A polypeptide transsalting device and methods of use thereof

Abstract

The application discloses a polypeptide salt conversion device and a use method thereof, and belongs to the technical field of polypeptide preparation; the device comprises an ion exchange salt conversion kettle and a detector, and the salt conversion kettle is provided with an upper cover, a pH meter, a stirring paddle, a lower sieve plate and a feed liquid inlet and outlet; the method comprises the following steps: filling ion exchange resin in the ion exchange salt conversion kettle, loading a polypeptide liquid after the polypeptide liquid is adjusted to below or above an isoelectric point, washing the polypeptide liquid with water to remove salt, then adding an acidic or alkaline ligand solution under the continuous stirring of the stirring paddle, stirring to make the resin fully contact with the ligand and promote the polypeptide to be quickly and uniformly converted, simultaneously monitoring the pH change of the system in real time by the pH meter to accurately control the reaction end point, and finally washing and removing to obtain the product. The detector monitors the cleaning of salt ions and the elution of a sample. The application can significantly improve the salt conversion efficiency and obtain a product with low impurity residue by stirring to strengthen mass transfer and pH linkage control.

Description

Technical Field

[0001] This invention relates to the field of polypeptide preparation technology, specifically to a polypeptide transfer device and its usage method. Background Technology

[0002] Peptide transsalting refers to the process of converting a peptide from one salt form to another. Typically, peptide products are obtained by purifying crude chemically synthesized peptides using reversed-phase liquid chromatography (RP-HPLC) to convert them into the target salt form, followed by drying. In the preparation of peptide products for pharmaceuticals or cosmetics, it is often necessary to convert them into low-toxicity, highly soluble salt forms such as acetate or sodium salts before subsequent drying. If the transsalting process contains many impurities, it may affect the activity of the peptide and even increase the risk of cytotoxicity. Therefore, the quality of transsalting is an indispensable step.

[0003] In peptide purification, an acidic mobile phase system is often used, and the peptides in the purified intermediates usually exist as salts of the corresponding acid. If the acid used is volatile (such as acetic acid or trifluoroacetic acid), post-processing is relatively simple. However, when a neutral or alkaline mobile phase system is used for purification, the obtained peptide intermediates are mostly sodium salts, ammonium salts, triethylamine salts, etc., with only some strongly basic peptides still existing as acidic salts, and a large amount of buffer salts often remain in the system. If the target product is a sodium or ammonium salt, the high pH value of sodium hydroxide or ammonia solution may degrade the chromatographic packing material and damage the peptide structure. Therefore, it is difficult to simultaneously achieve desalting and salt conversion in actual production.

[0004] For peptide products containing non-volatile acidic ligand salts, sodium salts, or ammonium salts, the commonly used processes currently include: 1) Solid-phase extraction: using porous resin or ODS adsorbent, after adsorption of the packing material, salt conversion and desalting are performed, followed by elution with a high proportion of organic phase. 2) Isoelectric point precipitation: adjusting the pH of the peptide purification intermediate to its isoelectric point to cause precipitation, washing the precipitate multiple times with water to desalt it, then redissolving the precipitate with sodium hydroxide or ammonia to obtain the corresponding peptide salt, and finally freeze-drying.

[0005] Each of the above methods has its own advantages and disadvantages in actual production: for example, 1) solid-phase extraction has a high organic phase content, which can easily lead to organic reagent residues and yield problems; 2) isoelectric point sedimentation and washing operations are cumbersome and time-consuming. Overall, existing peptide-to-salt conversion equipment or methods are costly, involve complicated steps, and are difficult to scale up. Therefore, developing an efficient and simple peptide-to-salt conversion device and its application method is of great significance. Summary of the Invention

[0006] To address the aforementioned issues, the present invention aims to provide a polypeptide transfer device and its usage method. By developing a complete set of devices based on ion exchange for organic solvent removal, desalting, and transfer, a high-concentration transfer concentrate is obtained, significantly reducing the number of operation steps, ensuring freeze-drying concentration, and significantly reducing organic solvent and salt ion residues, thereby improving the overall product yield and efficiency.

