Purification method of tilpotide

Abstract

The invention belongs to the technical field of purification of polypeptide drugs, and particularly relates to a purification method of tirpotide, which comprises the following steps: (a) preparing a tirpotide crude product solution by using an acetonitrile aqueous solution; (b) carrying out primary purification on the crude product solution of the tilpotide by selecting a specific stationary phase, a mobile phase and an elution gradient to obtain a first fraction; (c) performing secondary purification on the first fraction by selecting a specific stationary phase, a mobile phase and an elution gradient to obtain a second fraction; and (d) carrying out nanofiltration concentration, salt exchange, filtration sterilization and freeze-drying on the second fraction to obtain a tiall peptide product. The purity of the product obtained through the purification method is up to 99.5% or above, and the total recovery rate of purification is up to 80% or above. According to the method, the single-needle elution period is only 35-40 minutes, so that the use of organic solvents in the purification process of the tilpotide is greatly reduced, the purification cost is reduced, and the method tends to the long-term development of environmental protection.

Description

Technical Field

[0001] The invention belongs to the technical field of purification of polypeptide drugs, and particularly relates to a method for purifying telpotide. Background Art

[0002] The amino acid sequence of Tirzepatide is as follows: Tyr-{Aib}-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{Aib}-Leu-Asp-Lys-Ile-Ala-Gln-{diacid-C20-gamma-Glu-(AEEA)2-Lys}-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2, tepoxetine is a dual agonist of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptors. By activating these two receptors, it can regulate blood sugar levels, increase insulin secretion, inhibit glucagon secretion, and delay gastric emptying, thereby reducing food intake and weight loss. The original research company is Eli Lilly of the United States, and its main indications are the treatment of type 2 diabetes and weight loss. It is the first GIP / GLP dual receptor agonist approved by the FDA. Especially in the field of weight loss, it has an effect comparable to gastrectomy. It has an extremely broad market audience and can also improve the current widespread obesity and sub-health situation.

[0003] Since tirpotide contains non-natural amino acids, the product can only be obtained by artificial synthesis. The synthesis methods include all-solid phase synthesis and solid-liquid combined synthesis. However, the crude products obtained by both synthesis methods will contain racemic impurities, missing peptides, differential peptides, deamidation and other impurities that are similar to the structure of the main product. Many related structural impurities not only have no drug efficacy, but also have toxic side effects. In addition to being related to the pharmacological activity of the drug itself, the adverse reactions in the clinical use of drugs are sometimes also related to the impurities in the drug and must be strictly controlled. For example, 5-hydroxymethylfurfural in glucose infusion is harmful to human skeletal muscle and internal organs, and trace impurities in probucol can cause severe eye toxicity. Therefore, impurity control is a key element in drug safety assurance and an important manifestation of risk control awareness in drug research and development.

[0004] Currently, many methods for purifying telpotide have been developed. For example, although CN 117736273B discloses a method for purifying telpotide, it requires three purifications to obtain a high-quality product, and each purification cycle exceeds 60 minutes, resulting in the generation of a large amount of organic waste solvents, high costs, and is not suitable for the long-term development of environmental protection, and the total product recovery rate is low. Summary of the invention

[0005] In view of the problems existing in the prior art such as long purification cycle, high cost, environmental pollution, low purity and low total product recovery rate of crude telpotide, the present invention provides a purification method of telpotide, which has a short purification cycle, high efficiency, can obtain a high-purity product with a single impurity of less than 0.1%, has a high total purification recovery rate, low purification cost and can achieve green and sustainable development.

