A method for the purification of telopeptides from specific difficultly removed impurities

Abstract

This invention discloses a purification method for telpoide to remove specific difficult-to-remove impurities. The mobile phase used in this method can remove most of the impurities present in telpoide, especially for the missing peptide Ser. 11 Deletion of peptide Ile 12 This method effectively removes even the most difficult-to-remove impurities, reducing their concentration to less than 0.1%. Compared to other patents, this method only adds a 0.0004% sodium hydroxide solution with a pH of 10.00 to the solution during the concentration process, without affecting the purity or stability of the sample. Furthermore, a large amount of water is added and discharged during nanofiltration, carrying away most of the solvents used in the process and significantly reducing residual solvents in the finished product.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide synthesis and purification technology, specifically relating to a purification method for telpoeptide. Background Technology

[0002] Thiropeptide, a dual receptor agonist of GIP (gastric inhibitory polypeptide) and GLP-1 (glucagon-like peptide-1) developed by Eli Lilly, was approved by the US FDA in May 2022. It integrates the effects of two incretins into a single molecule to improve glycemic control in adults with type 2 diabetes. In November 2023, thiopeptide received FDA approval for weight management in obese and overweight adults. On May 21, 2024, the China National Medical Products Administration (NMPA) announced that Eli Lilly's application for marketing authorization of thiopeptide injection had been approved for the treatment of glycemic control in adults with type 2 diabetes. In addition, thiopeptide is also being investigated for indications such as NASH (non-alcoholic steatohepatitis) and heart failure. Thiropeptide is also the first dual-target agonist obesity treatment among similar marketed products. Both GIP and GLP-1 stimulate insulin secretion and lower blood glucose levels. In addition, GIP can increase energy expenditure. Therefore, telpolide can have a greater impact on blood sugar and weight. Its amino acid sequence is as follows: Tyr1-Aib-Glu-Gly-Thr5-Phe-Thr-Ser-Asp-Tyr10-Ser-Ile-Aib-Leu-Asp15-Lys-Ile-Ala-Gln-Lys20(AEEA-AEEA-γGlu-Eicosanedioic-acid)-Ala-Phe-Val-Gln-Trp25-Leu-Ile-Ala-Gly-Gly30-Pro-Ser-Ser-Gly-Ala35-Pro-Pro-Pro-Ser-NH2.

[0003] CN112661815A: This invention discloses a purification method for telpolide, in which crude telpolide is dissolved in purified water, and the pH is adjusted to 8.0 with ammonia until completely dissolved to obtain a crude peptide solution. The sample is then purified in two steps. However, the sample obtained by this invention has low purity, and Cl ions are introduced into the sample during the purification process.

[0004] CN117736273B: This invention discloses a purification method for telpolide, in which crude telpolide is dissolved in 50 mmol of ammonium bicarbonate, followed by three-step purification, salt conversion, concentration, and freeze-drying to obtain the sample. In this invention, after salt conversion, a sodium hydroxide solution with a pH as high as 12.70 is directly added to the purified fraction, which may affect the purity of the sample during subsequent scale-up production.

[0005] To date, the literature reports the following problems:

[0006] 1. In most patents, the solution after crude peptide dissolution is directly purified and eluted using a C8 reversed-phase column. After long-term operation, the life of the packing material will be greatly reduced, and the equipment maintenance will become more difficult.

[0007] 2. Some patented salt transfer operations either introduce new ions or may generate new impurities.

[0008] 3. Some solvent may remain during the concentration process, resulting in excessive residual solvent in the peptide. Summary of the Invention

[0009] To address the above drawbacks, this invention discloses a purification method for telpoeptide to remove specific difficult-to-remove impurities. The purification method includes the following steps:

[0010] Sample preparation: Dissolve the crude solid telpoeptide in a 5% sodium bicarbonate-acetonitrile solution, stir thoroughly to ensure complete dissolution, heat in a water bath, filter through a filter membrane, and collect the filtrate for later use.

