A method for purifying degarelix
A multi-step chromatographic purification process effectively addresses the impurity challenges in degarelix purification, achieving high purity and yield through the use of bonded silica gels and specific mobile phases, resulting in a uniform powdered product.
Patent Information
- Application Number
- CN202110347073.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2021-03-31
- Publication Date
- 2025-01-03
- Estimated Expiration
- 2041-03-31
AI Technical Summary
Existing methods for purifying degarelix, a peptide with 7 unnatural hydrophobic amino acids, result in high levels of byproducts and impurities, and there is a lack of effective separation and purification processes, particularly in recrystallization methods.
A multi-step chromatographic purification process using octaalkyl and octadecyl bonded silica gels with specific mobile phases and gradient elution, followed by column concentration and freeze-drying, to achieve high purity and yield of degarelix.
The method achieves a purity of over 99.5% and a higher yield compared to recrystallization, reducing gel-like precipitation and equipment loss, and results in a uniform powdered product.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of purification of crude degarelix, and particularly relates to a purification method of degarelix. Background Art
[0002] Degarelix, English name: Degarelix, its peptide sequence is: Ac-D-2-Nal-D-Phe(4-Cl)-D-3-Pal-Ser-4-Aph(L-Hor)-D-4-Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2. Its structural formula is shown in formula (1):
[0003]
[0004] Degarelix is a gonadotropin-releasing hormone (GnRH) receptor inhibitor drug that reversibly inhibits pituitary GnRH receptors to reduce gonadotropin release and subsequently inhibit testosterone release. It delays the growth and progression of prostate cancer by inhibiting testosterone, which is essential for the continued growth of prostate cancer. It was developed by Ferring and approved by the U.S. Food and Drug Administration (FDA) and launched in the U.S. on December 24, 2008 under the trade name Firmagon.
[0005] There are many methods for synthesizing degarelix at present. However, since the peptide sequence of degarelix contains 7 unnatural amino acids, and the amino acids are all hydrophobic amino acids, the crude degarelix obtained contains a lot of byproducts and crude peptide impurities, and the separation and purification process requirements are very high. Currently, there are few records of the separation and purification process of crude degarelix in the public literature, and most of them are separated and purified in the form of recrystallization. In this regard, the present invention hopes to provide a new method for purifying degarelix. Summary of the invention
[0006] The object of the present invention is to provide a method for purifying degarelix to solve the problems raised in the above background technology, and the specific scheme is as follows.
[0007] A method for purifying degarelix comprises the following steps:
[0008] 1. Dissolving: dissolving the crude degarelix peptide obtained by solid phase synthesis in an acetic acid aqueous solution, an acetonitrile aqueous solution or an ethanol aqueous solution;
[0009] 2. First purification: using octaalkyl bonded silica gel as a chromatographic column filler, the mobile phase is divided into A1 phase and B1 phase, the solution obtained in step 1 is subjected to gradient elution, a pure qualified fraction is collected, and an acetic acid aqueous solution, an acetonitrile aqueous solution or an ethanol aqueous solution is used to dilute the pure qualified fraction by more than one volume to obtain a pure diluted fraction; wherein the A1 phase is an acetic acid aqueous solution, and the B1 phase is a preparative grade acetonitrile;
[0010] 3. Second purification: using octadecyl bonded silica gel as the chromatographic column filler, the mobile phase is divided into A2 phase and B2 phase, gradient elution is performed on the first pure diluted fraction, and the second pure qualified fraction is collected, and acetic acid aqueous solution, acetonitrile aqueous solution or ethanol aqueous solution is used to dilute the second pure qualified fraction by more than one volume to obtain the second pure diluted fraction; wherein the A2 phase is trifluoroacetic acid-triethylamine buffer, and the B2 phase is preparative grade acetonitrile;
[0011] Fourth, the third purification: using octadecyl bonded silica gel as the chromatographic column filler, the mobile phase is divided into A3 phase and B3 phase, the second pure diluted fraction is subjected to gradient elution, the third pure qualified fraction is collected, and the third pure qualified fraction is diluted by more than one volume with acetic acid aqueous solution, acetonitrile aqueous solution or ethanol aqueous solution to obtain the third pure diluted fraction; wherein the A3 phase is ammonium acetate buffer, and the B3 phase is preparative grade acetonitrile;
[0012] 5. Concentrating the three pure diluted fractions by column concentration and vacuum rotary evaporation;
[0013] 6. Add an appropriate amount of tert-butyl alcohol aqueous solution to the peptide solution after reduced pressure concentration, filter the filtrate, and freeze-dry to obtain a powdered degarelix product.