[0006] The present invention provides a method for purifying telpotide, which comprises the following steps: (a) dissolving a crude telpotide raw material in an acetonitrile aqueous solution to obtain a crude telpotide solution; (b) purifying the crude tepoxetine solution for the first time, The first stationary phase is a reverse phase filler; The first mobile phase A is a phosphoric acid aqueous solution, the volume concentration of the phosphoric acid aqueous solution is 0.2% to 0.4%, and tetrabutylammonium hydrogen sulfate or triethylamine is added to adjust the pH to 2 to 4; The first mobile phase B is a mixed solution of acetonitrile and isopropanol in a volume ratio of 3:1; The elution gradient is 61% to 52% by volume of the first mobile phase A and 39% to 48% by volume of the first mobile phase B, and the linear gradient elution is performed for 35 to 40 minutes, and the fraction with a purity greater than 97% and a single impurity less than 1.5% is collected as the first fraction; (c) subjecting the first fraction to a second purification, The second stationary phase is a reverse phase filler; The second mobile phase A is an ammonium bicarbonate solution, and the concentration of the ammonium bicarbonate is 50-100 mM; The second mobile phase B is acetonitrile; The elution gradient is 65% to 60% by volume of the second mobile phase A and 35% to 40% by volume of the second mobile phase B, and the linear gradient elution is performed for 35 to 40 minutes, and the fraction with a purity greater than 99.5% and a single impurity less than 0.1% is collected as the second fraction; (d) performing nanofiltration concentration, salt exchange, and filtration sterilization on the second fraction to obtain a pre-lyophilization solution, and lyophilizing the pre-lyophilization solution to obtain tepol peptide.

[0007] Preferably, in the step (a), the crude telpotide raw material is dissolved in an acetonitrile aqueous solution having a volume concentration of 20-40%, stirred for 4 hours, and filtered to obtain the crude telpotide solution.

[0008] Preferably, in step (a), the solubility of the acetonitrile aqueous solution is 30%.

[0009] Preferably, in the step (b), the first mobile phase A is a phosphoric acid aqueous solution with a volume concentration of 0.3% and a pH of 3.0.

[0010] Preferably, in the step (c), the second mobile phase A is a 100 mM ammonium bicarbonate solution.

[0011] Preferably, the first reverse phase filler and the second reverse phase filler are independently selected from any one of tetraalkylsilane bonded silica filler, octaalkylsilane bonded silica filler and octadecylsilane bonded silica filler.

[0012] Preferably, the first reverse phase filler and the second reverse phase filler are both octadecylsilane bonded silica gel fillers, the first mobile phase A in step (b) is 0.3% by volume phosphoric acid water, and tetrabutylammonium hydrogen sulfate is added to adjust the pH to 3.0, and the second mobile phase A in step (c) is 100 mM ammonium bicarbonate solution.

[0013] Preferably, the concentration of the solution before freeze-drying is 70-100 mg / g, and the height of the solution before freeze-drying does not exceed 1.5 cm.

[0014] Preferably, in step (b), linear gradient elution is performed for 40 minutes according to the following parameters: , In step (c), linear gradient elution was performed for 35 minutes according to the following parameters: .

[0015] Preferably, in step (b), the first purification is performed according to the following parameters: , In the step (c), the second purification is performed according to the following parameters: .

[0016] The purification method of the present invention has the following advantages: 1) Proper addition of acetonitrile to the solvent used to dissolve the crude raw material of telpotide can accelerate the complete deacidification reaction of tryptophan, which is beneficial to the purification and separation and the improvement of recovery rate. The solubility of the crude raw material of telpotide is better, and the concentration can be as high as 50g / L. The obtained solution is acidic and has good stability, which solves the problem of large amount of solvent in crude solution and long purification cycle leading to deterioration of crude solution during large-scale production.

[0017] 2) The purification method of the present invention adopts the complementary method of acid and base for the two purifications, which can effectively remove impurities with different separation degrees under different properties, and can effectively separate deamidated impurities and racemic impurities close to the main peak. And by selecting different mobile phases and optimizing the elution gradient, the recovery rate and purity of the product are improved, and a high-purity product with a single impurity of less than 0.1% can be obtained.

