A method, system, and product for preparing high purity of deslorelin acetate soluble in water

Abstract

The present application relates to a method for purifying non-steroidal GnRH agonist polypeptide, in particular, a method, system and product for preparing high-purity deslorelin acetate soluble in water. The present application is prepared by purification of high-purity deslorelin acetate through preparative reverse phase chromatography, conversion of ion pairs in the column, optimization of salt conversion and freeze-drying pretreatment, to obtain a drug substance in the form of high acetate. The product has a purity of ≥98.5% by HPLC, an acetic acid content of about 13.9%~14.7% (w / w), a solubility in pure water of ≥120 mg / mL and a clarity maintained for 24 h; and a retention time consistent with the commercial reference. Compared with the comparative example, the yield of high-purity fraction is significantly improved, the impurities and non-target ion pair residues are reduced, and it is suitable for industrialization and preparation development.

Description

Technical Field

[0001] This invention relates to a method for purifying nonsteroidal GnRH agonist peptides, specifically a method, system, and product for preparing high-purity dilorelin acetate that is soluble in water. Background Technology

[0002] Delorelin is a nonsteroidal GnRH agonist. Like GnRH, it stimulates the pituitary gland to secrete FSH and LH. The release of FSH and LH is responsible for the subsequent production of sex hormones, and the release of FSH / LH is further regulated by the feedback mechanism of sex hormones. Under normal circumstances, GnRH is released in a pulsatile manner, which also causes a pulsatile release of FSH / LH. Continuous administration of GnRH can lead to desensitization and downregulation of GnRH receptors on gonadotropins, thereby inhibiting the release of gonadotropins and sex hormones. In veterinary clinical practice, repeated administration of a certain amount of delorelin to pets can inhibit normal gonadal function, achieving a certain contraceptive effect. Its long-acting sustained-release formulation can solve problems of uncontrolled breeding and related behaviors in pets, as well as the distress caused by spaying or neutering. It can also treat central precocious puberty in humans, as well as prostate and breast cancer, and other diseases and conditions that are beneficial in suppressing testosterone or estradiol levels.

[0003] As an analogue of GnRH (gonadotropin-releasing hormone), dilorelin has a chemical structure that differs from natural GnRH. It replaces the 6th glycine with D-tryptophan (D-Trp), removes the 10th glycine, and modifies the ethylamino group at the carboxyl terminus of the 9th proline. These structural changes reduce the hydrolytic activity of dilorelin by proteases and enhance its biological activity. In its synthesis process, our factory uses a solid-phase synthesis method with sequential coupling of Fmoc-protected amino acids to synthesize dilorelin: using 2-Cl-CTCResin (CTC resin) as a carrier, and with the participation of a condensing agent, Fmoc-Pro-OH, Fmoc-Arg(pbf)-OH, Fmoc-Leu-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp-(Boc)-OH, Fmoc-His(Trt)-OH, and pGlu-OH are sequentially linked to the resin. The peptides are then cleaved from the resin using a lysis buffer. Next, the C-terminus of proline is ethylamined in a liquid-phase reaction system, and finally, the side-chain protecting groups are removed to obtain crude dilorelin. This method has been disclosed in patent CN116199745A. However, the purity of crude dilorelin obtained by this method is about 80% to 90%, which cannot meet the purity requirements of the pharmaceutical industry for polypeptide drugs. Therefore, it cannot be used for the research and development and production of dilorelin formulations.

[0004] The crude des-Glycerin contains impurities with physicochemical properties similar to the main component, making it challenging to achieve high purity during purification. Additionally, the instructions for the commercial reference standard (product code: B-4066809, CAS number: 57773-65-6) purchased from BACHEM, Switzerland, indicate its English name as (Des-Glycerin). 10 D-Trp 6 Pro-NHEt 9 The compound is denoted as Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-PRO-NHEtHighacetatesalt. This indicates that dilorelin acetate is a high acetate-bound product, a characteristic not found in other GnRH or LHRH analogs or agonists. Dilorelin acetate not only possesses high acetate content but also hydrophobicity. The salt conversion and subsequent concentration steps are crucial in its preparation. Improper control of these steps can lead to a synergistic effect between the high acetate content and hydrophobicity, causing the product to easily precipitate from the solution. This precipitation directly affects the product's solubility; prior to this invention, the obtained product exhibited only minimal water solubility. Therefore, efficiently preparing high-purity, water-soluble high acetate dilorelin acetate has become a pressing technical challenge, and currently, there is no clear technical process available for reference.

