Purification method of tilpotide

Abstract

The invention relates to the technical field of polypeptide synthesis and purification, in particular to a purification method of tirpotide, which comprises the following steps: dissolving a tirpotide crude product in an ammonia water solution, and filtering to obtain a solution containing tirpotide; the method comprises the following steps: carrying out first purification on a solution containing tilpotitide by taking a reverse silica gel filler as a stationary phase, a polar organic solvent as a mobile phase A, an ammonium trifluoroacetate aqueous solution as a mobile phase B and purified water as a mobile phase C, so as to obtain a component of which the single impurity content is less than 0.5%; carrying out secondary purification on the obtained component by taking a reverse silica gel filler as a stationary phase, a polar organic solvent as a mobile phase A, an ammonium formate aqueous solution as a mobile phase B and purified water as a mobile phase C, so as to obtain a component of which the single impurity is less than 0.1%; and concentrating, filtering and drying the obtained components. The method has the advantages of short purification period, high impurity removal efficiency, high recovery rate and low environmental protection cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polypeptide synthesis and purification, and particularly relates to a purification method of tirzepatide. BACKGROUND

[0002] Tirzepatide is a glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) dual receptor agonist, and its molecular structure contains two unnatural amino acids and a long-chain fatty acid modification at position 20. Tirzepatide needs to be prepared by combining solid-phase synthesis and liquid-phase synthesis. During the synthesis process, impurities such as deletion peptides, cleavage impurities and oxidation impurities are easily produced, which have high polarity and solubility similar to the target peptide, thereby significantly increasing the difficulty of separation.

[0003] The existing tirzepatide purification process generally adopts a process of two-step purification and then separate salt conversion. However, the process has the following problems: (1) poor compatibility of the mobile phase system: the first step of purification mostly uses high-concentration ice acetic acid aqueous solution as an ion pair reagent. The strong acidity and high volatility not only cause serious corrosion to the HPLC equipment (pump body, sealing ring, pipeline), thereby shortening the service life of the equipment by more than 30%, but also cannot be used with mass spectrometry (MS) for online impurity identification, thereby making it difficult to trace the impurities during the process development stage and reducing the quality control efficiency. (2) risk of non-volatile salt residue: the second step of purification commonly uses non-volatile buffer salts such as ammonium dihydrogen phosphate and disodium hydrogen phosphate. The residual phosphate and ammonium ions in the product after purification need to be removed through a separate salt conversion step (such as reverse phase chromatography and isoelectric point precipitation), thereby increasing the process complexity and equipment investment. (3) low efficiency of the salt conversion step: the separate salt conversion needs to go through operations such as dilution, concentration, acid-base adjustment and centrifugal washing, thereby prolonging the process cycle of a single batch by 8-12 hours. In addition, the target peptide is prone to dead adsorption with the chromatographic filler or to loss due to uneven particle size during isoelectric point precipitation, and the overall yield is usually less than 85%. (4) product stability and environmental protection hidden danger: the pH value fluctuation (usually 4.0-8.0) during the salt conversion process easily causes the denaturation of tirzepatide, or affects the purity of the final product due to improper control of organic residues. At the same time, a large amount of high-salt organic waste liquid is generated during the salt conversion step, thereby increasing the treatment cost and not meeting the development trend of green pharmacy. SUMMARY

[0004] The purpose of the present application is to provide a purification method of tirzepatide, which has a short purification cycle, high impurity removal efficiency, high recovery rate and low environmental protection cost.