[0018] 3) The present invention adjusts the pH of the mobile phase by tetrabutylammonium hydrogen sulfate or triethylamine. Tetrabutylammonium hydrogen sulfate is used as an ion pair reagent to form ion pairs with anions in the sample. The formation of such ion pairs changes the retention behavior of the sample and the distribution state in the mobile phase. At the same time, tetrabutylammonium hydrogen sulfate can adjust the polarity of the mobile phase, making the polarity of the mobile phase more suitable for sample separation, reducing nonspecific adsorption between sample molecules and the stationary phase, and making the sample molecules have more suitable retention and elution behavior in the chromatographic column. Tetrabutylammonium hydrogen sulfate can react with silanol groups to reduce the activity of silanol groups, thereby reducing the tailing phenomenon of alkaline samples. Triethylamine can shield the silanol groups in the filler, reduce the interaction between the sample molecules and the silanol groups in the stationary phase, significantly improve the peak shape, and eliminate the tailing phenomenon.

[0019] 4) Compared with the common purification cycle of 100 minutes per injection, the elution cycle of the purification method of the present invention is only 35 to 40 minutes, which greatly shortens the purification cycle and significantly reduces the use of organic solvents in the purification process of telpotide, reduces the purification cost, and is more environmentally friendly in the long term.

[0020] In summary, the purification method of the present invention can improve the separation effect while reducing the purification cycle. The purity of the telpotide obtained by the purification method of the present invention is as high as 99.5% or more, the single impurity is less than 0.1%, and the total recovery rate of purification is as high as 80% or more. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the chromatogram of the crude tepoxetine in Example 1; Figure 2 This is the HPLC chromatogram of the first fraction obtained by the first purification of the crude teilpotide in Example 1; Figure 3 This is the HPLC chromatogram of the second fraction obtained by the second purification of the crude teilpotide in Example 1; Figure 4 This is the HPLC chromatogram of the first fraction obtained by the first purification of the crude teilpotide in Example 2; Figure 5 This is the HPLC chromatogram of the second fraction obtained by the second purification of the crude teilpotide in Example 2; Figure 6This is the HPLC chromatogram of the first fraction obtained by the first purification of the crude teilpotide in Example 5; Figure 7 This is the HPLC chromatogram of the second fraction obtained by the second purification of the crude teilpotide in Example 5; Figure 8 This is the HPLC chromatogram of the first fraction obtained by the first purification of the crude teilpotide in Example 6; Fig. 9 This is the HPLC chromatogram of the second fraction obtained by the second purification of the crude teilpotide in Example 6; Fig.10 This is the HPLC chromatogram of the first fraction obtained by the first purification of the crude teilpotide in Example 7; Fig.11 This is the HPLC chromatogram of the second fraction obtained by the second purification of the crude teilpotide in Example 7; Fig.12 is the HPLC chromatogram of the first fraction obtained by the first purification of the crude teilpotide in Comparative Example 1; Fig.13 is the HPLC chromatogram of the second fraction obtained by the second purification of the crude teilpotide in Comparative Example 1; Fig.14 is the HPLC chromatogram of the second fraction obtained by the first purification of the crude teilpotide in Comparative Example 2; Fig.15 is the HPLC chromatogram of the second fraction obtained by the second purification of the crude teilpotide in Comparative Example 2; Fig.16 is the HPLC chromatogram of the second fraction obtained after the first purification of the crude teilpotide in Comparative Example 3; Fig.17 This is the HPLC chromatogram of the second fraction obtained by the second purification of the crude teilpotide in Comparative Example 3. DETAILED DESCRIPTION

[0022] The technical scheme of the present invention is further described below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention. The methods used in the present invention are conventional production methods unless otherwise specified; the raw materials used are conventional commercial products unless otherwise specified.

[0023] The purification method of the present invention comprises four steps: (a) preparing a crude tepoxetine solution; (b) purifying the crude tepoxetine solution for the first time to obtain a first fraction; (c) purifying the first fraction for the second time to obtain a second fraction; and (d) performing nanofiltration concentration, salt exchange, filtration sterilization and freeze drying on the second fraction to obtain a tepoxetine product.

