Method for purifying tirzepatide
By employing a two-stage reversed-phase high-performance liquid chromatography purification and nanofiltration concentration freeze-drying process, the problems of long purification cycle, high cost, and low purity of telpopeptide have been solved, achieving efficient and environmentally friendly production of high-purity telpopeptide.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- FUJIAN GENOHOPE BIOTECH LTD
- Filing Date
- 2025-05-16
- Publication Date
- 2026-07-02
AI Technical Summary
Existing purification methods for telpoeptide are time-consuming, costly, environmentally unfriendly, have low purity, and low overall recovery rates, making it difficult to effectively remove racemic and deamidated impurities.
A two-stage reversed-phase high-performance liquid chromatography purification method was adopted, using an acid-base complementary mobile phase system, combined with tetrabutylammonium bisulfate and triethylamine to adjust the pH value, optimize the elution gradient, and combine nanofiltration concentration and lyophilization processes to achieve efficient separation and purification.
The purification cycle is shortened to 35-40 minutes, the purity is increased to over 99.5%, the single impurity is less than 0.1%, the total purification recovery rate is over 80%, the amount of organic solvent used is reduced, and it meets environmental protection requirements.
Smart Images
Figure CN2025095320_02072026_PF_FP_ABST
Abstract
Description
A purification method for telpoeptide Technical Field
[0001] This invention belongs to the field of peptide drug purification technology, specifically relating to a purification method for telpolide. Background Technology
[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. Tirzepatide is a dual agonist of glucose-dependent insulinotropic peptide (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. The original developer is Eli Lilly and Company, 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, and it has effects comparable to gastric bypass surgery, especially in the field of weight loss. It has an extremely broad market and can also improve the current prevalent obesity and sub-health status.
[0003] Because telpolide contains non-natural amino acids, it can only be obtained through artificial synthesis. Synthetic methods include all-solid-phase synthesis and solid-liquid combined synthesis. However, the crude products obtained by both methods often contain racemic impurities, deleted peptides, dimorphic peptides, and deamidated impurities that are difficult to remove, similar in structure to the main product. Many of these structural impurities not only lack therapeutic efficacy but also have toxic side effects. Adverse reactions in clinical use are sometimes related to impurities in drugs, in addition to their pharmacological activity, and must be strictly controlled. For example, 5-hydroxymethylfurfural in glucose infusions can damage skeletal muscle and internal organs, and trace impurities in probucol can cause severe ocular toxicity. Therefore, impurity control is a key element in ensuring drug safety and an important manifestation of risk control awareness in drug development.
[0004] Currently, numerous purification methods for telpolide have been developed. For example, although CN 117736273B discloses a purification method for telpolide, 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 solvent. The cost remains high and is not suitable for long-term environmental protection development, and the overall product recovery rate is low. Summary of the Invention
[0005] To address the problems of long purification cycles, high costs, environmental unfriendliness, low purity, and low overall product recovery rate of crude telpoeptide in existing technologies, this invention provides a purification method for telpoeptide that has a short purification cycle, high efficiency, can obtain high-purity products with less than 0.1% impurities, has a high overall purification recovery rate, low purification cost, and can achieve green and sustainable development.
[0006] This invention provides a method for purifying telpolide, the purification method comprising the following steps: (a) dissolving crude telpolide raw material in an acetonitrile aqueous solution to obtain a crude telpolide solution; (b) performing a first purification on the crude telpolide solution, wherein the first stationary phase is a reversed-phase packing material; the first mobile phase A is an aqueous phosphoric acid solution with a volume concentration of 0.2%–0.4%, and tetrabutylammonium hydrogen sulfate or triethylamine is added to adjust the pH to 2–4; the first mobile phase B is a mixed solution of acetonitrile and isopropanol in a volume ratio of 3:1; the elution gradient is such that the volume percentage of the first mobile phase A is 61%–52% and the volume percentage of the first mobile phase B is 39%–48%, and linear gradient elution is performed for 35–40 minutes, collecting the fraction with a purity greater than 97% and a single impurity of less than 1.5% as the first fraction; (c) performing a second purification on the first fraction, wherein the second stationary phase is a reversed-phase packing material. The second mobile phase A is an ammonium bicarbonate solution with a concentration of 50–100 mM; the second mobile phase B is acetonitrile; the elution gradient is 65%–60% by volume of the second mobile phase A and 35%–40% by volume of the second mobile phase B, and linear gradient elution is performed for 35–40 minutes. The fraction with a purity greater than 99.5% and a single impurity less than 0.1% is collected as the second fraction; (d) the second fraction is concentrated by nanofiltration, salt is replaced, and sterilized by filtration to obtain a solution before lyophilization. The solution before lyophilization is then lyophilized to obtain telpoeptide.
