Polypeptide salt conversion method

Abstract

The invention discloses a polypeptide salt conversion method, and belongs to the technical field of acidic long peptide preparation. The method comprises the following steps: carrying out reversed-phase liquid chromatography purification treatment on an acidic long peptide crude product twice to obtain a purified intermediate solution, then adding a deacidification agent into the purified intermediate solution to regulate pH, carrying out rotary evaporation concentration and centrifugal separation, retaining a salt-free solid, adding a sodium hydroxide solution, mixing, and carrying out freeze drying to obtain an acidic long peptide sodium salt product. By optimizing the deacidification step and the operation sequence, the acid regulator introduced in the purification process is efficiently removed, and the acid long peptide still keeps high concentration after deacidification, so that the freeze-drying concentration of the final acid long peptide sodium salt freeze-drying product is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of polypeptide preparation technology, and more specifically to a method for transsalting polypeptides. Background Technology

[0002] Peptide salt conversion refers to the process of transforming 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 freeze-drying or spray drying. In the preparation of peptide products for pharmaceuticals or cosmetics, after purification, the crude peptide product often needs to be converted into a low-toxicity, highly soluble salt form such as acetate or sodium salt before subsequent drying.

[0003] In peptide purification, acidic mobile phase systems are commonly used, such as trifluoroacetic acid, acetic acid, formic acid, citric acid, phosphoric acid, sulfuric acid, or hydrochloric acid. In the purified intermediates, the peptides typically form corresponding salts with the acid in the mobile phase, such as trifluoroacetic acid salts, acetates, or phosphates. If the acid used is a volatile acid such as acetic acid or trifluoroacetic acid, post-purification processing is relatively simple: the volatile acid is usually used as the final mobile phase for chromatographic purification or column concentration. After salt replacement and washing away excess buffer salts, elution is performed in the corresponding acidic system, and the resulting intermediate can be directly dried to obtain the target salt product. However, when purification is carried out in neutral or alkaline mobile phase systems such as ammonium acetate, triethylamine phosphate, ammonium phosphate, or ammonium sulfate, the obtained peptide intermediates are mostly sodium salts, ammonium salts, or triethylamine salts. Only some basic peptides retain their acidic salt form, and the solution often contains a large amount of buffer salts. When the target product is a sodium or ammonium salt, the high pH value of sodium hydroxide or ammonia solution may affect the chromatographic packing material and peptide structure, making it difficult to achieve desalting and salt conversion simultaneously through a single column chromatography in production.

[0004] For peptide products containing non-volatile acidic ligand salts, sodium salts, or ammonium salts, the commonly used processes currently include the following: 1) Reversed-phase chromatography: The peptide purification intermediate is desalted and converted using a reversed-phase column, then eluted with a high proportion of organic phase. The resulting eluent usually has a low ligand ion content, requiring rotary evaporation to remove the organic phase, replenishing the ligand ions, and finally lyophilizing. To meet batch lyophilization requirements, the solution after desalting often needs further concentration by vacuum concentration or nanofiltration. 2) Ion exchange chromatography: The peptide purification intermediate is adjusted to acidity, adsorbed using a cation exchange column, and then eluted with the corresponding sodium or ammonium salt solution. The eluent is desalted and concentrated by nanofiltration, and then lyophilized. 3) Isoelectric point precipitation: The pH of the peptide purification intermediate is adjusted to its isoelectric point to cause precipitation. The precipitate is washed multiple times with water to desalt it, then redissolved with sodium hydroxide or ammonia to obtain the corresponding peptide sodium or ammonium salt solution, and finally lyophilized.

[0005] Each of the above methods has its own advantages and disadvantages in actual production: nanofiltration desalination and concentration is time-consuming; vacuum dehydration and concentration, due to the high temperature, can easily lead to an increase in peptide impurities; isoelectric point sedimentation and washing operations are cumbersome and time-consuming. Overall, existing processes for converting peptides to sodium or ammonium salts are complex, time-consuming, and have low friendliness to the production environment and operators. Therefore, developing an efficient and simple method for peptide salt conversion is of great significance. Summary of the Invention

[0006] The purpose of this invention is to provide a method for transacidifying peptides, which significantly reduces the proportion of acid regulator in the final product by optimizing the deacidification steps and the order of operations, increases the freeze-drying concentration of acidic long peptide sodium salt products, maintains the stability of acidic long peptide sodium salt freeze-dried products, thereby ensuring product purity and improving the product yield and efficiency of the process.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows: A method for transsalting peptides includes synthesizing a crude acidic long peptide using a solid-phase synthesis method, purifying it twice using reversed-phase liquid chromatography to obtain a purified intermediate, adding a deacidifying agent to the purified intermediate to adjust the pH, concentrating by rotary evaporation and centrifuging, retaining the salt-free solid and mixing it with sodium hydroxide solution, and then freeze-drying it to obtain an acidic long peptide sodium salt product; in the second reversed-phase liquid chromatography, mobile phase A is an acid regulator, which includes at least one of citric acid, oxalic acid, tartaric acid, phosphoric acid and sulfuric acid; the freeze-drying concentration of the acidic long peptide sodium salt product is 95-108.38 mg / mL, the proportion of acid regulator is 0.02-0.14%, and the proportion of sodium ions is 1.35-1.39%.

