Process for the purification of the peptide etelcalcetide hydrochloride

Abstract

The application belongs to the technical field of eticaptide hydrochloride refining, and particularly relates to a refining method of eticaptide hydrochloride. The method comprises the following steps: dissolving eticaptide hydrochloride crude product in water and filtering; using DAC100 double columns and C18 reverse phase chromatography filler, using phosphate aqueous solution as first aqueous phase mobile phase A and methanol as mobile phase B to perform the purification part of the whole method, replacing the first aqueous phase mobile phase A with second aqueous phase mobile phase A' ammonium chloride aqueous solution to perform the purification and salt conversion in the later part of the method, and obtaining a sample solution; performing nanofiltration concentration on the sample solution, washing with purified water, finally washing with a low-concentration hydrochloric acid solution until the pH is adjusted, and then performing nanofiltration concentration; filtering the salt conversion aqueous solution and freeze-drying to obtain eticaptide hydrochloride pure product. The method is easy to implement, has a large sample loading amount, and can simultaneously perform purification and salt conversion on the double-column preparation liquid phase, and has high purity, high yield and less single impurities.

Description

Technical Field

[0001] This invention belongs to the field of itlactoide hydrochloride purification technology, specifically relating to a purification method for itlactoide hydrochloride. Background Technology

[0002] Iticatide hydrochloride, chemically named N-acetyl-D-cysteyl-S-(L-cysteine ​​disulfide)-D-alanyl-D-arginyl-D-arginyl-D-arginyl-D-alanyl-D-arginamide, hydrochloride, is a calcimimetic agent that synonymously regulates calcium-sensitive receptors (CaSRs). Iticatide binds to CaSRs and enhances receptor activation through extracellular calcium. Activation of CaSRs on parathyroid chief cells reduces parathyroid hormone (PTH) secretion. Its structural formula is as follows: , C 38 H 73 N 21 O 10 S²·xHCl (4≤x≤5) Etecalcetide (Parsabiv) is a calcium-sensitive receptor agonist used to treat secondary hyperparathyroidism (SHPT) following dialysis in adults with chronic kidney disease. This intravenous injection was developed by Kai Pharmaceuticals, Inc. (USA), which was acquired by Amgen Biopharmaceuticals, Inc. (USA), and Ono Pharmaceutical Co., Ltd. (Japan) acquired the rights to manufacture and sell it in Japan. The European Medicines Agency (EMA) first approved its marketing on November 11, 2016; the Pharmaceuticals and Medical Devices Agency (PMDA) of Japan approved its marketing in Japan in February 2017; and the U.S. Food and Drug Administration (FDA) approved its marketing in China on April 3, 2017. It was approved for import into China on May 6, 2023. A comparison of the international markets for cinacalcetide (sensipar / Mimpara) and etelacide (Parsabiv) is as follows: etelacide (Parsabiv) is considered the successor to cinacalcetide (sensipar / Mimpara) and has a promising market prospect.

[0003] Chinese invention patent CN108101959A, published on June 1, 2018, discloses a method for preparing high-purity peptides or their analogues. The purification process involves high-performance liquid chromatography (HPLC) to prepare and purify the peptides, collecting the target peak fraction. The stationary phase is C18, mobile phase A is an aqueous solution of phosphate, formate, or acetate, and mobile phase B is methanol or acetonitrile. Salt transfer is performed using an ion exchange resin, and the product is obtained by freeze-drying. This technique has a major drawback: the sample loading capacity is relatively small, making it difficult to apply to large-scale production.

[0004] Chinese invention patent CN112552376A, published on March 26, 2021, discloses a method for purifying verakatide. The purification process involves first treating the crude product by dissolving it in purified water, then adding an equal volume of acetonitrile and saturated saline solution to precipitate it. After dissolving the solid in water, a C18 reversed-phase chromatographic column is used with gradient elution using 0.2% hydrochloric acid as phase A and acetonitrile as phase B to obtain the pure product. Then, ammonium chloride solution is used as phase A, and after ion exchange in phase B, phase A is replaced with 0.1% hydrochloric acid aqueous solution to obtain a salt-converted eluent. This eluent is concentrated under reduced pressure and then lyophilized to obtain the product. This technique has drawbacks: the sample loading volume remains small, making it difficult to apply to large-scale production. Furthermore, separating purification and salt conversion leads to cumbersome operations and disrupts the continuous process, increasing production time and thus increasing risks.