[0014] The acetic acid in the mobile phase A1 in step 2 is 1‰-3‰ (V / V).
[0015] The gradient in step 2 is based on the volume percentage of mobile phase B1:
[0016] The pH of the trifluoroacetic acid-triethylamine buffer in the mobile phase A2 in step 3 is 3.0-4.0.
[0017] The gradient in step 3 is based on the volume percentage of the mobile phase B2:
[0018] The concentration of ammonium acetate in the mobile phase A3 in step 4 is 20 mmol / L-80 mmol / L.
[0019] The gradient in step 4 is based on the volume percentage of mobile phase B3:
[0020] During the three purification processes and the column concentration process, the column temperature of the chromatographic column was maintained at 30-40 degrees Celsius.
[0021] In the steps 1, 2, 3 and 4, the volume percentage of the solute corresponding to the acetic acid aqueous solution, the acetonitrile aqueous solution and the ethanol aqueous solution is 10-20%.
[0022] In the step six, tert-butanol aqueous solution is used for dilution. After dilution, the volume percentage of tert-butanol in the solution is 10-20%.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] (1) During the dissolution and purification of the crude product, by using acetic acid aqueous solution, acetonitrile aqueous solution or ethanol aqueous solution and taking measures to keep the column warm at a relatively high temperature, the gel-like precipitation of degarelix is reduced, and the loss of the gel precipitate to the purification equipment and the filler used for purification is reduced.
[0025] (2) In the multi-step purification process, an efficient and suitable mobile phase was used to obtain a refined peptide with a purity of more than 99.5%. At the same time, the overall yield was also higher than that of the general recrystallization method.
[0026] (3) Before freeze-drying, the gel solution is diluted with tert-butyl alcohol aqueous solution until it is clear. The resulting uniform solution can be quickly filtered and freeze-dried to obtain a powder with uniform content. The tert-butyl alcohol can be effectively removed by the freeze dryer. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 is the chromatogram of the crude degarelix in Example 1;
[0028] Figure 2 is a chromatogram of a pure qualified fraction in Example 1;
[0029] Figure 3 The chromatograms of the two pure qualified fractions in Example 1 are shown below:
[0030] Figure 4 The chromatograms of the three pure qualified fractions in Example 1 are shown below:
[0031] Figure 5 This is the chromatogram of the finished product of degarelix in Example 1. DETAILED DESCRIPTION
[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0033] See also Figure 1-Figure 5 , the present invention provides a method for purifying degarelix.
[0034] Example 1
[0035] Step 1: Dissolve
[0036] The synthesized degarelix was dissolved in a 20% by volume aqueous acetic acid solution (dissolution concentration was about 6 g / L), and the fully dissolved liquid was filtered through a polyethersulfone filter element, and the filtrate was kept for later use;
[0037] Step 2: First purification
[0038] The chromatographic column is an octaalkyl bonded silica gel reverse phase silica gel column, with a particle size of 50 μm, a pore size of 120A, a column diameter and length of 3 cm × 25 cm, and a column temperature of 40°C; the mobile phase A1 is a 3‰ acetic acid aqueous solution (V / V); the mobile phase B1 is preparative grade acetonitrile. Flow rate: 23 ml / min; detection wavelength 220 nm; elution gradient: The single needle injection volume is 1.0g.
[0039] Purification process: After the chromatographic column is rinsed with 80% acetonitrile by volume, the system is equilibrated with 10% acetonitrile for 10 minutes. The filtrate obtained in step 1 is injected (wherein the degarelix content is 1.0 g). Gradient elution is performed to collect the target peak, and this step is repeated to complete the treatment of the remaining filtrate in step 1 to obtain a pure qualified fraction.
[0040] Step 3: Second purification
[0041] The collected pure qualified fraction was diluted with 20% acetic acid aqueous solution to obtain a pure diluted fraction, and the pure diluted fraction was purified.