[0008] This invention employs two reversed-phase liquid chromatography purification steps combined with pH adjustment using a deacidifying agent. This efficiently removes the acid regulator introduced during the purification process, significantly reducing the residual proportion of the acid regulator in the final product. Simultaneously, this invention optimizes the deacidification steps and operational sequence, avoiding the dissolution and dilution of acidic long peptides in the aqueous phase caused by repeated washing in traditional isoelectric point sedimentation washing methods. This ensures that the acidic long peptides maintain a high concentration after deacidification, thereby significantly increasing the lyophilized concentration of the final acidic long peptide sodium salt freeze-dried product.

[0009] Preferably, the acidic long peptide includes smegglutide, telpogglutide, or retaloglutide.

[0010] Preferably, the purity of the purification intermediate is ≥90%.

[0011] Preferably, the deacidifying agent is calcium hydroxide or calcium carbonate.

[0012] Preferably, the calcium ion content of the acidic long peptide sodium salt product is 0.01-0.03%.

[0013] Preferably, the pH is 3-5. This invention regulates pH using a deacidifying agent, which not only influences the ionization state and solubility of the acid regulator, promoting its efficient separation from the product system under specific conditions, but also helps maintain the stability of acidic long peptides, reducing non-specific adsorption or co-precipitation losses during separation. This ensures product recovery and purity while removing impurities.

[0014] Preferably, the concentration of the sodium hydroxide solution is 4-5 g / L.

[0015] Preferably, the temperature for rotary evaporation concentration is 20-30℃.

[0016] Preferably, the concentration of citric acid is 20-40 mmol / L.

[0017] Preferably, the concentration of tartaric acid is 20-40 mmol / L.

[0018] Preferably, the polypeptide transsalting method is as follows: S1. Purification: The crude acidic long peptide was synthesized using a solid-phase polypeptide synthesis method and purified by reversed-phase liquid chromatography (RP-HPLC). The first purification HPLC conditions were as follows: a 40-60 mm diameter column; mobile phase A was a buffer solution; mobile phase B was acetonitrile; the elution gradient program was: the acetonitrile ratio increased from 25-30% to 35-40% over 50-70 min at a flow rate of 40-60 mL / min; the fraction with a purity ≥90% was collected. The second purification HPLC conditions were as follows: a 40-60 mm diameter column; mobile phase A was an acid adjuster; mobile phase B was acetonitrile; the elution gradient program was: the acetonitrile ratio increased from 30-40% to 45-55% over 50-70 min at a flow rate of 40-60 mL / min; the fraction with a purity ≥90% was collected as the purification intermediate.

[0019] S2, Deacidification: While stirring, add a deacidifying agent to the purification intermediate to adjust the pH to 3-5, and obtain the deacidified purification intermediate.

[0020] S3. Remove organic solvent: Under conditions of 20-30℃, the deacidified purification intermediate is rotary evaporated to remove acetonitrile, resulting in a salt-free suspension. After centrifugation, a salt-free solid is obtained.

[0021] S4. Salt formation: Add sodium hydroxide solution to the salt-free solid for redissolution, freeze-dry, and obtain sodium telpotassium salt.

[0022] More preferably, the acidic long peptide in step S1 includes smegglutinin, telpogglutinin, or retaliglutinin.

[0023] More preferably, the pH of the buffer solution in step S1 is 6-8.

[0024] More preferably, the concentration of the buffer solution in step S1 is 0.5-2%.

[0025] More preferably, the buffer solution in step S1 is an aqueous solution of ammonium formate or a solution of triethylamine phosphate. More preferably, the acid regulator in step S1 includes at least one of citric acid, oxalic acid, tartaric acid, phosphoric acid, and sulfuric acid.

[0026] More preferably, the concentration of citric acid is 20-30 mmol / L.

[0027] More preferably, the concentration of oxalic acid is 20-30 mmol / L.

[0028] More preferably, the concentration of tartaric acid is 20-30 mmol / L.

[0029] More preferably, the mass concentration of phosphoric acid is 0.05-0.2%.

