Method for reducing content of trifluoroacetic acid in polypeptide
Through anion exchange resin column treatment and freeze-drying technology, the problems of low efficiency and high cost in the existing methods are solved, and the TFA removal in high-efficiency and low-cost polypeptides are achieved, which is suitable for the industrial production of polypeptide products.
Patent Information
- Application Number
- CN202510613817.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-13
- Publication Date
- 2025-07-25
AI Technical Summary
Existing methods are inefficient, costly or have an impact on the biological activity of the polypeptide when reducing the content of trifluoroacetic acid (TFA) in a polypeptide.
The peptide solution was treated with anion exchange resin column, and the pH value was controlled to be neutral to weakly alkaline, with a flow rate of 0.5-2.0 mL/min. It was then freeze-dryed and used to use strong alkaline anion exchange resins such as Dowex 1X8, Amberlite IRA-910 or DOWEX MB-50 modified resin.
It effectively reduces the TFA content in the polypeptide to 0.01% to 0.08%, is easy to operate and low cost, has no significant impact on the biological activity of the polypeptide, and is suitable for industrial production.
Smart Images
Figure CN120365352A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a method for reducing residues in polypeptides, and particularly to a method for reducing the content of trifluoroacetic acid (TFA) in polypeptides. Background Art
[0002] During the polypeptide synthesis process, trifluoroacetic acid (TFA) is often used as a solvent or an acidic additive. However, the residue of TFA in polypeptide products will have an adverse impact on biological activity, stability, and safety. The existing methods for removing TFA mainly include precipitation method, extraction method, ion exchange method, etc., but these methods have problems such as low efficiency, high cost, or affecting the biological activity of polypeptides. Therefore, it is of great significance to develop an efficient, simple, and industrializable method for removing TFA. Summary of the Invention
[0003] The object of the present invention is to provide a method that can effectively reduce the content of trifluoroacetic acid in polypeptides, mainly solving the technical problems such as low efficiency, high cost, or affecting the biological activity of polypeptides in the existing methods. This method is simple to operate, low in cost, and suitable for large-scale production.
[0004] The technical solution of the present invention: A method for reducing the content of trifluoroacetic acid in polypeptides, comprising the following steps: (1) Dissolution step: Dissolve the polypeptide in water to form a polypeptide solution; (2) Ion exchange step: Pass the polypeptide solution through an anion exchange resin column, with the pH value of the polypeptide solution being neutral to weakly alkaline, and controlling the flow rate at 0.5 - 2.0 mL / min; (3) Collection step: Collect the polypeptide solution after being treated by the anion exchange resin column; (4) Freeze-drying step: Freeze-dry the collected polypeptide solution to obtain a polypeptide product with a low TFA content.
[0005] Preferably, the anion exchange resin column in step (2) is a strongly basic anion exchange resin column, selected from one of Dowex 1X8 anion exchange resin column, Amberlite IRA-910 anion exchange resin column or DOWEX MB-50 modified anion exchange resin column. The pH value of the polypeptide solution is adjusted to 7.0 - 8.5 by adding sodium hydroxide solution. More preferably, when the pH value of the polypeptide solution is adjusted to 7.2 - 7.8, the TFA removal efficiency is the highest. After being treated by the anion exchange resin column, the TFA content in the polypeptide solution is reduced to less than 0.1%. The temperature of freeze-drying in step (4) is -40°C to -60°C, and the vacuum degree is 10⁻² to 10⁻³ Pa. More preferably, the temperature of the freeze-drying is -55°C and the vacuum degree is 10⁻² Pa to ensure that the biological activity of the polypeptide is not affected. The low TFA content in step (4) is 0.01% to 0.08%.
[0006] The beneficial effects of the present invention are as follows: The method of the present invention effectively removes trifluoroacetate in the polypeptide through an anion exchange resin column, with simple operation and low cost. This method is applicable to a variety of polypeptide products and has no obvious impact on the biological activity of the polypeptide. The method of the present invention can achieve industrial production and has high application value. Description of the Drawings
[0007] Figure 1 It is the chromatogram of trifluoroacetate ion detection in the polypeptide of Example 1.