[0005] To achieve the above-mentioned purpose, the present application provides a purification method of tirzepatide, which comprises the following steps:

[0006] S1, dissolving tirzepatide crude product in an ammonia water solution, and obtaining a solution containing tirzepatide after filtration;

[0007] S2, the solution containing telopeptide obtained in S1 is subjected to first purification, with reverse silica gel filler as stationary phase, with polar organic solvent as mobile phase A phase, with trifluoroacetate ammonium aqueous solution as mobile phase B phase, and with purified water as mobile phase C phase, to obtain a component with single impurity less than 0.5%;

[0008] S3, the component obtained in S2 is subjected to second purification, with reverse silica gel filler as stationary phase, with polar organic solvent as mobile phase A phase, with formate ammonium aqueous solution as mobile phase B phase, and with purified water as mobile phase C phase, to obtain a component with single impurity less than 0.1%;

[0009] S4, the component obtained in S3 is subjected to concentration, filtration and drying.

[0010] Preferably, in S1, the pH of the ammonia solution is 9.1, and ultrasonic is used to assist dissolution, and 2‰ ammonia or 1% formic acid is used to fine-tune the pH to 9.0-9.3, and a 0.45 μm water filter membrane is used for filtration.

[0011] Preferably, in S2, the stationary phase of the first purification is Bonnasil HSC8 Pro reverse-phase silica gel filler, with a particle size of 10 μm and a pore size of 100 A.

[0012] Preferably, in S2, the concentration of the trifluoroacetate ammonium aqueous solution is 20 mM, and ammonia is used to adjust the pH to 3.0±0.1.

[0013] Preferably, in S2, the first purification is performed by linear gradient elution according to the following parameters:

[0014] Time Flow rate Phase A Phase B Phase C 0.00 min 480 mL / min 12％ 83％ 5％ 22.00 min 480 mL / min 28％ 67％ 5％ 78.00 min 480 mL / min 36％ 62％ 2％ 108.00 min 480 mL / min 43％ 55％ 2％ 108.01 min 480 mL / min 78％ 22％ 0％ 120.00 min 480 mL / min 12％ 83％ 5％ .

[0015] Preferably, in S3, the stationary phase of the second purification is UniSil 10-80 C18 reverse-phase silica gel filler, with a particle size of 5 μm and a pore size of 100 A.

[0016] Preferably, in S3, the concentration of the formate ammonium aqueous solution is 20 mM, and formic acid is used to adjust the pH to 6.5±0.1.

[0017] Preferably, in S3, the second purification is performed by linear gradient elution according to the following parameters:

[0018]

[0019]

[0020] Preferably, in S4, the concentration is to concentrate the components obtained in S3 in a water bath at 28-30 DEG C, under the condition of a cold trap at-15 DEG C, for 4-5 h to obtain a purified solution with a concentration of 200-250 g / L; the filtration is to sterilize and filter the purified solution through a 0.22 mu polyvinylidene fluoride membrane, and the drying is freeze drying.

[0021] Preferably, the freeze drying is to pre-freeze at-40 DEG C for 3 h, once dry at 10 DEG C for 15 h under a vacuum degree of 15 Pa, and twice dry at 25 DEG C for 10 h.

[0022] Therefore, the application has the following beneficial effects by using the above-mentioned purification method of telopeptide:

[0023] (1) The application uses volatile buffer salts ammonium trifluoroacetate and ammonium formate in the whole process, which can be directly used with HPLC-MS, realizes the identification of the first purified impure peptide and the online quality control of the second purified target peptide, improves the process development efficiency, and makes the impurity tracing more accurate.

[0024] (2) The second purification of the application directly forms telopeptide formate salt, omits the dilution-ultrafiltration-acid-base adjustment salt conversion step, shortens the single batch process cycle, reduces the equipment investment, avoids the pH fluctuation in the salt conversion process, reduces the risk of denaturation of the target peptide, and the formate salt form has higher water solubility than the traditional sodium salt, is stable, and is easy to store.

[0025] (3) The proportion of acetonitrile in the mobile phase of the application is the highest, which is 43% (first purification) and 40% (second purification), which is lower than the prior art (acetonitrile proportion ≥ 50%), reduces the generation of organic waste liquid, and the nanofiltration concentration process can realize the recovery of part of the solvent, and reduces the environmental protection treatment cost.