[0030] More preferably, the mass concentration of sulfuric acid is 0.05-0.2%.

[0031] More preferably, the deacidifying agent in step S2 is calcium carbonate or calcium hydroxide.

[0032] More preferably, the concentration of the sodium hydroxide solution in step S4 is 4-5 g / L.

[0033] The peptide-to-salt conversion method disclosed in this invention involves two reverse-phase liquid chromatography purification processes on an acidic crude long peptide to obtain a purified intermediate solution. A deacidifying agent is then added to the intermediate solution to adjust the pH. After rotary evaporation concentration and centrifugation, the salt-free solid is retained and mixed with sodium hydroxide solution. Finally, the product is freeze-dried to obtain the acidic long peptide sodium salt. This method offers the following advantages: It effectively controls residual impurities. The prepared acidic long peptide sodium salt product contains 0.02-0.14% acid regulator, 1.35-1.39% sodium ions, and 0.01-0.03% calcium ions, all meeting the specified quality requirements. Furthermore, it exhibits superior batch-to-batch stability. After 6 months of accelerated high-temperature treatment, the main component retention rate of the prepared acidic long peptide sodium salt is 96.3-96.8%, effectively improving product quality stability while ensuring compliance with quality standards. Simultaneously, this method reduces product loss in intermediate steps and significantly increases the freeze-drying concentration to 95-108.38 mg / mL. Therefore, this invention is a highly efficient method for deacidification, high product retention, and high stability of peptide transsalting. Attached Figure Description

[0034] Figure 1A1 represents the concentration of telpoeptide after deacidification, A2 represents the purified intermediate solution obtained in step S1 of Example 1, A3 represents the purified intermediate solution obtained in step S1 of Comparative Example 1, and A4 represents the supernatant obtained after deacidification treatment in step S2 of Comparative Example 1.

[0035] Figure 2 This refers to the freeze-drying concentration of the sodium thiopotassium salt freeze-dried product.

[0036] Figure 3 Main component retention rate. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0039] Example 1: Methods for salt transconjugation of telpolide include, S1. Purification: Crude telpoeptide was synthesized using a solid-phase polypeptide synthesis method and purified by reversed-phase liquid chromatography (RP-HPLC). The first purification HPLC conditions were as follows: a 50 mm diameter column; Unisil 10-100 C18 packing material; mobile phase A: 1% ammonium formate aqueous solution at pH 7.0; mobile phase B: acetonitrile; elution gradient program: acetonitrile concentration increased from 28% to 38% over 60 min at a flow rate of 50 mL / min; fraction with a purity ≥98% was collected. The second purification HPLC conditions were as follows: a 50 mm diameter column; Unisil 10-100 C18 packing material; mobile phase A: 30 mmol / L tartaric acid aqueous solution; mobile phase B: acetonitrile; elution gradient program: acetonitrile concentration increased from 35% to 50% over 60 min at a flow rate of 50 mL / min; fraction with a purity ≥99% was collected as the purification intermediate.

[0040] S2, Deacidification: While stirring, add calcium carbonate to the purification intermediate to adjust the pH to 4.0, and obtain the deacidified purification intermediate.

[0041] S3. Removal of organic solvent: At 25°C, the deacidified purification intermediate is rotary evaporated to remove acetonitrile, resulting in a salt-free suspension. The suspension is then centrifuged to obtain a salt-free solid.

[0042] S4. Salt formation: The salt-free solid was redissolved in sodium hydroxide solution, then freeze-dried to obtain sodium telpotassium salt. The concentration of the sodium hydroxide solution was 5 g / L.

[0043] Example 2: Methods for salt transconjugation of telpolide include, S1. Purification: Crude telpoeptide was synthesized using a solid-phase polypeptide synthesis method and purified by reversed-phase liquid chromatography (RP-HPLC). The first purification HPLC conditions were as follows: a 50 mm diameter column; Unisil 10-100 C18 packing material; mobile phase A: 1% ammonium formate aqueous solution at pH 7.0; mobile phase B: acetonitrile; elution gradient program: acetonitrile concentration increased from 28% to 38% over 60 min at a flow rate of 50 mL / min; fraction with a purity ≥98% was collected. The second purification HPLC conditions were as follows: a 50 mm diameter column; Unisil 10-100 C18 packing material; mobile phase A: 20 mmol / L tartaric acid aqueous solution; mobile phase B: acetonitrile; elution gradient program: acetonitrile concentration increased from 35% to 50% over 60 min at a flow rate of 50 mL / min; fraction with a purity ≥99% was collected as the purification intermediate.

[0044] S2, Deacidification: While stirring, add calcium carbonate to the purification intermediate to adjust the pH to 4.0, and obtain the deacidified purification intermediate.

