A method for purifying tilportide

Abstract

The present invention belongs to the technical field of purification of polypeptide drugs, and specifically relates to a method for purifying tilpotide, comprising: (a) preparing a crude tilpotide solution using an acetonitrile aqueous solution; (b) selecting a specific stationary phase, mobile phase, and elution gradient to purify the crude tilpotide solution for a first time to obtain a first fraction; (c) selecting a specific stationary phase, mobile phase, and elution gradient to purify the first fraction for a second time to obtain a second fraction; (d) performing nanofiltration concentration, salt exchange, filtration sterilization, and lyophilization on the second fraction to obtain the tilpotide product. The product obtained by the purification method of the present invention has a purity of more than 99.5%, and the total recovery rate of purification is more than 80%. The single-needle elution cycle of the present invention is only 35 to 40 minutes, which greatly reduces the use of organic solvents in the purification process of tilpotide, reduces purification costs, and is more environmentally friendly in the long term.

Description

Technical Field

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

[0002] The amino acid sequence of Tirzepatide is as follows:

[0003] 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. Tierpotide is a dual agonist of the glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptors. By activating these two receptors, it can regulate blood glucose levels, increase insulin secretion, inhibit glucagon secretion, and delay gastric emptying, thereby reducing food intake and weight loss. Developed by Eli Lilly and Company, its primary 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 audience market and can also improve the current widespread obesity and sub-health situation.

[0004] Because tilpotide contains unnatural amino acids, the product can only be obtained through artificial synthesis. 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, epipeptides, deamidated impurities, and other difficult-to-remove impurities with structures similar to the main product. Many related structural impurities not only lack drug efficacy but also have toxic side effects. Adverse reactions in the clinical use of drugs are not only related to the pharmacological activity of the drug itself, but are sometimes also related to 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 ocular toxicity. Therefore, impurity control is a key factor in ensuring drug safety and an important manifestation of risk control awareness in drug research and development.

[0005] Numerous methods for purifying tilpotide have been developed. For example, CN 117736273B discloses a method for purifying tilpotide. However, it requires three purification steps to obtain a high-quality product, and each purification cycle exceeds 60 minutes. This results in the generation of a large amount of organic waste solvent, resulting in high costs and being unsuitable for long-term environmental protection. Furthermore, the total product recovery rate is low. Summary of the Invention

[0006] In response to the problems existing in the prior art of crude tilpotide purification, such as long purification cycle, high cost, environmental pollution, low purity, and low total product recovery rate, the present invention provides a purification method for tilpotide, which has a short purification cycle, high efficiency, can obtain a high-purity product with a single impurity content of less than 0.1%, has a high total purification recovery rate, low purification cost, and can achieve green and sustainable development.

[0007] The present invention provides a method for purifying telpotide, which comprises the following steps:

[0008] (a) dissolving a crude tilpotide raw material in an acetonitrile aqueous solution to obtain a crude tilpotide solution;

[0009] (b) purifying the crude tilportide solution for the first time,

[0010] The first stationary phase is a reverse phase packing;

[0011] The first 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;

[0012] The first mobile phase B is a mixed solution of acetonitrile and isopropanol with a volume ratio of 3:1;

[0013] 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. 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;

[0014] (c) subjecting the first fraction to a second purification,

[0015] The second stationary phase is a reverse phase packing;

[0016] The second mobile phase A is an ammonium bicarbonate solution, and the concentration of the ammonium bicarbonate is 50-100 mM;

[0017] The second mobile phase B is acetonitrile;

[0018] 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 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;

[0019] (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.

[0020] Preferably, in the step (a), the crude tilpotide 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 tilpotide solution.

[0021] Preferably, in step (a), the solubility of the acetonitrile aqueous solution is 30%.

[0022] 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.

[0023] Preferably, in the step (c), the second mobile phase A is a 100 mM ammonium bicarbonate solution.

[0024] 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.

[0025] Preferably, the first reverse phase filler and the second reverse phase filler are both octadecylsilane bonded silica 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.

[0026] 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.

[0027] Preferably, in step (b), linear gradient elution is performed for 40 minutes according to the following parameters:

[0028] ,

[0029] In step (c), linear gradient elution was performed for 35 minutes according to the following parameters:

[0030] .