[0011] Pretreatment: A chromatographic column with polymer as the stationary phase; Phase A: 0.3% phosphate buffer (adjusted to pH 8.0 with ammonia); Phase B: acetonitrile;

[0012] One-phase: A chromatographic column with octaalkylsilane-bonded silica gel as the stationary phase; mobile phase: Phase A: 0.1% TFA aqueous solution; Phase B: acetonitrile.

[0013] Second-stage chromatography column: octaalkylsilane-bonded silica gel as stationary phase; mobile phase: Phase A: 50 mmol / L ammonium dihydrogen phosphate aqueous solution, pH adjusted to 7.0 with ammonia; Phase B: acetonitrile.

[0014] Salt conversion: A chromatographic column with octaalkylsilane-bonded silica gel as the stationary phase; mobile phase: phase A: water; phase B: acetonitrile;

[0015] Concentration and freeze-drying: The collected target peak fraction was concentrated by rotary evaporation under reduced pressure at a water temperature below 34 degrees Celsius until the acetonitrile content in the sample solution was reduced to a low level. Then, nanofiltration was used to continue the concentration, during which 0.0004% sodium hydroxide solution was added multiple times, followed by freeze-drying.

[0016] According to some of the technical solutions of the present invention, the volume ratio of the above-mentioned 5% sodium bicarbonate-acetonitrile solution is 9:1.

[0017] According to some technical solutions of the present invention, the chromatographic column specifications in the above pretreatment step are: 150mm × 250mm, flow rate: 400mL / min, detection wavelength: 230nm. Gradient: B%: 32% → 47%, 40min. Purification process: The chromatographic column is rinsed with 70% acetonitrile aqueous solution and then equilibrated with 95% A + 5% B as the mobile phase before loading the sample.

[0018] According to some of the technical solutions of the present invention, in the above-mentioned pure step, the chromatographic column specifications are: 150mm×250mm, flow rate: 400mL / min, detection wavelength: 230nm; gradient: B%: 37%→45%, 50min; the chromatographic column is rinsed with 70% acetonitrile aqueous solution and then equilibrated with 95%A+5%B as the mobile phase before loading the sample. After loading the sample, linear gradient elution is performed for 50min.

[0019] According to some of the technical solutions of the present invention, in the above-mentioned purification steps, the chromatographic column specifications are: 150mm×250mm, flow rate: 400mL / min, detection wavelength: 230nm, gradient: B%: 34%→44%, 50min; the chromatographic column is rinsed with 70% acetonitrile aqueous solution and then equilibrated with 95%A+5%B as the mobile phase before loading the sample. After the sample loading is completed, linear gradient elution is performed for 50min.

[0020] According to some of the technical solutions of the present invention, in the above-mentioned salt conversion step, the chromatographic column specifications are: 150mm×250mm, flow rate: 400mL / min, detection wavelength: 230nm, gradient: B%: 30%→50%, 30min; after rinsing the chromatographic column with 70% acetonitrile aqueous solution, equilibrate it with 95%A+5%B as the mobile phase, load the sample, and after the sample loading is completed, perform linear gradient elution for 30min.

[0021] According to some of the technical solutions of the present invention, in the above-mentioned concentration freeze-drying step, the collected target peak fraction is concentrated by vacuum rotary evaporation at a water temperature below 34 degrees Celsius, and further concentrated using nanofiltration equipment, during which the above-mentioned 0.0004% sodium hydroxide solution is added multiple times.

[0022] According to some of the technical solutions of the present invention, the purification method described above monitors the deletion peptide Ser. 11 and missing peptide Ile 12 .