[0005] Chinese patents CN114230637A, CN113968897A, CN110041407B, and CN116199745A detail the synthesis process of desloreilin, but none mention the details of purification or salt conversion. US patent US4218439 mentions ion exchange chromatography using a Sephadex G25 gel column on a CMC column, but does not mention purification details, efficiency, or relevant purification data (results). Furthermore, the Sephadex G25 gel column, based on molecular sieving, separates substances of different molecular weights through the pore size of the gel. This separation mechanism is effective for substances with large molecular weight differences, but less effective for substances with similar molecular weights. It is known that crude dilorelin contains impurities with similar physicochemical properties to the main component, so the Sephadex G25 gel column has certain limitations in the purification of crude dilorelin. Chinese patent CN105254701A mentions using a C18 reversed-phase column and a 0.1% TFA aqueous solution / acetonitrile system for purification, but does not mention how to combine with acetic acid to form a high acetate. Summary of the Invention

[0006] To achieve the above objectives, the present invention aims to provide a method for preparing high-purity dilorelin acetate soluble in water. The method involves purification of hydrophilic C18 via reversed-phase high-performance liquid chromatography (RP-HPLC) under different chromatographic conditions and salt conversion of different acetic acid aqueous solutions to form an acetate form. Finally, by controlling the concentration ratio, the mixture is filtered and lyophilized to obtain a high-purity dilorelin acetate raw material with a purity greater than 98.5% that is soluble in water.

[0007] To achieve the above technical objectives, the present invention adopts the following technical solution: A method for preparing high-purity dilorelin acetate soluble in water, the method comprising the following sequential steps: 1) Dissolution and pre-filtration: Dissolve crude diloxacin in purified water, stir, and filter; 2) Adsorption and gradient elution: A C18 packed column was used to load the filtered crude dilorelin peptide solution onto the column for sample adsorption; gradient elution was performed using phosphate buffer as mobile phase A and acetonitrile as mobile phase B. 3) Concentration: Evaporate the combined solutions to remove excess acetonitrile; 4) Salt conversion: Using a C18 packed column, load the concentrated dilorelin acetate solution onto the column, perform ion conversion with 15-25 mmol / L ammonium acetate solution for 1.5-5.0 column volumes, then equilibrate with 1.6%-2.0% (v / v) acetic acid aqueous solution for 4-10 column volumes, and finally elute with acetonitrile-acetic acid aqueous solution, collect and combine the pure products; 5) Concentrate and then freeze dry.

[0008] Preferably, in step 1), 1%-10% (preferably 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%) of acetonitrile is added to the purified water to aid dissolution, and the water is filtered through a 0.22μm~0.45μm microporous membrane.

[0009] As a preferred option, in step 2), the C18 packed column is a DAC300C18 column with a length of 50cm, a diameter of 30cm, and a packing particle size of 10μm. And / or, equilibrate 1.5–5.0 column volumes with phosphate buffer before use; And / or, sample flow rate 600-1000 ml / min, elution flow rate: 1200-2000 ml / min.

[0010] Preferably, in step 2), gradient elution is performed using triethylamine phosphate buffer as mobile phase A and acetonitrile as mobile phase B. The pH of mobile phase A is 2.3-5.0 (preferably 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, or 4.9), and the initial volume fraction of mobile phase B is 5-10% (preferably 6%, 7%, 8%, or 9%), and the final volume fraction is 26-30% (preferably 27%, 28%, or 29%).

[0011] Preferably, the gradient elution procedure in step 2) is as follows: Time: 0-4 min; Mobile phase A: 95%; Mobile phase B: 5% Time 4-6 min, mobile phase A 95%, mobile phase B 5%; Time: 6-64 min; Mobile phase A: 80%; Mobile phase B: 20% Time 64-100 min, mobile phase A 74%, mobile phase B 26%.

[0012] Preferably, in step 4), the C18 packed column is a DAC300C18 column with a length of 50cm, a diameter of 30cm, and a packing particle size of 10μm. And / or, equilibrate 1.5-5.0 column volumes with an aqueous acetic acid solution of 0.2%-2.5% (v / v) before use; And / or, perform ion conversion with 15-25 mmol / L ammonium acetate solution for 1.5-5.0 column volumes, and equilibrate with 1.6%-2.0% (v / v) aqueous acetic acid solution for 4-10 column volumes; preferably, use 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L, 21 mmol / L, 22 mmol / L, 23 mmol / L, 24 mmol / L or 25 mmol / L ammonium acetate solution; equilibrate with 1.6%, 1.7%, 1.8%, 1.9% or 2.0% (v / v) aqueous acetic acid solution for 4, 5, 6, 7, 8, 9 or 10 column volumes.

[0013] Preferably, the aqueous solution containing acetic acid and acetonitrile in step 4) includes 0.2%-2.5% by volume acetic acid and 40%-60% by volume acetonitrile.

[0014] Preferably, in step 5), the collected liquid is concentrated under reduced pressure to a peptide concentration of 30-100 mg / mL (preferably 40, 50, 60, 70, 80, or 90 mg / mL). After the acetonitrile is basically removed, it is filtered through a 0.22 μm to 0.45 μm filter membrane, dispensed into trays with a liquid layer thickness ≤10 mm, and then freeze-dried under vacuum after being kept at -35±1℃ for 1.5-3.0 h.