[0026] The technical solutions of the application will be further described in detail below by means of the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the liquid chromatogram of the telopeptide crude product of the application;

[0028] Figure 2 is the liquid chromatogram of the first purified fraction of example 1 of the application;

[0029] Figure 3 is the liquid chromatogram of the second purified fraction of example 1 of the application;

[0030] Figure 4 is the liquid chromatogram of the telopeptide formate salt fraction of example 1 of the application. DETAILED DESCRIPTION

[0031] The application will be further described below in conjunction with the accompanying drawings and embodiments. Unless otherwise defined, technical terms or scientific terms used in the present application shall have the commonly understood meanings as understood by one of ordinary skill in the art to which this application belongs. The features mentioned above or the features mentioned in the specific examples can be combined in any manner, and these specific examples are only used to illustrate the present application and not to limit the scope of the present application.

[0032] The present application provides a method for purifying tirzepatide, comprising the following steps:

[0033] S1, dissolving tirzepatide crude product in an ammonia solution, and filtering to obtain a solution containing tirzepatide;

[0034] S2, performing first purification on the solution containing tirzepatide obtained in S1, using reverse silica gel filler as the stationary phase, using a polar organic solvent as the mobile phase A phase, using an ammonium trifluoroacetate aqueous solution as the mobile phase B phase, and using purified water as the mobile phase C phase, to obtain a component with less than 0.5% single impurities;

[0035] S3, performing second purification on the component obtained in S2, using reverse silica gel filler as the stationary phase, using a polar organic solvent as the mobile phase A phase, using an ammonium formate aqueous solution as the mobile phase B phase, and using purified water as the mobile phase C phase, to obtain a component with less than 0.1% single impurities;

[0036] S4, concentrating, filtering, and drying the component obtained in S3.

[0037] Preferably, in S1, the pH of the ammonia solution is 9.1, and ultrasonic dissolution is used during dissolution, and the pH is adjusted to 9.0-9.3 using 2‰ ammonia or 1% formic acid, which can inhibit the deamidation reaction of tirzepatide, reduce the generation of impurities, and improve the solubility of tirzepatide in the aqueous solution.

[0038] 0.45 μm water filter membrane is used for filtration to remove undissolved particulate matter and mechanical impurities, and to avoid subsequent column blockage.

[0039] In some specific embodiments of the present application, the solid-liquid ratio of tirzepatide crude product and ammonia solution is 1:20 (g / mL); the ultrasonic power for ultrasonic dissolution is 300 W, the frequency is 20 kHz, and the dissolution time is 10 min. The present application improves the solubility of tirzepatide through ultrasonic dissolution, which can reduce the sample volume and improve the purification efficiency compared with conventional acid dissolution.

[0040] Preferably, in S2, the stationary phase for first purification is Bonnasil HS C8 Pro reverse-phase silica gel filler, the particle size is 10 μm, and the pore size is 100 A. The Bonnasil HSC8 Pro reverse-phase silica gel filler in the application has high specific surface area and good hydrophobicity, can realize preliminary separation of telopeptide and impurities through hydrophobic interaction, and has strong retention capacity for acidic impurities, thereby facilitating subsequent removal.

[0041] In some specific embodiments of the application, the chromatographic column used for the first purification is a DAC100 dynamic axial compression column (column length: 250 mm, inner diameter: 100 mm).

[0042] Preferably, in S2, the concentration of the aqueous ammonium trifluoroacetate solution is 20 mM; when the concentration of the aqueous ammonium trifluoroacetate solution is less than 10 mM, the ion pair effect is weakened, and the separation degree of the impurity peptide and the target peptide decreases; when the concentration of the aqueous ammonium trifluoroacetate solution is greater than 30 mM, the viscosity of the mobile phase increases, the column pressure exceeds 25 MPa, and the elution uniformity is affected.