[0007] Preferably, in step (a), the crude telpoeptide 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 telpoeptide solution.
[0008] Preferably, in step (a), the solubility of the acetonitrile aqueous solution is 30%.
[0009] Preferably, in 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 step (c), the second mobile phase A is a 100 mM ammonium bicarbonate solution.
[0011] Preferably, the first reversed-phase packing and the second reversed-phase packing are each independently selected from any one of tetraalkylsilane-bonded silica gel packing, octaalkylsilane-bonded silica gel packing, and octadecylsilane-bonded silica gel packing.
[0012] Preferably, both the first and second reversed-phase packing materials are octadecylsilane-bonded silica gel packing materials. In step (b), the first mobile phase A is 0.3% phosphoric acid water with added tetrabutylammonium bisulfate to adjust the pH to 3.0. In step (c), the second mobile phase A is a 100mM ammonium bicarbonate solution.
[0013] Preferably, the concentration of the solution before lyophilization is 70-100 mg / g, and the height of the solution before lyophilization is no more than 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 is 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 step (c), the second purification is performed according to the following parameters:
[0016] The purification method of this invention has the following advantages: 1) Appropriately adding acetonitrile to the solvent used to dissolve the crude telpoide raw material can accelerate the complete deacidification reaction of tryptophan, which is beneficial to purification, separation, and improved recovery rate. Furthermore, the solubility of the crude telpoide raw material is better, and the concentration can reach up to 50 g / L. The resulting solution is acidic and has good stability, solving the problem of crude solution deterioration caused by large solvent volumes and long purification cycles during large-scale production.
[0017] 2) The purification method of this invention employs a complementary acid-base approach in two purification processes, which effectively removes impurities with varying degrees of separation due to different properties, and effectively separates deamidated impurities and racemic impurities that are close to the main peak. Furthermore, by selecting different mobile phases and optimizing the elution gradient, the product recovery rate and purity are improved, resulting in a high-purity product with less than 0.1% single impurities.
[0018] 3) This invention adjusts the pH of the mobile phase using tetrabutylammonium bisulfate or triethylamine. Tetrabutylammonium bisulfate acts as an ion-pairing reagent, forming ion pairs with anions in the sample. This ion pair formation alters the sample's retention behavior and distribution within the mobile phase. Simultaneously, tetrabutylammonium bisulfate can adjust the polarity of the mobile phase, making it more suitable for sample separation, reducing non-specific adsorption between sample molecules and the stationary phase, and resulting in more suitable retention and elution behavior of sample molecules on the column. Tetrabutylammonium bisulfate can react with silanol groups, reducing their activity and thus reducing tailing in alkaline samples. Triethylamine can shield silanol groups in the packing material, reducing the interaction between sample molecules and silanol groups in the stationary phase, significantly improving peak shape and eliminating tailing.
[0019] 4) Compared to the common purification cycle of 100 minutes per injection, the purification method of this 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 telpoeptide, thereby reducing purification costs and moving towards a more environmentally friendly long-term development.
