. 9 shows the liquid phase spectrum of comparative example 2;

Figure 10 shows the liquid phase spectrum of Comparative Example 3;

FIG. 11 shows the liquid phase diagram of Comparative Example 4. FIG.

Detailed ways

The present invention discloses a preparation method of degarelix crude peptide, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

The invention provides a degarelix crude peptide precipitation method, the specific steps are as follows:

1) Cleavage degarelix peptide resin, filter to remove resin, obtain degarelix crude peptide lysate;

2) Concentrate degarelix crude peptide lysate, dilute with water, and adjust pH;

3) Settling and filtering to obtain a filter cake, and vacuuming for 15 minutes to obtain degarelix crude peptide.

Further, the degarelix peptide resin described in step 1) is obtained by the solid-phase synthesis method described in the applicant's patent (CN102329373A).

Further, in step 1), the lysing solution is TFA:H ₂ O=95:5.

Further, in step 1), the mass-volume ratio of the peptide resin to the lysing solution is 1:8.

Further, the volume of the concentrated lysate in step 2) is 10%-100% of the volume before concentration, preferably, the volume of the lysate after concentration is 30%-50% of the volume before concentration.

Further, the pressure during concentration under reduced pressure described in step 2) is 100hpa.

Further, the temperature during the concentration under reduced pressure in step 2) is 15°C to 45°C, preferably, the temperature during the concentration under reduced pressure is 20°C to 30°C.

Further, the volume of water added in step 2) is 2-10 times the volume of the lysate solution after concentration, preferably, the volume of water added is 3-6 times the volume of the lysate solution after concentration.

Further, the pH regulator described in step 2) can be selected from sodium hydroxide, potassium hydroxide, ammonia water, pyridine, triethylamine, sodium citrate, sodium bicarbonate, calcium bicarbonate, sodium hydrosulfide, sodium carbonate, carbonic acid Potassium hydrogen, disodium hydrogen phosphate, etc., preferably sodium hydroxide.

Further, the pH adjustment range in step 2) is 0-12, preferably, the pH adjustment range is 3-8.

Further, the temperature during pH adjustment in step 2) is -10°C to 20°C, preferably, the temperature during pH adjustment is 0°C to 15°C.

The present invention provides a novel degarelix crude peptide precipitation method. The organic solvent in the sedimentation filtration was replaced with water, the lysate was diluted with water, and the crude peptide was obtained by stirring and filtration. The method avoids the large use of organic solvents and repeated centrifugation steps, so it is simple, efficient, environmentally friendly and cheap; at the same time, the precipitation rate of this method is 97.2% ± 1%, which is comparable to the traditional anhydrous ether sedimentation centrifugal solid precipitation rate (this patent compares the implementation of 97.8% in Example 1) is comparable.

The raw materials and reagents used in the preparation method of the crude degarelix peptide provided by the present invention can be purchased from the market.

Abbreviations of the present invention and English meanings are as follows:

Abbreviations and English meaning GnRH Gonadotropin-releasing hormone Fmoc 9-Fluorenemethoxycarbonyl TFA Trifluoroacetate H₂O water N₂ nitrogen

Below in conjunction with embodiment, the present invention is further elaborated:

Example 1: Cleavage of degarelix resin

Take 20.0g of degarelix peptide resin obtained by the applicant's patent (CN 102329373A) solid-phase synthesis method and add it to a 500mL single-necked flask, and add a pre-prepared 160mL lysis solution (TFA:H ₂ O=95: 5), split at room temperature for 2 hours, filter the resin, wash with a small amount of TFA, combine the filtrates, and the filtrate volume is 160 mL.

Example 2:

Add 20 mL of degarelix lysate filtrate obtained in Example 1, pH=0 directly (without concentration, i.e. 100% lysate volume) into water (40mL) of 2 times the lysate filtrate volume to dilute, and separate out a mass Viscous solid, stirred with glass rod, the solid gradually dispersed in the transparent water phase in granular form, continued magnetic stirring, the solid particles became finer, and the solution was cloudy white slurry. The filter cake was obtained by filtration, because the crude peptide needed to be further dissolved in water and acetonitrile during the next purification, so the obtained filter cake was vacuumed for 15 minutes to obtain degarelix white solid crude peptide, wet weight 2.28g, after inspection, degarelix was obtained. The purity is 97.52% and the content is 49.2%, so the solid precipitation rate is 98.2%.

