CN115260293A - Purification method of ganirelix acetate - Google Patents

Purification method of ganirelix acetate Download PDF

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CN115260293A
CN115260293A CN202211005618.8A CN202211005618A CN115260293A CN 115260293 A CN115260293 A CN 115260293A CN 202211005618 A CN202211005618 A CN 202211005618A CN 115260293 A CN115260293 A CN 115260293A
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acetate
purification
mobile phase
acetonitrile
buffer solution
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CN115260293B (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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • 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 purification method of ganirelix acetate. The invention completes the purification of the crude peptide of ganirelix acetate by one-step reverse phase chromatography, and obtains the ganirelix acetate fine peptide sample with the purity of more than or equal to 99.9 percent, the maximum single impurity content of less than or equal to 0.05 percent, the content of acetate of 7.10 to 10.80 percent, the content of TFA ion of less than 0.05 percent, the content of chloride ion of less than 0.05 percent, the content of sulfate radical of less than 0.02 percent and the content of phosphate radical of less than 0.05 percent by salt conversion, rotary evaporation and freeze drying. The acetate-TFA buffer solution is adopted in the reverse phase chromatography, so that the defect that the ion pairing agent has no buffer capacity is overcome, the problem of poor reproducibility of an 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 the purification and salt conversion mobile phase system, the used materials are single, the introduction of other ions is reduced, the period of the salt conversion process is shortened, and the industrial scale-up production is facilitated.

Description

Purification method of ganirelix acetate
Technical Field
The invention relates to a method for purifying polypeptide, in particular to a purification mode of ganirelix acetate, and belongs to the technical field of biological medicine.
Background
Fertility disorders caused by premature luteinizing hormone spikes have reached 9% worldwide, so the use of gonadotropin releasing hormone GnRH antagonists has become a major clinical treatment in women receiving assisted reproductive technology controlled ovarian stimulation protocols.
The ganirelix acetate injection is developed and produced by Moshadong company, is approved to be marketed in China by the China national food and drug administration in 2013, and the active ingredient of the ganirelix acetate injection is ganirelix acetate. The ganirelix acetate is an artificially synthesized decapeptide compound, is obtained by substituting amino acids at 1 st, 2 nd, 3 rd, 6 th, 8 th and 10 th sites of endogenous GnRH, and has the amino acid sequence 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 on endogenous GnRH, the ganirelix acetate can be competitively combined on GnRH receptors of gonadotropin to rapidly and reversibly inhibit the release of Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) in vivo. In addition, the ganirelix acetate has the characteristics of less adverse reaction, high pregnancy rate, short treatment cycle and the like, so that the ganirelix acetate clinically occupies larger advantages and markets than similar medicines.
In the process of polypeptide synthesis, more impurities are easily generated, such as side reaction impurities, isomer impurities, racemization impurities, amino acid deletion or addition impurities, so that the purified crude ganirelix acetate after synthesis is mainly subjected to reverse phase chromatography with higher resolution. In patent CN 104371010B, reverse phase C18 packing is used as a stationary phase, 0.1% tfa/aqueous solution is used as a mobile phase a,0.1% tfa/acetonitrile aqueous solution is used as a mobile phase B, gradient elution is performed, and then the eluent is subjected to salt transfer and freeze drying to obtain a pure product of ganirelix acetate with purity of more than 99.7%, maximum single impurity known to be 0.09% at the maximum and unknown maximum single impurity 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, a 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 performed, then an eluent is subjected to salt transfer and freeze drying to obtain a ganirelix acetate pure product with the purity of 99.73% and the purity of less than 0.1% of single impurities, and the purification yield is 91.1%. Although the method has good purification effect and high yield, the method is not beneficial to industrial production, and sodium perchlorate is required in a purified mobile phase. The sodium perchlorate is an easily-exploded dangerous chemical, can cause explosion by rubbing or impacting with organic matters or contacting concentrated sulfuric acid, needs a large amount of sodium perchlorate in industrial amplification production, and has great potential safety hazard. And a plurality of other ions can be introduced by using the sodium perchlorate solution as a purification mobile phase, which brings great difficulty for subsequent salt conversion and detection, so that a certain problem still exists in the actual industrial production.
Based on the problems in the prior art, the purification process is beneficial to scale-up production on the premise of ensuring the purity and yield of the ganirelix acetate, and the problems to be solved by the invention are solved.
