CN115260293B - Method for purifying ganirelix acetate - Google Patents

Method for purifying ganirelix acetate Download PDF

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CN115260293B
CN115260293B CN202211005618.8A CN202211005618A CN115260293B CN 115260293 B CN115260293 B CN 115260293B CN 202211005618 A CN202211005618 A CN 202211005618A CN 115260293 B CN115260293 B CN 115260293B
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acetate
acetonitrile
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CN115260293A (en
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汤传根
刘彬
吕亮
杜慈
范晓阳
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Nanjing Hanxin Pharmaceutical Technology Co ltd
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Nanjing Hanxin Pharmaceutical Technology Co ltd
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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Abstract

The invention belongs to the technical field of purification methods, and discloses a method for purifying ganirelix acetate. The invention completes the purification of the ganirelix acetate crude peptide by one-step reverse phase chromatography, and then obtains the ganirelix acetate refined peptide sample with the purity more than or equal to 99.9 percent, the maximum single impurity content less than or equal to 0.05 percent, the acetate content of 7.10-10.80 percent, the TFA ion content less than 0.05 percent, the chloride ion content less than 0.05 percent, the sulfate radical content less than 0.02 percent and the phosphate radical content less than 0.05 percent through salt conversion, rotary evaporation and freeze drying. acetate-TFA buffer solution is adopted in reverse phase chromatography, so that the defect that the ion pairing agent has no buffer capacity is overcome, the problem of poor reproducibility of the ion pairing agent system is solved, the preparation peak shape is stable, and the impurity removal capacity is improved. Acetate and acetic acid are used in both the purification and salt transfer mobile phase systems, and the materials are single, so that the introduction of other ions is reduced, the period of the salt transfer process is shortened, and the industrial scale-up production is facilitated.

Description

Method for purifying ganirelix acetate
Technical Field
The invention relates to a purification method of polypeptide, in particular to a purification mode of ganirelix acetate, belonging to the technical field of biological medicine.
Background
The incidence of fertility disorders caused by premature luteinizing hormone peaks is up to 9% worldwide, so the use of gonadotropin releasing hormone GnRH antagonists in female receptive assisted reproductive technology controlled ovarian stimulation regimens has become a major clinical treatment modality.
The ganirelix acetate injection is developed and produced by the company moesadong, is approved by the national food and drug administration of China to be marketed in 2013, and the active ingredient is ganirelix acetate. Ganirelix acetate is an artificially synthesized decapeptide compound obtained by substituting amino acids at 1, 2, 3, 6, 8 and 10 sites of endogenous GnRH, and the amino acid sequence is as follows: ac-D-Nal-D-Cpa-D-Pal-Ser-Tyr-D-HArg (Et) 2 )-Leu-HArg(Et 2 )-Pro-D-Ala-NH 2 . Since ganirelix acetate has high antagonism to endogenous GnRH, it can competitively bind to GnRH receptor of gonadotropin, and inhibit Follicle Stimulating Hormone (FSH) and luteinizing hormone in vivo rapidly and reversiblyRelease of the hormone (LH). And the ganirelix acetate has the characteristics of less adverse reaction, high pregnancy rate, short treatment period and the like, so that the ganirelix acetate has greater advantages and market than similar medicaments in clinic.
In the process of polypeptide synthesis, more impurities such as side reaction impurities, isomer impurities, racemization impurities, amino acid deletion or addition impurities are easily generated, so that the purification of the synthesized ganirelix acetate crude peptide mainly adopts a reverse phase chromatography with higher resolution. In patent CN 104371010B, reversed-phase C18 filler is used as stationary phase, 0.1% tfa/aqueous solution is used as mobile phase a,0.1% tfa/acetonitrile aqueous solution is used as mobile phase B, gradient elution is performed, and then the eluent is subjected to salt transfer freeze-drying to obtain ganirelix acetate pure product with purity of more than 99.7%, the maximum single impurity is known to be 0.09%, and the maximum single impurity is unknown to be less than 0.1%, and the total yield is 65.7%. In patent CN 102993274B, octadecylsilane chemically bonded silica is used as a stationary phase, sodium perchlorate/phosphoric acid solution with a certain concentration is used as a mobile phase A, acetonitrile is used as a mobile phase B, gradient elution is carried out, and then the eluent is subjected to salt transfer freeze-drying to obtain a ganirelix acetate pure product with the purity of 99.73 percent and the purity of single impurities of less than 0.1 percent, and the purification yield is 91.1 percent. The method has good purification effect and high yield, but is unfavorable for industrial production, and sodium perchlorate is needed in the purification mobile phase. Sodium perchlorate is an easy-to-explosion dangerous chemical, and can cause explosion by friction or impact with organic matters or contact with concentrated sulfuric acid, so that a large amount of sodium perchlorate is needed in industrial amplification production, and great potential safety hazard exists. And the sodium perchlorate solution is used as a purification mobile phase to introduce a plurality of other ions, so that the subsequent salt conversion and detection are also more difficult, and therefore, a certain problem exists in the actual industrial production.