[0045] S3. Removal of organic solvent: At 25°C, the deacidified purification intermediate is rotary evaporated to remove acetonitrile, resulting in a salt-free suspension. The suspension is then centrifuged to obtain a salt-free solid.

[0046] S4. Salt formation: The salt-free solid was redissolved in sodium hydroxide solution, then freeze-dried to obtain sodium telpotassium salt. The concentration of the sodium hydroxide solution was 5 g / L.

[0047] Example 3: Methods for salt transconjugation of telpolide include, S1. Purification: Crude telpoeptide was synthesized using a solid-phase polypeptide synthesis method and purified by reversed-phase liquid chromatography (RP-HPLC). The first purification HPLC conditions were as follows: a 50 mm diameter column; Unisil 10-100 C18 packing material; mobile phase A: 1% ammonium formate aqueous solution at pH 7.0; mobile phase B: acetonitrile; elution gradient program: acetonitrile concentration increased from 28% to 38% over 60 min at a flow rate of 50 mL / min; fraction with a purity ≥98% was collected. The second purification HPLC conditions were as follows: a 50 mm diameter column; Unisil 10-100 C18 packing material; mobile phase A: 30 mmol / L citric acid aqueous solution; mobile phase B: acetonitrile; elution gradient program: acetonitrile concentration increased from 35% to 50% over 60 min at a flow rate of 50 mL / min; fraction with a purity ≥99% was collected as the purification intermediate.

[0048] S2, Deacidification: While stirring, add calcium carbonate to the purification intermediate to adjust the pH to 4.0, and obtain the deacidified purification intermediate.

[0049] S3. Removal of organic solvent: At 25°C, the deacidified purification intermediate is rotary evaporated to remove acetonitrile, resulting in a salt-free suspension. The suspension is then centrifuged to obtain a salt-free solid.

[0050] S4. Salt formation: The salt-free solid was redissolved in sodium hydroxide solution, then freeze-dried to obtain sodium telpotassium salt. The concentration of the sodium hydroxide solution was 5 g / L.

[0051] Example 4: Methods for salt transconjugation of telpolide include, S1. Purification: Crude telpoeptide was synthesized using a solid-phase polypeptide synthesis method and purified by reversed-phase liquid chromatography (RP-HPLC). The first purification HPLC conditions were as follows: a 50 mm diameter column; Unisil 10-100 C18 packing material; mobile phase A: 1% ammonium formate aqueous solution at pH 7.0; mobile phase B: acetonitrile; elution gradient program: acetonitrile concentration increased from 28% to 38% over 60 min at a flow rate of 50 mL / min; fraction with a purity ≥98% was collected. The second purification HPLC conditions were as follows: a 50 mm diameter column; Unisil 10-100 C18 packing material; mobile phase A: 30 mmol / L oxalic acid aqueous solution; mobile phase B: acetonitrile; elution gradient program: acetonitrile concentration increased from 35% to 50% over 60 min at a flow rate of 50 mL / min; fraction with a purity ≥99% was collected as the purification intermediate.

[0052] S2, Deacidification: While stirring, add calcium carbonate to the purification intermediate to adjust the pH to 4.0, and obtain the deacidified purification intermediate.

[0053] S3. Removal of organic solvent: At 25°C, the deacidified purification intermediate is rotary evaporated to remove acetonitrile, resulting in a salt-free suspension. The suspension is then centrifuged to obtain a salt-free solid.

[0054] S4. Salt formation: The salt-free solid was redissolved in sodium hydroxide solution, then freeze-dried to obtain sodium telpotassium salt. The concentration of the sodium hydroxide solution was 5 g / L.

[0055] Example 5: Methods for salt transfer of retaliglutide include, S1. Purification: Crude Retaglutide was synthesized using a solid-phase polypeptide synthesis method and purified by reversed-phase liquid chromatography (RP-HPLC). The first purification HPLC conditions were as follows: a 50 mm diameter column; Unisil 10-100 C18 packing material; mobile phase A: 1% ammonium formate aqueous solution at pH 7.0; mobile phase B: acetonitrile; elution gradient program: acetonitrile concentration increased from 28% to 38% over 60 min at a flow rate of 50 mL / min; fraction with a purity ≥90% was collected. The second purification HPLC conditions were as follows: a 50 mm diameter column; Unisil 10-100 C18 packing material; mobile phase A: 0.1% sulfuric acid solution; mobile phase B: acetonitrile; elution gradient program: acetonitrile concentration increased from 35% to 50% over 60 min at a flow rate of 50 mL / min; fraction with a purity ≥90% was collected as the purification intermediate.