[0005] As can be seen from the existing technologies, current purification methods for etakatide hydrochloride mainly involve high-pressure preparative HPLC purification followed by freeze-drying to obtain the product. The main problems with these methods are: firstly, the product has high polarity, limiting the adjustment range; secondly, the sample loading volume is small, making it unsuitable for large-scale production; and finally, the salt conversion process, performed via ion exchange or reverse HPLC, requires separate operation, increasing operation time and risk. Therefore, to overcome the shortcomings of existing technologies and improve the purity of etakatide hydrochloride, further research is needed on purification methods to enhance product purity. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a purification method for itcatide hydrochloride that is simple to operate and easy to implement.

[0007] The purification method for etakatide hydrochloride according to the present invention includes the following steps: (1) Dissolve crude itcatide hydrochloride in purified water and filter it through a 0.45 μm aqueous membrane to obtain a crude solution; (2) The chromatographic system was equilibrated using a first aqueous mobile phase A and an organic mobile phase B, wherein the first aqueous mobile phase A was an aqueous solution of hydrogen phosphate; the crude product solution was injected into a dual-column chromatographic system packed with C18 reversed-phase chromatographic packing material, and an automated purification and salt conversion program was performed to collect the solution containing etakatide hydrochloride: The chromatographic column after sample loading is eluted with a mixed solution of the first aqueous mobile phase A and mobile phase B to separate eticatrix hydrochloride from impurities. During the elution process, the first aqueous mobile phase A is switched to the second aqueous mobile phase A', which is an aqueous solution of ammonium chloride. This allows for in-situ ion exchange of eticatrix hydrochloride, which has been separated from impurities, on the chromatographic column, converting it into the form of hydrochloride salt. The fraction containing the target product eticatrix hydrochloride is then collected.

[0008] After the product undergoes a cycle of the method procedure, a qualified product solution flows out from sample port No. 2, with a purity greater than 99.00% and a single impurity of less than 0.2%.

[0009] The execution sequence of the automatic purification and salt conversion program in step (2) is as follows: a. From the start of system operation to the first minute, flush and equilibrate the system with mobile phase B; b. Starting from minute 1, inject the crude product solution into the chromatographic system at a preset flow rate for 1 minute; c. Starting from the 2nd minute, execute a gradient elution procedure: First, for the next 3.5 minutes, elute with an eluent consisting of a first aqueous mobile phase A and a mobile phase B in a volume ratio of 97:3; then, for the next 4 minutes, elute with an eluent whose composition changes linearly to an eluent with a first aqueous mobile phase A to mobile phase B volume ratio of 40:60. d. Starting from the 6th minute, the main purification cycle based on the real-time ultraviolet absorption signal is initiated: when the ultraviolet absorption signal value detected at a wavelength of 220 nm exceeds 2000, the system automatically switches the eluent to mobile phase B; when the signal value is below 2000, the system automatically switches the eluent to the first aqueous mobile phase A; this cycle continues until the total system running time reaches 41.8 minutes. e. Between 41.8 minutes and 42.4 minutes, the system is flushed sequentially with mobile phase B and the first aqueous mobile phase A; f. Between 42.4 and 42.9 minutes, a salt conversion step is performed: the system automatically replaces the first aqueous mobile phase A with the second aqueous mobile phase A', an ammonium chloride aqueous solution; g. Starting from minute 42.9, the final elution and collection procedure is executed: First, elution is performed for 4 minutes using an eluent composed of ammonium chloride solution and mobile phase B at a volume ratio of 40:60; then, the eluent composition is linearly changed to an ammonium chloride solution and mobile phase B at a volume ratio of 90:10 for another 4 minutes; finally, under this 90:10 elution condition, an automatic collection procedure based on the UV signal threshold is executed. When the signal value detected at a wavelength of 220 nm exceeds 1500, the system automatically switches the fraction collection port to the product collection position until the signal value is below 1500, completing the collection of the product solution. The acquisition process uses two wavelengths, 210 nm and 220 nm, respectively. The product tailing is different at the two wavelengths. The tailing is smaller at 220 nm, making it easier to achieve automatic switching by determining the threshold. 210 nm provides better monitoring of impurity removal during the process.