Based on the problems existing in the prior art, the purification process is beneficial to the amplified production on the premise of ensuring the purity and the yield of the ganirelix acetate, and the method is the problem to be solved.
Disclosure of Invention
The invention aims to provide a method for purifying ganirelix acetate. In the invention, the ganirelix acetate crude peptide is purified by adopting a one-step reversed phase chromatography, and then the ganirelix acetate refined peptide with the purity of more than or equal to 99.9 percent and the maximum single impurity content of less than or equal to 0.05 percent is obtained through salt conversion, rotary evaporation and freeze-drying.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a method for purifying ganirelix acetate, comprising the steps of:
1) Purifying: acidic buffer solution and organic solvent containing acetate and ion pairing agent are used as purified mobile phases (RP 1-A and RP 1-B), and reversed-phase filler is used as stationary phase;
2) Salt conversion: the acetate-containing buffer solution and the organic solvent are used as a salt transfer mobile phase (RP 2-A2), and the reverse phase filler is used as a stationary phase.
As an embodiment of the present invention, the acidic buffer containing acetate and ion pairing agent is selected from any one of ammonium acetate-trifluoroacetic acid (TFA) buffer, sodium acetate-trifluoroacetic acid buffer, potassium acetate-trifluoroacetic acid buffer.
Ion pairing agents can improve the retention capacity of the chromatographic column for the target compound and can improve the peak shape, but the ion pairing agents have no buffering capacity and are sensitive to pH, so that problems exist in the direct use of the ion pairing agents. The acid buffer solution containing acetate and the ion pairing agent is used in the invention, the acetate has stronger buffer capacity, can make up the defect that the ion pairing agent does not have buffer capacity, solves the problem of poor reproducibility of the preparation spectrum of the ion pairing agent system, and can also obviously improve the peak shape.
As an embodiment of the present invention, the mass concentration of TFA in the ammonium acetate-trifluoroacetic acid buffer, sodium acetate-trifluoroacetic acid buffer, potassium acetate-trifluoroacetic acid buffer is 0.001% to 5.0%, such as optionally 0.03% to 0.15%, such as further optionally 0.05%; the acetate concentration is 0.1-200mM, optionally 5-20mM, such as further optionally 10mM.
As one embodiment of the invention, the pH of the acetate-containing acidic buffer solution is less than or equal to 5.0, such as the pH is less than or equal to 3.0, such as the pH is further selected to be 2.3-3.0, and the pH is further selected to be 2.5; acetic acid is preferred to adjust the pH because it does not introduce new anions and does not destroy the buffering capacity of the system.
As one embodiment of the invention, the mobile phase is purified, a mixed solution of an acid buffer solution containing acetate and an ion pairing agent and acetonitrile is taken as a mobile phase RP1-A, an aqueous solution containing acetonitrile is taken as a mobile phase RP1-B, and linear gradient elution is carried out, wherein the initial gradient of the mobile phase RP1-B is 30% -38%, and optionally 34%; the termination gradient of mobile phase RP1-B is 40% -48%, optionally 44%.
As an embodiment of the present invention, the organic solvent used for the purification is an aqueous solution containing acetonitrile, such as an aqueous solution (mass concentration) optionally containing 10% -60% acetonitrile.
As one embodiment of the present invention, the acetate-containing buffer in the salt-transferred mobile phase (RP 2-A2) is selected from any one of ammonium acetate, potassium acetate and sodium acetate.