[0043] The pH is adjusted to 3.0±0.1 using ammonia water; at this pH, the trifluoroacetate (CF3COO-) can form a stable ion pair with the amino group of telopeptide, avoiding the hydrolysis of the peptide bond caused by pH<2.5 or the reduction of the ion pair efficiency caused by pH>3.5, enhancing the retention of telopeptide on the reverse-phase filler, and inhibiting the activity of the silanol group of the silica gel matrix, improving the peak shape, and reducing the tailing.

[0044] In some specific embodiments of the application, in S2, the polar organic solvent of the mobile phase A is acetonitrile (chromatographic purity, purity≥99.9%), which has moderate hydrophobicity, can effectively adjust the elution strength, and has good miscibility with water, and is not prone to generate bubbles.

[0045] The conductivity of the purified water of the mobile phase C is ≤1.0 μS / cm, and the TOC is ≤50 ppb, which is used to adjust the overall polarity of the mobile phase and optimize the elution gradient.

[0046] Preferably, in S2, the first purification is performed according to the following parameters for linear gradient elution, and the total elution period is 120 minutes (including the equilibration, elution, and regeneration steps):

[0047]

[0048] The collection condition is that the detection wavelength is 275 nm, which is the characteristic absorption wavelength of the Trp residue in the telopeptide molecule, has high sensitivity, and has little interference;

[0049] The collection standard is that the purity of the main peak is ≥99.0%, the single impurity is ≤0.5%, and the fraction is collected by online monitoring of HPLC (chromatographic column: Waters Xbridge C18, 5 μm, 4.6×250 mm; mobile phase: acetonitrile-0.1% phosphoric acid water), and the first purification fraction is obtained by combining.

[0050] In the present application, most of the low-hydrophobic and high-hydrophobic impurities are quickly removed by the first purification, thereby reducing the impurity load of subsequent fine purification.

[0051] Preferably, in S3, the stationary phase of the second purification is UniSil 10-80C18 reversed-phase silica gel packing with a particle size of 5 μm and a pore size of 120 A. In the present application, the UniSil 10-80C18 reversed-phase silica gel packing is a C18 long-chain bonded phase with strong hydrophobicity, small particle size, high theoretical plate number and better separation degree, and can effectively separate racemization and deamidation impurities with similar structures.

[0052] In some embodiments of the present application, the chromatographic column used for the second purification is a DAC100 dynamic axial compression column (with a column length of 250 mm and an inner diameter of 100 mm).

[0053] Preferably, in S3, the concentration of the ammonium formate aqueous solution is 20 mM. In the present application, the concentration is controlled at 20 mM to balance the buffer capacity and volatility. When the concentration is less than 15 mM, the buffer capacity is insufficient and the pH is prone to fluctuation. When the concentration is greater than 25 mM, the salt sublimation after lyophilization is incomplete and a small amount of ammonium formate may be left.

[0054] Formic acid is used to adjust the pH to 6.5±0.1. Ammonium formate is a weak acid salt. When the pH is 6.5, it is close to the isoelectric point (pI≈6.8) of telopeptide. At this time, the carboxyl group (-COOH) of the target peptide is combined with the ammonium ion (NH4 + ) in ammonium formate to directly form telopeptide formate (-COONH4), without subsequent salt conversion. The telopeptide formate molecule has weak electrification, and has stronger hydrophobic interaction with the C18 packing. Due to the charge difference, the retention behavior of the racemization and deamidation impurities is different from that of the main product, which is beneficial to separation.

[0055] In some embodiments of the present application, in S3, the polar organic solvent of the mobile phase A is acetonitrile (chromatographic purity, purity≥99.9%), which ensures consistency with the organic solvent of the first purification and reduces the peak shape fluctuation caused by solvent switching. The conductivity of the purified water of the mobile phase C is ≤1.0 μS / cm, and the TOC is ≤50 ppb, which is used to adjust the overall polarity of the mobile phase and optimize the elution gradient.