[0010] The first aqueous mobile phase A is an aqueous solution of hydrogen phosphate with a concentration of 40-50 mmol / L. The hydrogen phosphate includes potassium dihydrogen phosphate, sodium dihydrogen phosphate, and ammonium dihydrogen phosphate, with a pH of 2-4, preferably potassium dihydrogen phosphate with a pH of 3 and a concentration of 50 mmol / L.

[0011] In step (1), the crude etakatide hydrochloride is dissolved in purified water to a concentration of 100-150 mg / mL (i.e., etakatide hydrochloride is expressed in mg and purified water in mL). The sample volume is 45-60 g.

[0012] The injection volume of the solution containing crude etakatide hydrochloride in step (2) is 350 mL to 400 mL.

[0013] Following step (2), the method further includes: (3) The fraction containing the target product itcatide hydrochloride collected in step (2) is concentrated by nanofiltration and rinsed with purified water to desalinate. (4) After rinsing with a low-concentration hydrochloric acid solution to adjust the pH of the fraction, it is concentrated again by nanofiltration; (5) The solution after step (4) is freeze-dried to obtain purified itcatide hydrochloride.

[0014] The volume of purified water used for rinsing in step (3) should be no less than three times the volume of the fraction before nanofiltration, and it should be added in 3-5 batches.

[0015] The pH in step (4) is 3-4, preferably pH=3, and the hydrochloric acid mass concentration is 0.1%~0.3%, preferably 0.1%.

[0016] The freeze-drying process in step (5) involves freezing at -10°C for no less than 20 hours and sublimation at no more than 25°C.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The refining method described in this invention is simple to operate and easy to implement, and completes multiple operation steps in one system, avoiding the impact of multiple operation steps on the product.

[0018] (2) The purity of itcatide hydrochloride obtained by the purification method described in this invention is greater than 99.00%, the unknown single impurity is less than 0.2%, and the yield is 60-70%.

[0019] (3) The present invention achieves a better process sequence of "purification first and then salt conversion", which improves purification efficiency and product quality.

[0020] This invention, through precise program control, achieves effective separation of the target product from most impurities using an acidic phosphate system during chromatographic separation, followed by precise online salt conversion of the relatively pure target product peak that is about to elute. Compared to the "salt conversion first, then purification" approach, this "purification first, then salt conversion" logical sequence is more scientific, avoiding ineffective ion exchange of a large number of impurities, reducing the load on the chromatographic packing material, and potentially resulting in better peak shape and resolution, thereby further ensuring the purity of the final product.

[0021] (4) This invention provides a complete industrial production solution, solving the bottleneck of large-scale production. This invention creatively integrates the method with modern industrial unit operations such as the DAC dynamic axial compression chromatography system (solving the sample loading problem) and nanofiltration technology (for gentle and efficient concentration and desalting). This complete process package successfully transforms a sophisticated chromatographic separation technology into a stable, efficient, and high-throughput industrial production process, with yields and purity meeting commercial production requirements. Attached Figure Description

[0022] Figure 1 This is an HPLC chromatogram of the purified itcatide hydrochloride from Example 1.

[0023] Figure 2 This is an HPLC chromatogram of the purified itcatide hydrochloride from Example 2.

[0024] Figure 3 This is an HPLC chromatogram of crude etakatide hydrochloride used in Examples 1 and 2. Detailed Implementation

[0025] The present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0026] Unless otherwise specified, all raw materials used in the examples were commercially available.

[0027] The 0.45µm aqueous membrane was purchased from Shanghai Xinya Purification Device Factory.