As an embodiment of the invention, the acetate salt used in the trans-salt mobile phase (RP 2-A2) has a concentration of 5-1000mM, such as optionally 80-150mM, such as further optionally 100mM.
As an embodiment of the invention, the organic solvent used in the salt-transferred mobile phase (RP 2-A2) is an aqueous solution containing acetonitrile, such as an aqueous solution (mass concentration) optionally containing 5% -30% acetonitrile.
As one embodiment of the invention, after salt transfer, the elution mobile phases (RP 2-A3 and RP 2-B) are used for gradient elution, an aqueous solution containing acetic acid and acetonitrile is used as the mobile phases RP2-A3, and an aqueous solution containing acetonitrile is used as the mobile phases RP2-B; wherein the acetonitrile aqueous solution optionally contains 5% -60% acetonitrile (mass concentration); wherein the initial gradient of mobile phase RP2-A3 is 55% to 65%, such as optionally 60%; the termination gradient of mobile phase RP2-A3 is 65% to 75%, such as optionally 70%.
As an embodiment of the present invention, the mass concentration of acetic acid in the aqueous solution RP2-A3 containing acetic acid and acetonitrile in the eluting mobile phase used after the salt transfer may be selected to be 0.05% to 5%, such as further alternatively 0.5%.
As one embodiment of the invention, the mass concentration of acetonitrile in the aqueous solution RP2-B containing acetonitrile in the eluting mobile phase used after the salt transfer can be selected to be 5-60%.
As an embodiment of the present invention, the reverse phase filler used for purification and salt conversion is selected from a stationary phase made of porous silica particles or silica gel. For example, the stationary phase used in the present application may be made of porous silica particles having chemically bonded straight alkyl chains of 4 to 18 carbon atoms. For example a straight alkyl chain containing four (C4), eight (C8), twelve (C12) or eighteen (C18) carbon atoms, i.e. a butyl, octyl, dodecyl or octadecyl moiety. More specifically, it is octaalkyl-bonded silica gel or octadecyl-bonded silica gel, such as any filler having a size selected from the group consisting of Sepax BR-C18, unil 15-100C18, sepax Bio-C8 (2), sepax GP-C18, YMC-C8, HPLCONE8C18-100AA, HPLCONE-8C18D, HPLCONE-8C8K, HPLCONE-8C18K, and SP-200-8-C8-BIO, preferably HPLCONE8C18-100AA filler.
As an embodiment of the present invention, ganireliin acetate is isolated after purification by a method selected from one or a combination of lyophilization, addition of anti-solvent crystallization and isoelectric precipitation.
As a more specific embodiment of the present invention, a method for purifying ganirelix acetate by crude peptide pretreatment, one-step reverse phase chromatography purification and salt transfer, comprising the steps of:
(1) Pretreatment of crude peptide: dissolving ganirelix acetate crude peptide using an aqueous solution containing acetonitrile;
(2) Purifying: octadecylsilane chemically bonded silica filler is used as a stationary phase, an acidic buffer solution containing ammonium acetate-TFA and acetonitrile is used as a mobile phase RP1-A1, an acetonitrile solution is used as a mobile phase RP1-B, linear gradient elution is carried out, and main peak section components are collected;
(3) Salt conversion: octadecylsilane chemically bonded silica filler is used as a stationary phase, and buffer solution containing ammonium acetate and acetonitrile is used as a salt transfer mobile phase RP2-A2; and (3) taking an aqueous solution containing acetic acid and acetonitrile as an eluting mobile phase RP2-A3, taking an acetonitrile solution as an eluting mobile phase RP2-B, performing linear gradient elution, and collecting a main peak component.
As an embodiment of the present invention, the drying mode of the purified ganirelix acetate may be selected from freeze drying and spray drying.
In the invention, mobile phase, concentration, pH, gradient elution and the like are all obtained through experimental screening, and an optimal system is determined through comparing purity, yield, impurity removal effect, ion content and the like. The purification mobile phase system has an effect of removing impurities in crude peptide, and the yield is superior to other systems.