[0031] Preferably, in step (b), the first purification is performed according to the following parameters:

[0032] ,

[0033] In the step (c), the second purification is performed according to the following parameters:

[0034] .

[0035] The purification method of the present invention has the following advantages:

[0036] 1) Adding acetonitrile to the solvent used to dissolve crude telpotide can accelerate the complete deacidification reaction of tryptophan, facilitate purification and improve recovery. This also improves the solubility of the crude telpotide, allowing concentrations as high as 50 g / L. The resulting solution is acidic and stable, resolving the issue of large-scale production, which can lead to deterioration of the crude solution due to large solvent volumes and long purification cycles.

[0037] 2) The purification method of this invention utilizes complementary acid and base purification steps, effectively removing impurities with varying degrees of resolution, and effectively separating deamidated and racemic impurities located close to the main peak. Furthermore, by selecting different mobile phases and optimizing the elution gradient, product recovery and purity are improved, yielding a high-purity product with less than 0.1% of a single impurity.

[0038] 3) The present invention adjusts the pH of the mobile phase by using tetrabutylammonium hydrogen sulfate or triethylamine. Tetrabutylammonium hydrogen sulfate acts 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 its 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, reducing 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 sample molecules and silanol groups in the stationary phase, significantly improve the peak shape, and eliminate the tailing phenomenon.

[0039] 4) Compared to the common purification cycle of 100 minutes per injection, the purification method of the present invention has an elution cycle of only 35 to 40 minutes, which greatly shortens the purification cycle and significantly reduces the use of organic solvents in the purification process of telportide, reducing purification costs and tending to long-term environmentally friendly development.

[0040] In summary, the purification method of the present invention can improve the separation effect while reducing the purification cycle. The purity of telportin 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

[0041] Figure 1 is a chromatogram of the crude tilpoxetine product in Example 1;

[0042] Figure 2 This is an HPLC chromatogram of the first fraction obtained by the first purification of the crude teilpotide in Example 1;

[0043] Figure 3 This is an HPLC chromatogram of the second fraction obtained by the second purification of the crude teilpotide in Example 1;

[0044] Figure 4 This is an HPLC chromatogram of the first fraction obtained by the first purification of the crude tilpotide in Example 2;

[0045] Figure 5 This is an HPLC chromatogram of the second fraction obtained by the second purification of the crude tilpotide in Example 2;

[0046] Figure 6 This is the HPLC chromatogram of the first fraction obtained by the first purification of the crude tilpotide in Example 5;

[0047] Figure 7 This is an HPLC chromatogram of the second fraction obtained by the second purification of the crude tilpotide in Example 5;

[0048] Figure 8 This is the HPLC chromatogram of the first fraction obtained by the first purification of the crude tilpotide in Example 6;

[0049] Figure 9 This is an HPLC chromatogram of the second fraction obtained by the second purification of the crude tilpotide in Example 6;

[0050] Figure 10 This is the HPLC chromatogram of the first fraction obtained by the first purification of the crude teilpotide in Example 7;

[0051] Figure 11 This is an HPLC chromatogram of the second fraction obtained by the second purification of the crude teilpotide in Example 7;

[0052] Figure 12 This is the HPLC chromatogram of the first fraction obtained by the first purification of the crude tilpotide in Comparative Example 1;

[0053] Figure 13 This is the HPLC chromatogram of the second fraction obtained by the second purification of the crude tilpotide in Comparative Example 1;

[0054] Figure 14 This is the HPLC chromatogram of the second fraction obtained by the first purification of the crude tilpotide in Comparative Example 2;

[0055] Figure 15 This is the HPLC chromatogram of the second fraction obtained by the second purification of the crude tilpotide in Comparative Example 2;

[0056] Figure 16 This is the HPLC chromatogram of the second fraction obtained by the first purification of the crude tilpotide in Comparative Example 3;

[0057] Figure 17 This is the HPLC chromatogram of the second fraction obtained by the second purification of the crude tilpotide in Comparative Example 3. DETAILED DESCRIPTION

[0058] The technical solutions of the present invention are further described below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are intended only to illustrate and explain the present invention and are not intended to limit the present invention. The methods used in the present invention are all conventional production methods unless otherwise specified; the raw materials used are all conventional commercially available products unless otherwise specified.