[0042] The chromatographic column is an octadecyl bonded silica gel reverse phase silica gel column, with a particle size of 10 μm, a pore size of 120A, a column diameter and length of 3 cm × 25 cm, and a column temperature of 40°C; the mobile phase A2 is trifluoroacetic acid-triethylamine buffer, pH 3.5; the mobile phase B2 is preparative grade acetonitrile. Flow rate: 23 ml / min; detection wavelength 220 nm; elution gradient: the volume percentage of the mobile phase B2 is The single needle injection volume is 0.8g.
[0043] Purification process: After the chromatographic column is rinsed with 80% acetonitrile by volume, the system is equilibrated with 10% acetonitrile for 10 minutes. The first pure diluted fraction obtained in step 2 is injected (wherein the degarelix content is 0.8 g). Gradient elution is performed to collect the target peak, and this step is repeated to complete the treatment of the remaining first pure diluted fraction in step 2 to obtain the second pure qualified fraction.
[0044] Step 4: Third purification
[0045] The collected qualified second-pure fraction was diluted with 20% acetic acid aqueous solution by one volume to obtain a second-pure diluted fraction, and the second-pure diluted fraction was purified.
[0046] The chromatographic column is an octadecyl bonded silica gel reverse phase silica gel column, with a particle size of 10 μm, a pore size of 120A, a column diameter and length of 3 cm × 25 cm, and a column temperature of 40°C; the mobile phase A3 is 80 mmol / L ammonium acetate buffer; the mobile phase B3 is preparative grade acetonitrile. Flow rate: 23 ml / min; detection wavelength 220 nm; elution gradient: The single needle injection volume is 0.8g.
[0047] Purification process: After the chromatographic column is rinsed with 80% acetonitrile by volume, the system is equilibrated with 10% acetonitrile for 10 minutes. The second pure diluted fraction obtained in step 3 is injected (wherein the degarelix content is 0.8 g). Gradient elution is performed to collect the target peak, and this step is repeated to complete the treatment of the remaining second pure diluted fractions in step 3 to obtain the third pure qualified fraction.
[0048] Step 5: Column Concentration
[0049] The collected three pure qualified fractions were diluted with 20% acetic acid aqueous solution to obtain three pure diluted fractions, which were concentrated by column.
[0050] The chromatographic column is an octadecyl bonded silica gel reverse phase silica gel column, with a particle size of 10 μm, a pore size of 120A, a column diameter and length of 3 cm × 25 cm, and a column temperature of 40°C; the mobile phase A4 is a 1‰ acetic acid aqueous solution; the mobile phase B4 is preparative grade acetonitrile. Flow rate: 23 ml / min; detection wavelength 220 nm; elution gradient: The single needle injection volume is 1.0-2.0g.
[0051] Column concentration process: After the chromatographic column is rinsed with 80% acetonitrile by volume, the system is equilibrated with 10% acetonitrile for 10 minutes. The three pure diluted fractions obtained in step 4 are injected (wherein the degarelix content is 1.0-2.0 g). Gradient elution is performed to collect the target peak, and this step is repeated to complete the processing of the remaining three pure diluted fractions in step 4 to obtain the column concentration qualified fraction.
[0052] Step 6: Rotary evaporation and freeze drying
[0053] The qualified fractions from the column were concentrated under reduced pressure to 30 g / L, and the concentration temperature did not exceed 40° C. After the concentration was completed, a tert-butyl alcohol aqueous solution was added to account for 20% of the final sample solution by volume after dilution to redissolve the degarelix gel, and the concentration of degarelix was adjusted to 15 g / L-25 g / L, and then the filtrate was filtered and freeze-dried to obtain the finished degarelix product, with a purity of 99.65% and a total purification yield of 65.0%.
[0054] Example 2-3
[0055] Except that the volume fraction of acetic acid in step 2 is different from that in Example 1, the rest is the same as that in Example 1.
[0056]
[0057] Embodiment 4-5
[0058] Except that the pH of the trifluoroacetic acid-triethylamine buffer in step 3 is different from that in Example 1, the rest is the same as that in Example 1.
[0059]
[0060] Embodiment 6-7
[0061] Except that the ammonium acetate buffer in step 4 is different from that in Example 1, the rest is the same as that in Example 1.
[0062]
[0063] Embodiment 8-9
[0064] Except that the elution gradient in step 2 is different from that in Example 1, the rest is the same as that in Example 1.