Compared with the prior art, the purification method of ganirelix acetate provided by the invention has the following beneficial effects:
(1) The invention adopts one-step reversed phase chromatography purification and salt conversion, and finally obtains ganirelix acetate through rotary evaporation and freeze-drying, wherein the purity of the ganirelix acetate is more than or equal to 99.9 percent, if the purification further reaches 99.95 percent, the maximum single impurity content is less than or equal to 0.05, if the maximum single impurity content is further less than or equal to 0.03 percent, wherein the acetate content is 7.10-10.80 percent, the trifluoroacetate content is less than 0.05 percent (which is far lower than the central control standard of 0.1 percent), the chloride ion content is less than 0.05 percent, the sulfate radical content is less than 0.02 percent and the phosphate radical content is less than 0.05 percent, and the purified ganirelix acetate is at a higher level.
(2) The acetate-TFA buffer solution is adopted in the reversed phase chromatography purification of the invention, which overcomes the defect that the ion pairing agent has no buffer capacity, solves the problem of poor reproducibility of an ion pairing agent (TFA) system, ensures stable preparation peak shape and improves the impurity removal capacity.
(3) The reversed phase chromatography purification and salt conversion of the invention use the same acetate and acetic acid, the materials are single, and other ions are not introduced.
Drawings
FIG. 1 is an HPLC plot of a sample of ganirelix acetate purification in example 1.
FIG. 2 is a diagram of the purification preparation of ganirelix acetate in example 1.
FIG. 3 is an HPLC plot of a sample of ganirelix acetate purification in example 2.
FIG. 4 is an HPLC plot of a sample of ganirelix acetate purification in example 3.
FIG. 5 is an HPLC plot of a sample of ganirelix acetate purification in example 4.
Fig. 6 is an HPLC profile of a sample after ganirelix acetate transfer in example 5.
FIG. 7 is an HPLC plot of a sample of ganireliin acetate from example 5.
Fig. 8, 9 are HPLC profiles (repeated twice) of a sample purified from ganirelix acetate in comparative example 1.
Fig. 10 is a diagram of the preparation of ganirelix acetate in comparative example 1.
FIG. 11 is an HPLC plot of the sample after ganirelix acetate salt transfer in comparative example 2.
Fig. 12 is a graph of the ion content of the sample after ganirelix acetate transfer in comparative example 2.
Detailed Description
In order to make the technical features, objects and advantageous effects of the present invention more clearly understood, the technical solution of the present invention will be described in detail below with reference to the specific embodiments, but the protection content of the present invention is not limited to the following embodiments.
The ganirelix acetate crude peptide is derived from a ganirelix acetate sample obtained by a fragment solid phase synthesis method.
The formula of the RP1-A1 mobile phase is as follows: 10mM sodium acetate, 0.05% TFA,10% acetonitrile, pH 2.5 with acetic acid.
The formula of the RP1-A2 mobile phase is as follows: 10mM ammonium acetate, 0.10% TFA,10% acetonitrile, pH was adjusted to 2.5 with acetic acid.
The formula of the RP1-A3 mobile phase is as follows: 10mM ammonium acetate, 0.05% TFA,10% acetonitrile, pH 2.5 with acetic acid.
The formula of the RP1-A4 mobile phase is as follows: 10mM ammonium acetate, 0.05% TFA,10% acetonitrile, pH 3.0 with acetic acid.
The formula of the RP1-B is as follows: 60% acetonitrile.
The formula of the RP2-A1 mobile phase is as follows: 0.5% acetic acid, 10% acetonitrile.
The RP2-A2 mobile phase formula comprises: 100mM ammonium acetate, 10% acetonitrile.
The formula of the RP2-A3 mobile phase is as follows: 0.5% acetic acid, 50% acetonitrile.
The formula of the RP2-B mobile phase is as follows: 5% acetonitrile.
Example 1: purification of ganirelix acetate crude peptide
Sample treatment: 0.1g of ganirelix acetate crude peptide sample is weighed, 100 times of mass of 10% acetonitrile solution is added to dissolve the sample, and the mixture is filtered by a filter membrane with the pore diameter of 0.22 mu m and then purified.