[0059] The purification method of the present invention comprises four steps: (a) preparing a crude tilpotide solution; (b) performing a first purification on the crude tilpotide solution to obtain a first fraction; (c) performing a second purification on the first fraction to obtain a second fraction; and (d) performing nanofiltration concentration, salt exchange, filtration sterilization, and freeze-drying on the second fraction to obtain a tilpotide product.

[0060] In step (a), the present invention dissolves the crude telpotide raw material in an acetonitrile aqueous solution. A certain proportion of acetonitrile is added to the solvent used to dissolve the crude telpotide, which accelerates the complete deacidification reaction of tryptophan, facilitates purification and separation, and improves the recovery rate. The crude telpotide has better solubility, with a concentration of up to 50 g / L. The solution is acidic and has good stability, which solves the problem of large-scale production, such as the large amount of solvent in the crude solution and the long purification cycle, which causes the crude solution to deteriorate. In a preferred embodiment of the present invention, the volume concentration of the acetonitrile aqueous solution is 20-40%, and more preferably 30%. In a preferred embodiment of the present invention, the crude telpotide is dissolved in the acetonitrile aqueous solution and stirred for 4 hours to fully react, and then filtered, for example, using a 0.45 μm filter membrane to remove insoluble matter for subsequent purification.

[0061] After dissolution, telportide is purified twice (steps (b) and (c)). The two purifications are performed using a complementary acid and base method, which can effectively remove impurities with different properties and different separation degrees, and can effectively separate deamidated impurities and racemic impurities that are close to the main peak.

[0062] 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 a fraction with a purity greater than 97% and a single impurity less than 1.5% is collected as the first fraction. In a preferred embodiment, the 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 according to the following parameters for a linear gradient elution for 40 minutes:

[0063] .

[0064] In a preferred embodiment, the first purification is carried out according to the following parameters:

[0065] .

[0066] During the first purification process, the pH of mobile phase A is adjusted using tetrabutylammonium hydrogen sulfate or triethylamine. Tetrabutylammonium hydrogen sulfate is an ionic compound that acts as an ion-pairing agent, forming ion pairs with anions in the sample. This ion-pairing alters the sample's retention behavior and distribution within the mobile phase. Furthermore, tetrabutylammonium hydrogen sulfate adjusts the polarity of the mobile phase, making it more suitable for sample separation and reducing nonspecific adsorption between sample molecules and the stationary phase, resulting in more optimal retention and elution of sample molecules within the chromatographic column. Tetrabutylammonium hydrogen sulfate reacts with silanol groups, reducing their activity and thus reducing tailing in alkaline samples, improving purification efficiency and product purity. Triethylamine shields silanol groups in the packing material, reducing interactions between sample molecules and the stationary phase, significantly improving peak shape and eliminating tailing. The addition of isopropyl alcohol to mobile phase B improves the solubility of telportide, enhancing purification efficiency and product purity.

[0067] The second purification (step (c)) uses a reverse-phase packing as the stationary phase, mobile phase A is ammonium bicarbonate solution with a concentration of 50-100 mM, mobile phase B is acetonitrile, and the elution gradient is 65%-60% by volume of mobile phase A and 35%-40% by volume of mobile phase B. Elution is performed over a period of 35-40 minutes. The fraction with a purity greater than 99.5% and less than 0.1% impurity is collected as the second fraction. In a preferred embodiment, the second mobile phase A in step (c) is a 100 mM ammonium bicarbonate solution. In a preferred embodiment of the present invention, linear gradient elution is performed over a period of 35 minutes according to the following parameters:

[0068] .

[0069] In a preferred embodiment, the second purification is carried out according to the following parameters:

[0070] .

[0071] In a preferred embodiment, the stationary phases used in the first and second purifications may be the same or different, for example, selected from any one of tetraalkylsilane bonded silica gel, octadecylsilane bonded silica gel, and octadecylsilane bonded silica gel. Further preferably, both the first reversed-phase packing material and the second reversed-phase packing material are octadecylsilane bonded silica gel, and the first mobile phase A in step (b) is 0.3% by volume phosphoric acid in water, adjusted to pH 3.0 with the addition of tetrabutylammonium hydrogen sulfate, and the second mobile phase A in step (c) is 100 mM ammonium bicarbonate solution.