[0065]
[0066] Embodiment 10-11
[0067] Except that the elution gradient in step 3 is different from that in Example 1, the rest is the same as that in Example 1.
[0068]
[0069] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A method for purifying degarelix, characterized in that: is Method 1, Method 2, Method 3, Method 4, or Method 5; The specific steps of method one are:
1. Dissolving: dissolving the crude degarelix peptide obtained by solid phase synthesis in a 20% by volume acetic acid aqueous solution; 2. First purification: octaalkyl bonded silica gel is used as the chromatographic column filler, and the mobile phase is divided into A1 phase and B1 phase. The solution obtained in step 1 is subjected to gradient elution to collect a pure qualified fraction, and an acetic acid aqueous solution, an acetonitrile aqueous solution or an ethanol aqueous solution is used to dilute the pure qualified fraction by more than one volume to obtain a pure diluted fraction; wherein, A1 phase is acetic acid aqueous solution, and the B1 phase is preparative grade acetonitrile; the acetic acid in the mobile phase A1 is 1‰-3‰ V / V; by volume, the elution gradient of the mobile phase B1 is 3. Second purification: octadecyl bonded silica gel is used as the chromatographic column filler, and the mobile phase is divided into A2 phase and B2 phase. The first pure diluted fraction is subjected to gradient elution, and the second pure qualified fraction is collected. The second pure qualified fraction is diluted by more than one volume with acetic acid aqueous solution, acetonitrile aqueous solution or ethanol aqueous solution to obtain the second pure diluted fraction; wherein, the A2 phase is trifluoroacetic acid-triethylamine buffer, and the B2 phase is preparative grade acetonitrile; the mobile phase A2 phase is trifluoroacetic acid-triethylamine buffer, and the pH is 3.5; the elution gradient of the mobile phase B2 phase is calculated by volume.
4. First purification: octadecyl bonded silica gel is used as the chromatographic column filler, the mobile phase is divided into A3 phase and B3 phase, the second pure diluted fraction is subjected to gradient elution, the third pure qualified fraction is collected, and the third pure qualified fraction is diluted by more than one volume with acetic acid aqueous solution, acetonitrile aqueous solution or ethanol aqueous solution to obtain the third pure diluted fraction; wherein, the A3 phase is ammonium acetate buffer, and the B3 phase is preparative grade acetonitrile; the mobile phase A3 phase is 80mmol / L ammonium acetate buffer; by volume, the elution gradient of the mobile phase B3 phase is 5. Concentrating the three pure diluted fractions by column concentration and vacuum rotary evaporation; 6. adding the peptide solution concentrated under reduced pressure to a tert-butyl alcohol aqueous solution which accounts for 20% of the final sample solution by volume after dilution, filtering the filtrate, and freeze-drying the filtrate to obtain a powdered degarelix product; The specific steps of method 2 are as follows:
1. Dissolving: dissolving the crude degarelix peptide obtained by solid phase synthesis in a 20% by volume acetic acid aqueous solution; 2. First purification: octaalkyl bonded silica gel is used as the chromatographic column filler, and the mobile phase is divided into A1 phase and B1 phase. The solution obtained in step 1 is subjected to gradient elution to collect a pure qualified fraction, and an acetic acid aqueous solution, an acetonitrile aqueous solution or an ethanol aqueous solution is used to dilute the pure qualified fraction by more than one volume to obtain a pure diluted fraction; wherein, A1 phase is acetic acid aqueous solution, and the B1 phase is preparative grade acetonitrile; the acetic acid in the mobile phase A1 is 3‰ V / V; by volume, the elution gradient of the mobile phase B1 is 3. Second purification: octadecyl bonded silica gel is used as the chromatographic column filler, and the mobile phase is divided into A2 phase and B2 phase. The first pure diluted fraction is subjected to gradient elution, and the second pure qualified fraction is collected. The second pure qualified fraction is diluted by more than one volume with acetic acid aqueous solution, acetonitrile aqueous solution or ethanol aqueous solution to obtain the second pure diluted fraction; wherein, the A2 phase is trifluoroacetic acid-triethylamine buffer, and the B2 phase is preparative grade acetonitrile; the mobile phase A2 phase is trifluoroacetic acid-triethylamine buffer, and the pH is 3-4; the elution gradient of the mobile phase B2 phase is calculated by volume.