[0007] The present invention adopts the following technical solution to achieve the above objectives.

[0008] A peptide salt transfer device comprises an ion exchange salt transfer vessel and a detector. The ion exchange salt transfer vessel consists of a cylindrical container, a lower sieve plate, a top cover, a pH meter, a stirring paddle, and a feed inlet and outlet. The pH meter and the stirring paddle are used to control the sample loading and salt transfer process.

[0009] Preferably, the detector detects the washing of excess salt ions at a wavelength of 205-230 nm and the elution of the sample at a wavelength of 205-300 nm.

[0010] Preferably, the liquid inlet and outlet include a liquid inlet and a liquid outlet.

[0011] Preferably, the polypeptide transfer device further includes a pump, and the feed inlet and feed outlet can be connected to the pump for automated feed transfer.

[0012] Preferably, the feed outlet is connected to a detector to detect excess salt ions in the peptide solution after salt conversion and the sample elution status.

[0013] Preferably, the polypeptide includes an amphoteric polypeptide containing both acid and base groups.

[0014] Preferably, the polypeptide includes a GLP-1-like polypeptide.

[0015] Preferably, the polypeptide includes telpolide, smegglutide, retaglutide, survodutide, or liraglutide.

[0016] The peptide transsalting device has a detector for monitoring the washing process of excess salt ions and the elution process of the target sample. Each unit achieves automated feed transfer through pumps, which is beneficial for the large-scale transsalting production of GLP-1 similar peptides.

[0017] This invention also provides a method for using the aforementioned peptide transfer device. An ion exchange transfer vessel is filled with ion exchange resin. The intermediate peptide purification solution is adjusted to below or above its isoelectric point using a pH adjuster and then transferred to the ion exchange transfer vessel. Water washing removes organic solvents and desalts the peptide. Under stirring and pH monitoring conditions, an acidic or basic ligand solution is added to convert the peptide into a basic or acidic salt. Water washing yields an aqueous solution of the peptide's ligand salt. This method is simple to operate and highly integrated, completing the entire process of sample loading, desalting, transfer, and elution within a single vessel, avoiding losses caused by multiple material transfers. Real-time linkage control of stirring and pH ensures precise control of the transfer process and product uniformity. The water washing step effectively removes organic solvents and salt ion impurities, resulting in a high-purity product, making it particularly suitable for the efficient transfer of GLP-1-like peptides.

[0018] Preferably, the ion exchange resin is a cation exchange resin. The intermediate peptide purification solution is adjusted to below the isoelectric point with an acidic pH adjuster and loaded into the ion exchange salt conversion vessel. The organic solvent is removed and desalted by washing with water. An alkaline ligand solution is added under stirring. Under pH monitoring conditions, the peptide is converted into an alkaline salt and washed with water to obtain an aqueous solution of the alkaline ligand salt of the peptide.

[0019] Preferably, the polypeptide includes a GLP-1-like polypeptide.

[0020] Preferably, the cation exchange resin includes one or more of Amberlite IRC-50, Toyopearl GigaCap S-650M, and CM-Cellulose.

[0021] Preferably, the acidic pH adjuster is an acidic substance that can adjust the pH of the system and does not react with other components of the system, including organic acids and inorganic acids; more preferably, the acidic pH adjuster is at least one of citric acid, acetic acid or trifluoroacetic acid.

[0022] Preferably, the alkaline ligand solution is at least one of sodium hydroxide, potassium hydroxide, or ammonia.

[0023] Preferably, the pH monitoring condition is that the pH is adjusted to be 1 to 4 pH values ​​higher than the isoelectric point of the target peptide.

[0024] Preferably, the alkaline ligand salt aqueous solution of the polypeptide is directly freeze-dried or concentrated and then freeze-dried to obtain a polypeptide product with extremely low salt residue and organic solvent residue.