[0020] In summary, the purification method of the present invention can improve the separation effect while reducing the purification cycle. The purity of telpoeptide 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 purification recovery rate is as high as 80% or more. Attached Figure Description
[0021] Figure 1 is a chromatogram of crude telpolide in Example 1; Figure 2 is an HPLC chromatogram of the first fraction obtained after the first purification of crude telpolide in Example 1; Figure 3 is an HPLC chromatogram of the second fraction obtained after the second purification of crude telpolide in Example 1; Figure 4 is an HPLC chromatogram of the first fraction obtained after the first purification of crude telpolide in Example 2; Figure 5 is an HPLC chromatogram of the second fraction obtained after the second purification of crude telpolide in Example 2; Figure 6 is an HPLC chromatogram of the first fraction obtained after the first purification of crude telpolide in Example 5; Figure 7 is an HPLC chromatogram of the second fraction obtained after the second purification of crude telpolide in Example 5; Figure 8 is an HPLC chromatogram of the first fraction obtained after the first purification of crude telpolide in Example 6; Figure 9 is an HPLC chromatogram of the second fraction obtained after the second purification of crude telpolide in Example 6; Figure 10 is an HPLC chromatogram of the first fraction obtained after the first purification of crude telpolide in Example 7. Figure 11 is an HPLC chromatogram of the second fraction obtained from the second purification of crude telpolide in Example 7; Figure 12 is an HPLC chromatogram of the first fraction obtained from the first purification of crude telpolide in Comparative Example 1; Figure 13 is an HPLC chromatogram of the second fraction obtained from the second purification of crude telpolide in Comparative Example 1; Figure 14 is an HPLC chromatogram of the second fraction obtained from the first purification of crude telpolide in Comparative Example 2; Figure 15 is an HPLC chromatogram of the second fraction obtained from the second purification of crude telpolide in Comparative Example 2; Figure 16 is an HPLC chromatogram of the second fraction obtained from the first purification of crude telpolide in Comparative Example 3; Figure 17 is an HPLC chromatogram of the second fraction obtained from the second purification of crude telpolide in Comparative Example 3. Detailed Implementation
[0022] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention. Unless otherwise specified, the methods used in the present invention are conventional production methods; the raw materials used, unless otherwise specified, are conventional commercially available products.
[0023] The purification method of the present invention includes four steps: (a) preparing a crude solution of telpoeptide; (b) performing a first purification on the crude solution of telpoeptide 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 replacement, filtration sterilization, and freeze-drying on the second fraction to obtain the telpoeptide product.
[0024] In step (a), the present invention dissolves crude telpoide in an acetonitrile aqueous solution. Adding a certain proportion of acetonitrile to the solvent accelerates the complete deacidification reaction of tryptophan, facilitating purification, separation, and improved recovery rate. Furthermore, the crude telpoide exhibits better solubility, with a concentration up to 50 g / L. The solution is acidic and stable, solving the problem of crude solution deterioration caused by large solvent volumes and long purification cycles during large-scale production. In a preferred embodiment of the present invention, the volume concentration of the acetonitrile aqueous solution is 20-40%, more preferably 30%. In another preferred embodiment, after dissolving the crude telpoide in the acetonitrile aqueous solution, the mixture is stirred for 4 hours to allow for sufficient reaction, followed by filtration, for example using a 0.45 μm filter membrane to remove insoluble matter before subsequent purification.
[0025] After dissolving telpoeptide, it was purified twice (steps (b) and (c)). The two purifications were carried out using an acid-base complementary method, which can effectively remove impurities with different degrees of separation under different properties, and can effectively separate deamidated impurities and racemic impurities that are close to the main peak.
[0026] Specifically, in the first purification (step (b)), a reversed-phase packing is used as the stationary phase. Mobile phase A is a 0.2%–0.4% (v / v) aqueous solution of phosphoric acid, with tetrabutylammonium bisulfate or triethylamine added to adjust the pH to 2–4. Mobile phase B is a mixture of acetonitrile and isopropanol in a 3:1 (v / v) ratio. The elution gradient is 61%–52% (v / v) of mobile phase A and 39%–48% (v / v) of mobile phase B, and linear gradient elution is performed for 35–40 minutes. The 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, in step (b), mobile phase A is a 0.3% (v / v) aqueous solution of phosphoric acid with a pH of 3.0. In a preferred embodiment, the first purification is performed with linear gradient elution for 40 minutes according to the following parameters:
[0027] In a preferred embodiment, the first purification is performed according to the following parameters:
[0028] In the first purification process, the pH of mobile phase A was adjusted using tetrabutylammonium bisulfate (TBMS) or triethylamine. TBMS is an ionic compound that acts as an ion-pairing reagent, forming ion pairs with anions in the sample. This ion pairing alters the sample's retention behavior and distribution within the mobile phase. Simultaneously, TBMS can adjust the mobile phase polarity, making it more suitable for sample separation, reducing non-specific adsorption between sample molecules and the stationary phase, and resulting in more suitable retention and elution behavior of sample molecules on the column. TBMS can react with silanol groups, reducing their activity and thus minimizing tailing in alkaline samples, improving purification efficiency and product purity. Triethylamine can shield silanol groups in the packing material, reducing interactions between sample molecules and silanol groups in the stationary phase, significantly improving peak shape and eliminating tailing. Isopropanol was added to mobile phase B to improve the solubility of thiopeptide, further enhancing purification efficiency and product purity.