[0023] This invention uses a polymer column for purification and elution of the crude peptide solution before elution using a C8 column. This purification process effectively removes substances in the sample that could damage the reversed-phase packing material. The salt transfer operation uses only water and organic solvents, having minimal impact on sample purity and introducing no other ions. This significantly reduces residual solvent in the final product. The salt transfer is performed using water and an organic phase as the mobile phase. The qualified fractions are combined, thoroughly mixed, and then concentrated under reduced pressure with an appropriate amount of low-concentration sodium hydroxide solution. This is followed by multiple nanofiltration operations, during which an appropriate amount of low-concentration sodium hydroxide solution is added. Attached Figure Description

[0024] Figure 1 This is the liquid phase detection spectrum of Example 1 of the present invention;

[0025] Figure 2 This is the liquid phase detection spectrum of Example 1 of the present invention;

[0026] Figure 3 This is the liquid phase detection spectrum of Example 1 of the present invention;

[0027] Figure 4 This is the liquid phase detection spectrum of Example 1 of the present invention;

[0028] Figure 5 This is the liquid phase detection spectrum of Example 2 of the present invention;

[0029] Figure 6 This is the liquid phase detection spectrum of Example 2 of the present invention;

[0030] Figure 7 This is the liquid phase detection spectrum of Example 2 of the present invention;

[0031] Figure 8 This is the liquid phase detection spectrum of Example 2 of the present invention;

[0032] Figure 9 This is the liquid phase detection spectrum of Example 3 of the present invention;

[0033] Figure 10 This is the liquid phase detection spectrum of Example 3 of the present invention;

[0034] Figure 11 This is the liquid phase detection spectrum of Example 3 of the present invention;

[0035] Figure 12 This is the liquid phase detection spectrum of Example 3 of the present invention. Detailed Implementation

[0036] The following embodiments are for further illustration of some preferred embodiments of the present invention and are not all embodiments. Other embodiments based on the present invention made by those skilled in the art without inventive effort are all within the scope of protection of the present invention.

[0037] In this invention, unless otherwise specified, all abbreviations have the conventional meanings understood by those skilled in the art.

[0038] Example 1:

[0039] Sample processing: 24.88g of solid telpoeptide crude peptide (with attached...) Figure 1 Dissolve the sample in an appropriate amount of 5% sodium bicarbonate-acetonitrile (9:1), stir thoroughly to ensure complete dissolution, heat in a water bath at 38 degrees Celsius for 16 hours, filter through a 0.45 μm filter membrane, and collect the filtrate for later use.

[0040] 1. Pretreatment:

[0041] Purification conditions: Chromatographic column: Polymer-based stationary column, 150 mm × 250 mm. Mobile phase: Phase A: 0.3% phosphate buffer (pH adjusted to 8.0 with ammonia); Phase B: Acetonitrile, flow rate: 400 mL / min, detection wavelength: 230 nm. Gradient: B%: 32% → 47%, 40 min.

[0042] Purification process: The chromatographic column was rinsed with 70% acetonitrile aqueous solution, then equilibrated with 95% A + 5% B as the mobile phase before loading the sample (25g). After loading, elution was performed using a linear gradient for 40 minutes. The target peak fraction was collected. The collected target peak fraction was diluted by half with an equal volume of purified water to obtain a pure sample for loading.

[0043] 2. One Pure:

[0044] Purification conditions: Column: Octylsilane-bonded silica gel column, 150 mm × 250 mm. Mobile phase: Phase A: 0.1% TFA aqueous solution; Phase B: acetonitrile; flow rate: 400 mL / min; detection wavelength: 230 nm. Gradient: B%: 37% → 45%, 50 min.

[0045] Purification process: The chromatographic column was rinsed thoroughly with 70% acetonitrile aqueous solution, then equilibrated using 95% A + 5% B as the mobile phase before loading the sample. After sample loading, linear gradient elution was performed for 50 minutes. The target peak was collected, yielding a fraction with a purity greater than 97% (see attached image). Figure 2 ), in which the peptide Ser is missing 11 It accounts for 0.360%, and the missing peptide Ile 12It accounts for 0.548%. The collected target peak fraction was diluted by half with an equal volume of purified water and used as the sample for secondary purification.

[0046] 3. Two pure ones:

[0047] Purification conditions: Chromatographic column: 150 mm × 250 mm column with octaalkylsilane-bonded silica gel as the stationary phase. Mobile phase: Phase A: 50 mmol / L ammonium dihydrogen phosphate aqueous solution, pH adjusted to 7.0 with ammonia; Phase B: acetonitrile, flow rate: 400 mL / min, detection wavelength: 230 nm. Gradient: B%: 34% → 44%, 50 min.