[0056] Preferably, in S3, the second purification is performed by linear gradient elution according to the following parameters, and the total elution period is 90 minutes (including the equilibration, elution and regeneration steps).

[0057]

[0058] Collection condition: detection wavelength 275 nm, same as S2, to ensure consistency of detection.

[0059] Collection criteria: The main peak purity ≥ 99.5%, single impurity ≤ 0.1%, acetonitrile residue < 0.5% were collected by HPLC online monitoring (chromatographic column: Waters Xbridge C18, 5 μm, 4.6 x 250 mm; mobile phase: acetonitrile-0.1% phosphoric acid water), the second purification fraction was obtained by combining, the formate content was 1.2%-1.5% by ion chromatography detection, and the tirzepatide formate fraction was directly obtained.

[0060] The present application utilizes the high separation degree of C18 filler and the mobile phase condition of pH 6.5 in the second purification to enlarge the retention difference of tirzepatide and racemic, deamidated impurities, so that the difficult-to-separate impurities are completely removed.

[0061] Preferably, in S4, the concentration is to transfer the component obtained in S3 into a rotary evaporator under reduced pressure, concentrate under the condition of 28-30℃ water bath and-15℃ cold trap for 4-5h to obtain a purified solution with a concentration of 200-250g / L; the filtration is to filter the purified solution through a 0.22μm polyvinylidene fluoride membrane to remove possible bacteria, mycoplasma and other microorganisms, and ensure the sterility of the product. The drying is freeze drying.

[0062] Preferably, the freeze drying is to completely freeze the solution after pre-freezing at-40℃ for 3h to prevent ice crystal growth from damaging the molecular structure. A vacuum degree of 15Pa is maintained, and the first drying is performed at 10℃ for 15h, and the second drying is performed at 25℃ for 10h to obtain tirzepatide formate freeze-dried powder.

[0063] Example 1

[0064] The present application provides a purification method of tirzepatide, comprising the following steps:

[0065] S1, taking solid-phase synthesized tirzepatide crude product (10g, net peptide content 5.2g, HPLC detection purity 72.3%, single impurity highest 2.8%), adding 200mL 2‰ ammonia water solution (pH 9.1), ultrasonic (300W, 20kHz) assisted dissolution for 10min, adjusting pH to 9.2 with 1% formic acid, filtering with 0.45μm water filter membrane, and collecting the sample solution, as shown in Figure 1 .

[0066] S2, the solution containing tirzepatide in S1 is subjected to first purification

[0067] DAC100 dynamic axial compression column (Hanbang Technology) is used, and Bonnasil HSC8 Pro reversed-phase silica gel filler (10μm, ) is filled, and the column bed height is 250mm; an HPLC detection system is connected, and the detection wavelength is 275nm.

[0068] The mobile phase is prepared as follows:

[0069] Mobile phase A phase: chromatographically pure acetonitrile (purity 99.9%).

[0070] Mobile phase B phase: weigh 2.31 g of ammonium trifluoroacetate, dissolve in purified water and make up to 1000 mL to obtain a 20 mM ammonium trifluoroacetate aqueous solution, and adjust the pH to 3.0 with concentrated ammonia water.

[0071] Mobile phase C phase: purified water (conductivity 0.8 μS / cm, TOC 35 ppb).

[0072] Loading and elution: load all the crude product solution obtained in S1, and perform linear elution according to the following gradient parameters, with a total elution period of 120 minutes (including equilibration, elution, and regeneration steps):

[0073] Table 1 Linear elution gradient parameters for the first purification of Example 1

[0074]

[0075] Collection conditions: detection wavelength 275 nm, which is the characteristic absorption wavelength of the Trp residue in the Telopeptide molecule, high sensitivity and less interference;

[0076] Collection criteria: online monitoring by HPLC (chromatographic column: Waters Xbridge C18, 5 μm, 4.6 x 250 mm; mobile phase: acetonitrile-0.1% phosphoric acid water), collecting fractions with a main peak purity ≥ 99.0% and single impurities ≤ 0.5%, and combining to obtain the first purification fraction, as shown in Figure 2 .