[0072] By selecting different mobile phases and optimizing the elution gradient during the two purification steps, the present invention improves the product's purification recovery rate and purity, reducing the product's single impurity content to less than 0.1%. Furthermore, compared to the common purification cycle of 100 minutes per injection, the present invention provides an elution cycle of 35 to 40 minutes, significantly shortening the purification cycle to well below conventional levels. This significantly reduces the use of organic solvents during the purification of crude telportide, lowering purification costs and contributing to a more environmentally friendly long-term development.

[0073] After the two purifications are completed, the second fraction is subjected to nanofiltration concentration, salt exchange, filtration sterilization, and lyophilization to obtain tilportide. In a preferred embodiment, the second fraction is subjected to nanofiltration concentration, salt exchange, and filtration sterilization to obtain a pre-lyophilization solution, wherein the concentration of the pre-lyophilization solution is 70-100 mg / g, and the height of the pre-lyophilization solution does not exceed 1.5 cm. The height of the pre-lyophilization solution affects the moisture content; if the height is too high, the moisture content is relatively high. The present invention reduces the moisture content of the product by controlling the height of the pre-lyophilization solution to no more than 1.5 cm.

[0074] The purification method of the present invention can greatly shorten the purification cycle and obtain a high-purity product, wherein the purity of the telportide product reaches more than 99.5%, the single impurity is controlled below 0.1%, and the total purification recovery rate is as high as more than 80%. Example 1

[0075] The crude tilpotide was dissolved, purified for the first time, and purified for the second time using the following steps. The crude tilpotide was prepared by the applicant through a solid-phase total synthesis method, and the net peptide content was calibrated using a standard substance using a method commonly used in the art.

[0076] (a) 24.0 g of crude tilpoitide (net peptide content: 13.2 g) was accurately weighed and dissolved in 400 mL of 30% by volume acetonitrile aqueous solution. The mixture was stirred for 4 hours to obtain a crude tilpoitide solution. HPLC (Waters, 2695) was used to determine the complete decarboxylation of tryptophan. The purity of the crude product was determined to be 73.95%. The HPLC spectrum is shown below. Figure 1 shown.

[0077] (b) First purification

[0078] The crude tilpoxetine solution obtained in step (a) is subjected to a first purification, and the purification conditions are as follows:

[0079] MPLC-DAC50 preparation system (Hanbang Technology);

[0080] Stationary phase: octaalkylsilane bonded silica gel packing (Nanomicron Technology);

[0081] Flow rate: 50 mL / min;

[0082] Detection wavelength: 275nm;

[0083] Mobile phase A: 0.2% by volume phosphoric acid aqueous solution, with tetrabutylammonium hydrogen sulfate added to adjust the pH to 3.5 (phosphoric acid: Sinopharm Chemical; tetrabutylammonium hydrogen sulfate: Sinopharm Chemical);

[0084] Mobile phase B: acetonitrile / isopropanol = 3 / 1 (v / v) (acetonitrile: Shanghai Xingke; isopropanol: Xilong Science);

[0085] Loading capacity: The crude sample loading amount is 8 g;

[0086] 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 time of 40 min.

[0087] Table 1 (Balance before injection - linear elution - column regeneration - balance after injection)

[0088] .

[0089] 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 less than 1.5%. The HPLC spectrum of the first fraction was as follows: Figure 2 shown.

[0090] (c) Second purification

[0091] The first fraction obtained in step (b) is purified for the second time under the following purification conditions:

[0092] MPLC-DAC50 preparation system (Hanbang Technology);

[0093] Stationary phase: octaalkylsilane bonded silica gel packing (Nanomicron Technology);

[0094] Flow rate: 50 mL / min;

[0095] Detection wavelength: 275nm;

[0096] Mobile phase A: 100 mM ammonium bicarbonate solution (ammonium bicarbonate: Sinopharm Chemical);

[0097] Mobile phase B: acetonitrile (Shanghai Xingke);

[0098] Loading capacity: The sample load is 8 g;

[0099] Purification was performed according to the parameters listed in Table 2 (including pre-injection equilibration, linear elution, column regeneration, and post-injection equilibration), with a linear gradient elution time of 35 min.

[0100] Table 2 (Balance before injection - linear elution - column regeneration - balance after injection)

[0101] .