The purification process comprises the following steps: the processed ganirelix acetate crude peptide is used as a sample, octadecylsilane chemically bonded silica filler with the size of HPLCONE8C18-100AA is used as a stationary phase, the flow rate is 200cm/hr, and the detection wavelength is 280nm. 2 CVs in the column were equilibrated with RP1-A1 mobile phase; loading according to the loading amount of 10g/L resin of total protein of the loaded sample; after loading, the column was equilibrated with RP1-A1 mobile phase for 2 CVs; and finally, carrying out linear gradient elution by using RP1-A1 and RP1-B (the RP1-B is eluted for 45min from 34% -44%), wherein the collected main peak section is the ganirelix acetate purified sample. The purity of a purified sample was 99.95%, the maximum single impurity content was 0.05% and the purification yield was 70.2% by HPLC detection. The HPLC profile is shown in FIG. 1. The purification preparation is shown in FIG. 2.
Example 2: purification of ganirelix acetate crude peptide
Sample treatment: 0.1g of ganirelix acetate crude peptide sample is weighed, 100 times of mass of 10% acetonitrile solution is added to dissolve the sample, and the mixture is filtered by a filter membrane with the pore diameter of 0.22 mu m and then purified.
The purification process comprises the following steps: the processed ganirelix acetate crude peptide is used as a sample, octadecylsilane chemically bonded silica filler with the size of HPLCONE8C18-100AA is used as a stationary phase, the flow rate is 200cm/hr, and the detection wavelength is 280nm. 2 CVs in the column were equilibrated with RP1-A2 mobile phase; loading according to the loading amount of 10g/L resin of total protein of the loaded sample; after loading, the column was equilibrated with RP1-A2 mobile phase for 2 CVs; and finally, carrying out linear gradient elution by using RP1-A2 and RP1-B (the RP1-B is eluted for 45min from 34% -44%), wherein the collected main peak section is the ganirelix acetate purified sample. The purity of a purified sample was 99.91%, the maximum single impurity content was 0.04%, and the purification yield was 71.7% by HPLC detection. The HPLC profile is shown in FIG. 3.
Example 3: purification of ganirelix acetate crude peptide
Sample treatment: 0.1g of ganirelix acetate crude peptide sample is weighed, 100 times of mass of 10% acetonitrile solution is added to dissolve the sample, and the mixture is filtered by a filter membrane with the pore diameter of 0.22 mu m and then purified.
The purification process comprises the following steps: the processed ganirelix acetate crude peptide is used as a sample, octadecylsilane chemically bonded silica filler with the size of HPLCONE8C18-100AA is used as a stationary phase, the flow rate is 200cm/hr, and the detection wavelength is 280nm. 2 CVs in the column were equilibrated with RP1-A3 mobile phase; loading according to the loading amount of 10g/L resin of total protein of the loaded sample; after loading, the column was equilibrated with RP1-A3 mobile phase for 2 CVs; and finally, carrying out linear gradient elution by using RP1-A3 and RP1-B (the RP1-B is eluted for 45min from 34% -44%), wherein the collected main peak section is the ganirelix acetate purified sample. The purity of a purified sample was 99.93%, the maximum single impurity content was 0.03%, and the purification yield was 71.0% by HPLC detection. The HPLC profile is shown in FIG. 4.
Example 4: purification of ganirelix acetate crude peptide
Sample treatment: 0.1g of ganirelix acetate crude peptide sample is weighed, 100 times of mass of 10% acetonitrile solution is added to dissolve the sample, and the mixture is filtered by a filter membrane with the pore diameter of 0.22 mu m and then purified.
The purification process comprises the following steps: the processed ganirelix acetate crude peptide is used as a sample, octadecylsilane chemically bonded silica filler with the size of HPLCONE8C18-100AA is used as a stationary phase, the flow rate is 200cm/hr, and the detection wavelength is 280nm. 2 CVs in the column were equilibrated with RP1-A4 mobile phase; loading according to the loading amount of 10g/L resin of total protein of the loaded sample; after loading, the column was equilibrated with RP1-A4 mobile phase for 2 CVs; and finally, carrying out linear gradient elution by using RP1-A4 and RP1-B (the RP1-B is eluted for 45min from 34% -44%), wherein the collected main peak section is the ganirelix acetate purified sample. The purity of a purified sample was 99.90%, the maximum single impurity content was 0.03%, and the purification yield was 73.2% by HPLC detection. The HPLC profile is shown in FIG. 5.
Example 5: transferring salt, rotary steaming and freeze-drying of purified eluent of ganirelix acetate
Sample treatment: the ganirelix acetate purified one eluent obtained in example 1 was diluted with one volume of purified water to be converted into salt.