[0056] S2, Deacidification: While stirring, add calcium hydroxide to the purification intermediate to adjust the pH to 4.38, and obtain the deacidified purification intermediate.

[0057] S3. Removal of organic solvent: At 25°C, the deacidified purification intermediate is rotary evaporated to remove acetonitrile, resulting in a salt-free suspension. The suspension is then centrifuged to obtain a salt-free solid.

[0058] S4. Salt formation: The salt-free solid was redissolved in sodium hydroxide solution, then freeze-dried to obtain sodium retaliptide. The concentration of the sodium hydroxide solution was 5 g / L.

[0059] Example 6: Methods for salt transfer of smegglutinin include, S1. Purification: Crude smegglutinin was synthesized using a solid-phase polypeptide synthesis method and purified by reversed-phase liquid chromatography (RP-HPLC). The first purification HPLC conditions were as follows: a 50 mm diameter column; Unisil 10-100 C18 packing material; mobile phase A: 1% triethylamine phosphate aqueous solution (pH 7.0); mobile phase B: acetonitrile; elution gradient program: acetonitrile concentration increased from 25% to 35% over 60 min at a flow rate of 50 mL / min; fraction with a purity ≥96% was collected. The second purification HPLC conditions were as follows: a 50 mm diameter column; Unisil 10-100 C18 packing material; mobile phase A: 0.1% phosphoric acid aqueous solution; mobile phase B: acetonitrile; elution gradient program: acetonitrile concentration increased from 30% to 45% over 60 min at a flow rate of 50 mL / min; fraction with a purity ≥98% was collected as the purification intermediate.

[0060] S2, Deacidification: While stirring, add calcium hydroxide to the purification intermediate to adjust the pH to 4.5, and obtain the deacidified purification intermediate.

[0061] S3. Removal of organic solvent: At 25°C, the deacidified purification intermediate is rotary evaporated to remove acetonitrile, resulting in a salt-free suspension. The suspension is then centrifuged to obtain a salt-free solid.

[0062] S4. Salt formation: The salt-free solid was redissolved in sodium hydroxide solution and freeze-dried to obtain sodium smegglutide. The concentration of the sodium hydroxide solution was 4 g / L.

[0063] Comparative Example 1: Methods for salt transconjugation of telpolide include, S1. Purification: Crude telpoeptide was synthesized using a solid-phase polypeptide synthesis method and purified by reversed-phase liquid chromatography (RP-HPLC). The first purification HPLC conditions were as follows: a 50 mm diameter column; Unisil 10-100 C18 packing material; mobile phase A: 1% ammonium formate aqueous solution at pH 7.0; mobile phase B: acetonitrile; elution gradient program: acetonitrile concentration increased from 28% to 38% over 60 min at a flow rate of 50 mL / min; fraction with a purity ≥98% was collected. The second purification HPLC conditions were as follows: a 50 mm diameter column; Unisil 10-100 C18 packing material; mobile phase A: 30 mmol / L tartaric acid aqueous solution; mobile phase B: acetonitrile; elution gradient program: acetonitrile concentration increased from 35% to 50% over 60 min at a flow rate of 50 mL / min; fraction with a purity ≥99% was collected as the purification intermediate.

[0064] S2, Isoelectric point sedimentation and washing: The solvent in the purified intermediate liquid was removed by rotary evaporation. The pH was adjusted to 4.0 by adding 0.5 mmol / L sodium hydroxide solution. The solid precipitate was collected by centrifugation. Water was added and stirred. The solid precipitate was collected by centrifugation again. The water-centrifugation operation was repeated 3 times to obtain a salt-free solid. The mass of water was 3 times the mass of the solid precipitate.

[0065] S3. Salt formation: The salt-free solid is reconstituted with sodium hydroxide solution, then freeze-dried to obtain sodium thiopotate. The concentration of the sodium hydroxide solution is 5 g / L.

[0066] Comparative Example 2: The salt transfer method for telpoide is the same as that of Comparative Example 1, except that the concentration of tartaric acid aqueous solution in step S1 is changed to 20 mmol / L and the mass of water in step S2 is changed to twice the mass of solid precipitate.

[0067] Comparative Example 3: The salt conversion method for telpolide is the same as in Example 1, except that in step S1, only the first reversed-phase liquid chromatography purification is performed and the second reversed-phase liquid chromatography purification is not performed.

[0068] Comparative Example 4: The salt transfer method for telpolide is the same as that in Example 1, except that step S2 deacidification treatment is not performed.

[0069] Experimental example: 1. Product quality inspection The proportions of tartaric acid, sodium ions, and calcium ions in the sodium telpoide salt products prepared in Example 1 and Comparative Example 1 were determined by high performance liquid chromatography (HPLC). Three batches of experiments were conducted for each group. The specifications for acidic long peptide salt products require an acid regulator proportion ≤0.5%, a sodium ion proportion ≤4.0%, and a calcium ion proportion ≤0.5%.