[0025] Preferably, the ion exchange resin is an anion exchange resin. The intermediate peptide purification solution is adjusted to above the isoelectric point with an alkaline pH adjuster and transferred to the ion exchange salt conversion vessel. The organic solvent is removed and desalted by washing with water. An acidic ligand solution is added under stirring. Under pH monitoring conditions, the peptide is converted into an acidic salt and washed with water to obtain an aqueous solution of the acidic ligand salt of the peptide.

[0026] Preferably, the alkaline pH adjuster is an alkaline substance that can adjust the pH of the system and does not react with other components of the system; more preferably, the alkaline pH adjuster is sodium carbonate.

[0027] The acidic ligand solution is at least one of acetic acid, sulfuric acid, phosphoric acid, and hydrochloric acid.

[0028] Preferably, the stirring rate is 100~300 rpm.

[0029] Preferably, the stirring time is 5 to 30 minutes.

[0030] Preferably, the polypeptide includes a GLP-1-like polypeptide.

[0031] Preferably, the anion exchange resin includes one or more of DEAE Agarose 6FF, WorkBeads 40 DEAE, and Toyopearl NH2-750F.

[0032] Preferably, the pH monitoring condition is that the pH is adjusted to be 1 to 4 pH values ​​below the isoelectric point of the target peptide.

[0033] Preferably, the acidic ligand salt aqueous solution of the polypeptide is directly freeze-dried or concentrated and then freeze-dried to obtain a polypeptide product with extremely low salt residue and organic solvent residue.

[0034] Beneficial effects This invention provides a peptide transsalting device and its usage method. The core of the device lies in using anion or cation exchange resin as the solid-phase adsorption medium and employing a pH adjuster to precisely control the pH of the crude peptide solution to below or above the isoelectric point of the target peptide, thereby achieving selective and efficient adsorption of amphoteric peptides. By introducing pump-driven automated sample injection, dynamic mixing with a stirring paddle, and real-time feedback control via an online pH meter, the device can monitor the adsorption and washing process throughout, effectively avoiding peptide degradation and product loss caused by localized over-alkaliness. It is particularly suitable for easily degradable amphoteric peptide systems such as GLP-1 analogs. Subsequently, organic solvents and salts are removed by washing with water, followed by the addition of a ligand solution to complete the transsalting process, and finally, the product is obtained by freeze-drying. This method combines isoelectric point adsorption, real-time pH control, and automated operation, significantly improving product yield and purity, avoiding the problems of multiple and cumbersome purification steps in existing technologies using two high-performance liquid chromatography (HPLC) processes, simplifying the purification process, obtaining products with extremely low salt and organic solvent residues, enhancing process stability and product safety, and providing a feasible strategy for the efficient industrial transsalting of easily degradable peptides. Attached Figure Description

[0035] The applicant will further describe the invention in detail with reference to the accompanying drawings.

[0036] Figure 1 A schematic diagram showing the polypeptide transfer device provided by the present invention; Figure 2 The image shows the liquid phase elution diagram of Example 2 provided by the present invention (220 nm and 280 nm are the detection wavelengths of high performance liquid chromatography, used to monitor the main peak of the peptide and impurity peaks, the same below). Figure 3 This shows the liquid phase elution diagram of Example 3 provided by the present invention; Figure 4 This shows the liquid phase elution diagram of Example 4 provided by the present invention; Figure 5 This shows the liquid phase elution diagram of Example 5 provided by the present invention; Figure 6 This shows the liquid phase elution diagram of Example 6 provided by the present invention; Figure 7 This shows the liquid phase elution diagram of Comparative Example 1 provided by the present invention; Figure 8 This shows the liquid phase elution diagram of Comparative Example 2 provided by the present invention. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0039] Example 1: A polypeptide transfer device, such as Figure 1 As shown, it consists of an ion exchange salt transfer vessel and a detector. The ion exchange salt transfer vessel is composed of a cylindrical container, a lower sieve plate, a top cover, a pH meter, a stirring paddle, and a feed inlet and outlet. The pH meter and stirring paddle are used to control the sample loading and salt transfer process. The detector detects the washing of excess salt ions at a wavelength of 205~220nm, and can also detect the elution of the sample at a wavelength of 205~300nm.