[0024] In step (a), the present invention dissolves the crude raw material of tepote in an acetonitrile aqueous solution, and adds a certain proportion of acetonitrile to the solvent for dissolving the crude tepote, which accelerates the complete deacidification reaction of tryptophan, and is conducive to the improvement of purification separation and recovery rate. And the crude tepote has better solubility, the concentration can be as high as 50g / L, the solution is acidic, and the stability is good, which solves the problem that the crude solution has a large amount of solvent and a long purification cycle during large-scale production, resulting in the deterioration of the crude solution. In a preferred embodiment of the present invention, the volume concentration of the acetonitrile aqueous solution is 20-40%, and the more preferred volume concentration is 30%. In a preferred embodiment of the present invention, the crude tepote is dissolved in the acetonitrile aqueous solution and stirred for 4 hours to fully react, and then filtered, for example, using a 0.45μm filter membrane to remove insoluble matter for subsequent purification.

[0025] After dissolution, telportipeptide is purified twice (steps (b) and (c)). The two purifications are performed in an acid-base complementary manner, which can effectively remove impurities with different separation degrees under different properties, and can effectively separate deamidated impurities and racemic impurities that are close to the main peak.

[0026] Specifically, the first purification (step (b)) uses a reverse phase filler as the stationary phase, mobile phase A is a phosphoric acid aqueous solution with a volume concentration of 0.2% to 0.4%, and tetrabutylammonium hydrogen sulfate or triethylamine is added to adjust the pH to 2 to 4, mobile phase B is a mixed solution of acetonitrile and isopropanol with a volume ratio of 3:1, and the elution gradient is 61% to 52% by volume of the mobile phase A and 39% to 48% by volume of the mobile phase B. Linear gradient elution is performed for 35 to 40 minutes, and fractions with a purity greater than 97% and a single impurity less than 1.5% are collected as the first fraction. In a preferred embodiment, mobile phase A in step (b) is a phosphoric acid aqueous solution with a volume concentration of 0.3% and a pH of 3.0. In a preferred embodiment, the first purification is performed with a linear gradient elution for 40 minutes according to the following parameters: .

[0027] In a preferred embodiment, the first purification is carried out according to the following parameters: .

[0028] During the first purification process, the pH value of mobile phase A is adjusted by tetrabutylammonium hydrogen sulfate or triethylamine. Tetrabutylammonium hydrogen sulfate is a class of ionic compounds. As an ion pair reagent, it forms ion pairs with anions in the sample. The formation of such ion pairs changes the retention behavior of the sample and the distribution state in the mobile phase. At the same time, tetrabutylammonium hydrogen sulfate can adjust the polarity of the mobile phase, making the polarity of the mobile phase more suitable for sample separation, reducing the nonspecific adsorption between the sample molecules and the stationary phase, and making the sample molecules have more suitable retention and elution behavior in the chromatographic column. Tetrabutylammonium hydrogen sulfate can react with silanol groups to reduce the activity of silanol groups, thereby reducing the tailing phenomenon of alkaline samples and improving the purification efficiency and product purity. Triethylamine can shield the silanol groups in the filler, reduce the interaction between the sample molecules and the silanol groups in the stationary phase, significantly improve the peak shape, and eliminate the tailing phenomenon. Isopropanol is added to mobile phase B to improve the solubility of telportide, improve the purification efficiency and product purity.

[0029] The second purification (step (c)) uses a reverse phase filler as the stationary phase, mobile phase A is an ammonium bicarbonate solution, the concentration of the ammonium bicarbonate is 50-100mM, mobile phase B is acetonitrile, and the elution gradient is 65% to 60% by volume of the mobile phase A and 35% to 40% by volume of the mobile phase B. The linear gradient elution is performed for 35 to 40 minutes, and the fraction with a purity greater than 99.5% / single impurity less than 0.1% is collected as the second fraction. In a preferred embodiment, the second mobile phase A in step (c) is a 100mM ammonium bicarbonate solution. In a preferred embodiment of the present invention, the linear gradient elution is performed for 35 minutes according to the following parameters: .

[0030] In a preferred embodiment, the second purification is carried out according to the following parameters: .