Salt transfer process: the diluted ganirelix acetate purified eluent is used as a sample, octadecylsilane chemically bonded silica filler with the size of HPLCONE8C18-100AA is used as a stationary phase, the flow rate is 200cm/hr, and the detection wavelength is 280nm. 2 CVs in the column were equilibrated with RP2-A1 mobile phase; loading according to the loading capacity of 30g/L resin of the total protein of the loaded sample; after loading, the column was equilibrated with RP2-A1 mobile phase for 2 CVs; 4 CVs of RP2-A2 mobile phase equilibrium chromatographic column are used for salt conversion; washing with 3 CV of RP2-B mobile phase equilibrium chromatographic column; and finally, carrying out linear gradient elution by using RP2-A3 and RP2-B (the RP2-A is eluted for 30min from 60% -70%), wherein the collected main peak section is the sample after ganirelix acetate salt conversion. The purity of the sample after salt conversion is 99.96 percent, the maximum single impurity content is 0.04 percent, and the salt conversion yield is 93 percent through HPLC detection. The HPLC profile is shown in FIG. 6.
And (3) rotary steaming: the salt-converted sample is put into a rotary evaporator, the temperature is set to 25 ℃, the rotating speed is 100rpm, and the sample is subjected to rotary evaporation until no obvious liquid drips out from a condenser and the mass of the sample is about 50% of that of the initial sample.
And (3) freeze-drying: and (3) pre-freezing the sample subjected to rotary steaming in a refrigerator at the temperature of minus 80 ℃, and then, putting the pre-frozen sample into a freeze dryer for freeze drying to obtain the ganirelix acetate. Through detection, the purity of the ganirelix acetate is 99.95%, the maximum single impurity content is 0.03%, the acetate content is 9.18%, the trifluoroacetate ion content is 0.038%, and the contents of chloride ion, sulfate radical and phosphate radical are respectively 0.029%, 0.004% and 0.017%. The HPLC profile is shown in FIG. 7.
Example 6
The ganirelix acetate purified eluate obtained in examples 2 to 4 was subjected to the same post-treatment as in example 5, by rotary evaporation and lyophilization, respectively.
The purity of the sample after the ganirelix acetate purification-eluent salt conversion in example 2 is 99.95%, the maximum single impurity content is 0.05%, and the salt conversion yield is 92%. The purity of the ganirelix acetate after freeze-drying is 99.94%, the maximum single impurity content is 0.05%, the acetate content is 9.23%, the trifluoroacetate content is 0.040%, and the contents of chloride ions, sulfate radicals and phosphate radicals are respectively 0.046%, 0.007% and 0.023%.
The purity of the sample after the ganirelix acetate purification-eluent salt conversion in example 3 is 99.92%, the maximum single impurity content is 0.04%, and the salt conversion yield is 94%. The purity of the ganirelix acetate after freeze-drying is 99.93%, the maximum single impurity content is 0.03%, wherein the acetate content is 9.40%, the trifluoroacetate content is 0.041%, and the contents of chloride ions, sulfate radicals and phosphate radicals are respectively 0.044%, 0.012% and 0.034%.
The purity of the sample after the ganirelix acetate purification-eluent salt conversion in example 4 is 99.92%, the maximum single impurity content is 0.03%, and the salt conversion yield is 93%. The purity of the ganirelix acetate after freeze-drying is 99.92%, the maximum single impurity content is 0.03%, wherein the acetate content is 9.24%, the trifluoroacetate content is 0.037%, and the contents of chloride ions, sulfate radicals and phosphate radicals are respectively 0.037%, 0.004% and 0.040%.
Comparative example 1
Referring to example 1, 10mM ammonium acetate was removed from mobile phase RP1-A1, and the other conditions and steps were repeated twice as in the purification of example 1 to examine the effect of ammonium acetate in the mobile phase on ganirelix acetate purity, impurities, yield, preparation scheme, etc. HPLC detection results are shown in figures 8 and 9, the purity of the eluent obtained in the first time is 99.79%, the maximum single impurity content is 0.13%, the yield is 63%, and the peak shape of the purification preparation diagram is basically the same as that of figure 2; the purity of the eluent obtained by repeating the second time was 99.01%, the maximum single impurity content was 0.21%, the yield was 51%, and the purification preparation chart is shown in fig. 10. Compared with examples 1-4, when ammonium acetate is added into the TFA ion pairing agent system, the prepared peak shape is stable, and the purity and the yield are obviously improved; however, the purification preparation is poor in reproducibility without ammonium acetate, and the probability of 50% is similar to that of FIG. 10, namely, the peak shape is not sharp, and the tail of the peak rear end is serious.