[0048] Purification process: The chromatographic column was rinsed thoroughly with 70% acetonitrile aqueous solution, then equilibrated using 95% A + 5% B as the mobile phase before loading the sample. After sample loading, linear gradient elution was performed for 50 min. The target peak was collected, yielding a fraction with a purity greater than 99.3% and a single impurity content less than 0.15% (see attached image). Figure 3 ), in which the peptide Ser is missing 11 It accounts for 0.329%, and the missing peptide Ile 12 It accounts for 0.029%. The collected target peak fraction was diluted by half with an equal volume of purified water and used as the sample for salt transfer and loading.

[0049] 4. Salt conversion:

[0050] Purification conditions: Chromatographic column: 150 mm × 250 mm column with octaalkylsilane-bonded silica gel as the stationary phase. Mobile phase: Phase A: water; Phase B: acetonitrile; flow rate: 400 mL / min; detection wavelength: 230 nm. Gradient: B%: 30% → 50%, 30 min.

[0051] Purification process: The chromatographic column was rinsed thoroughly with 70% acetonitrile aqueous solution, then equilibrated using 95% A + 5% B as the mobile phase before loading the sample. After sample loading, linear gradient elution was performed for 30 minutes. The target peak was collected, yielding a fraction with a purity greater than 99.5% and less than 0.1% of single impurities (see attached image). Figure 4 ), in which the peptide Ser is missing 11 It accounts for 0.080%, lacking peptide Ile 12 It accounts for 0.037%.

[0052] 5. Concentrated freeze-drying:

[0053] The collected target peak fraction was concentrated by rotary evaporation under reduced pressure at a water temperature below 34°C until the acetonitrile content in the sample solution was low. Further concentration was then achieved using nanofiltration, with 0.0004% sodium hydroxide solution added multiple times during this process. Once the sample concentration reached approximately 20 mg / mL, it was freeze-dried to obtain 7.14 g of telpolide with a purity of 99.78%, representing an overall purification yield of 69.6%.

[0054] Example 2

[0055] Sample processing: 20.15g of solid telpoeptide crude peptide (with attached...) Figure 5 Dissolve the sample in an appropriate amount of 5% sodium bicarbonate-acetonitrile (9:1), stir thoroughly to ensure complete dissolution, heat in a water bath at 38 degrees Celsius for 16 hours, filter through a 0.45 μm filter membrane, and collect the filtrate for later use.

[0056] 1. Pretreatment:

[0057] Purification conditions: Chromatographic column: Polymer-based stationary column, 150 mm × 250 mm. Mobile phase: Phase A: 0.3% phosphate buffer (pH adjusted to 8.0 with ammonia); Phase B: Acetonitrile, flow rate: 400 mL / min, detection wavelength: 230 nm. Gradient: B%: 32% → 47%, 40 min.

[0058] Purification process: The chromatographic column was rinsed with 70% acetonitrile aqueous solution, then equilibrated with 95% A + 5% B as the mobile phase before loading the sample (25g). After loading, elution was performed using a linear gradient for 40 minutes. The target peak fraction was collected. The collected target peak fraction was diluted by half with an equal volume of purified water to obtain a pure sample for loading.

[0059] 2. One Pure:

[0060] Purification conditions: Column: Octylsilane-bonded silica gel column, 150 mm × 250 mm. Mobile phase: Phase A: 0.1% TFA aqueous solution; Phase B: acetonitrile; flow rate: 400 mL / min; detection wavelength: 230 nm. Gradient: B%: 37% → 45%, 50 min.

[0061] Purification process: The chromatographic column was rinsed thoroughly with 70% acetonitrile aqueous solution, then equilibrated using 95% A + 5% B as the mobile phase before loading the sample. After sample loading, linear gradient elution was performed for 50 minutes. The target peak was collected, yielding a fraction with a purity greater than 97% (see attached image). Figure 6 ), in which the peptide Ser is missing 11 It accounts for 0.351%, and the missing peptide Ile 12 It accounts for 0.615%. The collected target peak fraction was diluted by half with an equal volume of purified water and used as the sample for secondary purification.