4. First purification: octadecyl bonded silica gel is used as the chromatographic column filler, the mobile phase is divided into A3 phase and B3 phase, the second pure diluted fraction is subjected to gradient elution, the third pure qualified fraction is collected, and the third pure qualified fraction is diluted by more than one volume with acetic acid aqueous solution, acetonitrile aqueous solution or ethanol aqueous solution to obtain the third pure diluted fraction; wherein, the A3 phase is ammonium acetate buffer, and the B3 phase is preparative grade acetonitrile; the mobile phase A3 phase is 80mmol / L ammonium acetate buffer; by volume, the elution gradient of the mobile phase B3 phase is 5. Concentrating the three pure diluted fractions by column concentration and vacuum rotary evaporation; 6. adding the peptide solution concentrated under reduced pressure to a tert-butyl alcohol aqueous solution which accounts for 20% of the final sample solution by volume after dilution, filtering the filtrate, and freeze-drying the filtrate to obtain a powdered degarelix product; The specific steps of method three are as follows:
1. Dissolving: dissolving the crude degarelix peptide obtained by solid phase synthesis in a 20% by volume acetic acid aqueous solution; 2. First purification: octaalkyl bonded silica gel is used as the chromatographic column filler, and the mobile phase is divided into A1 phase and B1 phase. The solution obtained in step 1 is subjected to gradient elution to collect a pure qualified fraction, and an acetic acid aqueous solution, an acetonitrile aqueous solution or an ethanol aqueous solution is used to dilute the pure qualified fraction by more than one volume to obtain a pure diluted fraction; wherein, A1 phase is acetic acid aqueous solution, and the B1 phase is preparative grade acetonitrile; the acetic acid in the mobile phase A1 is 3‰ V / V; by volume, the elution gradient of the mobile phase B1 is 3. Second purification: octadecyl bonded silica gel is used as the chromatographic column filler, and the mobile phase is divided into A2 phase and B2 phase. The first pure diluted fraction is subjected to gradient elution, and the second pure qualified fraction is collected. The second pure qualified fraction is diluted by more than one volume with acetic acid aqueous solution, acetonitrile aqueous solution or ethanol aqueous solution to obtain the second pure diluted fraction; wherein, the A2 phase is trifluoroacetic acid-triethylamine buffer, and the B2 phase is preparative grade acetonitrile; the mobile phase A2 phase is trifluoroacetic acid-triethylamine buffer, and the pH is 3.5; the elution gradient of the mobile phase B2 phase is calculated by volume.
4. First purification: octadecyl bonded silica gel is used as the chromatographic column filler, the mobile phase is divided into A3 phase and B3 phase, the second pure diluted fraction is subjected to gradient elution, the third pure qualified fraction is collected, and the third pure qualified fraction is diluted by more than one volume with acetic acid aqueous solution, acetonitrile aqueous solution or ethanol aqueous solution to obtain the third pure diluted fraction; wherein the A3 phase is ammonium acetate buffer, and the B3 phase is preparative grade acetonitrile; the mobile phase A3 phase is 20-80mmol / L ammonium acetate buffer; by volume, the elution gradient of the mobile phase B3 phase is 5. Concentrating the three pure diluted fractions by column concentration and vacuum rotary evaporation; 6. adding the peptide solution concentrated under reduced pressure to a tert-butyl alcohol aqueous solution which accounts for 20% of the final sample solution by volume after dilution, filtering the filtrate, and freeze-drying the filtrate to obtain a powdered degarelix product; The specific steps of method 4 are as follows:
1. Dissolving: dissolving the crude degarelix peptide obtained by solid phase synthesis in a 20% by volume acetic acid aqueous solution; 2. First purification: octaalkyl bonded silica gel is used as the chromatographic column filler, and the mobile phase is divided into A1 phase and B1 phase. The solution obtained in step 1 is subjected to gradient elution to collect a pure qualified fraction, and an acetic acid aqueous solution, an acetonitrile aqueous solution or an ethanol aqueous solution is used to dilute the pure qualified fraction by more than one volume to obtain a pure diluted fraction; wherein, A1 phase is acetic acid aqueous solution, and the B1 phase is preparative grade acetonitrile; the acetic acid in the mobile phase A1 is 3‰ V / V; by volume, the elution gradient of the mobile phase B1 is 3. Second purification: octadecyl bonded silica gel is used as the chromatographic column filler, and the mobile phase is divided into A2 phase and B2 phase. The first pure diluted fraction is subjected to gradient elution, and the second pure qualified fraction is collected. The second pure qualified fraction is diluted by more than one volume with acetic acid aqueous solution, acetonitrile aqueous solution or ethanol aqueous solution to obtain the second pure diluted fraction; wherein, the A2 phase is trifluoroacetic acid-triethylamine buffer, and the B2 phase is preparative grade acetonitrile; the mobile phase A2 phase is trifluoroacetic acid-triethylamine buffer, and the pH is 3.5; the elution gradient of the mobile phase B2 phase is calculated by volume.