[0031] In a preferred embodiment, the stationary phases in the first purification and the second purification can be the same or different, for example, selected from any one of tetraalkylsilane bonded silica gel filler, octadecylsilane bonded silica gel filler, and octadecylsilane bonded silica gel filler. Further preferably, the first reverse phase filler and the second reverse phase filler are both octadecylsilane bonded silica gel fillers, and the first mobile phase A in the step (b) is 0.3% by volume phosphoric acid water, and tetrabutylammonium hydrogen sulfate is added to adjust the pH to 3.0, and the second mobile phase A in the step (c) is 100 mM ammonium bicarbonate solution.

[0032] The present invention improves the purification recovery rate and purity of the product by selecting different mobile phases in two purifications and optimizing the elution gradient, so that the single impurity content in the product is less than 0.1%. In addition, compared with the common purification cycle of 100 minutes per injection, the present invention provides an elution cycle of 35 to 40 minutes, which greatly shortens the purification cycle, making the purification cycle far lower than the conventional level, greatly reducing the use of organic solvents in the purification process of crude telportin, reducing the purification cost and tending to long-term environmental protection.

[0033] After the two purifications are completed, the second fraction is subjected to nanofiltration concentration, salt exchange, filtration sterilization, and freeze-drying to obtain tepol peptide. In a preferred embodiment, the second fraction is subjected to nanofiltration concentration, salt exchange, and filtration sterilization to obtain a pre-lyophilized solution, the concentration of the pre-lyophilized solution is 70-100 mg / g, and the height of the pre-lyophilized solution does not exceed 1.5 cm. The height of the pre-lyophilized solution will affect the moisture content. If the height is too high, the moisture content is relatively large. The present invention reduces the moisture content of the product by controlling the height of the pre-lyophilized solution to not exceed 1.5 cm.

[0034] The purification method of the present invention can greatly shorten the purification cycle and obtain a high-purity product, wherein the purity of the telpotide product reaches more than 99.5%, the single impurity is controlled below 0.1%, and the total recovery rate of purification is as high as more than 80%. Example 1

[0035] The following steps are used to dissolve, purify for the first time and purify for the second time the crude telpotide, wherein the crude telpotide is prepared by the applicant through a solid phase total synthesis method, and the net peptide content thereof is calibrated using a standard substance by a method commonly used in the art.

[0036] (a) 24.0 g of crude telpotide (net peptide content: 13.2 g) was accurately weighed and dissolved in 400 mL of acetonitrile aqueous solution with a volume concentration of 30%, and stirred for 4 hours to obtain a crude telpotide solution. HPLC (Waters, 2695) was used to detect that the decarboxylation conversion of tryptophan was complete, and the purity of the crude product was determined to be 73.95%. The HPLC spectrum is shown in the figure below. Figure 1 shown.

[0037] (b) First purification The crude telpotide solution obtained in step (a) is purified for the first time under the following purification conditions: MPLC-DAC50 preparation system (Hanbang Technology); Stationary phase: octadecylsilane bonded silica filler (Nanomicron Technology); Flow rate: 50mL / min; Detection wavelength: 275nm; Mobile phase A: 0.2% volume concentration of phosphoric acid aqueous solution, add tetrabutylammonium hydrogen sulfate to adjust the pH to 3.5 (phosphoric acid: Sinopharm Chemical; tetrabutylammonium hydrogen sulfate: Sinopharm Chemical); Mobile phase B: acetonitrile / isopropanol = 3 / 1 (v / v) (acetonitrile: Shanghai Xingke; isopropanol: Xilong Science); Loading capacity: 8 g crude sample; After the sample was loaded, purification was performed according to the parameters in Table 1 (including pre-injection equilibration, linear elution, column regeneration and post-injection equilibration), with a linear gradient elution of 40 min.

[0038] Table 1 (Balance before injection - linear elution - column regeneration - balance after injection) .

[0039] The target peak was collected, and the fraction with a purity greater than 97% and a single impurity less than 1.5% was collected as the first fraction. The purity of the first fraction was 97.35%, and the single impurity was less than 1.5%. The HPLC spectrum of the first fraction was as follows: Figure 2 shown.