[0102] 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 less than 0.1%. The HPLC spectrum of the second fraction was as follows: Figure 3 shown. Example 2

[0103] Except for the following conditions, everything else is the same as Example 1:

[0104] (b) First purification:

[0105] Stationary phase: octadecylsilane bonded silica gel packing (Nanomicron Technology);

[0106] Mobile phase A: 0.3% phosphoric acid in water, adjusted to pH 3.0 by adding tetrabutylammonium hydrogen sulfate;

[0107] Mobile phase B: acetonitrile / isopropanol = 3 / 1 (v / v);

[0108] The components with a purity greater than 97% were collected as the first fraction. The purity of the first fraction was 97.90%, and its HPLC spectrum was as follows: Figure 4 shown.

[0109] (c) Second purification:

[0110] Stationary phase: octadecylsilane bonded silica gel packing (Nanomicron Technology);

[0111] 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 content was less than 0.1%. Its HPLC spectrum was as follows: Figure 5 shown. Example 3

[0112] The second fraction obtained in Example 1 was loaded into the concentration reflux tank of a multifunctional membrane nanofiltration machine (Hangzhou Ruina Membrane Engineering Co., Ltd.). The frequency converter was set to 30 Hz, and the membrane outlet pressure was adjusted to 1.5 MPa. Continuous nanofiltration and concentration were performed using the multifunctional membrane nanofiltration machine. When the volume of the refluxed product liquid reached 10% of the original volume, 0.02% sodium hydroxide solution (sodium hydroxide: Sinopharm Chemical) was continuously added to replace the product for at least five volumes to achieve salt conversion. The product was then filtered through a 0.22 μm filter membrane to obtain a pre-lyophilization solution with a concentration of 75 mg / ml. The solution was placed in a lyophilization tray, with the solution height controlled to no more than 1.5 cm. Lyophilization was performed for 50 hours to obtain 10.7 g of finished product. The calculated total recovery rate for purification was 81%, where total recovery rate = weight of lyophilized product / net peptide content before purification x 100%. Example 4

[0113] The second fraction obtained in Example 2 was concentrated by nanofiltration and desalted in the same manner as in Example 3. The fraction was then filtered through a 0.22 μm filter membrane to obtain a pre-lyophilized solution with a concentration of 72 mg / ml. The solution was placed in a lyophilization tray, with the height of the solution controlled to not exceed 1.5 cm, and lyophilized for 50 hours to obtain 11.2 g of the finished product, with a total recovery rate of 84.8%. Example 5

[0114] 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 was 97.53%, and its HPLC spectrum was as follows: Figure 6 As shown, the purity of the second fraction is 99.80%, and the single impurity is less than 0.1%. Its HPLC spectrum is as shown Figure 7 shown. Example 6

[0115] Except that the concentration of ammonium bicarbonate in the second mobile phase A in the second purification step was 50 mM, all other steps were the same as in Example 5. The purity of the first fraction obtained was 97.53%. The HPLC spectrum thereof is shown in FIG. Figure 8 As shown, the purity of the second fraction is 99.83%, and the single impurity is less than 0.1%. Its HPLC spectrum is as shown Figure 9 shown. Example 7

[0116] Except that the concentration of ammonium bicarbonate in the second mobile phase A in the second purification step was 75 mM, all other steps were the same as in Example 5. The purity of the first fraction obtained was 97.53%. The HPLC spectrum thereof is shown in FIG. Figure 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 shown Figure 11 shown. Example 8

[0117] The second fraction obtained in Example 5 was concentrated by nanofiltration and desalted in the same manner as in Example 3. The fraction was then filtered through a 0.22 μm filter membrane to obtain a pre-lyophilized solution with a concentration of 70 mg / ml. The solution was placed in a lyophilization tray, with the height of the solution controlled to not exceed 1.5 cm, and lyophilized for 50 hours to obtain 11.4 g of the finished product, with an overall recovery rate of 86.3%. Example 9

[0118] The second fraction obtained in Example 6 was concentrated by nanofiltration and desalted in the same manner as in Example 3. The fraction was then filtered through a 0.22 μm filter membrane to obtain a pre-lyophilized solution with a concentration of 71 mg / ml. The solution was placed in a lyophilization tray, with the height of the solution controlled to not exceed 1.5 cm, and lyophilized for 50 hours to obtain 11.3 g of the finished product, with an overall recovery rate of 85.6%. Example 10

[0119] The second fraction obtained in Example 7 was concentrated by nanofiltration and desalted in the same manner as in Example 3. The fraction was then filtered through a 0.22 μm filter membrane to obtain a pre-lyophilized solution with a concentration of 71 mg / ml. The solution was placed in a lyophilization tray, with the height of the solution controlled to not exceed 1.5 cm, and lyophilized for 50 hours to obtain 11.3 g of the finished product, with an overall purification recovery of 85.6%.