[0015] Furthermore, the present invention also discloses a system for implementing the method, the system comprising: A liquid chromatography unit equipped with a DAC-prepared C18 column; Solvent delivery and online switching module for phosphate buffer, acetonitrile, acetic acid solution, and ammonium acetate solution; Ultraviolet detector; Fractionation collector; and, The unit consists of a vacuum concentration unit and a vacuum freeze-drying unit.

[0016] Furthermore, the present invention also discloses the method for preparing high-purity dilorelin acetate, wherein the dilorelin has a purity of 98.5% or higher and is soluble in water.

[0017] This invention, by employing the aforementioned technical solutions, achieves simultaneous improvement in the key quality attributes of diloxacin acetate: First, the purity of the finished product consistently reaches and exceeds ≥98.5%; second, it forms a high acetate state, enabling the active pharmaceutical ingredient to reach ≥120 mg / mL in pure water and remain clear without precipitation for 24 hours, significantly superior to the turbidity, colloidalization, or coagulation commonly observed in comparative studies; third, while ensuring a substantially equivalent overall yield, the yield of the high-purity fraction is significantly improved, and tests show no significant fluctuation in purity before and after salt conversion, indicating that the impurity migration and salt form establishment process has good stability and selectivity for the main component; fourth, residual non-target ions / additives are significantly reduced, which is beneficial for subsequent formulation development and regulatory consistency; fifth, the product exhibits excellent batch-to-batch reproducibility and scale-up stability, with small fluctuations and strong controllability in release indicators such as solubility, clarity, and salt content, thus achieving a synergistic technical effect in terms of "high purity, high water solubility, and stability." Attached Figure Description

[0018] Figure 1 The effects of dissolving and filtering the crude product in Examples 1 and 2 of this invention are shown; wherein... Figure 1 In Example A, the crude product solution before filtration is from Example 1 and Comparative Example 1. Figure 1 Image B shows the effect after filtration in Example 2. Figure 1 C represents the effect after filtering in Example 1.

[0019] Figure 2 This is a graph showing the online monitoring of purification in Example 3.

[0020] Figure 3 This is an online monitoring graph for purification in Example 4.

[0021] Figure 4 This is a comparison chart of the water solubility of Comparative Example 3 and Example 7.

[0022] Figure 5 For Comparative Example 4, the white solids that precipitated out after salt concentration and filtration were the upper layer filtered out.

[0023] Figure 6 To compare example 6, after salt concentration and precipitation, 300 ml of acetic acid was added to each sample. Figure 6 (A) and 450ml ( Figure 6 (B) Solution that was not filtered after dissolution.

[0024] Figure 7 Comparative Example 7: Solution after salt concentration (unfiltered).

[0025] Figure 8 Comparative Example 8: Solution after salt concentration (unfiltered).

[0026] Figure 9 This is the HPLC chromatogram of crude dilorelin.

[0027] Figure 10 This is the mass spectrum of diloxacin acetate. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present invention.

[0029] This invention provides a method for purifying and converting dilorelin acetate to salts, comprising the following steps: 1. Preparation instruments and conditions Instrument: Cyvax liquid chromatography system, equipped with an ultraviolet detector; Preparation column: DAC300C18 column, approximately 50cm in length and 30cm in diameter, with a packing particle size of 10μm. Mobile phase (purification): Phase A is the aqueous phase (triethylamine phosphate buffer), and Phase B is the organic phase (acetonitrile). Mobile phase (salt conversion): Phase A is the aqueous phase (1.8% volume percentage acetic acid aqueous solution), Phase B is the organic phase (acetonitrile), and Phase C is the 20 mmol / L ammonium acetate aqueous solution; Flow rate: 1600 ml / min; Detection wavelength: 220nm; Temperature: Room temperature.

[0030] (a) Sample preparation: The above crude diloxacin (HPLC chromatogram as shown) was prepared. Figure 9 Add approximately 100g of the sample to 5L of pure water while stirring, add an appropriate amount of acetonitrile to aid dissolution (approximately 5% acetonitrile), filter through a 0.22μm microporous membrane, and use for sample loading.

[0031] (b) Purification: A C18 packed column was used at a wavelength of 220 nm. Two column volumes were equilibrated with phosphate buffer; the filtered crude dilorelin peptide solution was loaded onto the column to allow sample adsorption; the sample was eluted with phosphate buffer and acetonitrile, collected, and detected by HPLC.

[0032] (c) Concentration: The combined solutions are rotary evaporated to remove excess acetonitrile.

[0033] (d) Salt conversion: Equilibrate with acetic acid aqueous solution for 2 column volumes. Load the concentrated dilorelin acetate solution onto the column, perform ion conversion with 20 mmol / L ammonium acetate solution for 2 column volumes, equilibrate with acetic acid aqueous solution for 5 column volumes, and finally collect and combine the pure products to obtain a pure dilorelin acetate solution.

[0034] (e) Concentration before lyophilization: Take the pure dilorelin acetate solution obtained after desalting and combining the pure products, and add it to a clean concentration vessel. Concentrate to a peptide concentration of approximately 60 mg / ml.