[0070] Table 1 Product Quality Inspection

[0071] The results are shown in Table 1. In both Example 1 and Comparative Example 1, the proportions of tartaric acid, sodium ions, and calcium ions in the sodium telpotetate prepared according to the specifications for the finished product met the requirements. A small amount of calcium ions was detected in the sodium telpotetate in Comparative Example 1, which may have originated from the water source used in the preparation process. Natural water or ordinary industrial water typically contains inorganic ions such as calcium. These inorganic ions may be introduced into the system during water treatment or material contact and may not be completely removed during subsequent purification, thus remaining in trace amounts in the final sodium telpotetate product.

[0072] Compared to Comparative Example 1, the sodium and calcium ion ratios of the telpoide sodium salt product obtained in Example 1 were similar to those in Comparative Example 1, while the tartaric acid ratio was slightly lower. Furthermore, the content of each component exhibited superior batch-to-batch stability. This indicates that using the process of the present invention for peptide transsalting not only ensures that the finished product quality meets relevant regulatory requirements but also effectively improves the stability of product quality.

[0073] 2. pH control during deacidification treatment The experiment was conducted following the salt transamination method for retaglutide in Example 5. First, purification was performed using reversed-phase liquid chromatography (RP-HPLC). Then, calcium hydroxide was added to adjust the pH for deacidification, with five pH gradients set at 3.88, 4.06, 4.21, 4.38, and 4.58. After deacidification, the reaction solution was filtered and lyophilized in a hanging bottle. The sulfate ion content in the lyophilized sample was determined using high-performance liquid chromatography (HPLC).

[0074] Table 2 Sulfate ion content

[0075] The results are shown in Table 2. With the addition of calcium hydroxide, the sulfate ion content gradually decreased. This result indicates that precise pH control during the deacidification process can effectively reduce the residual acid regulator content in the product.

[0076] 3. Deacidification effect The content and percentage of acid regulators in the samples were determined by high-performance liquid chromatography (HPLC). The samples included the purified intermediate solution obtained in step S1 of Example 1, the supernatant filtrate obtained after deacidification treatment in step S2 of Example 1, the purified intermediate solution obtained in step S1 of Example 2, the supernatant filtrate obtained after deacidification treatment in step S2 of Example 2, the purified intermediate solution obtained in step S1 of Comparative Example 1, the supernatant filtrate obtained after isoelectric point sedimentation and washing treatment in step S2 of Comparative Example 1, a 30 mmol / L citric acid standard solution, and a 30 mmol / L tartaric acid standard solution. The supernatant filtrate obtained after isoelectric point sedimentation and washing treatment in step S2 of Comparative Example 1 was obtained after three cycles of water addition and centrifugation, followed by filtration of the supernatant. Tartaric acid was used as the acid regulator in Example 1 and Comparative Example 1, and citric acid was used as the acid regulator in Example 2. The percentage of acid regulators (%) was calculated as: acid regulator content / total sample volume × 100%.

[0077] Table 3 Deacidification effect

[0078] The results are shown in Table 3. Compared with the 30 mmol / L citric acid standard solution and the 30 mmol / L tartaric acid standard solution, the acid regulator content in the purification intermediate solution of Step S1 in Examples 1-2 and Comparative Example 1 was significantly reduced. Specifically, the acid regulator content in Examples 1 and Comparative Example 1 was 3.06 mg / mL, accounting for 34.74% of the total; in Example 2, the acid regulator content was 2.89 mg / mL, accounting for 23.56%. However, the proportion of acid regulator in the purification intermediate solutions of Examples 1-2 and Comparative Example 1 was still far higher than the specified upper limit of ≤0.5% for the final product. This indicates that reversed-phase liquid chromatography alone cannot completely remove the acid regulator from the system, resulting in a still high residual amount of acid regulator in the purification intermediate solution. Therefore, to obtain a higher purity acidic long peptide product, further optimization of the subsequent processing technology is needed to reduce the acid regulator content in the product.

[0079] Compared with the purification intermediate solution in step S1 of Examples 1-2, the acid regulator content in the supernatant filtrate obtained after deacidification treatment in step S2 was significantly reduced. Specifically, in Example 1, the acid regulator content decreased to 0.02 mg / mL, and the acid regulator ratio decreased to 0.29%; in Example 2, the acid regulator content decreased to 0.04 mg / mL, and the acid regulator ratio decreased to 0.29%. This result indicates that introducing a deacidification treatment step after reversed-phase liquid chromatography purification can efficiently remove acid regulators from the sample, enabling the product to meet the requirement that the acid regulator ratio in the finished product is ≤0.5%, laying a key foundation for obtaining high-concentration acidic long peptide sodium salt products.