[0062] 3. Two pure ones:

[0063] Purification conditions: Chromatographic column: 150 mm × 250 mm column with octaalkylsilane-bonded silica gel as the stationary phase. Mobile phase: Phase A: 50 mmol / L ammonium dihydrogen phosphate aqueous solution, pH adjusted to 7.0 with ammonia; Phase B: acetonitrile, flow rate: 400 mL / min, detection wavelength: 230 nm. Gradient: B%: 34% → 44%, 50 min.

[0064] Purification process: The chromatographic column was rinsed thoroughly with 70% acetonitrile aqueous solution, then equilibrated using 95% A + 5% B as the mobile phase before loading the sample. After sample loading, linear gradient elution was performed for 50 min. The target peak was collected, yielding a fraction with a purity greater than 99.3% and a single impurity content less than 0.15% (see attached image). Figure 7 ), in which the peptide Ser is missing 11 It accounts for 0.314%, and the missing peptide Ile 12 Not detected. The collected target peak fraction was diluted by half with an equal volume of purified water and used as the sample for salt transfer loading.

[0065] 4. Salt conversion:

[0066] Purification conditions: Chromatographic column: 150 mm × 250 mm column with octaalkylsilane-bonded silica gel as the stationary phase. Mobile phase: Phase A: water; Phase B: acetonitrile; flow rate: 400 mL / min; detection wavelength: 230 nm. Gradient: B%: 30% → 50%, 30 min.

[0067] Purification process: The chromatographic column was rinsed thoroughly with 70% acetonitrile aqueous solution, then equilibrated using 95% A + 5% B as the mobile phase before loading the sample. After sample loading, linear gradient elution was performed for 30 minutes. The target peak was collected, yielding a fraction with a purity greater than 99.5% and less than 0.1% of single impurities (see attached image). Figure 8 ), in which the peptide Ser is missing 11 It accounts for 0.082%, lacking the peptide Ile. 12 It accounts for 0.036%.

[0068] 5. Concentrated freeze-drying:

[0069] The collected target peak fraction was concentrated by rotary evaporation under reduced pressure at a water temperature below 34°C until the acetonitrile content in the sample solution was low. Further concentration was then achieved using nanofiltration, with 0.0004% sodium hydroxide solution added multiple times during this process. Once the sample concentration reached approximately 20 mg / mL, it was freeze-dried to obtain 5.89 g of telpolide with a purity of 99.83%, representing an overall purification yield of 70.9%.

[0070] Example 3

[0071] Sample preparation: Dissolve 25.23g of solid telpoeptide crude peptide in an appropriate amount of 5% sodium bicarbonate-acetonitrile (9:1), stir thoroughly to ensure complete dissolution, heat in a water bath at 38 degrees Celsius for 16 hours, filter through a 0.45μm filter membrane, and collect the filtrate for later use.

[0072] 1. Pretreatment:

[0073] Purification conditions: Chromatographic column: Polymer-based stationary column, 150 mm × 250 mm. Mobile phase: Phase A: 0.3% phosphate buffer (pH adjusted to 8.0 with ammonia); Phase B: Acetonitrile, flow rate: 400 mL / min, detection wavelength: 230 nm. Gradient: B%: 32% → 47%, 40 min.

[0074] Purification process: The chromatographic column was rinsed thoroughly with 70% acetonitrile aqueous solution, then equilibrated with 95% A + 5% B as the mobile phase before loading the sample (25g). After loading, elution was performed using a linear gradient elution for 40 minutes. The target peak fraction was collected (see attached image). Figure 9 The collected target peak fraction was diluted by half with an equal volume of purified water to obtain a pure sample for loading.