Disclosure of Invention
The invention aims to provide a purification method of ganirelix acetate. In the invention, the crude ganirelix acetate peptide is purified by adopting a one-step reverse phase chromatography, and then the ganirelix acetate fine 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 by 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) And (3) purification: taking an acidic buffer solution containing acetate and an ion pairing agent and an organic solvent as purification mobile phases (RP 1-A and RP 1-B), and taking a reversed-phase filler as a stationary phase;
2) Salt conversion: the acetate-containing buffer solution and organic solvent are used as a salt-transfer mobile phase (RP 2-A2), and the reversed-phase filler is used as a stationary phase.
As an embodiment of the present invention, the acidic buffer containing acetate and an ion pairing agent is selected from any one of an ammonium acetate-trifluoroacetic acid (TFA) buffer, a sodium acetate-trifluoroacetic acid buffer, and a potassium acetate-trifluoroacetic acid buffer.
The ion pairing agent can improve the retention capacity of the chromatographic column on a target compound and can improve the peak shape, but the ion pairing agent has no buffer capacity and is sensitive to pH, so that the direct use of the ion pairing agent has some problems. According to the invention, the acidic buffer solution containing acetate and the ion pairing agent is used, the acetate has strong buffering capacity, the defect that the ion pairing agent has no buffering capacity can be overcome, the problem of poor reproducibility of a preparation map of an ion pairing agent system is solved, and simultaneously, the peak shape can be obviously improved.
As an embodiment of the present invention, the mass concentration of TFA in the ammonium acetate-trifluoroacetic acid buffer, the sodium acetate-trifluoroacetic acid buffer, and the potassium acetate-trifluoroacetic acid buffer is 0.001% to 5.0%, such as 0.03% to 0.15%, such as 0.05%; the acetate is present at a concentration of 0.1-200mM, optionally 5-20mM, and further optionally 10mM.
In one embodiment of the present invention, the pH of the acidic buffer containing acetate is less than or equal to 5.0, such as less than or equal to 3.0, such as 2.3-3.0, such as 2.5; acetic acid is preferably used to adjust the pH as it does not introduce new anions and does not destroy the buffering capacity of the system.
In the purification mobile phase, a mixed solution of an acidic buffer solution containing acetate and an ion pairing agent and acetonitrile is used as a mobile phase RP1-A, an aqueous solution containing acetonitrile is used 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 can be selected as 34%; the mobile phase RP1-B has a terminal gradient of 40% to 48%, optionally 44%.
In one embodiment of the present invention, the organic solvent used for the purification is an aqueous solution containing acetonitrile, for example, an aqueous solution (mass concentration) optionally containing 10% to 60% acetonitrile.
In one embodiment of the present invention, the acetate-containing buffer in the salt-transfer 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 is used in the saline-converted mobile phase (RP 2-A2) in a concentration of 5-1000mM, such as optionally 80-150mM, such as further optionally 100mM.
In one embodiment of the present invention, the organic solvent used in the mobile phase for salt inversion (RP 2-A2) is an aqueous solution containing acetonitrile, for example, an aqueous solution (mass concentration) optionally containing 5% to 30% acetonitrile.
As an embodiment of the invention, after the salt transfer, gradient elution is carried out by using elution mobile phases (RP 2-A3 and RP 2-B), an aqueous solution containing acetic acid and acetonitrile is used as the mobile phase RP2-A3, and an aqueous solution containing acetonitrile is used as the mobile phase RP2-B; wherein the acetonitrile water solution can optionally contain 5-60% of acetonitrile (mass concentration); wherein the initial gradient of the mobile phase RP2-A3 is 55% -65%, and optionally 60%; the mobile phase RP2-A3 has a termination gradient of 65% to 75%, optionally 70%.
In one embodiment of the present invention, in the elution mobile phase used after the salt conversion, the mass concentration of acetic acid in the aqueous solution RP2-A3 containing acetic acid and acetonitrile may be optionally 0.05% to 5%, such as further 0.5%.
In one embodiment of the present invention, the elution mobile phase used after the salt conversion may be an aqueous solution RP2-B containing acetonitrile, wherein the acetonitrile concentration is selected from 5% to 60% by mass.