[0028] Crude peptide of itcatide hydrochloride synthesized by solid-phase synthesis: Using 3 kg of Rink amide resin (degree of substitution 0.6-0.8 mmol / g) as the stationary phase, The protecting group was removed by stirring in 15 L of 20% DBLK solution (20% piperidine / DMF solution, volume percentage). After washing with DMF 6 times, condensation was carried out with 6 eq of 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide as condensing agents, and 3 eq of Fmoc-Pbf-D-arginine was added. After the reaction was detected by ninhydrin colorimetric reagent, the reaction was blocked with 6 eq of acetic anhydride and 6 eq of N,N-diisopropylethylamine, and washed three times. Subsequently, the protection was removed sequentially using 20% ​​DBLK solution, and Fmoc-D-alanine, Fmoc-Pbf-D-arginine, Fmoc-Pbf-D-arginine, Fmoc-Pbf-D-arginine, Fmoc-D-alanine, and Fmoc-S-triphenylmethyl-D-cysteine ​​were condensed in sequence using 6 eq of 1-hydroxybenzotriazole and N,N-diisopropylethylamine as condensing agents, with a total amino acid amount of 3 eq. After condensation, the C-terminus of the amino acid sequence was acetylated using 6 eq of acetic anhydride and 6 eq of N,N-diisopropylethylamine. The obtained resin was cleaved using a cleavage reagent (trifluoroacetic acid: triisopropylsilane: m-phenylenediamine: water = 90:2.5:2.5:5, volume ratio) at 10-20℃. The resulting lysate was added to 10 times its volume of isopropyl ether and stirred to induce crystallization. After centrifugation at high speed, the crystals were dried to obtain the amino acid backbone. The amino acid backbone was then activated with dithiodipyridine in purified water to activate the thiol groups. After the reaction was complete, the mixture was extracted six times with isopropyl ether. Cysteine ​​hydrochloride was added to the aqueous phase and stirred at 0-5℃ for 4 hours. The mixture was then added dropwise to 10 times its volume of isopropanol and stirred to induce crystallization. After centrifugation at high speed, the crystals were dried to obtain crude itlactide hydrochloride.

[0029] Example 1 The purification method of etakatide hydrochloride in this embodiment includes the following steps: (1) Dissolve 45g (8.46mmol, based on 5 trifluoroacetates, 30% content) of crude itcatide hydrochloride obtained by solid-phase synthesis in 400mL of purified water and filter using a 0.45um aqueous membrane to obtain crude product solution. (2) The crude solution from step (1) was eluted using a DAC100 dual-column system (C18 reversed-phase chromatography packing material) with the first aqueous mobile phase A adjusted to pH 2 using 50 mmol / L potassium dihydrogen phosphate aqueous solution, and methanol as mobile phase B. The first aqueous mobile phase A was replaced with the second aqueous mobile phase A' and 50 mmol / L ammonium chloride aqueous solution in the 13B section. The specific purification and salt conversion procedure execution sequence is shown in Table 1 below: Table 1. Execution sequence of purification and salt conversion procedures

[0030] The sample solution is collected through port 2 of the fraction collector.

[0031] (3) The sample solution obtained above was concentrated to 500 mL by nanofiltration and then rinsed with 4 L of purified water three times.

[0032] (4) Finally, adjust the pH to 3.1 with 10 mL of 0.1 wt% hydrochloric acid solution and concentrate to 500 mL.

[0033] (5) Freeze-dry the solution after step (4) at -45°C for 8 hours. After freezing, turn on the vacuum and keep the vacuum level less than 50Pa for drying. Increase the temperature by 5°C every 3 hours. After keeping the temperature at 0°C for 10 hours, increase the temperature by 5°C every 2 hours. Keep the temperature constant at 20°C until the product is freeze-dried to obtain refined itcatide hydrochloride.

[0034] The HPLC peak table of the lyophilized itlactoide hydrochloride in this embodiment is shown in Table 2, and the HPLC chromatogram is shown in Figure 3. Figure 1 As shown. The yield (calculated as recovery rate) of this embodiment is 65%, the purity is 99.605%, and the maximum unknown single impurity is 0.078%.