[0029] The second purification (step (c)) uses a reversed-phase packing as the stationary phase, with mobile phase A being an ammonium bicarbonate solution at a concentration of 50–100 mM, and mobile phase B being acetonitrile. The elution gradient is 65%–60% by volume for mobile phase A and 35%–40% by volume for mobile phase B. Linear gradient elution is performed for 35–40 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. 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 invention, linear gradient elution is performed for 35 minutes according to the following parameters:
[0030] In a preferred embodiment, the second purification is performed according to the following parameters:
[0031] In a preferred embodiment, the stationary phases in the first and second purification processes can be the same or different, for example, selected from any one of tetraalkylsilane-bonded silica gel packing, octaalkylsilane-bonded silica gel packing, and octadecylsilane-bonded silica gel packing. More preferably, both the first and second reversed-phase packings are octadecylsilane-bonded silica gel packing, and in step (b), the first mobile phase A is 0.3% phosphoric acid aqueous solution with tetrabutylammonium bisulfate added to adjust the pH to 3.0; and in step (c), the second mobile phase A is a 100mM ammonium bicarbonate solution.
[0032] This invention improves the purification recovery rate and purity of the product by selecting different mobile phases and optimizing the elution gradient in two purification processes, resulting in a single impurity content of less than 0.1%. Furthermore, compared to the common 100-minute purification cycle per injection, this invention provides an elution cycle of 35-40 minutes, significantly shortening the purification cycle to a much lower level than conventional methods. This substantially reduces the use of organic solvents in the purification of crude telpolide, lowering purification costs and promoting a more environmentally friendly long-term development.
[0033] After two purification processes, the second fraction is concentrated by nanofiltration, salt replaced, filtered for sterilization, and lyophilized to obtain telpoeptide. In a preferred embodiment, the second fraction is concentrated by nanofiltration, salt replaced, and filtered for sterilization to obtain a solution before lyophilization. The concentration of the solution before lyophilization is 70–100 mg / g, and the height of the solution before lyophilization does not exceed 1.5 cm. The height of the solution before lyophilization affects the moisture content; if the height is too high, the moisture content will be high. This invention reduces the moisture content of the product by controlling the height of the solution before lyophilization to not exceed 1.5 cm.
[0034] The purification method of this invention can significantly shorten the purification cycle and obtain high-purity products. The purity of the telpoeptide product reaches over 99.5%, with single impurities controlled below 0.1%, and the total purification recovery rate is over 80%. Example 1
[0035] The crude telpoeptide was dissolved, purified for the first time, and purified for the second time using the following steps. The crude telpoeptide was prepared by the applicant using a solid-phase total synthesis method, and the net peptide content was determined using a standard according to methods commonly used in the art.
[0036] (a) Accurately weigh 24.0 g of crude telpoide (net peptide content 13.2 g), dissolve it in 400 mL of acetonitrile aqueous solution with a volume concentration of 30%, stir for 4 hours to obtain crude telpoide solution. HPLC (Waters, 2695) showed that the decarboxylation of tryptophan was complete, and the purity of crude product was determined to be 73.95%. Its HPLC chromatogram is shown in Figure 1.