[0040] The liquid inlet and outlet include a liquid inlet and a liquid outlet, which are respectively connected to pumps for automated liquid transfer.

[0041] The feed outlet is connected to a detector to detect excess salt ions in the peptide solution after salt conversion and the sample elution status.

[0042] When the polypeptide conversion device is used for polypeptide conversion, firstly, ion exchange packing is filled into the ion exchange conversion vessel, and then the polypeptide solution to be converted is injected into the vessel through a pump. After the polypeptide solution is adjusted to below or above the isoelectric point with an acidic pH adjuster or an alkaline pH adjuster, the stirring paddle is started for stirring, and then the system is rinsed with pure water. After rinsing, add a suitable acidic or alkaline ligand solution and monitor the pH value in the reactor in real time. Adjust the pH to 1-4 pH values ​​higher or lower than the isoelectric point of the target peptide (for peptides with an isoelectric point less than 7, the salt can be converted to either an acidic or basic salt; if conversion to an acidic salt is required, use anion exchange packing material and lower the pH during elution; otherwise, use a cation exchange packing material and raise the pH during elution. Peptides with an isoelectric point greater than 7 can only be converted to acidic salts), and stop adding the solution when it stabilizes. Maintain stirring, and finally elute the target peptide with water to obtain an aqueous solution of the peptide's ligand salt. The solution can also be directly freeze-dried or concentrated and then freeze-dried to obtain a peptide product with extremely low salt and organic solvent residues.

[0043] Example 2: Based on the above embodiments, the specific steps for transferring telpoide using the aforementioned polypeptide transfer device include: S1. Sample Loading: The ion exchange transfer vessel is loaded with cation exchange packing material. The intermediate solution of telpolide, prepared by reversed-phase liquid chromatography with a concentration of 16 mg / mL, contains 40% (v / v) acetonitrile and water. Citric acid is added to adjust the pH to 2.9, and the solution is loaded into the ion exchange transfer vessel via a pump.

[0044] S2, Desalting and removing organic solvents: Wash the packing material after sample loading with 5 column volumes of water.

[0045] S3. Salt Transfer: With the impeller rotating at 150 rpm, slowly add 0.5M sodium hydroxide solution while monitoring the pH in the reactor in real time. Stop adding alkali when the pH reaches 6.9. Continue stirring for 10 minutes to allow the peptide to completely desorb from the cation exchange resin and bind with sodium ions to form telpoide sodium salt. Maintain the pH at 7.0–7.2 during this process to avoid localized over-alkalinity.

[0046] S4. Elution: Elute the converted sodium telpotassium salt from the packing material with water to obtain an aqueous solution of sodium telpotassium salt with a concentration of 76 mg / mL (sodium telpotassium salt yield 98.6%). Figure 2 The elution effect of sodium telpotassium salt on the liquid phase was shown to be good, with no tailing. The lyophilized sample was tested and found to have a citric acid residue of 0.058%, which is far below the limit of 0.5%; and a acetonitrile residue of 13 ppm, which is far below the limit of 410 ppm.

[0047] Freeze-drying (subsequent examples and comparative examples follow this method): The collected liquid was filtered through a 0.22 μm filter membrane, dispensed into freeze-drying trays, and freeze-dried (pre-freeze at -40°C for 4 h, first dry at -20°C / 0.2 mbar for 24 h, second dry at 25°C / 0.05 mbar for 8 h). The content of telpoeptide was determined (subsequent examples and comparative examples followed this method): reversed-phase high-performance liquid chromatography (RP-HPLC) was used with a C18 column (250 mm in length, 4.6 mm in inner diameter, and 5 µm in particle size). Mobile phase A was 0.1% trifluoroacetic acid (TFA) aqueous solution, and mobile phase B was 0.1% trifluoroacetic acid acetonitrile solution. The gradient elution program started at 20% B and linearly increased to 90% B within 12 minutes. The flow rate was 0.3–1.0 mL / min, the column temperature was 35 °C, the detection wavelength was 220 nm, and the injection volume was 5 µL. The content of each component was calculated based on the curve of the telpoeptide quasi-sample.