[0080] After isoelectric point sedimentation and washing, the acid regulator content in the supernatant of Comparative Example 1 was 0.13 mg / mL. Although this value is much lower than that in the intermediate purified solution of step S1, it is still higher than that in the supernatant obtained after deacidification treatment in Step S2 of Examples 1-2. The difference between the processes of Examples 1-2 and Comparative Example 1 is that in Step S2 of Comparative Example 1, the solvent was removed by rotary evaporation first, then the pH was adjusted to 4.0, followed by three water-centrifugation washes to remove the acid regulator; while in Step S2 of Examples 1-2, the pH was directly adjusted to 4.0 without rotary evaporation to remove the solvent or subsequent water-centrifugation washes. This result indicates that the deacidification treatment method of the present invention is more effective in removing the acid regulator from the sample compared to the traditional isoelectric point sedimentation and washing method. This may be because traditional isoelectric point sedimentation washing methods may cause some acid regulators to co-precipitate or adsorb onto the solid surface during rotary evaporation, making them difficult to remove completely in subsequent washing. While multiple centrifugal washing aims to remove acid regulators, it may also cause secondary dissolution or redistribution, thus limiting the overall deacidification effect. The present invention, by directly adjusting pH, may avoid the encapsulation or retention effects caused by phase transitions and recrystallization, thereby achieving more efficient separation of acid regulators from the product.

[0081] 4. Concentration of telpoeptide after deacidification The content of telpoide in the samples was determined by high performance liquid chromatography (HPLC). The samples included the purified intermediate solution obtained in step S1 of Example 1, the supernatant obtained after deacidification treatment in step S2 of Example 1, the purified intermediate solution obtained in step S1 of Comparative Example 1, and the supernatant obtained after isoelectric point precipitation and washing treatment in step S2 of Comparative Example 1. The supernatant obtained after isoelectric point precipitation and washing treatment in step S2 of Comparative Example 1 was the sample after being filtered following three cycles of water addition and centrifugation.

[0082] Figure 1 A1 represents the concentration of telpoeptide after deacidification, A2 represents the purified intermediate solution obtained in step S1 of Example 1, A3 represents the purified intermediate solution obtained in step S1 of Comparative Example 1, and A4 represents the supernatant obtained after deacidification treatment in step S2 of Comparative Example 1.

[0083] The results are as follows Figure 1 As shown, after purification by reversed-phase liquid chromatography, the concentration of telpoeptide in the intermediate purified solutions obtained in Example 1 and Comparative Example 1 was 8.79 mg / mL. Subsequently, after isoelectric point sedimentation washing in Comparative Example 1, the concentration of telpoeptide in the supernatant was 1.42 mg / mL, significantly lower than the concentration after deacidification treatment in Example 1. This may be because during the isoelectric point sedimentation washing process in Comparative Example 1, the acidic long peptide was repeatedly washed in precipitate form, resulting in significant dissolution loss in the aqueous phase and dilution. Therefore, compared with the traditional isoelectric point sedimentation washing method, the deacidification process of this invention can not only remove acid regulators more efficiently, but also better retain the concentration of the target product.

[0084] 5. Freeze-drying concentration The lyophilization concentration of the sodium thiopotassium salt lyophilized products prepared in Examples 1-2 and Comparative Examples 1-2 was determined by high performance liquid chromatography.

[0085] Figure 2 The lyophilization concentration of the sodium thiopotassium salt lyophilized product is shown in the results. Figure 2It can be seen that the lyophilized concentration of Examples 1-2 is significantly higher than that of Comparative Example 1. This is due to the difference in the steps of the peptide transsalting method. In Comparative Example 1, after purifying the crude telpoide by reversed-phase liquid chromatography, the solvent was first removed by rotary evaporation, then the pH was adjusted to 4.0, followed by three cycles of water addition and centrifugation washing, and finally, sodium hydroxide solution was added and mixed before lyophilization to obtain the lyophilized sodium telpoide product. In contrast, in Examples 1-2, after purifying the crude telpoide by reversed-phase liquid chromatography, the pH was first adjusted to 4.0, then the solvent was removed by rotary evaporation, followed by the addition of sodium hydroxide solution and lyophilization to obtain the lyophilized sodium telpoide product, without any water addition and centrifugation washing operation. This may be because in Comparative Example 1, during the isoelectric point sedimentation washing process, the acidic long peptide was repeatedly washed in the form of a precipitate, resulting in a significant loss of dissolution in the aqueous phase and dilution. This indicates that using the process method of the present invention for peptide transsalting can effectively reduce product loss in intermediate steps, thereby increasing the lyophilized concentration of the final product.