4. First purification: octadecyl bonded silica gel is used as the chromatographic column filler, the mobile phase is divided into A3 phase and B3 phase, the second pure diluted fraction is subjected to gradient elution, the third pure qualified fraction is collected, and the third pure qualified fraction is diluted by more than one volume with acetic acid aqueous solution, acetonitrile aqueous solution or ethanol aqueous solution to obtain the third pure diluted fraction; wherein, the A3 phase is ammonium acetate buffer, and the B3 phase is preparative grade acetonitrile; the mobile phase A3 phase is 80mmol / L ammonium acetate buffer; by volume, the elution gradient of the mobile phase B3 phase is 5. Concentrating the three pure diluted fractions by column concentration and vacuum rotary evaporation; 6. adding the peptide solution concentrated under reduced pressure to a tert-butyl alcohol aqueous solution which accounts for 20% of the final sample solution by volume after dilution, filtering the filtrate, and freeze-drying the filtrate to obtain a powdered degarelix product; Among them, the specific steps of method five are:
1. Dissolving: dissolving the crude degarelix peptide obtained by solid phase synthesis in a 20% by volume acetic acid aqueous solution; 2. First purification: octaalkyl bonded silica gel is used as the chromatographic column filler, and the mobile phase is divided into A1 phase and B1 phase. The solution obtained in step 1 is subjected to gradient elution to collect a pure qualified fraction, and an acetic acid aqueous solution, an acetonitrile aqueous solution or an ethanol aqueous solution is used to dilute the pure qualified fraction by more than one volume to obtain a pure diluted fraction; wherein, A1 phase is acetic acid aqueous solution, and the B1 phase is preparative grade acetonitrile; the acetic acid in the mobile phase A1 is 3‰ V / V; by volume, the elution gradient of the mobile phase B1 is 3. Second purification: octadecyl bonded silica gel is used as the chromatographic column filler, and the mobile phase is divided into A2 phase and B2 phase. The first pure diluted fraction is subjected to gradient elution, and the second pure qualified fraction is collected. The second pure qualified fraction is diluted by more than one volume with acetic acid aqueous solution, acetonitrile aqueous solution or ethanol aqueous solution to obtain the second pure diluted fraction; wherein, the A2 phase is trifluoroacetic acid-triethylamine buffer, and the B2 phase is preparative grade acetonitrile; the mobile phase A2 phase is trifluoroacetic acid-triethylamine buffer, and the pH is 3.5; the elution gradient of the mobile phase B2 phase is calculated by volume.
4. First purification: octadecyl bonded silica gel is used as the chromatographic column filler, the mobile phase is divided into A3 phase and B3 phase, the second pure diluted fraction is subjected to gradient elution, the third pure qualified fraction is collected, and the third pure qualified fraction is diluted by more than one volume with acetic acid aqueous solution, acetonitrile aqueous solution or ethanol aqueous solution to obtain the third pure diluted fraction; wherein, the A3 phase is ammonium acetate buffer, and the B3 phase is preparative grade acetonitrile; the mobile phase A3 phase is 80mmol / L ammonium acetate buffer; by volume, the elution gradient of the mobile phase B3 phase is 5. Concentrating the three pure diluted fractions by column concentration and vacuum rotary evaporation; 6. The peptide solution after reduced pressure concentration is added to the tert-butyl alcohol aqueous solution which accounts for 20% of the final sample solution by volume after dilution, and the filtrate is filtered and freeze-dried to obtain a powdered degarelix product.
2. A method for purifying degarelix according to claim 1, characterized in that: During the three purification processes and the column concentration process, the column temperature of the chromatographic column was maintained at 30-40 degrees Celsius.
Citation Information
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