[0035] (f) Freeze-drying: After filtering the concentrated solution of diloxacin acetate obtained in the above steps through a microporous membrane, the solution is evenly distributed into stainless steel trays, and the liquid layer thickness shall not exceed 1 cm. The product is frozen to -35±1℃ and kept at that temperature for 2 hours, and then freeze-dried under vacuum.

[0036] Example 1: Dissolution of crude diloxacin before purification Sample preparation: Add about 100g of the crude dilorelin to 5L of pure water under stirring, add an appropriate amount of acetonitrile to aid dissolution (about 5% acetonitrile), filter with a 0.22μm microporous membrane, and use for sample loading.

[0037] Example 2: Dissolution of crude dilorelin peptide before purification Sample preparation: Add about 100g of the crude dilorelin to 5L of pure water under stirring, add an appropriate amount of acetonitrile to aid dissolution (about 5% acetonitrile), filter through a 0.45μm microporous membrane, and use for sample loading.

[0038] Example 1: Dissolution of crude dilorelin peptide before purification Table 1 below shows the results of HPLC analysis of the purity and crude peptide content of crude dilorelin before and after filtration following dissolution using the crude products of Example 1 and Comparative Example 1 of this invention. Table 1 Comparison of purity and content of crude dilorelin before and after filtration

[0039] Figure 1 The effects of crude product dissolution and filtration before and after Example 1 and Example 2 of this invention are given. Table 1 shows that the purity and content of crude diloxacin did not change significantly before and after filtration after dissolution. Figure 1 A comparison of Examples 1 and 2 clearly shows that Example 1 is the preferred method for filtering crude dilorelin after dissolution. Without affecting the purity and crude peptide content, the solution becomes clear, which can effectively protect the service life of the preparation liquid phase and the preparation column packing.

[0040] Example 3: Purification of crude diloxacin (a) Sample preparation: Same as in Example 1.

[0041] (b) Purification: A C18 packed column was used in the Cyvax HPLC system. The wavelength was 220 nm, the loading flow rate was 800 mL / min, and the elution flow rate was 1600 mL / min. Two column volumes were equilibrated with triethylamine phosphate buffer (pH 2.3). The filtered crude dilorelin peptide solution was loaded onto the column to allow sample adsorption. Elution was performed with triethylamine phosphate buffer (pH 2.3) and acetonitrile using the following gradient elution, collection, and HPLC detection. The purification online monitoring graph is shown below. Figure 2 As shown.

[0042] Purification gradient 1

[0043] Example 4: Purification of crude diloxacillin (a) Sample preparation: Same as in Example 1.

[0044] (b) Purification: A Cyvax HPLC system with a C18 packed column was used. The wavelength was 220 nm, the loading flow rate was 800 mL / min, and the elution flow rate was 1600 mL / min. Two column volumes were equilibrated with triethylamine phosphate buffer (pH 5.0). The filtered crude dilorelin peptide solution was loaded onto the column to allow sample adsorption. Elution was performed with triethylamine phosphate buffer (pH 5.0) and acetonitrile using the following gradient elution, collection, and HPLC detection. The purification online monitoring graph is shown below. Figure 3 As shown.

[0045] Purification gradient 1

[0046] Example 5: Purification of crude diloxacin acetate (a) Sample preparation: Same as in Example 1.

[0047] (b) Purification: A Cyvax liquid chromatography system with a C18 packed column was used. The wavelength was 220 nm, the loading flow rate was 800 mL / min, and the elution flow rate was 1600 mL / min. Approximately two column volumes were equilibrated with triethylamine phosphate buffer (pH 5.0). The filtered crude dilorelin peptide solution was loaded onto the column to allow sample adsorption. Elution was performed with triethylamine phosphate buffer (pH 5.0) and acetonitrile according to the following gradient, followed by collection and HPLC detection.

[0048] Purification gradient 4

[0049] Example 6: Purification of crude diloxacin acetate (a) Sample preparation: Same as in Example 1.

[0050] (b) Purification: A Cyvax liquid chromatography system with a C18 packed column was used. The wavelength was 220 nm, the loading flow rate was 800 mL / min, and the elution flow rate was 1600 mL / min. Two column volumes were equilibrated with triethylamine phosphate buffer (pH 2.3). The filtered crude dilorelin peptide solution was loaded onto the column to allow sample adsorption. Elution was performed with triethylamine phosphate buffer (pH 2.3) and acetonitrile according to the following gradient, followed by collection and HPLC detection.

[0051] Purification gradient 4

[0052] Comparative Example 1: Purification of crude delorelin (reproducing the conditions of patent CN105254701A) (a) Sample preparation: Same as in Example 1.

[0053] (b) Purification: The conditions of patent CN105254701A were reproduced. Mobile phase A: 0.1% TFA, mobile phase B: acetonitrile, and the elution flow rate was converted according to the column volume of our chromatographic column. The gradient elution, collection and HPLC detection were the same as those of patent CN105254701A.