[0086] Based on the lyophilization concentration data from Examples 1-2 and Comparative Examples 1-2, it can be seen that using an acid adjuster of appropriate concentration in reverse liquid chromatography purification helps to increase the lyophilization concentration of the final product.

[0087] 6. Freeze-drying stability The acidic long peptide sodium salts prepared in Examples 1-6 and Comparative Examples 1-4 were lyophilized and subjected to an accelerated testing at 60°C for 6 months. Samples were taken on day 0 and month 6, and the content of acidic long peptide sodium salts was determined by high performance liquid chromatography. The content of acidic long peptide sodium salts on day 0 was recorded as C0, and the content of acidic long peptide sodium salts in month 6 was recorded as C1. The retention rate of the principal component was calculated according to the following formula: Principal component retention rate (%) = C1 / C0 × 100%.

[0088] The results are as follows Figure 3As shown, compared with Comparative Example 1, the retention rate of the main components in Examples 1-2 was significantly improved. This was due to the difference in the steps of the peptide transsalting method. In Comparative Example 1, the crude acidic long peptide was purified by reversed-phase liquid chromatography, the solvent was removed by rotary evaporation, the pH was adjusted to 4.0, and then it was washed three times with water and centrifuged. Finally, sodium hydroxide solution was added and mixed, and then freeze-dried to obtain the lyophilized product of the sodium salt of the acidic long peptide. In contrast, in Examples 1-2, the crude acidic long peptide was purified by reversed-phase liquid chromatography, the pH was adjusted to 4.0, the solvent was removed by rotary evaporation, and then sodium hydroxide solution was added and mixed, and then freeze-dried to obtain the lyophilized product of the sodium salt of the acidic long peptide. No water-centrifugation washing operation was performed throughout the process. Compared with Comparative Examples 3-4, the retention rate of the main components in Examples 1-2 was significantly improved. This was due to the difference in the steps of the peptide transsalting method. In Examples 1-2, an acid adjuster was first used for reversed-phase liquid chromatography purification, and then a deacidifying agent was used for deacidification. In contrast, Comparative Example 3 only used an acid adjuster for reversed-phase liquid chromatography purification, and Comparative Example 4 only used a deacidifying agent for deacidification. This demonstrates that using the process method of the present invention for peptide salt conversion can improve the retention rate of the main components in the final product and maintain the stability of the lyophilized acidic long peptide sodium salt.

[0089] The conventional operations in the operation steps of this invention are well known to those skilled in the art and will not be described in detail here.

[0090] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any changes and modifications made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for transconverting polypeptides to salts, characterized in that, The process includes synthesizing crude acidic long peptides using a solid-phase synthesis method, followed by two reversed-phase liquid chromatography (RP-LC) purification processes to obtain a purified intermediate solution. A deacidifying agent is then added to the purified intermediate solution to adjust the pH. After rotary evaporation concentration and centrifugation, the salt-free solid is retained and mixed with sodium hydroxide solution. Finally, the product is freeze-dried to obtain the acidic long peptide sodium salt product. In the second RPC, mobile phase A is an acid regulator, which includes at least one of citric acid, oxalic acid, tartaric acid, phosphoric acid, and sulfuric acid. The freeze-dried concentration of the acidic long peptide sodium salt product is 95-108.38 mg / mL, the acid regulator content is 0.02-0.14%, and the sodium ion content is 1.35-1.39%.

2. The polypeptide transsalting method according to claim 1, characterized in that, The acidic long peptides include smegglutinin, telpogglutinin, or retaliglutinin.

3. The polypeptide transsalting method according to claim 1, characterized in that, The purity of the purification intermediate is ≥90%.

4. The polypeptide transsalting method according to claim 1, characterized in that, The deacidifying agent is calcium hydroxide or calcium carbonate.

5. The polypeptide transsalting method according to claim 1, characterized in that, The calcium ion content of the acidic long peptide sodium salt product is 0.01-0.03%.

6. The polypeptide transsalting method according to claim 1, characterized in that, The pH is 3-5.

7. The polypeptide transsalting method according to claim 1, characterized in that, The concentration of the sodium hydroxide solution is 4-5 g / L.

8. The polypeptide transsalting method according to claim 1, characterized in that, The temperature for rotary evaporation concentration is 20-30℃.

9. The polypeptide transsalting method according to claim 1, characterized in that, The concentration of citric acid is 20-40 mmol / L.

10. The polypeptide transsalting method according to claim 1, characterized in that, The concentration of tartaric acid is 20-40 mmol / L.

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