[0075] 2. One Pure:

[0076] Purification conditions: Column: Octylsilane-bonded silica gel column, 150 mm × 250 mm. Mobile phase: Phase A: 0.1% TFA aqueous solution; Phase B: acetonitrile; flow rate: 400 mL / min; detection wavelength: 230 nm. Gradient: B%: 37% → 45%, 50 min.

[0077] Purification process: The chromatographic column was rinsed thoroughly with 70% acetonitrile aqueous solution, then equilibrated using 95% A + 5% B as the mobile phase before loading the sample. After sample loading, linear gradient elution was performed for 50 minutes. The target peak was collected, yielding a fraction with a purity greater than 97% (see attached image). Figure 10 ), in which the peptide Ser is missing 11 It accounts for 0.370%, and the missing peptide Ile 12 It accounts for 0.576%. The collected target peak fraction was diluted by half with an equal volume of purified water and used as the sample for secondary purification.

[0078] 3. Two pure ones:

[0079] Purification conditions: Chromatographic column: 150 mm × 250 mm column with octaalkylsilane-bonded silica gel as the stationary phase. Mobile phase: Phase A: 50 mmol / L ammonium dihydrogen phosphate aqueous solution, pH adjusted to 7.0 with ammonia; Phase B: acetonitrile, flow rate: 400 mL / min, detection wavelength: 230 nm. Gradient: B%: 34% → 44%, 50 min.

[0080] Purification process: The chromatographic column was rinsed thoroughly with 70% acetonitrile aqueous solution, then equilibrated using 95% A + 5% B as the mobile phase before loading the sample. After sample loading, linear gradient elution was performed for 50 min. The target peak was collected, yielding a fraction with a purity greater than 99.3% and a single impurity content less than 0.15% (see attached image). Figure 11 ), in which the peptide Ser is missing 11 It accounts for 0.337%, and the missing peptide Ile 12 It accounts for 0.027%. The collected target peak fraction was diluted by half with an equal volume of purified water and used as the sample for salt transfer and loading.

[0081] 4. Salt conversion:

[0082] Purification conditions: Chromatographic column: 150 mm × 250 mm column with octaalkylsilane-bonded silica gel as the stationary phase. Mobile phase: Phase A: water; Phase B: acetonitrile; flow rate: 400 mL / min; detection wavelength: 230 nm. Gradient: B%: 30% → 50%, 30 min.

[0083] Purification process: The chromatographic column was rinsed thoroughly with 70% acetonitrile aqueous solution, then equilibrated using 95% A + 5% B as the mobile phase before loading the sample. After sample loading, linear gradient elution was performed for 30 minutes. The target peak was collected, yielding a fraction with a purity greater than 99.5% and less than 0.1% of single impurities (see attached image). Figure 12 ), in which the peptide Ser is missing 11 It accounts for 0.083%, lacking peptide Ile 12 It accounts for 0.029%.

[0084] 5. Concentrated freeze-drying:

[0085] The collected target peak fraction was concentrated by rotary evaporation under reduced pressure at a water temperature below 34°C until the acetonitrile content in the sample solution was low. Further concentration was then achieved using nanofiltration, with 0.0004% sodium hydroxide solution added multiple times during this process. Once the sample concentration reached approximately 20 mg / mL, it was freeze-dried to obtain 7.28 g of telpolide with a purity of 99.83%, resulting in an overall purification yield of 70.0%.

[0086] Based on the above three batches of experiments, the mobile phase used in this method can remove most of the impurities present in telpolide, especially for the missing peptide Ser. 11 Deletion of peptide Ile12 This method effectively removes even the most difficult-to-remove impurities, reducing their concentration to less than 0.1%. Compared to other patents, this method only adds a 0.0004% sodium hydroxide solution with a pH of 10.00 to the solution during the concentration process, without affecting the purity or stability of the sample. Furthermore, a large amount of water is added and discharged during nanofiltration, carrying away most of the solvents used in the process and significantly reducing residual solvents in the finished product.