[0037] (b) First purification: The crude telpoide solution obtained in step (a) was subjected to the first purification under the following conditions: MPLC-DAC50 preparation system (Hanbang Technology); Stationary phase: octaalkylsilane-bonded silica gel packing (Nanomicro Technology); Flow rate: 50 mL / min; Detection wavelength: 275 nm; Mobile phase A: 0.2% (v / v) aqueous phosphoric acid solution, with tetrabutylammonium bisulfate added to adjust the pH to 3.5 (phosphoric acid: Sinopharm Chemical; tetrabutylammonium bisulfate: Sinopharm Chemical); Mobile phase B: acetonitrile / isopropanol = 3 / 1 (v / v) (acetonitrile: Shanghai Xingke; isopropanol: Xilong Scientific); Loading capacity: 8 g of crude product was loaded; After loading, purification was performed according to the parameters in Table 1 below (including pre-injection equilibration, linear elution, column regeneration, and post-injection equilibration), with linear gradient elution for 40 min.
[0038] Table 1 (Pre-injection equilibration - linear elution - column regeneration - post-injection equilibration)
[0039] The target peak was collected, and the fraction with a purity greater than 97% and a single impurity of less than 1.5% was collected as the first fraction. The purity of the first fraction was detected to be 97.35% and the single impurity was less than 1.5%. Its HPLC chromatogram is shown in Figure 2.
[0040] (c) Second purification: The first fraction obtained in step (b) was purified a second time under the following conditions: MPLC-DAC50 preparation system (Hanbang Technology); Stationary phase: octylsilane-bonded silica gel packing (Nanomicro Technology); Flow rate: 50 mL / min; Detection wavelength: 275 nm; Mobile phase A: 100 mM ammonium bicarbonate solution (ammonium bicarbonate: Sinopharm Chemical); Mobile phase B: acetonitrile (Shanghai Xingke); Loading capacity: 8 g; Purification was performed according to the parameters in Table 2 below (including pre-injection equilibration, linear elution, column regeneration, and post-injection equilibration), with linear gradient elution for 35 min.
[0041] Table 2 (Pre-injection equilibration - linear elution - column regeneration - post-injection equilibration)
[0042] The target peak was collected, and the fraction with a purity greater than 99.5% and a single impurity of 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 chromatogram is shown in Figure 3. Example 2
[0043] Except for the following conditions, all other conditions were the same as in Example 1: (b) First purification: Stationary phase: octadecylsilane-bonded silica gel packing (Nanomicro Technology); Mobile phase A: 0.3% aqueous phosphoric acid solution, with tetrabutylammonium bisulfate added to adjust the pH to 3.0; Mobile phase B: acetonitrile / isopropanol = 3 / 1 (v / v); The fraction with a purity greater than 97% was collected as the first fraction, with a purity of 97.90%, and its HPLC chromatogram is shown in Figure 4.
[0044] (c) Second purification: Stationary phase: octadecylsilane-bonded silica gel packing material (NanoMicro Technology); The fraction with a purity greater than 99.5% was collected as the second fraction, which had a purity of 99.88% and a single impurity of less than 0.1%. Its HPLC chromatogram is shown in Figure 5. Example 3
[0045] 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 adjusted to 30Hz, and the membrane pressure was adjusted to 1.5MPa. Continuous nanofiltration concentration was performed using the multifunctional membrane nanofiltration machine. When the volume of the reflux product liquid reached 10% of the original volume, 0.02% sodium hydroxide solution (sodium hydroxide: Sinopharm Chemical) was continuously added to replace at least 5 volumes to achieve salt conversion. After filtration through a 0.22μm filter membrane, a solution with a concentration of 75mg / ml before lyophilization was obtained. This solution was placed in a lyophilization tray, with the solution height controlled to not exceed 1.5cm, and lyophilized for 50 hours to obtain 10.7g of finished product. The total purification recovery rate was calculated to be 81%, where the total purification recovery rate = weight of lyophilized finished product / net peptide content before purification x 100%. Example 4