[0040] (c) Second purification The first fraction obtained in step (b) is purified for the second time, and the purification conditions are as follows: MPLC-DAC50 preparation system (Hanbang Technology); Stationary phase: octadecylsilane bonded silica filler (Nanomicron Technology); Flow rate: 50mL / min; Detection wavelength: 275nm; Mobile phase A: 100 mM ammonium bicarbonate solution (ammonium bicarbonate: Sinopharm Chemical); Mobile phase B: acetonitrile (Shanghai Xingke); Loading capacity: The loading amount is 8g; Purification was performed according to the parameters in Table 2 (including pre-injection equilibration, linear elution, column regeneration and post-injection equilibration), with a linear gradient elution of 35 min.

[0041] Table 2 (Balance before injection - linear elution - column regeneration - balance after injection) .

[0042] The target peak was collected, and the fraction with a purity greater than 99.5% and a single impurity less than 0.1% was collected as the second fraction. The purity of the second fraction was 99.75%, and the single impurity was less than 0.1%. Its HPLC spectrum was as follows: Figure 3 shown. Example 2

[0043] Except for the following conditions, everything else is the same as Example 1: (b) First purification: Stationary phase: octadecylsilane bonded silica filler (Nanomicron Technology); Mobile phase A: 0.3% phosphoric acid in water, add tetrabutylammonium hydrogen sulfate to adjust the pH to 3.0; Mobile phase B: acetonitrile / isopropanol = 3 / 1 (v / v); The components with a purity greater than 97% were collected as the first fraction. The purity of the first fraction was 97.90%. Its HPLC spectrum was as follows: Figure 4 shown.

[0044] (c) Second purification: Stationary phase: octadecylsilane bonded silica filler (Nanomicron Technology); The components with a purity greater than 99.5% were collected as the second fraction. The purity of the second fraction was 99.88%, and the single impurity was less than 0.1%. Its HPLC spectrum was as follows: Figure 5 shown. Example 3

[0045] The second fraction obtained in Example 1 was loaded into the concentration reflux device tank of a multifunctional membrane nanofilter (Hangzhou Ruina Membrane Engineering Co., Ltd.), the frequency converter was adjusted to 30 Hz, and the membrane pressure was adjusted to 1.5 MPa. The multifunctional membrane nanofilter was used for continuous nanofiltration and concentration. When the volume of the reflux product liquid reached 10% of the original volume, 0.02% sodium hydroxide water (sodium hydroxide: Sinopharm Chemical) was continuously added to replace at least 5 volumes to achieve salt conversion. Then, the solution was filtered through a 0.22 μm filter membrane to obtain a pre-lyophilized solution with a concentration of 75 mg / ml, which was placed in a lyophilization tray, and the solution height was controlled to be no more than 1.5 cm. The solution was lyophilized for 50 hours to obtain 10.7 grams of finished product, and the total recovery rate of purification was calculated to be 81%, wherein the total recovery rate of purification = weight of lyophilized finished product / net peptide content before purification x 100%. Example 4

[0046] The second fraction obtained in Example 2 was subjected to nanofiltration concentration and salt conversion in the same manner as in Example 3. It was then filtered through a 0.22 um filter membrane to obtain a pre-lyophilized solution with a concentration of 72 mg / ml, which was placed in a lyophilization tray with the solution height controlled to be no more than 1.5 cm. The lyophilization was performed for 50 hours to obtain 11.2 g of a finished product, wherein the total recovery rate of purification was 84.8%. Example 5

[0047] Except for the pH adjustment by adding triethylamine to mobile phase A in the first purification step, the other steps were the same as in Example 1. The purity of the first fraction obtained was 97.53%, and its HPLC spectrum was as follows: Figure 6As shown, the purity of the second fraction obtained is 99.80%, and the single impurity is less than 0.1%. Its HPLC spectrum is as follows Figure 7 shown. Example 6

[0048] Except that the concentration of ammonium bicarbonate in the second mobile phase A in the second purification step was 50 mM, everything else was the same as in Example 5. The purity of the first fraction obtained was 97.53%. Its HPLC spectrum is shown in FIG. Figure 8 As shown, the purity of the second fraction obtained is 99.83%, and the single impurity is less than 0.1%. Its HPLC spectrum is as follows Fig. 9 shown. Example 7