Example 3:

20 mL of the degarelix lysate filtrate obtained in Example 1 was concentrated under reduced pressure at 15° C. to 20° C. The volume after concentration was 12 mL (that is, the volume of the lysate solution after concentration was 60% of that before concentration). 4 times the volume of water (48 mL) was added to the concentrated lysate filtrate to dilute, and the filtrate was white and turbid. Cool the filtrate to 0 °C, adjust pH=1 with 1 mmol/L potassium hydroxide, control the temperature at 0 °C to 5 °C, stand for filtration to obtain a filter cake, because the crude peptide needs to be further dissolved in water and acetonitrile in the next step of purification, Therefore, the obtained filter cake was vacuumed for 15 minutes to obtain degarelix white solid crude peptide with a wet weight of 2.30g. After inspection, the purity of degarelix was 96.85% and the content was 48.3%, so the solid precipitation rate was 97.8%.

Example 4:

20 mL of the degarelix lysate filtrate obtained in Example 1 was concentrated under reduced pressure at 20° C. to 25° C. The volume after concentration was 10 mL (that is, the volume of the lysate solution after concentration was 50% of that before concentration). Three times the volume of water (30 mL) was added to the concentrated lysate filtrate to dilute, and the filtrate was white and turbid. Cool the filtrate to 5 °C, adjust pH=3 with 1 mmol/L sodium hydroxide, control the temperature at 5 °C to 10 °C, stand for filtration to obtain a filter cake, because the crude peptide needs to be further dissolved in water and acetonitrile in the next step of purification, Therefore, the obtained filter cake was evacuated for 15 minutes to obtain degarelix white solid crude peptide, wet weight 2.22g, after inspection, the purity of degarelix was 97.97% and the content was 49.2%, so the solid precipitation rate was 98.2%.

Example 5:

20 mL of the degarelix lysate filtrate obtained in Example 1 was concentrated under reduced pressure at 25°C to 35°C, and the volume after concentration was 2 mL (that is, the volume of the lysate solution after concentration was 10% of that before concentration), and the concentrated solution 10 times the volume of water (20 mL) was added to the concentrated lysate filtrate to dilute, and the filtrate was white and turbid. Cool the filtrate to 10°C, adjust the pH to 5 with sodium bicarbonate, control the temperature at 10°C to 20°C, and filter by standing to obtain a filter cake. Because the crude peptide needs to be further dissolved in water and acetonitrile in the next step of purification, it will be obtained. The filter cake was vacuumed for 15 minutes to obtain degarelix white solid crude peptide with a wet weight of 2.25g. After inspection, the purity of degarelix was 95.63% and the content was 49.4%, so the solid precipitation rate was 97.5%.

Example 6:

Concentrate 20 mL of the degarelix lysate filtrate obtained in Example 1 under reduced pressure at 35° C. to 45° C. The volume after concentration is 8 mL (that is, the volume of the lysate solution after concentration is 40% of that before concentration). 6 times the volume of water (48 mL) was added to the concentrated lysate filtrate to dilute, and the filtrate was white and turbid. Cool the filtrate to -10 °C, adjust pH=8 with 1 mmol/L sodium hydroxide, control the temperature at -10 °C ~ 5 °C, stand for filtration to obtain a filter cake, because the crude peptide needs further water and acetonitrile in the next step of purification Dissolved, so the obtained filter cake was vacuumed for 15min to get degarelix white solid crude peptide, wet weight 2.29g, after inspection, the purity of degarelix was 95.50%, and the content was 48.6%, so the solid precipitation rate was 97.6% .

Example 7:

20 mL of the degarelix lysate filtrate obtained in Example 1 was concentrated under reduced pressure at 20° C. to 25° C. The volume after concentration was 4 mL (that is, the volume of the lysate solution after concentration was 20% of that before concentration). 8 times the volume of water (32 mL) was added to the concentrated lysate filtrate to dilute, and the filtrate was white and turbid. Cool the filtrate to 0°C, adjust pH=10 with sodium citrate, control the temperature at 0°C to 10°C, stand for filtration to obtain a filter cake, because the crude peptide needs to be further dissolved in water and acetonitrile during the next purification, so the obtained The filter cake was vacuumed for 15 minutes to obtain degarelix white solid crude peptide with a wet weight of 2.21 g. After inspection, the purity of degarelix was 92.57% and the content was 47.6%, so the solid precipitation rate was 92.4%.

Example 8:

20 mL of the degarelix lysate filtrate obtained in Example 1 was concentrated under reduced pressure at 15° C. to 20° C. The volume after concentration was 6 mL (that is, the volume of the lysate solution after concentration was 30% of that before concentration). 5 times the volume of water (30 mL) was diluted to medium, and a mass of viscous solid was precipitated, which was stirred with a glass rod, and the solid gradually dispersed in the transparent water phase in the form of granules. Cool the filtrate to -5°C, adjust pH=11 with 1mmol/L sodium hydroxide, control the temperature at -5°C-5°C, stand for filtration to obtain a filter cake, because the crude peptide needs further water and acetonitrile in the next step of purification Dissolved, so the obtained filter cake was vacuumed for 15min to obtain degarelix white solid crude peptide, wet weight 2.26g, after inspection, the purity of degarelix was 91.72%, and the content was 46.6%, so the solid precipitation rate was 91.8% .