[0048] Acetonitrile residue determination (subsequent examples and comparative examples follow this method): The test sample was sealed in a headspace vial and heated to evaporate the acetonitrile into the gas phase. The headspace gas was injected into the gas chromatograph (FID detector), and quantified using the external standard method. The instrument conditions were: DB-624 column (300 mm × 0.32 mm × 1.8 μm), column temperature 40 °C, injector / detector temperature 200–250 °C, headspace equilibrium temperature 100 °C for 30–45 min. Approximately 100 mg of the test sample was accurately weighed into a headspace vial and dissolved in 1.0 mL of DMSO or water. At the same time, a series of acetonitrile reference solutions of different concentrations were prepared to establish a standard curve. The acetonitrile peak area measured in the test sample was substituted into the standard curve to obtain the concentration C (μg / mL). The acetonitrile residue (ppm) was calculated according to the formula: acetonitrile residue (ppm) = (C × V) / m × 1000 (V is the final volume in mL, and m is the sample weight in mg). The limit requirement is acetonitrile ≤ 410 ppm (compliant with ICH Q3C).

[0049] Citric acid residue (subsequent examples and comparative examples follow the same method): A C18 reversed-phase column (4.6 × 250 mm, 5 μm) was used. The mobile phase consisted of a 95:5 mixture of 0.01 mol / L potassium dihydrogen phosphate buffer (pH 2.5) and methanol. The flow rate was 1.0 mL / min, the column temperature was 30 °C, and the detection wavelength was 210 nm. Samples were appropriately diluted with water or buffer and filtered before injection. The external standard method was used to quantify the residue based on peak area.

[0050] Example 3: Based on the above embodiments, the transfer of smegglutinin using the above-described polypeptide transfer device includes, S1. Sample Loading: The ion exchange transfer vessel is loaded with cation exchange packing material. The smegglutinin intermediate solution, obtained by reverse-phase preparative liquid chromatography purification, has a smegglutinin concentration of 8 mg / mL and contains 30% by volume acetonitrile and water. HAc (acetic acid) is added to adjust the pH to 2.9, and then the solution is loaded into the ion exchange transfer vessel.

[0051] S2, Desalting and removing organic solvents: Wash the packing material after sample loading with 5 column volumes of water.

[0052] S3. Salt Transfer: With the impeller rotating at 150 rpm, slowly add 0.3M sodium hydroxide solution while monitoring the pH in the reactor in real time. Stop adding alkali when the pH reaches 6.9. Continue stirring for 10 minutes to allow the peptide to completely desorb from the cation exchange resin and bind with sodium ions to form smegglutide sodium salt. Maintain the pH between 7.0 and 7.2 during this process to avoid localized over-alkalinity.

[0053] S4. Elution: Elute the converted sodium sematropin from the packing material with water to obtain an aqueous solution of sodium sematropin with a concentration of 45 mg / mL (sodium sematropin yield 98.12%). Figure 3 The elution effect of sodium smegglutide on the liquid phase was shown to be good, with no tailing. HAc was not detected in the lyophilized sample, and was far below the limit of 0.5%. Acetonitrile residue was not detected, and was far below the limit of 410 ppm.

[0054] Example 4: Based on the above embodiments, the method for transferring retaglutide using the above-described polypeptide transfer device includes, S1. Sample Loading: The ion exchange transfer vessel is loaded with cation exchange packing material. The intermediate solution of retaliutide, obtained by reverse-phase preparative liquid chromatography purification, has a retaliutide concentration of 17 mg / mL and contains 40% by volume acetonitrile and water. Citric acid is added to adjust the pH to 3.0, and the solution is loaded into the ion exchange transfer vessel via a pump.

[0055] S2, Desalting and removing organic solvents: Wash the packing material after sample loading with 5 column volumes of water.