As an embodiment of the invention, the reversed phase filler used for purification and salt transformation 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 linear alkyl chains of 4 to 18 carbon atoms. For example, a linear 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 preferably octaalkyl-bonded silica gel or octadecyl-bonded silica gel, such as any filler selected from Sepax BR-C18, unisil 15-100C18, sepax Bio-C8 (2), sepax GP-C18, YMC-C8, HPLCONE8C18-100AA, HPLCONE8C 18D, HPLCONE-8C8K, HPLCONE-8C18K or SP-200-8-C8-BIO, preferably HPLCONE8C18-100AA filler.
As an embodiment of the present invention, the ganirelix acetate peptide is isolated after purification by one or a combination of lyophilization, crystallization with the addition of an anti-solvent, and isoelectric precipitation.
As a more specific embodiment of the invention, a purification method of ganirelix acetate, which comprises the steps of crude peptide pretreatment, one-step reverse phase chromatography purification and salt transfer, comprises the following steps:
(1) Pretreatment of crude peptide: dissolving the crude peptide ganirelix acetate by using an aqueous solution containing acetonitrile;
(2) And (3) purification: performing linear gradient elution by using octadecylsilane chemically bonded silica filler as a stationary phase, an acidic buffer solution containing ammonium acetate-TFA and acetonitrile as a mobile phase RP1-A1 and an acetonitrile solution as a mobile phase RP1-B, and collecting a main peak section component;
(3) Salt conversion: using octadecylsilane chemically bonded silica filler as a stationary phase, and using a buffer solution containing ammonium acetate and acetonitrile as a salt-transfer mobile phase RP2-A2; and (3) performing linear gradient elution by taking an aqueous solution containing acetic acid and acetonitrile as an eluting mobile phase RP2-A3 and taking an acetonitrile solution as an eluting mobile phase RP2-B, and collecting main peak section components.
As an embodiment of the present invention, the drying method of the purified ganirelix acetate can be freeze drying or spray drying.
In the invention, the mobile phase, the concentration, the pH value, the gradient elution and the like are obtained by experimental screening, and an optimal system is determined by comparing the purity, the yield, the removal effect on impurities, the ion content and the like. The purification mobile phase system in the invention has better effect of removing impurities in the crude peptide and better yield than 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 reverse phase chromatography purification and salt conversion, and finally, the purity of the ganirelix acetate refined peptide obtained by rotary evaporation and freeze drying is more than or equal to 99.9 percent, if the purification can further reach 99.95 percent, the maximum single impurity content is less than or equal to 0.05 percent, if the maximum single impurity content is further less than or equal to 0.03 percent, wherein the content of acetate is 7.10 to 10.80 percent, the content of trifluoroacetate is less than 0.05 percent (far lower than the control standard of 0.1 percent), the content of chloride ions is less than 0.05 percent, the content of sulfate radicals is less than 0.02 percent, and the content of phosphate radicals is less than 0.05 percent, and the purification level is higher.
(2) The acetate-TFA buffer solution is adopted in the reversed-phase chromatographic purification, so that the defect that the ion-pairing agent has no buffering capacity is overcome, the problem of poor reproducibility of the ion-pairing agent (TFA) system is solved, the preparation peak shape is stable, and the impurity removal capacity is improved.
(3) The reverse phase chromatography purification and the salt conversion both use the same acetate and acetic acid, the used materials are single, and other ions are not introduced.
Drawings
FIG. 1 is an HPLC profile of a sample of the ganirelix acetate purification of example 1.
FIG. 2 is a diagram of the purification preparation of ganirelix acetate in example 1.
FIG. 3 is an HPLC chromatogram of a sample of the ganirelix acetate purification of example 2.
FIG. 4 is an HPLC chromatogram of a sample of the ganirelix acetate purification of example 3.
FIG. 5 is an HPLC chromatogram of a sample of the purified ganirelix acetate of example 4.
FIG. 6 is an HPLC profile of a sample of the sample of example 5 after the salt transfer of ganirelix acetate.
FIG. 7 is an HPLC chromatogram of a sample of ganirelix acetate peptide from example 5.
FIGS. 8 and 9 show HPLC profiles (duplicate) of a sample of the purified ganirelix acetate of comparative example 1.
FIG. 10 is a diagram showing the purification preparation of ganirelix acetate in comparative example 1.
FIG. 11 is an HPLC chromatogram of a sample after the trans-salting of ganirelix acetate in comparative example 2.
FIG. 12 is a graph of the ion content of the sample after the salt transfer of ganirelix acetate in comparative example 2.