Comparative example 2
Referring to example 5, mobile phase RP2-A1 was exchanged for 20mM ammonium acetate, the remaining conditions and steps were the same as in the salt transfer process of example 5. The effect of buffer salt concentration in the mobile phase on ganirelix acetate purity, impurities, TFA ion content in the sample, etc. was examined. As shown in FIG. 11, the purity of the sample was 99.95%, the maximum single impurity content was 0.02%, the acetate content was 8.55%, the trifluoroacetate content was 0.767%, and the chloride, sulfate and phosphate contents were 0.080%, 0.030% and 0.023%, respectively. In comparison with example 5, the change of ammonium acetate concentration in comparative example 2 has little effect on purity, but the trifluoroacetate ion content is much higher than 0.10%, and as shown in fig. 12, does not meet the central control standard.

Claims (4)

1. The method for purifying ganirelix acetate is characterized by comprising the following steps of:
(1) Purifying: performing linear gradient elution by taking reversed-phase packing as a stationary phase and RP1-A and RP1-B as purified mobile phases; the purified mobile phase RP1-A is an acidic buffer solution of acetate, an ion pairing agent and acetonitrile; the purified mobile phase RP1-B is an aqueous solution of acetonitrile;
the acid buffer solution of the acetate and the ion pairing agent is any one of an ammonium acetate-trifluoroacetic acid buffer solution, a sodium acetate-trifluoroacetic acid buffer solution and a potassium acetate-trifluoroacetic acid buffer solution; wherein the acetate concentration is 0.1-10mM, and the trifluoroacetic acid mass concentration is 0.001% -5.0%; the pH value of the acidic buffer solution of the acetate and the ion pairing agent is less than or equal to 5.0;
(2) Salt conversion: reversed-phase filler is used as a stationary phase, and aqueous solution of acetate and acetonitrile is used as a salt transfer mobile phase RP2-A2;
the acetate is any one of ammonium acetate, potassium acetate and sodium acetate; wherein the concentration of the acetate is 80-1000mM;
(3) Washing and elution: washing by using acetonitrile water solution as eluting mobile phase RP2-B; and carrying out linear gradient elution by taking an aqueous solution of acetic acid and acetonitrile as a mobile phase RP2-A3 and taking an aqueous solution of acetonitrile as a mobile phase RP 2-B.
2. The purification method according to claim 1, wherein: in the step (3), an aqueous solution of acetic acid and acetonitrile is used as a mobile phase RP2-A3 in the flushing and elution, wherein the mass concentration of the acetic acid is 0.05% -5%; the acetonitrile water solution is used as mobile phase RP2-B, and the mass concentration of acetonitrile is 5%.
3. The purification method according to claim 1, wherein: the reversed phase filler used for purification and salt conversion is selected from a stationary phase made of porous silica particles or silica gel.
4. The purification method according to claim 1, wherein: purification by crude peptide pretreatment, one-step reverse phase chromatography purification and salt transfer, comprising the steps of:
(1) Pretreatment of crude peptide: dissolving ganirelix acetate crude peptide with an aqueous solution of acetonitrile;
(2) Purifying: performing linear gradient elution by taking octadecylsilane chemically bonded silica filler as a stationary phase, ammonium acetate-TFA and an acidic buffer solution of acetonitrile as a mobile phase RP1-A1, and an aqueous solution of acetonitrile as a mobile phase RP 1-B;
(3) Salt conversion: octadecylsilane chemically bonded silica filler is used as a stationary phase, and an aqueous solution of ammonium acetate and acetonitrile is used as a salt transfer mobile phase RP2-A2; and (3) performing linear gradient elution by taking an aqueous solution of acetic acid and acetonitrile as an eluting mobile phase RP2-A3 and an aqueous solution of acetonitrile as an eluting mobile phase RP 2-B.
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