[0035] Table 2. HPLC peak table of itlactoide hydrochloride

[0036] Example 2 The purification method of etakatide hydrochloride in this embodiment includes the following steps: (1) Dissolve 60g (11.28mmol, based on 5 trifluoroacetates, 30% content) of crude itcatide hydrochloride obtained by solid-phase synthesis in 400mL of purified water and filter using a 0.45um aqueous membrane to obtain crude product solution. (2) The crude solution from step (1) was eluted using a DAC100 dual-column system (C18 reversed-phase chromatography packing material) with the first aqueous mobile phase A adjusted to pH 2 using 50 mmol / L potassium dihydrogen phosphate aqueous solution, and methanol as mobile phase B. Elution was performed according to the established method. In the 13B section, the first aqueous mobile phase A was replaced with the second aqueous mobile phase A' and 50 mmol / L ammonium chloride aqueous solution. The specific purification and salt conversion procedure execution sequence is shown in Table 3 below: Table 3. Execution sequence of purification and salt conversion procedures

[0037] The sample solution is collected through port 2 of the fraction collector.

[0038] (3) The sample solution obtained above was concentrated to 500 mL by nanofiltration and then rinsed with 1.5 L of purified water three times.

[0039] (4) Finally, adjust the pH to 3.6 with 10 mL of 0.3 wt% hydrochloric acid solution and concentrate to 500 mL.

[0040] (5) Freeze-dry the solution after step (4) at -45℃ for 10 hours. After freezing, turn on the vacuum and keep the vacuum level less than 50Pa for drying. Increase the temperature by 5℃ every 3 hours. After keeping the temperature at 0℃ for 10 hours, increase the temperature by 5℃ every 2 hours. Keep the temperature at 20℃ until the product is freeze-dried to obtain refined itcatide hydrochloride.

[0041] The HPLC peak table of purified itlactoide hydrochloride in this embodiment is shown in Table 4, signal: DAD1B, Sig=210,4, Ref=360,100. The HPLC chromatogram is shown below. Figure 2 As shown. The yield (calculated as recovery rate) of this embodiment is 68%, the purity is 99.613%, and the maximum unknown single impurity is 0.117%.

[0042] Table 4. HPLC peak table of itlactoide hydrochloride

[0043] The difference between Example 2 and Example 1 is that the injection volume is increased from 45g to 60g, the buffer salt is changed from potassium dihydrogen phosphate to sodium dihydrogen phosphate, and the hydrochloric acid mass concentration is increased to 0.3%.

[0044] Example 2 and Example 1 used the same batch of crude product. The HPLC peak table of the crude product is shown in Table 5, and the HPLC chromatograms are shown in Figure 1. Figure 3 As shown.

[0045] Table 5. HPLC Peak Table of Crude Product

[0046] As can be seen from the comparison between Example 2 and Example 1, increasing the injection volume and adjusting the buffer salt and hydrochloric acid will lead to an increase in the content of some impurities in the product, but 60g can still meet the limit requirements, and further increasing the injection volume will have the risk of exceeding the limit.

Claims

1. A method for purifying etakatide hydrochloride, characterized in that, Includes the following steps: (1) Dissolve crude itcatide hydrochloride in purified water and filter to obtain crude solution; (2) The chromatographic system was equilibrated using a first aqueous mobile phase A and an organic mobile phase B, wherein the first aqueous mobile phase A was an aqueous solution of hydrogen phosphate; the crude product solution was injected into a dual-column chromatographic system packed with C18 reversed-phase chromatographic packing material, and an automated purification and salt conversion program was performed to collect the solution containing etakatide hydrochloride: The chromatographic column after sample loading is eluted with a mixed solution of the first aqueous mobile phase A and mobile phase B to separate eticatrix hydrochloride from impurities. During the elution process, the first aqueous mobile phase A is switched to the second aqueous mobile phase A', which is an aqueous solution of ammonium chloride. This allows for in-situ ion exchange of eticatrix hydrochloride, which has been separated from impurities, on the chromatographic column, converting it into the form of hydrochloride salt. The fraction containing the target product eticatrix hydrochloride is then collected.