[0049] Except that the concentration of ammonium bicarbonate in the second mobile phase A in the second purification step was 75 mM, everything else was the same as in Example 5. The purity of the first fraction obtained was 97.53%. Its HPLC spectrum is shown in FIG. Fig.10 As shown, the purity of the second fraction obtained is 99.81%, and the single impurity is less than 0.1%. Its HPLC spectrum is as follows Fig.11 shown. Example 8

[0050] The second fraction obtained in Example 5 was subjected to nanofiltration concentration and salt conversion in the same manner as in Example 3. It was then filtered through a 0.22 um filter membrane to obtain a pre-lyophilized solution with a concentration of 70 mg / ml, which was placed in a lyophilization tray with the solution height controlled to be no more than 1.5 cm. The lyophilization was performed for 50 hours to obtain 11.4 g of a finished product, wherein the total recovery rate of purification was 86.3%. Example 9

[0051] The second fraction obtained in Example 6 was subjected to nanofiltration concentration and salt conversion in the same manner as in Example 3. It was then filtered through a 0.22 um filter membrane to obtain a pre-lyophilized solution with a concentration of 71 mg / ml, which was placed in a lyophilization tray with the solution height controlled to be no more than 1.5 cm. The lyophilization was performed for 50 hours to obtain 11.3 g of a finished product, wherein the total recovery rate of purification was 85.6%. Example 10

[0052] The second fraction obtained in Example 7 was subjected to nanofiltration concentration and salt conversion in the same manner as in Example 3. It was then filtered through a 0.22 um filter membrane to obtain a pre-lyophilized solution with a concentration of 71 mg / ml, which was placed in a lyophilization tray with the solution height controlled to be no more than 1.5 cm. The lyophilization was performed for 50 hours to obtain 11.3 g of a finished product, wherein the total recovery rate of purification was 85.6%.

[0053] Comparative Example 1 Except that ammonia water was used to adjust the pH of mobile phase A in the first purification step, the other steps were the same as in Example 1. The purity of the first fraction obtained was 94.20%, and its HPLC spectrum was as follows: Fig.12 As shown, the purity of the second fraction was 99.42%, and there was more than 0.1% of a single impurity. Its HPLC spectrum was as follows Fig.13 shown.

[0054] Comparative Example 2 Except that the mobile phase B in the first purification step was 100% by volume acetonitrile, everything else was the same as in Example 1. The purity of the first fraction obtained was 95.49%. Its HPLC spectrum is shown in FIG. Fig.14 The purity of the second fraction was 99.48%, and there was more than 0.1% of a single impurity. Its HPLC spectrum was as shown in Fig.15 shown.

[0055] Comparative Example 3 Except that the elution gradient in the first purification step was 60% to 51% by volume of mobile phase A and 40% to 49% by volume of mobile phase B, the rest was the same as in Example 1. The purity of the first fraction obtained was 94.53%, and its HPLC spectrum was as follows: Fig.16 The purity of the second fraction was 99.40%, and there was more than 0.1% of a single impurity, and its HPLC spectrum was shown in Figure 17.

[0056] Table 3 (“—” means the same as the previous one) .

[0057] The results of the above examples and comparative examples are shown in Table 3. In Comparative Example 1, the pH value of the mobile phase A in the first purification step was adjusted by adding ammonia water, and the purity of the first and second fractions obtained were lower than that of the present invention, and there was more than 0.1% of a single impurity. In Comparative Example 2, the first mobile phase B in the first purification step was acetonitrile, and no isopropanol was added. The purity of the product obtained was lower than that of the present invention, and there was more than 0.1% of a single impurity. The elution gradient of the first purification in Comparative Example 3 was different from that of the present invention, and the purity of the first fraction obtained was low. According to the present invention, acetonitrile is appropriately added to the solvent for dissolving the crude telpotide raw material, so as to accelerate the tryptophan deacidification reaction and improve the solubility of the crude telpotide raw material, and the purity and recovery rate of the product are improved by selecting different mobile phases and optimizing the elution gradient, so that the content of single impurities in the product is controlled to be less than 0.1%, and the tailing phenomenon is improved by adding tetrabutylammonium hydrogen sulfate or triethylamine to the first mobile phase A, and the solubility of telpotide is improved by adding isopropanol to the first mobile phase B, so as to improve the purification efficiency, thereby optimizing the entire purification process, that is, the purification cycle can be greatly shortened, the use of purification reagents is reduced, the purification cost is reduced, and the purity and recovery rate of the product can be improved, so that the content of single impurities in the product is less than 0.1%, and green and sustainable development is achieved.