Comparative Example 1:

After the lysate obtained from our patent (CN 102329373A), the lysate was poured into 10 times the volume of pre-cooled anhydrous ether for sedimentation and centrifugation to obtain the crude peptide, which was dried under N ₂ . After inspection, the purity of degarelix was 99.42% and the yield was 92.0 %, and the solid precipitation rate was 97.8%.

Comparative Example 2:

20 mL of the degarelix lysate filtrate obtained in Example 1 was concentrated under reduced pressure at 20°C to 25°C, the volume after concentration was 5 mL, added to 40 mL of methyl tertiary ether, filtered under nitrogen protection, and the filter cake was filtered with a small amount of methyl ether. The tertiary ether was washed twice, and the filter cake was blown dry with nitrogen to obtain 1.04 g of white fine powder with a crude peptide purity of 92.57% and a solid precipitation rate of 91.2%.

Comparative Example 3:

20 mL of the degarelix lysate filtrate obtained in Example 1 was added to 200 mL of methyl tertiary ether, filtered under nitrogen protection, and the filtration was difficult. cake to obtain 0.82 g of white fine powder, the purity of crude peptide is 90.34%, and the solid precipitation rate is 71.9%.

Comparative Example 4:

20 mL of the degarelix lysate filtrate obtained in Example 1 was concentrated under reduced pressure at 20°C-25°C, and the volume after concentration was 10 mL (that is, the volume of the lysate after concentration was 50% of that before concentration), and the concentrated Three times the volume of water (30 mL) was added to the concentrated lysate filtrate to dilute, and the filtrate was white and turbid. Cool the filtrate to 0 °C, adjust pH=14 with 1 mmol/L sodium hydroxide, control the temperature at 0 °C-10 °C, stand for filtration to obtain a filter cake, because the crude peptide needs to be further dissolved in water and acetonitrile in the next step of purification, Therefore, the obtained filter cake was vacuumed for 15 minutes to obtain degarelix white solid crude peptide with a wet weight of 3.06g. After inspection, the purity of degarelix was 88.27% and the content was 43.2%, so the solid precipitation rate was 89.2%.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (10)

1. the preparation method of degarelix crude peptide, is characterized in that, comprises the steps: Step 1: prepare and obtain degarelix peptide resin, crack to obtain degarelix crude peptide lysate; Step 2: Take the degarelix crude peptide lysate obtained in step 1, concentrate under reduced pressure to obtain a concentrated solution, mix with water, adjust the pH value to 0-12, settle and filter to collect the filter cake, and prepare the Rick crude peptide; No organic solvent is used in step 2. 2 . The preparation method according to claim 1 , wherein the pH value in step 2 is 3-8. 3 . 3. The preparation method according to claim 1 or 2, wherein the temperature for adjusting the pH value in step 2 is -10°C to 20°C. 4. The preparation method according to any one of claims 1 to 3, wherein the pH adjusting agent for adjusting pH value described in step 2 comprises sodium hydroxide, potassium hydroxide, ammoniacal liquor, pyridine, triethylamine, One or more mixtures of sodium citrate, sodium bicarbonate, calcium bicarbonate, sodium hydrosulfide, sodium carbonate, potassium bicarbonate and disodium hydrogen phosphate. 5. The preparation method according to any one of claims 1 to 4, wherein the volume ratio of water to the concentrated solution in step 2 is (2-10):1. 6. The preparation method according to any one of claims 1 to 5, wherein in step 2, the pressure of concentration under reduced pressure is 100 hpa, and the temperature is 15°C to 45°C. 7. The preparation method according to any one of claims 1 to 6, wherein the volume of the concentrated solution in step 2 is 10% to 100% of the volume of the degarelix crude peptide lysate before concentration . 8. The preparation method according to any one of claims 1 to 7, characterized in that, after collecting the filter cake in step 2, it also comprises a step of vacuuming for 15 min. 9. The preparation method according to any one of claims 1 to 8, wherein the lysing solution used in the cleavage in step 1 is TFA:H ₂ O=95:5. 10. The preparation method of claim 9, wherein, in g/mL, the mass-volume ratio of the degarelix peptide resin to the lysing solution in step 1 is 1:8.

Images