[0046] The second fraction obtained in Example 2 was concentrated by nanofiltration and converted to salt using the same method as in Example 3. After filtration through a 0.22 μm filter membrane, a solution with a concentration of 72 mg / ml was obtained before lyophilization. This solution was placed in a lyophilization tray, with the solution height controlled to not exceed 1.5 cm, and lyophilized for 50 hours to obtain 11.2 g of the final product, with a total purification recovery rate of 84.8%. Example 5
[0047] Except for the first purification step, which involved adjusting the pH by adding triethylamine to mobile phase A, everything else was the same as in Example 1. The purity of the first fraction was 97.53%, and its HPLC chromatogram is shown in Figure 6. The purity of the second fraction was 99.80%, with a single impurity of less than 0.1%, and its HPLC chromatogram is shown in Figure 7. Example 6
[0048] Except for the concentration of ammonium bicarbonate in the second mobile phase A being 50 mM in the second purification step, everything else was the same as in Example 5. The purity of the first fraction obtained was 97.53%, and its HPLC chromatogram is shown in Figure 8. The purity of the second fraction obtained was 99.83%, with a single impurity of less than 0.1%, and its HPLC chromatogram is shown in Figure 9. Example 7
[0049] Except for the concentration of ammonium bicarbonate in the second mobile phase A being 75 mM in the second purification step, everything else was the same as in Example 5. The purity of the first fraction obtained was 97.53%, and its HPLC chromatogram is shown in Figure 10. The purity of the second fraction obtained was 99.81%, with a single impurity of less than 0.1%, and its HPLC chromatogram is shown in Figure 11. Example 8
[0050] The second fraction obtained in Example 5 was concentrated by nanofiltration and converted to salt using the same method as in Example 3. After filtration through a 0.22 μm filter membrane, a solution with a concentration of 70 mg / ml was obtained before lyophilization. This solution was placed in a lyophilization tray, with the solution height controlled to not exceed 1.5 cm, and lyophilized for 50 hours to obtain 11.4 g of the final product, with a total purification recovery rate of 86.3%. Example 9
[0051] The second fraction obtained in Example 6 was concentrated by nanofiltration and converted to salt using the same method as in Example 3. After filtration through a 0.22 μm filter membrane, a solution with a concentration of 71 mg / ml was obtained before lyophilization. This solution was placed in a lyophilization tray, with the solution height controlled to not exceed 1.5 cm, and lyophilized for 50 hours to obtain 11.3 g of the final product, with a total purification recovery rate of 85.6%. Example 10
[0052] The second fraction obtained in Example 7 was concentrated by nanofiltration and converted to salt using the same method as in Example 3. It was then filtered through a 0.22 μm filter membrane to obtain a solution before lyophilization with a concentration of 71 mg / ml. This solution was placed in a lyophilization tray, with the solution height controlled to not exceed 1.5 cm, and lyophilized for 50 hours to obtain 11.3 g of the final product, with a total purification recovery rate of 85.6%.
[0053] Comparative Example 1 was identical to Example 1 except that ammonia was used to adjust the pH of mobile phase A in the first purification step. The purity of the first fraction was 94.20%, and its HPLC chromatogram is shown in Figure 12. The purity of the second fraction was 99.42%, and it contained more than 0.1% of a single impurity, and its HPLC chromatogram is shown in Figure 13.
[0054] Comparative Example 2 was identical to Example 1 except that the mobile phase B in the first purification step was 100% acetonitrile (v / v). The purity of the first fraction obtained was 95.49%, and its HPLC chromatogram is shown in Figure 14. The purity of the second fraction obtained was 99.48%, and it contained more than 0.1% of a single impurity, and its HPLC chromatogram is shown in Figure 15.
[0055] Comparative Example 3 was identical to Example 1 except that the elution gradient in the first purification step was 60%–51% mobile phase A and 40%–49% mobile phase B by volume. The purity of the first fraction obtained was 94.53%, and its HPLC chromatogram is shown in Figure 16. The purity of the second fraction obtained was 99.40%, and it contained more than 0.1% single impurities, and its HPLC chromatogram is shown in Figure 17.