Detailed Description
In order to clearly understand the technical features, objects and advantages of the present invention, the following detailed description is provided for the technical solutions of the present invention with reference to the specific embodiments, but the present invention is not limited to the following embodiments.
The crude ganirelix acetate peptide is derived from ganirelix acetate samples obtained by utilizing a fragment solid-phase synthesis method.
The RP1-A1 mobile phase formula is as follows: 10mM sodium acetate, 0.05% TFA,10% acetonitrile, pH adjusted to 2.5 with acetic acid.
The RP1-A2 mobile phase formula comprises: 10mM ammonium acetate, 0.10% TFA,10% acetonitrile, pH adjusted to 2.5 with acetic acid.
The RP1-A3 mobile phase formula comprises: 10mM ammonium acetate, 0.05% TFA,10% acetonitrile, pH adjusted to 2.5 with acetic acid.
The RP1-A4 mobile phase formula comprises: 10mM ammonium acetate, 0.05% TFA,10% acetonitrile, pH adjusted to 3.0 with acetic acid.
The RP1-B formula comprises: 60% acetonitrile.
The RP2-A1 mobile phase formula is as follows: 0.5% acetic acid, 10% acetonitrile.
The RP2-A2 mobile phase formula comprises: 100mM ammonium acetate, 10% acetonitrile.
The RP2-A3 mobile phase formula is as follows: 0.5% acetic acid, 50% acetonitrile.
The RP2-B mobile phase formula comprises: 5% acetonitrile.
Example 1: purification of crude ganirelix acetate
Sample treatment: weighing 0.1g of crude peptide sample of ganirelix acetate, adding 100 times of 10% acetonitrile solution by mass to dissolve the sample, filtering by using a filter membrane with the pore diameter of 0.22 mu m, and then waiting for purification.
And (3) purification process: taking the processed crude ganirelix acetate as a sample, taking an octadecylsilane chemically bonded silica filler of HPLCONE8C18-100AA type as a stationary phase, wherein the flow rate is 200cm/hr, and the detection wavelength is 280nm. Equilibrating the column with 2 CVs using RP1-A1 mobile phase; loading according to the loading capacity of 10g/L of total protein of the loaded sample; after loading, balancing 2 CVs on the chromatographic column by using RP1-A1 mobile phase; and finally, performing linear gradient elution by using RP1-A1 and RP1-B (the RP1-B is eluted for 45min from 34% to 44%), and collecting a main peak section which is a sample for purifying the ganirelix acetate. The purity of a purified sample is 99.95 percent, the maximum single impurity content is 0.05 percent, and the purification yield is 70.2 percent through HPLC detection. The HPLC profile is shown in FIG. 1. The purification scheme is shown in FIG. 2.
Example 2: purification of crude ganirelix acetate
Sample treatment: weighing 0.1g of crude peptide sample of ganirelix acetate, adding 100 times of 10% acetonitrile solution by mass to dissolve the sample, filtering by using a filter membrane with the pore diameter of 0.22 mu m, and then purifying.
And (3) purification process: taking the treated crude ganirelix acetate as a sample, taking an octadecyl silane bonded silica gel filler of HPLCONE8C18-100AA type as a stationary phase, wherein the flow rate is 200cm/hr, and the detection wavelength is 280nm. Equilibrating 2 CVs on the column with RP1-A2 mobile phase; loading according to the loading capacity of 10g/L of total protein of the loaded sample; after loading, balancing 2 CVs on the chromatographic column by using RP1-A2 mobile phase; and finally, performing linear gradient elution by using RP1-A2 and RP1-B (the RP1-B is eluted for 45min from 34% to 44%), and collecting a main peak section which is a sample for purifying the ganirelix acetate. HPLC detection shows that the purity of a purified sample is 99.91%, the maximum single impurity content is 0.04%, and the purification yield is 71.7%. The HPLC profile is shown in FIG. 3.
Example 3: purification of crude ganirelix acetate
Sample treatment: weighing 0.1g of crude peptide sample of ganirelix acetate, adding 100 times of 10% acetonitrile solution by mass to dissolve the sample, filtering by using a filter membrane with the pore diameter of 0.22 mu m, and then waiting for purification.