[0008] Figure 2 It is the chromatogram of trifluoroacetate ion detection in the polypeptide of Example 2.
[0009] Figure 3 It is the chromatogram of trifluoroacetate ion detection in the polypeptide of Example 3.
[0010] Figure 4 It is the chromatogram of trifluoroacetate ion detection in the polypeptide of Example 4.
[0011] Figure 5 It is the chromatogram of trifluoroacetate ion detection in the polypeptide of Example 5.
[0012] Figure 6 It is the chromatogram of trifluoroacetate ion detection in the polypeptide of Example 6. Detailed Embodiments Example 1
[0013] 1. Dissolution step: Dissolve 100 mg of polypeptide in 10 mL of water to form a polypeptide solution.
[0014] 2. Ion exchange step: Using a Dowex 1X8 anion exchange resin column, adjust the pH value of the polypeptide solution to 7.5 and pass it through the resin column at a flow rate of 1.0 mL / min.
[0015] 3. Collection step: Collect the polypeptide solution after passing through the resin column and wait for freeze-drying.
[0016] 4. Freeze-drying step: Freeze-dry the collected polypeptide solution at a temperature of -55°C and a vacuum of 10⁻² Pa to obtain a polypeptide product with a low TFA content. Detect the TFA content, and the result is 0.02%. The chromatogram of trifluoroacetate ions in the polypeptide is shown in Figure 1 . Example 2
[0017] 1. Dissolution step: Dissolve 200 mg of polypeptide in 20 mL of water to form a polypeptide solution.
[0018] 2. Ion exchange step: Using an Amberlite IRA-910 anion exchange resin column, adjust the pH value of the polypeptide solution to 8.0 and pass it through the resin column at a flow rate of 1.5 mL / min.
[0019] 3. Collection step: Collect the polypeptide solution after passing through the resin column and wait for freeze-drying.
[0020] 4. Freeze-drying step: Freeze-dry the collected polypeptide solution at a temperature of -45°C and a vacuum of 10⁻³ Pa to obtain a polypeptide product with a low TFA content. Detect the TFA content, and the result is 0.03%. The chromatogram of trifluoroacetate ions in the polypeptide is shown in Figure 2 . Example 3
[0021] 1. Dissolution step: Dissolve 150 mg of polypeptide in 15 mL of water to form a polypeptide solution.
[0022] 2. Ion exchange step: Using a DOWEX MB-50 modified anion exchange resin column, adjust the pH value of the polypeptide solution to 7.2 and pass it through the resin column at a flow rate of 1.2 mL / min.
[0023] 3. Collection step: Collect the polypeptide solution after passing through the resin column and wait for freeze-drying.
[0024] 4. Freeze-drying step: Freeze-dry the collected polypeptide solution at a temperature of -55°C and a vacuum of 10⁻² Pa to obtain a polypeptide product with a low TFA content. Detect the TFA content, and the result is 0.01%. The chromatogram of trifluoroacetate ions in the polypeptide is shown in Figure 3 . Example 4
[0025] 1. Dissolution step: Dissolve 250 mg of the polypeptide in 25 mL of water to form a polypeptide solution.
[0026] 2. Pretreatment step: Add nano-activated carbon (0.5 g / L) to the polypeptide solution and stir for 30 minutes to initially adsorb TFA.
[0027] 3. Ion exchange step: Use a Dowex 1X8 anion exchange resin column to adjust the pH value of the polypeptide solution to 7.8 and pass it through the resin column at a flow rate of 1.0 mL / min.
[0028] 4. Collection step: Collect the polypeptide solution after treatment with the resin column and wait for lyophilization.
[0029] 5. Freeze-drying step: Lyophilize the collected polypeptide solution at a temperature of -50 °C and a vacuum degree of 10⁻² Pa to obtain a polypeptide product with a low TFA content. Detect the TFA content, and the result is 0.01%. The chromatogram of trifluoroacetate ions in the polypeptide is shown in Figure 4 . Example 5
[0030] 1. Dissolution step: Dissolve 300 mg of the polypeptide in 30 mL of water to form a polypeptide solution.