[0087] It should be noted that the above preferred embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for purifying telpoide to remove specific difficult-to-remove impurities, characterized in that, The purification method includes the following steps: Sample preparation: Dissolve the crude solid telpoeptide in a 5% sodium bicarbonate-acetonitrile solution, stir thoroughly to ensure complete dissolution, heat in a water bath, filter through a filter membrane, and collect the filtrate for later use. Pretreatment: A chromatographic column with polymer as the stationary phase; Phase A: 0.3% phosphate buffer (adjusted to pH 8.0 with ammonia); Phase B: acetonitrile; One-phase: A chromatographic column with octaalkylsilane-bonded silica gel as the stationary phase; mobile phase: Phase A: 0.1% TFA aqueous solution; Phase B: acetonitrile. Second-stage chromatography column: octaalkylsilane-bonded silica gel as stationary phase; mobile phase: Phase A: 50 mmol / L ammonium dihydrogen phosphate aqueous solution, pH adjusted to 7.0 with ammonia; Phase B: acetonitrile. Salt conversion: Chromatographic column with octaalkylsilane-bonded silica gel as stationary phase, mobile phase: phase A: water; Phase B: Acetonitrile; Concentration and freeze-drying: The collected target peak fraction was concentrated by rotary evaporation under reduced pressure at a water temperature below 34 degrees Celsius until the acetonitrile content in the sample solution decreased. Then, nanofiltration was used to continue the concentration process, during which 0.0004% sodium hydroxide solution was added multiple times, followed by freeze-drying.

2. The purification method for telpoide to remove specific difficult-to-remove impurities according to claim 1, characterized in that, The volume ratio of the 5% sodium bicarbonate-acetonitrile solution is 9:

1.

3. The purification method for telpoeptide to remove specific difficult-to-remove impurities according to claim 2, characterized in that, The pretreatment step uses a chromatographic column with dimensions of 150 mm × 250 mm, a flow rate of 400 mL / min, and a detection wavelength of 230 nm. The gradient is B% from 32% to 47% over 40 minutes. The purification process involves rinsing the column with a 70% acetonitrile aqueous solution, then equilibrating it with a mobile phase of 95% A + 5% B before loading the sample.

4. The purification method for telpoeptide to remove specific difficult-to-remove impurities according to claim 3, characterized in that, In the first pure step, the chromatographic column specifications are: 150mm×250mm, flow rate: 400mL / min, detection wavelength: 230nm; gradient: B%: 37%→45%, 50min; after rinsing the chromatographic column with 70% acetonitrile aqueous solution, equilibrate it with 95%A+5%B as the mobile phase, load the sample, and after loading the sample, perform linear gradient elution for 50min.

5. The purification method for telpoeptide to remove specific difficult-to-remove impurities according to claim 4, characterized in that, In the two purification steps, the chromatographic column specifications are: 150mm×250mm, flow rate: 400mL / min, detection wavelength: 230nm, gradient: B%: 34%→44%, 50min; after rinsing the chromatographic column with 70% acetonitrile aqueous solution, it is equilibrated with 95%A+5%B as the mobile phase, and then the sample is loaded. After the sample is loaded, linear gradient elution is performed for 50min.

6. The purification method for telpoeptide to remove specific difficult-to-remove impurities according to claim 5, characterized in that, In the salt conversion step, the chromatographic column specifications are: 150mm×250mm, flow rate: 400mL / min, detection wavelength: 230nm, gradient: B%: 30%→50%, 30min; after rinsing the chromatographic column with 70% acetonitrile aqueous solution, equilibrate it with 95%A+5%B as the mobile phase, load the sample, and after loading the sample, perform linear gradient elution for 30min.

7. The purification method for telpoeptide to remove specific difficult-to-remove impurities according to claim 6, characterized in that, In the concentration and freeze-drying step, the collected target peak fraction is concentrated by rotary evaporation under reduced pressure at a water temperature below 34 degrees Celsius, and further concentrated using nanofiltration equipment, during which the 0.0004% sodium hydroxide solution is added multiple times.

8. The purification method for telpoeptide to remove specific difficult-to-remove impurities according to claim 7, characterized in that, The purification method monitors the missing peptide Ser. 11 and missing peptide Ile 12 .

Images