And (3) purification process: taking the treated crude ganirelix acetate as a sample, taking an octadecyl silane bonded silica gel filler of HPLCONE8C18-100AA type as a stationary phase, wherein the flow rate is 200cm/hr, and the detection wavelength is 280nm. Equilibrating the column with 2 CVs using RP1-A3 mobile phase; loading according to the loading capacity of 10g/L of total protein of the loaded sample; after loading, the RP1-A3 mobile phase is used for balancing 2 CVs on the chromatographic column; and finally, performing linear gradient elution by using RP1-A3 and RP1-B (the RP1-B is eluted for 45min from 34% to 44%), and collecting a main peak section which is a sample for purifying the ganirelix acetate. HPLC detection shows that the purity of a purified sample is 99.93%, the maximum single impurity content is 0.03%, and the purification yield is 71.0%. The HPLC profile is shown in FIG. 4.
Example 4: purification of crude Ganirelix acetate
Sample treatment: weighing 0.1g of crude peptide sample of ganirelix acetate, adding 100 times of 10% acetonitrile solution by mass to dissolve the sample, filtering by using a filter membrane with the pore diameter of 0.22 mu m, and then waiting for purification.
And (3) purification process: taking the processed crude ganirelix acetate as a sample, taking an octadecylsilane chemically bonded silica filler of HPLCONE8C18-100AA type as a stationary phase, wherein the flow rate is 200cm/hr, and the detection wavelength is 280nm. Equilibrating the column with 2 CVs using RP1-A4 mobile phase; loading according to the loading capacity of 10g/L of total protein of the loaded sample; after loading, the RP1-A4 mobile phase is used for balancing 2 CVs on the chromatographic column; and finally, performing linear gradient elution by using RP1-A4 and RP1-B (the RP1-B is eluted for 45min from 34% to 44%), and collecting a main peak section which is a sample for purifying the ganirelix acetate. HPLC detection shows that the purity of a purified sample is 99.90%, the maximum single impurity content is 0.03%, and the purification yield is 73.2%. The HPLC profile is shown in FIG. 5.
Example 5: purification of ganirelix acetate by desalting, rotary evaporation and freeze-drying of an eluate
Sample treatment: the purified ganirelix acetate eluate obtained in example 1 was diluted with one volume of purified water to be desalted.
A salt conversion process: diluted ganirelix acetate purified eluent is used as a sample, octadecylsilane chemically bonded silica filler of HPLCONE8C18-100AA type is used as a stationary phase, the flow rate is 200cm/hr, and the detection wavelength is 280nm. Equilibrating the column with 2 CVs using RP2-A1 mobile phase; loading the sample according to the loading capacity of 30g/L of total protein of the loaded sample; after loading, the column is equilibrated with RP2-A1 mobile phase for 2 CVs; performing salt conversion by using RP2-A2 mobile phase equilibrium chromatographic column with 4 CVs; washing with RP2-B mobile phase equilibrium chromatographic column with 3 CV; and finally, performing linear gradient elution by using RP2-A3 and RP2-B (the RP2-A is eluted for 30min from 60% to 70%), and collecting a main peak section which is a sample after the ganirelix acetate is subjected to salt transfer. HPLC detection shows that the purity of the sample after salt conversion is 99.96%, the maximum single impurity content is 0.04%, and the salt conversion yield is 93%. The HPLC profile is shown in FIG. 6.
And (3) rotary steaming process: and (3) putting the sample subjected to salt conversion into a rotary evaporator, setting the temperature at 25 ℃, setting the rotating speed at 100rpm, and carrying out rotary evaporation until no obvious liquid drips out of the condenser and the mass of the sample is about 50% of that of the initial sample.
And (3) freeze-drying: and (3) placing the sample subjected to rotary evaporation into a refrigerator at the temperature of-80 ℃ for pre-freezing, and then placing the sample into a freeze dryer for freeze-drying after the pre-freezing is finished to obtain the ganirelix acetate refined peptide. Through detection, the purity of the ganirelix acetate refined peptide is 99.95%, the maximum single impurity content is 0.03%, wherein the content of acetate is 9.18%, the content of trifluoroacetate ions is 0.038%, and the contents of chloride ions, sulfate radicals and phosphate radicals are 0.029%, 0.004% and 0.017% respectively. The HPLC profile is shown in FIG. 7.
Example 6
The purified and eluted solutions of ganirelix acetate obtained in examples 2-4 were subjected to the same post-treatments of trans-salting, rotary evaporation and lyophilization as in example 5.