[0120] Comparative Example 1

[0121] Except that ammonia water was used to adjust the pH of mobile phase A in the first purification step, all 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: Figure 12As 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 shown Figure 13 shown.

[0122] Comparative Example 2

[0123] Except that the mobile phase B in the first purification step was 100% acetonitrile by volume, all other steps were the same as in Example 1. The purity of the first fraction obtained was 95.49%, and its HPLC spectrum was as shown in FIG. Figure 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. Figure 15 shown.

[0124] Comparative Example 3

[0125] 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: Figure 16 The purity of the second fraction was 99.40%, and there was more than 0.1% of a single impurity. The HPLC spectrum of the second fraction is shown in Figure 17.

[0126] Table 3 (“—” means the same as the previous one)

[0127] .

[0128] The results of the above examples and comparative examples are shown in Table 3. In Comparative Example 1, the pH of mobile phase A in the first purification step was adjusted by adding aqueous ammonia. The purity of the first and second fractions obtained were lower than those of the present invention, and greater than 0.1% of a single impurity was present. In Comparative Example 2, the first mobile phase B in the first purification step was acetonitrile without isopropanol. The purity of the resulting product was lower than that of the present invention, and greater than 0.1% of a single impurity was present. In Comparative Example 3, the elution gradient used in the first purification step was different from that of the present invention, resulting in a low purity of the first fraction. The present invention accelerates the tryptophan deacidification reaction and improves the solubility of the crude tilpotide raw material by appropriately adding acetonitrile to the solvent for dissolving the crude tilpotide raw material, improves the purity and recovery rate of the product by selecting different mobile phases and optimizing the elution gradient, controls the single impurity content in the product to be less than 0.1%, improves the tailing phenomenon by adding tetrabutylammonium hydrogen sulfate or triethylamine to the first mobile phase A, and improves the solubility of tilpotide by adding isopropyl alcohol to the first mobile phase B, thereby improving the purification efficiency, thereby optimizing the entire purification process, significantly shortening the purification cycle, reducing the use of purification reagents, lowering the purification cost, and achieving improved product purity and recovery rate, so that the single impurity content in the product is less than 0.1%, thereby achieving green and sustainable development.

[0129] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. 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 may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, and all of these should be included in the scope of the claims of the present invention.

Claims

1. A method for purifying tilpoxetine, characterized in that: The purification method comprises the following steps: (a) dissolving a crude tilpotide raw material in an acetonitrile aqueous solution to obtain a crude tilpotide solution; (b) purifying the crude tilportide solution for the first time, The first stationary phase is a reverse phase packing; The first 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; The first mobile phase B is a mixed solution of acetonitrile and isopropanol with a volume ratio of 3:1; The first purification was performed by gradient elution according to the following parameters: ， The fraction with a purity greater than 97% and a single impurity less than 1.5% was collected as the first fraction; (c) subjecting the first fraction to a second purification, The second stationary phase is a reverse phase packing; 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 second purification was performed by gradient elution according to the following parameters: ， The fraction with a purity greater than 99.5% and a single impurity less than 0.1% was 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, wherein In the step (a), the crude tilpotide 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 tilpotide 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, wherein 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, wherein In the step (c), the second mobile phase A is a 100 mM ammonium bicarbonate solution.

6. The purification method according to claim 1, wherein The reverse phase filler of the first stationary phase and the reverse phase filler of the second stationary phase 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 reverse phase filler of the first stationary phase and the reverse phase filler of the second stationary phase are both octadecylsilane bonded silica gel fillers. In the step (b), the first mobile phase A 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. In the step (c), the second mobile phase A is an ammonium bicarbonate solution with a concentration of 100 mM.

8. The purification method according to claim 1, wherein 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.

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