[0056] S3. Salt Transfer: With the impeller rotating at 150 rpm, slowly add 0.3M sodium hydroxide solution while monitoring the pH in the reactor in real time. Stop adding alkali when the pH reaches 7.0. Continue stirring for 10 minutes to allow the peptide to completely desorb from the cation exchange resin and bind with sodium ions to form retaloupeptide sodium salt. Maintain the pH between 7.0 and 7.2 during this process to avoid localized over-alkalinity.

[0057] S4. Elution: Elute the converted sodium retalidinate from the packing material with water to obtain an aqueous solution of sodium retalidinate at a concentration of 55 mg / mL (sodium retalidinate yield 98.36%). Figure 4 The elution effect of sodium retaliptide on the liquid phase was shown to be good, with no tailing. The lyophilized sample was tested and found to have a citric acid residue of 0.017%, which is far below the limit of 0.5%; and a acetonitrile residue of 18 ppm, which is far below the limit of 410 ppm.

[0058] Example 5: Based on the above embodiments, the method for transferring Survodutide using the above-described peptide transfer device includes, S1. Sample loading: The ion exchange transfer vessel is loaded with cation exchange packing material. The Survodutide intermediate solution, obtained by reverse-phase preparative liquid chromatography purification, has a Survodutide concentration of 18 mg / mL and contains 38% by volume acetonitrile and water. TFA (trifluoroacetic acid) is added to adjust the pH to 2.9, and the solution is loaded into the ion exchange transfer vessel via a pump.

[0059] S2, Desalting and removing organic solvents: Wash the packing material after sample loading with 5 column volumes of water.

[0060] S3. Salt Transfer: With the stirrer rotating at 150 rpm, slowly add 0.3M sodium hydroxide solution while monitoring the pH in the reactor in real time. Stop adding alkali when the pH reaches 8.0. Continue stirring for 10 minutes to allow the peptides to completely desorb from the cation exchange resin and bind with sodium ions to form Survodutide sodium salt. Maintain the pH between 8.0 and 8.2 during this process to avoid localized over-alkalinity.

[0061] S4. Elution: Elute the converted sodium Survodutide from the packing material with water to obtain an aqueous solution of sodium Survodutide with a concentration of 48 mg / mL (sodium Survodutide yield 97.68%). Figure 5 The elution effect of sodium Survodutide on the liquid phase was shown to be good, with no tailing. The lyophilized sample was tested and found to have a TFA residue of 0.010%, which is far below the limit of 0.5%; and an acetonitrile residue of 13 ppm, which is far below the limit of 410 ppm.

[0062] TFA Residue Rate: An anion exchange column (Dionex IonPac AS11-HC, 4×250mm) was used with a suppressed conductivity detector; isocratic elution was performed using 20 mmol / L potassium hydroxide solution as the eluent at a flow rate of 1.0 mL / min and a column temperature of 30℃; the sample was diluted with deionized water to a suitable concentration, filtered through a 0.22 μm filter membrane, and injected in a volume of 25 μL; the external standard method was used to quantify the TFA residue rate in the sample based on the peak area of ​​trifluoroacetic acid.

[0063] Example 6: Based on the above embodiments, the method for transferring liraglutide using the above-described polypeptide transfer device includes, S1. Sample Loading: The ion exchange transfer vessel is loaded with cation exchange packing material. The liraglutide intermediate solution, obtained by reverse-phase preparative liquid chromatography purification, has a liraglutide concentration of 15 mg / mL and contains 37% by volume acetonitrile and water. Acetic acid is added to adjust the pH to 3.0, and the solution is loaded into the ion exchange transfer vessel via a pump.

[0064] S2, Desalting and removing organic solvents: Wash the packing material after sample loading with 5 column volumes of water.

[0065] S3. Salt Transfer: With the impeller rotating at 150 rpm, slowly add 0.3M sodium hydroxide solution while monitoring the pH in the reactor in real time. Stop adding alkali when the pH reaches 7.0. Continue stirring for 10 minutes to allow the peptide to completely desorb from the cation exchange resin and bind with sodium ions to form liraglutide sodium salt. Maintain the pH between 7.0 and 7.2 during this process to avoid localized over-alkalinity.