The purification of ganirelix acetate-the sample purity after the salt transfer of the eluent of example 2 was 99.95%, the maximum single impurity content was 0.05%, and the salt transfer yield was 92%. After freeze-drying, the purity of the ganirelix acetate refined peptide is 99.94%, the maximum single impurity content is 0.05%, wherein the content of acetate is 9.23%, the content of trifluoroacetate is 0.040%, and the contents of chloride ions, sulfate radicals and phosphate radicals are 0.046%, 0.007% and 0.023% respectively.
The purification of ganirelix acetate-eluent in example 3 was found to have a purity of 99.92%, a maximum single impurity content of 0.04%, and a trans-salt yield of 94%. After freeze-drying, the purity of the ganirelix acetate refined peptide is 99.93%, the maximum single impurity content is 0.03%, wherein the content of acetate is 9.40%, the content of trifluoroacetate is 0.041%, and the contents of chloride ions, sulfate radicals and phosphate radicals are 0.044%, 0.012% and 0.034% respectively.
The purification of ganirelix acetate-eluent in example 4 was found to have a purity of 99.92%, a maximum single impurity content of 0.03%, and a trans-salt yield of 93%. The purity of the ganirelix acetate refined peptide after freeze-drying is 99.92 percent, the maximum single impurity content is 0.03 percent, wherein the content of acetate is 9.24 percent, the content of trifluoroacetate is 0.037 percent, and the contents of chloride ions, sulfate radicals and phosphate radicals are 0.037 percent, 0.004 percent and 0.040 percent respectively.
Comparative example 1
Referring to example 1, the mobile phase RP1-A1 was purified by removing 10mM ammonium acetate, and the remaining conditions and procedures were the same as in example 1, and repeated twice to examine the effect of ammonium acetate in the mobile phase on the purity, impurities, yield, preparation profile, etc. of ganirelix acetate. The HPLC detection results are shown in FIGS. 8 and 9, the purity of the eluate obtained by repeating 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 substantially the same as that of FIG. 2; the purity of the eluent obtained in the second time is 99.01%, the maximum single impurity content is 0.21%, the yield is 51%, and the purification preparation diagram is shown in FIG. 10. Compared with examples 1-4, when ammonium acetate is added into a TFA ion-pairing agent system, the purification preparation peak shape is stable, and the purity and the yield are obviously improved; without the addition of ammonium acetate, the reproducibility of the purified preparation chart was poor, and a case similar to that of fig. 10 was found with a probability of about 50%, that is, the peak shape was not sharp, and the tailing of the rear end of the peak was severe.
Comparative example 2
Referring to example 5, 100mM ammonium acetate was replaced with 20mM ammonium acetate in mobile phase RP2-A1, and the other conditions and procedures were the same as in the salt conversion process of example 5. The influence of the concentration of the buffer salt in the mobile phase on the purity of ganirelix acetate, impurities, the content of TFA ions in the sample and the like is examined. The detection result is shown in fig. 11, the purity of the sample is 99.95%, the maximum single impurity content is 0.02%, wherein the acetate content is 8.55%, the trifluoroacetate content is 0.767%, and the contents of chloride, sulfate and phosphate are 0.080%, 0.030% and 0.023%, respectively. Compared 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%, as shown in fig. 12, and does not meet the central control standard.

Claims (10)

1. A purification method of ganirelix acetate is characterized by comprising the following steps:
(1) And (3) purification: taking an acidic buffer solution containing acetate and an ion pairing agent and an organic solvent as purification mobile phases RP1-A and RP1-B, and taking a reversed-phase filler as a stationary phase;
(2) Salt conversion: the acetate-containing buffer solution and organic solvent are used as a salt-transfer mobile phase RP2-A2, and the reversed-phase filler is used as a stationary phase.
2. The purification method according to claim 1, characterized in that: the acidic buffer solution containing acetate and ion pairing agent is any one selected from ammonium acetate-trifluoroacetic acid buffer solution, sodium acetate-trifluoroacetic acid buffer solution and potassium acetate-trifluoroacetic acid buffer solution.
3. The purification method according to claim 2, characterized in that: the mass concentration of trifluoroacetic acid in the ammonium acetate-trifluoroacetic acid buffer solution, the sodium acetate-trifluoroacetic acid buffer solution and the potassium acetate-trifluoroacetic acid buffer solution is 0.001% -5.0%; the concentration of the acetate is 0.1-200mM.