[0058] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for purifying tepote, characterized in that: The purification method comprises the following steps: (a) dissolving a crude telpotide raw material in an acetonitrile aqueous solution to obtain a crude telpotide solution; (b) subjecting the crude tebuconazole solution to a first purification, The first stationary phase is a reverse phase filler; The first mobile phase A is a phosphoric acid aqueous solution, the volume concentration of the phosphoric acid aqueous solution is 0.2% to 0.4%, and tetrabutylammonium hydrogen sulfate or triethylamine is added to adjust the pH to 2 to 4; The first mobile phase B is a mixed solution of acetonitrile and isopropanol in a volume ratio of 3:1; The elution gradient is 61% to 52% by volume of the first mobile phase A and 39% to 48% by volume of the first mobile phase B, and the linear gradient elution is performed for 35 to 40 minutes, and the fraction with a purity greater than 97% and a single impurity less than 1.5% is collected as the first fraction; (c) subjecting the first fraction to a second purification, The second stationary phase is a reverse phase filler; The second mobile phase A is an ammonium bicarbonate solution, and the concentration of the ammonium bicarbonate is 50-100 mM; The second mobile phase B is acetonitrile; The elution gradient is 65% to 60% by volume of the second mobile phase A and 35% to 40% by volume of the second mobile phase B, and the linear gradient elution is performed for 35 to 40 minutes, and the fraction with a purity greater than 99.5% and a single impurity less than 0.1% is collected as the second fraction; (d) performing nanofiltration concentration, salt exchange, and filtration sterilization on the second fraction to obtain a pre-lyophilization solution, and lyophilizing the pre-lyophilization solution to obtain tepol peptide.

2. The purification method according to claim 1, characterized in that In the step (a), the crude telpotide raw material is dissolved in an acetonitrile aqueous solution with a volume concentration of 20-40%, stirred for 4 hours, and filtered to obtain the crude telpotide solution.

3. The purification method according to claim 2, characterized in that: In the step (a), the solubility of the acetonitrile aqueous solution is 30%.

4. The purification method according to claim 1, characterized in that In the step (b), the first mobile phase A is a phosphoric acid aqueous solution with a volume concentration of 0.3% and a pH of 3.

0.

5. The purification method according to claim 1, characterized in that In the step (c), the second mobile phase A is a 100 mM ammonium bicarbonate solution.

6. The purification method according to claim 1, characterized in that The first reverse phase filler and the second reverse phase filler are independently selected from any one of tetraalkylsilane bonded silica filler, octaalkylsilane bonded silica filler and octadecylsilane bonded silica filler.

7. The purification method according to claim 6, characterized in that The first reverse phase filler and the second reverse phase filler are both octadecylsilane bonded silica gel fillers, the first mobile phase A in the step (b) is a phosphoric acid aqueous solution with a volume concentration of 0.3%, and tetrabutylammonium hydrogen sulfate is added to adjust the pH to 3.0, and the second mobile phase A in the step (c) is an ammonium bicarbonate solution with a concentration of 100 mM.

8. The purification method according to claim 1, characterized in that The concentration of the solution before freeze-drying is 70-100 mg / g, and the height of the solution before freeze-drying does not exceed 1.5 cm.

9. The purification method according to claim 1, characterized in that In the step (b), linear gradient elution was performed for 40 minutes according to the following parameters: In step (c), linear gradient elution was performed for 35 minutes according to the following parameters: 。 10. The purification method according to claim 9, characterized in that In the step (b), the first purification is performed according to the following parameters: ， In the step (c), the second purification is performed according to the following parameters: 。

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