[0054] Purification gradient 2 (patent CN105254701A)

[0055] Comparative Example 2: Purification of crude diloxacin acetate (a) Sample preparation: Same as in Example 1.

[0056] (b) Purification: A Cyvax liquid chromatography system with a C18 packed column was used. The wavelength was 220 nm, the loading flow rate was 800 ml / min, and the elution flow rate was 1600 ml / min. Two column volumes were equilibrated with triethylamine phosphate buffer (pH 5.0). The filtered crude dilorelin peptide solution was loaded onto the column to allow sample adsorption. Elution was performed with triethylamine phosphate buffer (pH 5.0) and acetonitrile according to the following gradient, followed by collection and HPLC detection.

[0057] Purification gradient 3

[0058] Table 2 below shows the HPLC analysis results of crude dilorelin obtained from Examples 3-6 and Comparative Examples 1-2 of this invention, prepared using the same column, and then purified and collected under different chromatographic conditions (different buffer salts, pH changes and gradient adjustments of the same buffer salt). Table 2. Purification results of crude diloxacin acetate

[0059] A comparison of the experimental data from Examples 3, 4, 5, and 6 with Comparative Examples 1 and 2 in Table 2 clearly shows that the overall yield of peptides purified from Comparative Examples 1-2 and Examples 3-6 is not significantly different. The conditions for Comparative Example 1 (patent CN105254701A) are basically the same as those for Comparative Example 2, making it difficult to achieve the target purity of over 98.5%. Even with similarly insignificant differences in overall yield, Examples 3 and 4 show significantly higher yields of peptides with a purity of over 98.5%. While Examples 5 and 6 are slightly lower, yields of peptides with a purity of over 98.5% can be achieved through multiple operations. (See also...) Figure 2 , Figure 3 It can be seen that, when the yields of different purities are not significantly different between Example 3 and Example 4, Example 3 has a faster peak elution time (about 20 minutes faster). Considering the timeliness and the economy of the organic solvent used, Example 3 is the optimal example.

[0060] Example 7: Salt conversion of dilorelin acetate and pretreatment before lyophilization (a) Take approximately 100g of the purified dilorelin solution (containing pure peptide) with a purity of ≥98.5% and combine the fractions. Rotary evaporate the solution to remove excess acetonitrile. Equilibrate with 1.8% acetic acid aqueous solution for 2 column volumes. Load the concentrated dilorelin solution onto the column and perform ion conversion with 20 mmol / L ammonium acetate solution for 2 column volumes. Equilibrate with 1.8% acetic acid aqueous solution for 5 column volumes. Finally, elute with 50% acetonitrile-acetic acid aqueous solution containing 1.8% acetic acid according to the following gradient. Collect and combine the pure products to obtain a pure dilorelin acetate solution.

[0061]

[0062] (b) Concentration: Take the pure diloxaline acetate solution obtained after desalting and combining the pure products, and add it to a clean concentration vessel. Concentrate to approximately 60 mg / ml.

[0063] (c) Freeze-drying: The concentrated solution of diloxacin acetate obtained in the above steps is filtered through a 0.22 μm filter membrane and then evenly dispensed into stainless steel trays. The liquid layer thickness shall not exceed 1 cm. The product is frozen to -35±1℃ and held at that temperature for 2 hours, then freeze-dried under vacuum.

[0064] Example 8: Salt conversion of dilorelin acetate and pretreatment before lyophilization (a) Take approximately 100g of the purified dilorelin solution (containing pure peptide) with a purity of ≥98.5% and combine the fractions. Rotary evaporate the solution to remove excess acetonitrile. Equilibrate with 1.8% acetic acid aqueous solution for 2 column volumes. Load the concentrated dilorelin solution onto the column and perform ion conversion with 20 mmol / L ammonium acetate solution for 2 column volumes. Equilibrate with 1.8% acetic acid aqueous solution for 5 column volumes. Finally, elute with 50% acetonitrile-acetic acid aqueous solution containing 1.8% acetic acid according to the following gradient. Collect and combine the pure products to obtain a pure dilorelin acetate solution.

[0065]

[0066] (b) Concentration: Take the pure diloxaline acetate solution obtained after desalting and combining the pure products, and add it to a clean concentration vessel. Concentrate to approximately 60 mg / ml.

[0067] (c) Freeze-drying: Dissolve the pure diloxaline acetate obtained in the above steps into a concentrated solution and dispense it evenly into stainless steel trays. The liquid layer thickness should not exceed 1 cm. Freeze the product to -35±1℃ for 2 hours, and then freeze-dry it under vacuum.