4. The purification process according to claim 1, characterized in that: the pH value of the acid buffer solution containing the acetate is less than or equal to 5.0.
5. The purification method according to claim 1, characterized in that: and (3) purifying the mobile phase, and performing linear gradient elution by taking a mixed solution of an acid buffer solution containing acetate and an ion pairing agent and acetonitrile as a mobile phase RP1-A and taking an aqueous solution containing acetonitrile as a mobile phase RP 1-B.
6. The purification method according to claim 1, characterized in that: the buffer solution containing acetate in the salt-transfer mobile phase is selected from any one of ammonium acetate, potassium acetate and sodium acetate, and the concentration of the acetate is 5-1000mM.
7. The purification method according to claim 1, characterized in that: the organic solvent used for purification and the organic solvent used in the salt-converted mobile phase are both aqueous solutions containing acetonitrile.
8. The purification process according to claim 1, characterized in that: after the salt conversion, performing gradient elution by using an elution mobile phase, and taking an aqueous solution containing acetic acid and acetonitrile as a mobile phase RP2-A3, wherein the mass concentration of the acetic acid is 0.05-5%; and taking an acetonitrile-containing aqueous solution as a mobile phase RP2-B, wherein the mass concentration of the acetonitrile is 5-60%.
9. The purification process according to claim 1, characterized in that: the reversed phase filler used for purification and salt conversion is selected from a stationary phase made of porous silica particles or silica gel.
10. The purification process according to claim 1, characterized in that: purification by crude peptide pretreatment, one-step reverse phase chromatographic purification and salt transfer, comprising the steps of:
(1) Pretreatment of crude peptide: dissolving the crude peptide ganirelix acetate by using an aqueous solution containing acetonitrile;
(2) And (3) purification: performing linear gradient elution by using octadecylsilane chemically bonded silica filler as a stationary phase, an acidic buffer solution containing ammonium acetate-TFA and acetonitrile as a mobile phase RP1-A1 and an acetonitrile solution as a mobile phase RP 1-B;
(3) Salt conversion: using octadecylsilane chemically bonded silica filler as a stationary phase, and using a buffer solution containing ammonium acetate and acetonitrile as a salt-transfer mobile phase RP2-A2; and (3) performing linear gradient elution by using an aqueous solution containing acetic acid and acetonitrile as an eluting mobile phase RP2-A3 and using an acetonitrile solution as an eluting mobile phase RP 2-B.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102993274A (en) * 2012-11-30 2013-03-27 深圳翰宇药业股份有限公司 Purification method of ganirelix acetate
CN104844694A (en) * 2014-02-17 2015-08-19 深圳翰宇药业股份有限公司 Ganirelix acetate preparation method
CN105693844A (en) * 2016-03-10 2016-06-22 泰州施美康多肽药物技术有限公司 Preparation method of gonadotrophin-releasing hormone analogue acetate
CN107056894A (en) * 2017-05-26 2017-08-18 济南康和医药科技有限公司 A kind of method of fragment method synthesis in solid state ganirelix acetate
CN109879938A (en) * 2017-12-06 2019-06-14 正大天晴药业集团股份有限公司 A kind of preparation method of ganirelix acetate
CN114478708A (en) * 2021-12-29 2022-05-13 南京汉欣医药科技有限公司 Solid phase synthesis method of ganirelix fragment

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102993274A (en) * 2012-11-30 2013-03-27 深圳翰宇药业股份有限公司 Purification method of ganirelix acetate
CN104844694A (en) * 2014-02-17 2015-08-19 深圳翰宇药业股份有限公司 Ganirelix acetate preparation method
CN105693844A (en) * 2016-03-10 2016-06-22 泰州施美康多肽药物技术有限公司 Preparation method of gonadotrophin-releasing hormone analogue acetate
CN107056894A (en) * 2017-05-26 2017-08-18 济南康和医药科技有限公司 A kind of method of fragment method synthesis in solid state ganirelix acetate
CN109879938A (en) * 2017-12-06 2019-06-14 正大天晴药业集团股份有限公司 A kind of preparation method of ganirelix acetate
CN114478708A (en) * 2021-12-29 2022-05-13 南京汉欣医药科技有限公司 Solid phase synthesis method of ganirelix fragment

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KUMARSWAMY UMMITI等: "Determination of Amino Acid Composition of Ganirelix Acetate in an Injectable Formulation by Pre-column Derivatization with 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate" *

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