[0077] Fraction collection: online monitoring by HPLC, collecting fractions in the main peak elution period (95-105 min), and after combining, the purity was 99.2% and the highest single impurity was 0.45%, to obtain the first purification fraction.

[0078] S3, Second Purification of Telopeptide

[0079] The first purification fraction obtained in S2 was directly used as the loading solution.

[0080] Another DAC100 dynamic axial compression column was used, filled with UniSil 10-80 C18 reversed-phase silica gel filler (5 μm, Nanometer Microtechnology), with a column bed height of 200 mm; the detection system was the same as in S2.

[0081] Mobile phase preparation:

[0082] Mobile phase A phase: same as S2 (chromatographically pure acetonitrile);

[0083] ​Mobile phase B phase: 1.58 g of ammonium formate was weighed, dissolved with purified water and constant volume to 1000 mL to obtain 20 mM ammonium formate aqueous solution, and the pH was adjusted to 6.5 with formic acid.

[0084] Mobile phase C phase: same as S2 (purified water);

[0085] Loading and elution: all the first purification fraction obtained by S2 was loaded, and linear elution was carried out according to the following gradient parameters, and the total elution period was 90 minutes (including equilibration, elution, regeneration steps):

[0086] Table 2 Linear elution gradient parameters of the second purification of Example 2

[0087]

[0088]

[0089] Collection conditions: detection wavelength 275 nm, same as S2, to ensure consistency of detection.

[0090] Collection criteria: online monitoring by HPLC (chromatographic column: Waters Xbridge C18, 5 μm, 4.6 x 250 mm; mobile phase: acetonitrile-0.1% phosphoric acid water), collecting fractions with main peak purity ≥ 99.5%, single impurity ≤ 0.1%, acetonitrile residue < 0.5%, and combining to obtain the second purification fraction, as shown in Figure 3 The ion chromatography detected the formate content to be 1.2%-1.5%, and the tirzepatide formate fraction was directly obtained.

[0091] Fraction collection: online monitoring by HPLC, collecting the fraction in the main peak period (65-72 min), and after combining, the purity was 99.8%, the single impurity was ≤ 0.1%, and the second purification fraction was obtained.

[0092] S4, subsequent treatment and lyophilization

[0093] Concentration: the second purification fraction of S3 was transferred to a rotary evaporator under reduced pressure, concentrated for 4.5 h under the condition of 29°C water bath and -15°C cold trap, and the purified liquid with a concentration of 245 g / L was obtained.

[0094] Sterile filtration: the purified liquid was filtered through a 0.22 μm PVDF membrane, and the filtrate was collected.

[0095] Freeze-drying: The obtained collected filtrate was pre-frozen at -40℃ for 3h, then the solution was completely frozen to prevent ice crystal growth from destroying the molecular structure. Maintaining a vacuum degree of 15Pa, the solution was dried at 10℃ for 15h and at 25℃ for 10h, obtaining 4.37g of tirzepatide formate lyophilized product, with a calculated total recovery of 84.0% (4.37g / 5.2g x 100%); the purity of the finished product was 99.8%, the single impurity was ≤0.1%, and the moisture content was 0.4%, as shown in Table 1. Figure 4

[0096] Example 2

[0097] The difference from Example 1 is that the crude tirzepatide in S1 (10g, net peptide content 6.5g, HPLC purity 70.5%, single impurity up to 3.1%); the pH of mobile phase B in S2 was adjusted to 3.1, and the pH of mobile phase B in S3 was adjusted to 6.6; the rest were the same as Example 1. The purity of the first purified fraction was 99.1%, and the single impurity was 0.48%; the purity of the second purified fraction was 99.7%, and the single impurity was ≤0.1%; finally, 5.33g of finished product was obtained, with a total recovery of 82.0% (5.33g / 6.5g x 100%); the purity of the finished product was 99.8%, the single impurity was ≤0.1%, and the moisture content of the finished product was 0.45%.