[0068] Example 9: Salt conversion of dilorelin acetate and pretreatment before lyophilization (a) Take approximately 100g of the purified dilorelin solution (containing pure peptide) with a purity of ≥98.5% and combine the fractions. Rotary evaporate the solution to remove excess acetonitrile. Equilibrate with 1.8% acetic acid aqueous solution for 2 column volumes. Load the concentrated dilorelin solution onto the column and perform ion conversion with 20 mmol / L ammonium acetate solution for 2 column volumes. Equilibrate with 1.8% acetic acid aqueous solution for 10 column volumes. Finally, elute with 50% acetonitrile-acetic acid aqueous solution containing 1.8% acetic acid according to the following gradient. Collect and combine the pure products to obtain a pure dilorelin acetate solution.

[0069]

[0070] (b) Concentration: Take the pure diloxaline acetate solution obtained after desalting and combining the pure products, and add it to a clean concentration vessel. Concentrate to approximately 60 mg / ml.

[0071] (c) Freeze-drying: The concentrated solution of diloxacin acetate obtained in the above steps is filtered through a 0.22 μm filter membrane and then evenly dispensed into stainless steel trays. The liquid layer thickness shall not exceed 1 cm. The product is frozen to -35±1℃ and held at that temperature for 2 hours, then freeze-dried under vacuum.

[0072] Comparative Example 3: Salt Conversion of Diloxaline Acetate and Pretreatment Before Lyophilization (a) Take approximately 100g of the purified dilorelin solution (containing pure peptide) with a purity of ≥98.5% and combine the fractions. Rotary evaporate the solution to remove excess acetonitrile. Equilibrate with 0.2% acetic acid aqueous solution for 2 column volumes. Load the concentrated dilorelin solution onto the column and perform ion conversion with 20 mmol / L ammonium acetate solution for 2 column volumes. Equilibrate with 0.2% acetic acid aqueous solution for 5 column volumes. Finally, elute with 50% acetonitrile-acetic acid aqueous solution containing 0.2% acetic acid according to the following gradient. Collect and combine the pure products to obtain a pure dilorelin acetate solution.

[0073]

[0074] (b) Concentration: Take the pure diloxaline acetate solution obtained after desalting and combining the pure products, and add it to a clean concentration vessel. Concentrate to approximately 60 mg / ml.

[0075] (c) Freeze-drying: The concentrated, yogurt-like solution of dilorelin acetate obtained in the above steps was evenly dispensed into stainless steel trays, with the liquid layer thickness not exceeding 1 cm. The product was frozen to -35±1℃ for 2 hours, followed by vacuum freeze-drying. A comparison of the water solubility of Comparative Example 3 and Example 7 is shown in the figure. Figure 4 As shown.

[0076] Comparative Example 4: Salt Conversion of Diloxaline Acetate and Pretreatment Before Lyophilization (a) Take approximately 100g of the purified dilorelin solution (containing pure peptide) with a purity of ≥98.5% and combine the fractions. Rotary evaporate the solution to remove excess acetonitrile. Equilibrate with 0.2% acetic acid aqueous solution for 2 column volumes. Load the concentrated dilorelin solution onto the column and perform ion conversion with 20 mmol / L ammonium acetate solution for 2 column volumes. Equilibrate with 0.2% acetic acid aqueous solution for 5 column volumes. Finally, elute with 50% acetonitrile-acetic acid aqueous solution containing 0.2% acetic acid according to the following gradient. Collect and combine the pure products to obtain a pure dilorelin acetate solution.

[0077]

[0078] (b) Concentration: Take the pure diloxaline acetate solution obtained after desalting and combining the pure products, and add it to a clean concentration vessel. Concentrate to approximately 60 mg / ml.

[0079] (c) Freeze-drying: The pure diloxacin acetate obtained in the above steps is severely precipitated into a concentrated yogurt-like solution (e.g., Figure 5 (As shown) After filtration through a 0.22μm filter membrane, the product is evenly distributed into stainless steel trays, with the liquid layer thickness not exceeding 1 cm. The product is then frozen to -35±1℃ for 2 hours, followed by vacuum freeze-drying.

[0080] Comparative Example 5: Salt Conversion of Diloxaline Acetate and Pretreatment Before Lyophilization (a) Take approximately 100g of the purified dilorelin solution (containing pure peptide) with a purity of ≥98.5% and combine the fractions. Rotary evaporate the solution to remove excess acetonitrile. Equilibrate with approximately 2 column volumes of 0.5% acetic acid aqueous solution. Load the concentrated dilorelin solution onto the column and perform ion conversion with 20 mmol / L ammonium acetate solution for 2 column volumes. Equilibrate with 5 column volumes of 0.5% acetic acid aqueous solution. Finally, elute with a 50% acetonitrile-acetic acid aqueous solution containing 0.5% acetic acid according to the following gradient. Collect and combine the pure products to obtain a pure dilorelin acetate solution.

[0081]

[0082] (b) Concentration: Take the pure diloxaline acetate solution obtained after desalting and combining the pure products, and add it to a clean concentration vessel. Concentrate to approximately 30 mg / ml.

[0083] (c) Freeze-drying: Dispense the concentrated dilorelin acetate solution obtained in the above steps evenly into stainless steel trays, ensuring the liquid layer thickness does not exceed 1 cm. Freeze the product to -35±1℃ for 2 hours, then freeze-dry under vacuum.