2. The purification method for iticatide hydrochloride according to claim 1, characterized in that, The execution sequence of the automatic purification and salt conversion program in step (2) is as follows: a. From the start of system operation to the first minute, flush and equilibrate the system with mobile phase B; b. Starting from minute 1, inject the crude product solution into the chromatographic system at a preset flow rate for 1 minute; c. Starting from the 2nd minute, execute a gradient elution procedure: First, for the next 3.5 minutes, elute with an eluent consisting of a first aqueous mobile phase A and a mobile phase B in a volume ratio of 97:3; then, for the next 4 minutes, elute with an eluent whose composition changes linearly to an eluent with a first aqueous mobile phase A to mobile phase B volume ratio of 40:

60. d. Starting from the 6th minute, the main purification cycle based on the real-time ultraviolet absorption signal is initiated: when the ultraviolet absorption signal value detected at a wavelength of 220 nm exceeds 2000, the system automatically switches the eluent to mobile phase B; when the signal value is below 2000, the system automatically switches the eluent to the first aqueous mobile phase A; this cycle continues until the total system running time reaches 41.8 minutes. e. Between 41.8 minutes and 42.4 minutes, the system is flushed sequentially with mobile phase B and the first aqueous mobile phase A; f. Between 42.4 and 42.9 minutes, a salt conversion step is performed: the system automatically replaces the first aqueous mobile phase A with the second aqueous mobile phase A', an ammonium chloride aqueous solution; g. Starting from minute 42.9, the final elution and collection procedure is executed: First, elution is performed for 4 minutes using an eluent composed of ammonium chloride solution and mobile phase B at a volume ratio of 40:60; then, the eluent composition is linearly changed to an ammonium chloride solution and mobile phase B at a volume ratio of 90:10 for 4 minutes; finally, under this 90:10 elution condition, an automatic collection procedure based on the ultraviolet signal threshold is executed. When the signal value detected at a wavelength of 220 nm exceeds 1500, the system automatically switches the fraction collection port to the product collection position until the signal value is below 1500, completing the collection of the product solution.

3. The method according to claim 1, characterized in that: The first aqueous mobile phase A is a 40-50 mmol / L aqueous solution of hydrogen phosphate with a pH of 2-4. The hydrogen phosphate includes potassium dihydrogen phosphate, sodium dihydrogen phosphate, and ammonium dihydrogen phosphate.

4. The purification method for iticatide hydrochloride according to claim 1, characterized in that, In step (1), the crude etakatide hydrochloride is dissolved in purified water to a concentration of 100-150 mg / mL.

5. The purification method for iticatide hydrochloride according to claim 4, characterized in that, The injection volume of the crude solution containing etakatide hydrochloride in step (2) is 350 mL to 400 mL.

6. The purification method for iticatide hydrochloride according to claim 1, characterized in that, Following step (2), the method further includes: (3) The fraction containing the target product itcatide hydrochloride collected in step (2) is concentrated by nanofiltration and rinsed with purified water to desalinate. (4) After rinsing with a low-concentration hydrochloric acid solution to adjust the pH of the fraction, it is concentrated again by nanofiltration; (5) The solution after step (4) is freeze-dried to obtain purified itcatide hydrochloride.

7. The purification method for iticatide hydrochloride according to claim 6, characterized in that, The volume of purified water used for rinsing in step (3) should be no less than three times the volume of the fraction before nanofiltration, and it should be added in 3-5 batches.

8. The purification method for iticatide hydrochloride according to claim 6, characterized in that, The pH value in step (4) is 3-4.

9. The purification method for iticatide hydrochloride according to claim 6, characterized in that, The hydrochloric acid concentration in step (4) is 0.1% to 0.3%.

10. The purification method of itlactoide hydrochloride according to claim 6, characterized in that, The freeze-drying process described in step (5) involves freezing at temperatures of -10°C and below for a time of not less than 20 hours and sublimation at temperatures not higher than 25°C.

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