[0031] 2. Ion exchange step: Use an Amberlite IRA-910 anion exchange resin column to adjust the pH value of the polypeptide solution to 7.5.
[0032] 3. Dynamic flow rate control: Dynamically adjust the flow rate according to the initial content of TFA in the polypeptide solution (detected as 0.5%): the initial flow rate is 0.5 mL / min, and when the TFA content drops to 0.2%, the flow rate is increased to 1.5 mL / min.
[0033] 4. Collection step: Collect the polypeptide solution after treatment with the resin column and wait for lyophilization.
[0034] 5. Freeze-drying step: Lyophilize the collected polypeptide solution at a temperature of -45 °C and a vacuum degree of 10⁻³ Pa to obtain a polypeptide product with a low TFA content. Detect the TFA content, and the result is 0.02%. The chromatogram of trifluoroacetate ions in the polypeptide is shown in Figure 5 . Example 6
[0035] 1. Dissolution step: Dissolve 180 mg of the polypeptide in 18 mL of water to form a polypeptide solution.
[0036] 2. Ion exchange step: Using a Dowex 1X8 anion exchange resin column, adjust the pH value of the polypeptide solution to 7.3 and pass it through the resin column at a flow rate of 1.1 mL / min.
[0037] 3. Collection step: Collect the polypeptide solution after being treated by the resin column and wait for lyophilization.
[0038] 4. Freeze-drying step: Lyophilize the collected polypeptide solution at a temperature of -50°C and a vacuum degree of 10⁻² Pa to obtain a polypeptide product with a low TFA content. Detect the TFA content, and the result is 0.08%. The chromatogram of trifluoroacetate ions in the polypeptide is shown in Figure 6 .
Claims
1. A method for reducing the content of trifluoroacetic acid in a polypeptide, characterized in that, It includes the following steps: (1) Dissolve the polypeptide in water to form a polypeptide solution; (2) Pass the polypeptide solution through an anion exchange resin column, with the pH value of the polypeptide solution being neutral to weakly alkaline, and control the flow rate at 0.5 - 2.0 mL / min; (3) Collect the polypeptide solution after being treated by the anion exchange resin column; (4) Freeze-dry the collected polypeptide solution to obtain a polypeptide product with a low TFA content.
2. The method according to claim 1, characterized in that The anion exchange resin column in step (2) is a strongly basic anion exchange resin column, selected from one of Dowex 1X8 anion exchange resin column, Amberlite IRA-910 anion exchange resin column or DOWEX MB-50 modified anion exchange resin column.
3. The method according to claim 1, characterized in that, The pH value of the polypeptide solution in step (2) is adjusted to 7.0 - 8.5 by adding sodium hydroxide solution.
4. The method according to claim 1, characterized in that The temperature of the freeze-drying in step (4) is -40°C to -60°C, and the vacuum degree is 10⁻² to 10⁻³ Pa.
5. The method according to claim 1, characterized in that, In the polypeptide solution after being treated by the anion exchange resin column in step (2), the TFA content is reduced to less than 0.1%.
6. The method according to claim 1, wherein Before passing through the anion exchange resin column in step (2), the polypeptide solution is pretreated by nano-activated carbon.
7. The method according to claim 1, characterized in that, The flow rate of the anion exchange resin column in step (2) is dynamically adjusted from 0.5 to 1.5 mL / min according to the initial TFA content in the polypeptide solution.
8. The method according to claim 3, wherein The pH value of the polypeptide solution in step (2) is adjusted to 7.2 - 7.
8.
9. The method according to claim 4, wherein The temperature of the freeze-drying in step (4) is -55°C, and the vacuum degree is 10⁻² Pa.
10. The method according to claim 1, characterized in that, The low TFA content in step (4) is 0.01% to 0.08%.