[0084] Comparative Example 6: Salt Conversion of Diloxaline Acetate and Pre-treatment Before Lyophilization (a) Take approximately 100g of the purified dilorelin solution (containing pure peptide) with a purity of ≥98.5% and combine the fractions. Rotary evaporate the solution to remove excess acetonitrile. Equilibrate with 0.5% acetic acid aqueous solution for 2 column volumes. Load the concentrated dilorelin solution onto the column and perform ion conversion with 20 mmol / L ammonium acetate solution for 2 column volumes. Equilibrate with 0.5% acetic acid aqueous solution for 5 column volumes. Finally, elute with 50% acetonitrile-acetic acid aqueous solution containing 0.5% acetic acid according to the following gradient. Collect and combine the pure products to obtain a pure dilorelin acetate solution.

[0085]

[0086] (b) Concentration: Take the pure diloxaline acetate solution obtained after desalting and combining the pure products, and add it to a clean concentration vessel. Concentrate to approximately 60 mg / ml.

[0087] (c) Freeze-drying: Add approximately 450 ml (e.g., to the concentrated, yogurt-like solution of diloxacin acetate obtained in the above steps) to the solution. Figure 6 (As shown in Figure B) Dissolve the product in acetic acid, then filter it through a 0.22μm filter membrane and evenly distribute it into stainless steel trays. The liquid layer thickness should not exceed 1 cm. Freeze the product to -35±1℃ for 2 hours, then freeze-dry it under vacuum.

[0088] Comparative Example 7: Salt Conversion of Diloxaline Acetate and Pre-treatment Before Lyophilization (a) Take approximately 100g of the purified dilorelin solution (containing pure peptide) with a purity of ≥98.5% and combine the fractions. Rotary evaporate the solution to remove excess acetonitrile. Equilibrate with 1.3% acetic acid aqueous solution for 2 column volumes. Load the concentrated dilorelin solution onto the column and perform ion conversion with 20 mmol / L ammonium acetate solution for 2 column volumes. Equilibrate with 1.3% acetic acid aqueous solution for 5 column volumes. Finally, elute with 50% acetonitrile-acetic acid aqueous solution containing 1.3% acetic acid according to the following gradient. Collect and combine the pure products to obtain a pure dilorelin acetate solution.

[0089]

[0090] (b) Concentration: Take the desalted and combined pure product solution of dilorelin acetate and add it to a clean concentration vessel. Concentrate to approximately 60 mg / ml, the solution as follows: Figure 7 As shown.

[0091] (c) Freeze-drying: The concentrated solution of diloxacin acetate obtained in the above steps is filtered through a 0.22 μm filter membrane and then evenly dispensed into stainless steel trays. The liquid layer thickness shall not exceed 1 cm. The product is frozen to -35±1℃ and held at that temperature for 2 hours, then freeze-dried under vacuum.

[0092] Comparative Example 8: Salt Conversion of Diloxaline Acetate and Pre-treatment Before Lyophilization (a) Take approximately 100g of the purified dilorelin solution (containing pure peptide) with a purity of ≥98.5% and combine the fractions. Rotary evaporate the solution to remove excess acetonitrile. Equilibrate with approximately 2 column volumes of 1.5% acetic acid aqueous solution. Load the concentrated dilorelin solution onto the column and perform ion conversion with 20 mmol / L ammonium acetate solution for 2 column volumes. Equilibrate with 5 column volumes of 1.5% acetic acid aqueous solution. Finally, elute with a 50% acetonitrile-acetic acid aqueous solution containing 1.5% acetic acid according to the following gradient. Collect and combine the pure products to obtain a pure dilorelin acetate solution.

[0093]

[0094] (b) Concentration: Take the desalted and combined pure product solution of dilorelin acetate and add it to a clean concentration vessel. Concentrate to approximately 60 mg / ml, the solution as follows: Figure 8 As shown.

[0095] (c) Freeze-drying: The concentrated solution of diloxacin acetate obtained in the above steps is filtered through a 0.22 μm filter membrane and then evenly dispensed into stainless steel trays. The liquid layer thickness shall not exceed 1 cm. The product is frozen to -35±1℃ and held at that temperature for 2 hours, then freeze-dried under vacuum.

[0096] Table 3 below shows the dilorelin acetate salt conversion and pre-freeze-drying treatments of Examples 6-8 and Comparative Examples 3-8 of this invention. The final freeze-drying was achieved through adjustments to different acetic acid concentrations, comparisons of post-equilibration times, and control of the concentration ratio before freeze-drying. The analysis also includes comparisons of purity before and after salt conversion, changes in acetic acid content, precipitation during the concentration process before freeze-drying, and the solubility of the final product. Specific analytical results are shown in Table 3. Table 3. Diloxaline acetate salt conversion and pre-lyophilization treatment

[0097] Prior to this invention, following the salt conversion method of Comparative Example 3, the product was concentrated and then freeze-dried, resulting in extremely low water solubility. A comparison of the experimental data from Examples 7-9 and Comparative Examples 3-8 in Table 2 clearly shows that dilorelin acetate requires combination with high-concentration acetic acid during preparation. In Table 3, neither the comparative examples nor the examples show significant impact on purity before and after salt conversion. However, the concentration of acetic acid during salt conversion and the concentration ratio before freeze-drying both affect precipitation. Examples 7, 8, and 9 all achieve the final objective of this invention. The main difference between Examples 7 and 9 is the length of the equilibration time after salt conversion; five volumes of equilibration are sufficient. Considering factors such as product stability and timeliness, Example 7 is the optimal example.