[0066] S4. Elution: Elute the converted liraglutide sodium salt from the packing material with water to obtain an aqueous solution of liraglutide sodium salt with a concentration of 53 mg / mL (liraglutide sodium salt yield 97.22%). Figure 6 The elution effect of liraglutide sodium salt on the liquid phase was shown to be good. The lyophilized sample without tailing was tested and found to have acetic acid residue of 0.011%, which is far below the limit of 0.5%; and acetonitrile residue of 1 ppm, which is far below the limit of 410 ppm.

[0067] Acetic acid residue was determined by gas chromatography (GC): a polyethylene glycol (PEG) modified capillary column (HP-INNOWAX, 30m × 0.32mm × 0.25μm) equipped with a flame ionization detector (FID), using nitrogen or helium as the carrier gas. The sample was accurately weighed and placed in a headspace vial, dissolved in water, and sealed. After equilibration at 80℃ for 20 min, 1.0 mL was injected into the headspace. The temperature program was as follows: initial temperature 35℃, held for 5 min, then increased at 30℃ / min to 120℃, held for 2 min. The acetic acid content was calculated based on the peak area using the internal standard method (isopropanol as the internal standard), thus determining the residue rate.

[0068] Comparative Example 1: Based on the above embodiments, the method for transsalting telpoide using the aforementioned peptide transsalting device differs from Example 2 in that the transsalting device is a conventional ion-exchange chromatography column and a protein purification instrument, and the process is a conventional ion-exchange preparation process. Compared to Example 2, since it is static elution, damage to the sample from high concentrations of sodium hydroxide must be avoided, and the salt used for transsalting must be a buffer salt containing sodium ions. Specific steps include… S1. Sample loading: The ion exchange column is loaded with cation exchange packing material. The intermediate solution of telpolide, obtained by reverse-phase preparative liquid chromatography with a concentration of 16 mg / mL, is added with citric acid, 40% (v / v) acetonitrile, and water.

[0069] S2, Desalting and removing organic solvents: Wash the packing material after sample loading with 5 column volumes of water.

[0070] S3, Salt elution: Elute with 0.2M sodium bicarbonate aqueous solution to obtain an aqueous solution of telpoprit sodium salt containing sodium bicarbonate. The elution peaks show severe tailing. Figure 7 The concentration of the collected solution was 7 mg / mL (yield 43.2%), which was low. The residual citric acid was 0.12%, which was far below the limit of 0.5%; the residual acetonitrile was 440 ppm, which was slightly above the limit of 410 ppm.

[0071] Comparative Example 2: Based on the above embodiments, the method for transsalting telpoide using the aforementioned peptide transsalting device differs from Example 2 in that the transsalting device is a conventional ion-exchange chromatography column and a protein purification instrument, and the process is a conventional ion-exchange preparation process. Compared to Example 2, since it is static elution, damage to the sample from high concentrations of sodium hydroxide must be avoided, and the salt used for transsalting must be a buffer salt containing sodium ions. Specific steps include… S1. Sample loading: The ion exchange column is loaded with cation exchange packing material. The intermediate solution of telpolide, obtained by reverse-phase preparative liquid chromatography with a concentration of 16 mg / mL, is added with citric acid, 40% (v / v) acetonitrile, and water.

[0072] S2, Desalting and removing organic solvents: Wash the packing material after sample loading with 5 column volumes of water.

[0073] S3, Salt conversion elution: Elute with 1M disodium hydrogen phosphate aqueous solution to obtain an aqueous solution of telpoide sodium salt containing disodium hydrogen phosphate. The elution peak tailing is severe. Figure 8 The concentration of the collected solution was 10 mg / mL (yield 42.31%), which was low. The residual citric acid was 0.10%, far below the limit of 0.5%; the residual acetonitrile was 460 ppm, slightly above the limit of 410 ppm. Compared with Comparative Example 1, increasing the concentration of the salt transfer buffer did not significantly improve the elution effect.