[0098] Comparative Example 1

[0099] The same batch of crude tirzepatide (10g, net peptide content 5.2g, HPLC purity 72.3%, single impurity up to 2.8%) was purified using the prior art:

[0100] First purification: C8 column (10μm, ), mobile phase A (acetonitrile), B (2% ice acetic acid solution), the first purification was linear gradient elution according to the following parameters,

[0101] Table 3 Linear elution gradient parameters of the first purification of Comparative Example 1

[0102]

[0103] The collection conditions and collection criteria were the same as Example 1, and the first purified fraction was obtained.

[0104] Second purification: C18 column (10μm, ), mobile phase A (acetonitrile), B (0.02mol / L ammonium dihydrogen phosphate, pH 6.5), the second purification was linear gradient elution according to the following parameters:

[0105] Table 4 Linear elution gradient parameters of the second purification of Comparative Example 1

[0106] ​

[0107] The collection conditions and collection criteria are the same as in Example 1, and a second purified fraction is obtained.

[0108] Salt exchange step: the second purified stream is diluted 2 times, then concentrated by 3KD ultrafiltration membrane, precipitated by adjusting pH to 4.8 using 1M hydrochloric acid, centrifuged and washed after being refrigerated at 4°C for 3h, then freeze-dried after adjusting pH to 8.0 using 1M sodium hydroxide, and the freeze-drying procedure is the same as in Example 1. A freeze-dried powder of telopeptide sodium salt 4.0g is obtained, with a total yield of 77.6%, a test product purity of 83.8%, a single impurity of ≤0.1%, and a moisture content of 0.8%.

[0109] Exploratory experiment

[0110] On the basis of Example 1, only the pH of the second purification B phase is changed, and the rest of the conditions remain unchanged, to explore the effect of the pH of the mobile phase on the second purification. The results are as follows:

[0111] Table 5 Effect of different pH of second purification B phase on second purification

[0112]

[0113]

[0114] As shown in Table 5, when the pH is 6.5, the binding efficiency of the target peptide with ammonium formate is the highest, and the purity, yield and formate content are all optimal. Deviation of the pH can easily lead to incomplete separation of impurities or excessive residual formate.

[0115] On the basis of Example 1, only the concentration of ammonium trifluoroacetate in the first purification B phase is changed, and the rest of the conditions remain unchanged, to explore the effect of the salt concentration of the mobile phase on the first purification. The results are as follows:

[0116] Table 6 Effect of different concentrations of ammonium trifluoroacetate in the first purification B phase on the first purification

[0117]

[0118] As shown in Table 6, when the concentration of ammonium trifluoroacetate is 20mM, the column pressure can be controlled within 25MPa (the safety threshold of the equipment) while ensuring the resolution (≥2.5) and purity. When the concentration is 10mM, the ion pair effect is insufficient, and the impurities and target peptides are not completely separated. When the concentration is 30mM, the column pressure is excessive, which can easily lead to collapse of the chromatographic column filler and affect the stability of the process.

[0119] On the basis of Example 1, only the pore size of the C18 filler in the second purification is changed, and the rest of the conditions remain unchanged, to explore the effect of the pore size of the stationary phase in the second purification on the removal of impurities. The results are as follows:

[0120] Table 7 Effect of different pore sizes of C18 filler in the second purification on the removal of impurities

[0121]

[0122] As shown in Table 7, C18 packing material with varying pore sizes is suitable for telpoide-formate complexes (molecular diameter approximately...). The steric repulsion selectivity is optimal, enabling efficient removal of oxidative impurities and long-chain fatty acid impurities; pore size The target peptide is easily confined by the pore size of the packing material, and excessive retention leads to a decrease in yield; pore size The screening effect on small molecule impurities weakens, and the impurity removal rate decreases.