[0098] In summary, based on the analytical results, high-purity, water-soluble dilorelin acetate can be efficiently prepared according to Examples 1, 3, and 7. The prepared dilorelin acetate product was then subjected to full-sequence peptide analysis by mass spectrometry by a third party. The mass spectrometry chromatogram is shown below. Figure 10 .

[0099] The test results prove that the relative molecular mass of dilorelin acetate prepared by our factory is consistent with the theoretical value. The sample was analyzed by liquid chromatography-mass spectrometry (LC-MS), using primary and secondary ion data, combined with theoretical sequence and configuration library construction for matching analysis. The full sequence identification result was: Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-NHEt. Simultaneously, the prepared dilorelin acetate product and a commercial reference standard purchased from BACHEM (product code: B-4066809, CAS number: 57773-65-6) were analyzed by HPLC: the purity of the BACHEM commercial reference standard was 99.54%, and the purity of the dilorelin acetate test sample prepared by our factory was 99.50%, with both having the same retention time.

[0100] The foregoing description of embodiments of the present invention, through which those skilled in the art are able to implement or use the present invention, will be readily apparent to those skilled in the art. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novelty disclosed herein.

Claims

1. A method for preparing high-purity dilorelin acetate soluble in water, characterized in that, The method includes the following sequential steps: 1) Dissolution and pre-filtration: Dissolve crude diloxacin in purified water, stir, and filter; add 1%-10% (v / v) acetonitrile to the purified water to aid dissolution; 2) Adsorption and gradient elution: A C18 packed column was used. The filtered crude dilorelin peptide solution was loaded onto the column for sample adsorption. Gradient elution was performed using triethylamine phosphate buffer as mobile phase A and acetonitrile as mobile phase B. The pH of mobile phase A was 2.3–5.0, the sample flow rate was 600–1000 ml / min, and the elution flow rate was 1200–2000 ml / min. The gradient elution procedure was as follows: Time: 0-4 min; Mobile phase A: 95%; Mobile phase B: 5% Time 4-6 min, mobile phase A 95%, mobile phase B 5%; Time: 6-64 min; Mobile phase A: 80%; Mobile phase B: 20% Time: 64-100 min; Mobile phase A: 74%; Mobile phase B: 26% 3) Concentration: Evaporate the combined solutions to remove excess acetonitrile; 4) Salt conversion: Using a C18 packed column, load the concentrated dilorelin acetate solution onto the column, perform ion conversion with 15-25 mmol / L ammonium acetate solution for 1.5-5.0 column volumes, then equilibrate with 1.6%-2.0% (v / v) acetic acid aqueous solution for 4-10 column volumes, and finally elute with acetonitrile-acetic acid aqueous solution, collect and combine the pure products; 5) Concentrate and then freeze dry.

2. The method according to claim 1, characterized in that, Step 1) uses a 0.22μm to 0.45μm microporous membrane for filtration.

3. The method according to claim 1, characterized in that, In step 2), the C18 packed column used is a DAC300C18 column with a length of 50cm, a diameter of 30cm, and a packing particle size of 10μm. And / or, equilibrate 1.5–5.0 column volumes with phosphate buffer before use.

4. The method according to claim 1, characterized in that, In step 4), the C18 packed column used is a DAC300C18 column with a length of 50cm, a diameter of 30cm, and a packing particle size of 10μm. And / or, equilibrate 1.5-5.0 column volumes with an aqueous acetic acid solution of 0.2%-2.5% (v / v) before use; And / or, perform ion conversion with 15-25 mmol / L ammonium acetate solution for 1.5-5.0 column volumes, and equilibrate with 1.5%-3.0% (v / v) aqueous acetic acid solution for 4-10 column volumes.

5. The method according to claim 1, characterized in that, Step 4) contains an aqueous solution of acetic acid and acetonitrile containing 0.2%-2.5% by volume acetic acid and 40%-60% by volume acetonitrile.

6. The method according to claim 1, characterized in that, In step 5), the collected liquid is concentrated to a peptide concentration of 30–100 mg / mL under reduced pressure. After removing acetonitrile, it is filtered through a 0.22 μm to 0.45 μm filter membrane, dispensed into trays with a liquid layer thickness ≤10 mm, and then freeze-dried under vacuum after being kept at -35±1℃ for 1.5–3.0 h.

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