Preparation method of degarelix crude peptide

Abstract

The invention relates to the field of polypeptide synthesis, in particular to a preparation method of degarelix crude peptide. The method comprises the following steps: replacing an organic solvent in sedimentation and filtration with water, adding water to dilute a lysate, and stirring and filtering to obtain crude peptide; the method avoids mass use of an organic solvent and repeated operation of a centrifugation step, so that the method is simple, efficient, environment-friendly and low in cost; meanwhile, the precipitation rate of the method is 97.2% + / -1%, which is almost the same as the solid precipitation rate (97.8% in the embodiment 1 of the patent) of the traditional absolute ether precipitation centrifugation.

Description

technical field

[0001] The present invention relates to the field of polypeptide synthesis, in particular to a preparation method of crude degarelix peptide. Background technique

[0002] 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:

[0003]

[0004] The amino acid abbreviated peptide sequence is as follows:

[0005] Ac-D-Nal-D-Cpa-D-pal-Ser-Aph(Hor)-D-Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH 2

[0006] The synthesis process of degarelix is ​​mainly Fmoc solid-phase synthesis, and the resin peptide of solid-phase synthesis...

Examples

preparation example Construction

[0049]The 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.

[0050] The invention provides a degarelix crude peptide precipitation method, the specific steps are as follows:

[0051] 1) Cleavage degarelix peptide resin, filter to remove resin, obtain dega...

Embodiment 1

[0069] Example 1: Cleavage of degarelix resin

[0070] Take 20.0 g of degarelix peptide resin obtained by the solid-phase synthesis method of the applicant's patent (CN 102329373A) and add it to a 500 mL single-neck flask, and add 160 mL of pre-prepared lysis solution (TFA:H 2 O=95:5), cracked at room temperature for 2 hours, filtered the resin, washed with a small amount of TFA, combined the filtrates, and the filtrate volume was 160 mL.

Embodiment 2

[0072] 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%.

technical field

[0001] The present invention relates to the field of polypeptide synthesis, in particular to a preparation method of crude degarelix peptide. Background technique

[0002] 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:

[0003]

[0004] The amino acid abbreviated peptide sequence is as follows:

[0005] Ac-D-Nal-D-Cpa-D-pal-Ser-Aph(Hor)-D-Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH 2

[0006] The synthesis process of degarelix is ​​mainly Fmoc solid-phase synthesis, and the resin peptide of solid-phase synthesis...

Examples

preparation example Construction

[0049]The 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.

[0050] The invention provides a degarelix crude peptide precipitation method, the specific steps are as follows:

[0051] 1) Cleavage degarelix peptide resin, filter to remove resin, obtain dega...

Embodiment 1

[0069] Example 1: Cleavage of degarelix resin

[0070] Take 20.0 g of degarelix peptide resin obtained by the solid-phase synthesis method of the applicant's patent (CN 102329373A) and add it to a 500 mL single-neck flask, and add 160 mL of pre-prepared lysis solution (TFA:H 2 O=95:5), cracked at room temperature for 2 hours, filtered the resin, washed with a small amount of TFA, combined the filtrates, and the filtrate volume was 160 mL.

Embodiment 2

[0072] 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%.

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