[0074] Compared to the static elution strategies used in Comparative Examples 1 and 2, which are limited by the inability to use sodium hydroxide for salt conversion and can only use disodium hydrogen phosphate or sodium bicarbonate buffer solutions, resulting in low concentrations of the salt conversion solution and severe tailing of chromatographic elution peaks, significantly affecting product purity and subsequent processing efficiency, Examples 2-6 employ a dynamic elution mode using sodium hydroxide as the salt conversion reagent and combining it with online real-time pH monitoring under stirring conditions. This achieves highly efficient and controllable salt conversion of peptides. This dynamic process not only significantly increases the concentration of the salt conversion product and effectively eliminates elution peak tailing, ensuring superior overall product quality, but also has good scalability, is suitable for large-scale production, and greatly reduces the intensity of manual operation and human intervention errors, thereby effectively reducing overall production costs while improving production efficiency.

Claims

1. A polypeptide transfer device, characterized in that, The device includes an ion exchange salt transfer vessel and a detector. The ion exchange salt transfer vessel includes a top cover, a pH meter, a stirrer, a lower sieve plate, and a feed inlet and outlet. The pH meter and the stirrer are used to control the sample loading and salt transfer process.

2. The polypeptide transfer device according to claim 1, characterized in that, The detector detects the cleaning of excess salt ions at a wavelength of 205-230 nm and the elution of samples at a wavelength of 205-300 nm.

3. The polypeptide transfer device according to claim 1, characterized in that, The polypeptides include amphoteric polypeptides that contain both acid and base groups.

4. A polypeptide transfer device according to claim 1 or 3, characterized in that, The polypeptides include GLP-1-like polypeptides.

5. The method of using the polypeptide transsalting device according to any one of claims 1 to 4, characterized in that, Ion exchange transfer vessel is filled with ion exchange resin. The intermediate solution of peptide purification is adjusted to below or above the isoelectric point with a pH adjuster and then transferred to the ion exchange transfer vessel. The organic solvent is removed and desalted by washing with water. Acidic or basic ligand solution is added under stirring and pH monitoring conditions to convert the peptide into basic or acidic salt. The peptide is then washed with water to obtain an aqueous solution of ligand salt.

6. The method of using the polypeptide transfer device according to claim 5, characterized in that, The ion exchange resin is a cation exchange resin. The intermediate peptide purification solution is adjusted to below the isoelectric point with an acidic pH adjuster and transferred to the ion exchange salt transfer vessel. The organic solvent is removed and desalted by washing with water. An alkaline ligand solution is added under stirring. Under pH monitoring conditions, the peptide is converted into an alkaline salt and washed with water to obtain an aqueous solution of the alkaline ligand salt of the peptide.

7. The method of using the polypeptide transfer device according to claim 6, characterized in that, The acidic pH adjuster is an acidic substance that can adjust the pH of the system and does not react with other components of the system; The alkaline ligand solution is at least one of sodium hydroxide, potassium hydroxide, or ammonia.

8. The method of using the polypeptide transfer device according to claim 5, characterized in that, The ion exchange resin is an anion exchange resin. The intermediate solution of peptide purification is adjusted to above the isoelectric point with an alkaline pH adjuster and transferred to the ion exchange salt transfer vessel. The organic solvent is removed and desalted by washing with water. An acidic ligand solution is added under stirring. Under pH monitoring conditions, the peptide is converted into an acidic salt and washed with water to obtain an aqueous solution of the acidic ligand salt of the peptide.

9. The method of using the polypeptide transfer device according to claim 8, characterized in that, The alkaline pH adjuster is an alkaline substance that can adjust the pH of the system and does not react with other components of the system; The acidic ligand solution is at least one of acetic acid, sulfuric acid, phosphoric acid, and hydrochloric acid.

10. The method of using the polypeptide transfer device according to any one of claims 5 to 9, characterized in that, The stirring speed is 100~300 rpm.

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