[0123] Based on Example 1, the drying temperature was changed once while other conditions remained the same. The freeze-dried product was stored at 4°C and 25°C for 6 months, and the change in purity was detected to investigate the effect of the freeze-drying process on product stability. The results are shown in the table below:

[0124] Table 8. Effects of different freeze-drying processes on product stability

[0125]

[0126] As shown in Table 8, the initial purity and storage stability of the product are optimal when the drying temperature is 10℃. The purity decreases by only 0.1% after 6 months of storage at 4℃ and by 0.3% at 25℃. Too low a temperature (5℃) will prolong the freeze-drying time (increase by 2 hours) and the moisture removal will be incomplete. Too high a temperature (15℃) will easily cause partial denaturation of telpoeptide, and the purity will decrease significantly during storage.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for purifying telpolide, characterized in that, Includes the following steps: S1. Dissolve crude telpoeptide in an ammonia solution, filter, and obtain a solution containing telpoeptide. S2. The solution containing telpoeptide obtained in S1 is subjected to a first purification using reverse silica gel packing as the stationary phase, a polar organic solvent as the mobile phase A, an ammonium trifluoroacetate aqueous solution as the mobile phase B, and purified water as the mobile phase C, to obtain a component with less than 0.5% single impurities. S3. The component obtained in S2 is subjected to a second purification using reverse silica gel packing as the stationary phase, a polar organic solvent as the mobile phase A, an ammonium formate aqueous solution as the mobile phase B, and purified water as the mobile phase C, to obtain a component with less than 0.1% impurities. S4. Concentrate, filter, and dry the components obtained in S3.

2. The purification method for telpolide according to claim 1, characterized in that: In S1, the pH of the ammonia solution is 9.

1. Ultrasonic dissolution is used to aid dissolution. The pH is finely adjusted to 9.0-9.3 with 2‰ ammonia or 1% formic acid. Filtration is performed using a 0.45μm aqueous filter membrane.

3. The purification method for telpolide according to claim 1, characterized in that: In S2, the first purified stationary phase was Bonnasil HSC8Pro reversed-phase silica gel packing material with a particle size of 10 μm and a pore size of [missing information].

4. The purification method for telpolide according to claim 1, characterized in that: In S2, the concentration of ammonium trifluoroacetate aqueous solution is 20 mM, and the pH is adjusted to 3.0 ± 0.1 using ammonia.

5. The purification method for telpoeptide according to claim 1, characterized in that: In S2, the first purification process involves linear gradient elution according to the following parameters: 。 6. The purification method for telpolide according to claim 1, characterized in that: In S3, the stationary phase for the second purification was UniSil 10-80C18 reversed-phase silica gel packing material with a particle size of 5 μm and a pore size of [missing information].

7. The purification method for telpolide according to claim 1, characterized in that: In S3, the concentration of ammonium formate aqueous solution is 20 mM, and the pH is adjusted to 6.5 ± 0.1 using formic acid.

8. The purification method for telpolide according to claim 1, characterized in that: In S3, the second purification process involves linear gradient elution with the following parameters:

9. The purification method for telpolide according to claim 1, characterized in that: In S4, the component obtained in S3 is concentrated by concentrating it in a water bath at 28-30℃ and a cold trap at -15℃ for 4-5 hours to obtain a purified solution with a concentration of 200-250 g / L. Filtration was performed by sterilizing the purified solution through a 0.22 μm polyvinylidene fluoride membrane, and drying was performed by freeze drying.

10. The purification method of telpolide according to claim 9, characterized in that: Freeze-drying involves pre-freezing at -40℃ for 3 hours, followed by drying at 10℃ for 15 hours under a vacuum of 15 Pa, and then drying again at 25℃ for 10 hours.

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