[0056] Table 3 ("-" indicates the same as the previous one)
[0057] The results of the above embodiments and comparative examples are shown in Table 3. In Comparative Example 1, the pH value of mobile phase A in the first purification step was adjusted by adding ammonia. The purity of the first and second fractions obtained was lower than that of the present invention, and there was a single impurity greater than 0.1%. 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 a single impurity greater than 0.1%. In Comparative Example 3, the elution gradient in the first purification step was different from that of the present invention, and the purity of the first fraction obtained was low. This invention accelerates the deacidification reaction of tryptophan and improves the solubility of crude telpoide by appropriately adding acetonitrile to the solvent in which it is dissolved. By selecting different mobile phases and optimizing the elution gradient, the purity and recovery rate of the product are improved, and the content of single impurities in the product is controlled to below 0.1%. Furthermore, the tailing phenomenon is improved by adding tetrabutylammonium bisulfate or triethylamine to the first mobile phase A, and the solubility of telpoide is improved by adding isopropanol to the first mobile phase B, thus increasing purification efficiency. This optimizes the entire purification process, significantly shortening the purification cycle, reducing the use of purification reagents, lowering purification costs, and simultaneously improving product purity and recovery rate, ensuring that the content of single impurities in the product is less than 0.1%, achieving green and sustainable development.
[0058] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. 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 be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.
Claims
1. A method for purifying telopeptides, characterized in that, The purification method includes the following steps: (a) The crude telpoide raw material was dissolved in an aqueous acetonitrile solution to obtain a crude telpoide solution; (b) The crude telpoeptide solution was subjected to a first purification. The first stationary phase is a reverse-phase packing; The first mobile phase A is an aqueous phosphoric acid 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 in a volume ratio of 3:1; The elution gradient is 61%–52% by volume of the first mobile phase A and 39%–48% by volume of the first mobile phase B. Linear gradient elution is performed for 35–40 minutes, and the fraction with a purity greater than 97% and a single impurity of less than 1.5% is collected as the first fraction. (c) The first fraction is purified a second time. The second stationary phase is a reverse-phase packing; The second mobile phase A is an ammonium bicarbonate solution, wherein the concentration of ammonium bicarbonate is 50-100 mM; The second mobile phase B is acetonitrile; The elution gradient is such that the volume percentage of the second mobile phase A is 65%–60% and the volume percentage of the second mobile phase B is 35%–40%. Linear gradient elution is performed for 35–40 minutes, and the fraction with a purity greater than 99.5% and a single impurity of less than 0.1% is collected as the second fraction. (d) The second fraction is concentrated by nanofiltration, salt is replaced, and sterilized by filtration to obtain a solution before lyophilization. The solution before lyophilization is then lyophilized to obtain telpoeptide.
2. The purification method of claim 1, wherein, In step (a), the crude telpoeptide 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 telpoeptide solution.
3. The purification method according to claim 2, characterized in that, In step (a), the solubility of the acetonitrile aqueous solution is 30%.
4. The purification method of claim 1, wherein, In 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 of claim 1, wherein, In step (c), the second mobile phase A is a 100 mM ammonium bicarbonate solution.
6. The purification method of claim 1, wherein, The first reversed-phase packing and the second reversed-phase packing are each independently selected from any one of tetraalkylsilane-bonded silica gel packing, octaalkylsilane-bonded silica gel packing, and octadecylsilane-bonded silica gel packing.
7. The purification method of claim 6, wherein, Both the first and second reversed-phase packing materials are octadecylsilane-bonded silica gel packing materials. In step (b), the first mobile phase A is a 0.3% volume concentration aqueous solution of phosphoric acid, and tetrabutylammonium bisulfate is added to adjust the pH to 3.
0. In step (c), the second mobile phase A is a 100mM ammonium bicarbonate solution.
8. The purification method of claim 1, wherein, The concentration of the solution before lyophilization is 70-100 mg / g, and the height of the solution before lyophilization does not exceed 1.5 cm.
9. The purification method of claim 1, wherein, In said step (b), linear gradient elution is performed for 40 minutes according to the following parameters: In said step (c), linear gradient elution is performed for 35 minutes according to the following parameters:
10. The purification method of claim 9, wherein, In said step (b), the first purification is performed according to the following parameters: In said step (c), said second purification is performed according to the following parameters: