A transsalting process containing a plurality of arginine polypeptides

Abstract

This invention discloses a method for salt transfer of peptides containing multiple arginine residues, solving key technical problems such as denaturation and degradation, low purity, and unstable yield in the salt transfer process of peptides containing multiple arginine residues. This invention employs a synergistic strategy of segmented acid adjustment and gradient crystallization. Targeting the multi-level basic sites and structural sensitivity of peptides containing multiple arginine residues, this method achieves stepwise protonation salt transfer of peptide residues and regular crystal growth by progressively adjusting the pH value in three stages (7.5-8.0 → 5.5-6.0 → 3.5-4.0) combined with gradient cooling crystallization (first decreasing to 8-12℃ at 0.5-1℃ / min, then decreasing to 2-5℃ at 0.2-0.5℃ / min).

Description

Technical Field

[0001] This invention relates to the field of peptide transsalting technology, and specifically to a transsalting method for peptides containing multiple arginine residues. Background Technology

[0002] Polypeptides containing multiple arginine residues are a class of biomolecules with special physiological activities and have extremely high application value in the fields of medicine, biopharmaceuticals, and cosmetics. These polypeptide molecules contain multiple basic arginine residues, and their salt forms (such as polypeptide hydrochloride and polypeptide phosphate) have better water solubility, stability, and storage performance compared to free polypeptides. They are also easier to formulate into various dosage forms such as injections and tablets. Therefore, polypeptide salt conversion is a key step in its industrialization process.

[0003] In actual production, peptides containing multiple arginines often suffer from uneven purity and easy aggregation due to limitations in the preparation process, posing challenges to the salt transfer process. Efficient salt transfer of these peptides to prepare high-purity peptide salt products is a core requirement for improving product quality and expanding application scenarios. However, current technology has not yet found an effective salt transfer method suitable for the characteristics of peptides containing multiple arginines.

[0004] In existing technologies, peptide transsalting methods often borrow from the transsalting processes of single amino acids or small molecule peptides, failing to fully consider the unique characteristics of peptides containing multiple arginine residues. Firstly, these peptides have large molecular weights and complex structures, containing multiple basic arginine residues with different pKa values. They are highly sensitive to sudden changes in temperature, pH, and oxidative environments. Traditional high-temperature dissolution (typically 40-60℃) and one-step acid-adjusting processes easily lead to destruction of the peptide's spatial structure, main chain breakage, or side chain modification, causing peptide denaturation and degradation, and significantly reducing product activity. Secondly, peptide molecules easily form aggregates through hydrogen bonds, hydrophobic interactions, and electrostatic attraction. Rapid cooling crystallization (typically at a cooling rate >2℃ / min) causes these aggregates and impurities to precipitate rapidly, resulting in low product purity (typically 95%-97.5%), irregular crystal forms, and unstable yields (batch-to-batch variations can reach 5%-10%). Furthermore, existing methods generally lack antioxidant protection measures, making peptides susceptible to oxidation by dissolved oxygen during transsalting, further affecting product quality. Simultaneously, the use of large amounts of organic solvents increases production costs and environmental pressures. Current technology has not yet found that the synergistic treatment of peptides containing multiple arginines through segmented acid adjustment and gradient crystallization can produce significant technical effects.

[0005] In summary, existing technologies have not yet solved the key technical problems encountered in the salt conversion of peptides containing multiple arginines, such as denaturation and degradation, low purity, and unstable yield. Therefore, developing a salt conversion method adapted to the characteristics of peptides containing multiple arginines, avoiding peptide denaturation and oxidation, and producing high-purity, stable-yield products suitable for industrial production, has become an urgent and practically significant technical problem to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for the salt transfer of peptides containing multiple arginine residues. Unlike the one-step acid adjustment and rapid crystallization approach of existing technologies, this invention, through extensive creative experiments, has for the first time discovered that the synergistic treatment of peptides containing multiple arginine residues using segmented acid adjustment and gradient crystallization can produce significant technical effects: segmented acid adjustment enables the gradual protonation of multiple basic sites in the peptide, avoiding structural damage caused by sudden pH changes; gradient crystallization promotes regular crystal growth and reduces the co-precipitation of aggregates and impurities. This invention achieves highly efficient salt transfer of peptides by optimizing the process parameters of each step, resulting in products with high purity, stable yield, and good activity retention. The technical solution of this invention is not a conventional improvement easily conceived by those skilled in the art based on existing technology, but rather a non-obvious technical achievement obtained through creative labor.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for salt transfer containing multiple arginine peptides includes four steps: raw material pretreatment, segmented acid adjustment, gradient crystallization, and separation and purification.

[0008] In the raw material pretreatment step, a mixed solvent consisting of water and a polar organic solvent is used to dissolve raw materials containing multiple arginine peptides. The mixed solvent is a mixture of water and a polar organic solvent at a volume ratio of 2:1-4:1 (preferably 3:1). Compared to a single solvent, the mixed solvent can balance the water solubility and dispersibility of the peptides, reducing molecular aggregation. The dissolution temperature is controlled within a mild range of 15-30℃ (preferably 18-25℃) to avoid peptide denaturation due to high temperatures. The mass ratio of the raw materials containing multiple arginine peptides to the mixed solvent is controlled at 1:5-1:10 (preferably 1:6-1:8). A ratio that is too low will result in insufficient dissolution due to insufficient solvent, while a ratio that is too high will increase the energy consumption for subsequent crystallization. The mixture is stirred at 150-250 r / min for 20-30 min (preferably 25 min) and filtered through a 0.1-0.22 μm microfiltration membrane. This removes mechanical impurities and retains large molecular aggregates, providing a homogeneous raw material solution for subsequent salt conversion.

[0009] If the raw material contains multiple arginine peptides and contains hydrophobic impurities, it should be pretreated by centrifugation after stirring and dissolving. The centrifugation speed should be 1500-2000 r / min and the centrifugation time should be 8-10 min, and then filtered through a microfiltration membrane.

[0010] The segmented pH adjustment step is one of the core innovative steps of this invention. Targeting the multi-level basicity of polypeptides containing multiple arginine residues (terminal arginine residue pKa approximately 9.0-9.5, middle arginine residue pKa approximately 8.5-9.0, and side-chain guanidinyl pKa approximately 12.5), this invention, for the first time, employs a three-stage, stepwise pH adjustment strategy to avoid pH abrupt changes that could disrupt the polypeptide's spatial structure: the first stage, pH 7.5-8.0 (preferably 7.8), initially protonates the more basic terminal arginine residues; the second stage, pH 5.5-6.0 (preferably 5.8), progressively protonates the middle arginine residues; and the third stage, pH 3.5-4.0 (preferably 3.9), completes the protonation and salt transfer of all arginine residues. After each adjustment, the mixture is stirred at a constant temperature for 20-30 minutes to ensure uniform and complete salt transfer. The three pH ranges and corresponding isothermal stirring temperatures (first range 20-25℃ (preferably 22℃), second range 18-22℃ (preferably 20℃), third range 15-18℃ (preferably 16℃)) were determined through extensive creative experiments. Deviations from these ranges can lead to incomplete salt transfer or peptide denaturation. Simultaneously, the acid solution concentration is reduced to 0.2-1 mol / L (preferably 0.5-0.8 mol / L), and the dropping rate is slowed to 0.5-2 mL / min (preferably 1 mL / min), effectively reducing damage to the peptides caused by excessively high local acid concentrations. Adding 0.1%-0.3% by weight of an antioxidant, such as vitamin C or sodium bisulfite, effectively inhibits peptide oxidative degradation and ensures product activity.

[0011] The gradient crystallization step is another core innovation of this invention. Addressing the tendency of multiple arginine-containing peptides to aggregate, this invention, for the first time, employs a gradient cooling strategy of "first slow cooling for initial crystallization, then slow cooling for full growth": First, the temperature is lowered to 8-12℃ at a rate of 0.5-1℃ / min, and crystallization is maintained at this temperature for 3-4 hours, allowing the peptide salts to slowly form crystal nuclei and avoiding aggregate formation caused by rapid cooling; then, the temperature is lowered to 2-5℃ at a rate of 0.2-0.5℃ / min, and crystallization continues for 6-8 hours, allowing the crystal nuclei to fully grow into regular crystals and reducing impurity encapsulation. This range of cooling rates and crystallization temperatures was determined through extensive creative experiments. A faster cooling rate leads to smaller crystals and reduced purity, while a slower cooling rate reduces production efficiency. During this process, low-speed intermittent stirring (30-80 r / min, stirring for 40 min and stopping for 60 min) is used, which promotes uniform crystal distribution and avoids continuous stirring from damaging the crystal structure or causing secondary aggregation.

[0012] The preferred gradient cooling process is as follows: first, the temperature is lowered to 10℃ at a rate of 0.8℃ / min, and crystallization is maintained at this temperature for 3.5 hours with intermittent stirring at a speed of 50 r / min; then, the temperature is lowered to 3℃ at a rate of 0.3℃ / min, and crystallization continues for 7 hours.

[0013] In the separation and purification steps, the centrifugation speed is reduced to 2000-4000 r / min, and the centrifugation time is extended to 15-20 min to avoid damage to the crystal structure caused by high speed. A gradient washing strategy is adopted, first washing with a mixed solvent with high water content to remove surface polar impurities, and then dehydrating with an anhydrous solvent to reduce crystal dissolution loss during the washing process. Finally, vacuum freeze drying is adopted, drying in a low temperature vacuum environment of -40 to -30℃ to completely avoid peptide denaturation caused by high temperature, while retaining the loose crystal structure and improving product solubility.

[0014] The preferred centrifugation speed is 3000 r / min, and the centrifugation time is 18 min. During washing, the volume ratio of the mixed solvent to water is 1:1.5, and the washing is performed 3 times. For washing with anhydrous polar organic solvent, the washing is performed 2 times. The vacuum freeze-drying temperature is -35℃, the vacuum degree is 5 Pa, and the drying time is 10 h.

[0015] The arginine-containing polypeptide is one of Arg-Arg, Ly-Arg-Arg, and Arg-Gly-Ap-Arg; the polar organic solvent is one of ethanol, acetone, and acetonitrile; the acid solution is one of hydrochloric acid, phosphoric acid, and formic acid (preferably hydrochloric acid), and an antioxidant with a mass percentage of 0.1%-0.3% (preferably 0.2%) is added to the acid solution, wherein the antioxidant is vitamin C or sodium bisulfite (preferably vitamin C).

[0016] The beneficial effects of this invention are as follows: 1. Innovative Achievement of Highly Efficient Salt Transfer: Unlike existing one-step acidification processes, this invention, targeting the multi-level basicity sites and structural sensitivity of peptides containing multiple arginine residues, employs a segmented acidification strategy for the first time. Combined with a gentle acid concentration and dropping rate, this achieves stepwise protonation and salt transfer of peptide residues, effectively avoiding peptide denaturation and degradation caused by sudden pH changes. Simultaneously, antioxidants are added to inhibit oxidation reactions and ensure product activity. The synergistic effect of this technology is beyond the expectations of those skilled in the art based on existing techniques. Using this method, the peptide degradation rate is below the detection limit of high-performance liquid chromatography (HPLC), while the peptide degradation rate of traditional one-step acidification methods is typically 1%-3%.

[0017] 2. Significantly Improved Product Purity: Unlike existing rapid crystallization processes, this invention employs a synergistic strategy of low-rate gradient crystallization and low-speed intermittent stirring to promote the regular growth of polypeptide salt crystals and effectively reduce molecular aggregation and impurity encapsulation. The effect of this synergistic technology is unpredictable by those skilled in the art based on existing techniques. Product purity can reach over 99.6%, an improvement of more than 2 percentage points compared to traditional methods (purity approximately 97.5%). The product exhibits regular crystal form and uniform particle size distribution (D50 of 50-100 μm), while products obtained by traditional methods have irregular crystal forms and a wide particle size distribution (D50 of 10-200 μm).

[0018] 3. Stable Yield and Suitable for Industrial Production: This invention achieves high process repeatability by optimizing the process parameters of each step. The product yield is consistently above 85%, with batch-to-batch variation less than 2%, while traditional methods exhibit larger yield fluctuations (75%-85%) and batch-to-batch variations reaching 5%-10%. The entire process utilizes a mixed solvent system and vacuum freeze-drying, balancing dissolution efficiency and product stability. Solvent recovery rates exceed 90%, resulting in minimal waste emissions and low production costs. The process conditions of this invention are mild and controllable (maximum temperature not exceeding 30℃), with low equipment requirements, and stable industrial production has been achieved, demonstrating excellent practicality and repeatability. Attached Figure Description

[0019] Figure 1 This is the high-performance liquid chromatography (HPLC) chromatogram of the product from Example 1. Figure 2 This is the high-performance liquid chromatography (HPLC) chromatogram of the product from Example 2; Figure 3 This is the high-performance liquid chromatography (HPLC) chromatogram of the product in Example 3; Figure 4 This is a high-performance liquid chromatography (HPLC) chromatogram of the comparative product. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the content described. Example 1

[0021] The polypeptide raw material processed in this embodiment is Arg-Arg (diarginine), and the specific salt conversion method is as follows: 1. Raw material pretreatment: Mix 50g of Arg-Arg peptide raw material with 350g of mixed solvent (water to ethanol volume ratio 3:1), stir at 200r / min at 22℃ for 25min to dissolve, and obtain raw material solution; filter the raw material solution with a 0.22μm microfiltration membrane to remove mechanical impurities and aggregates; 2. Staged pH adjustment: Slowly add a 0.5 mol / L hydrochloric acid solution (containing 0.2% vitamin C antioxidant by mass) dropwise to the filtered raw material solution at a rate of 1 mL / min; in the first stage, adjust the pH to 7.8 and stir at 22℃ for 25 min; in the second stage, adjust the pH to 5.8 and stir at 20℃ for 25 min; in the third stage, adjust the pH to 3.9 and stir at 16℃ for 25 min. 3. Gradient crystallization: The adjusted system was placed in a crystallization tank and first cooled to 10°C at a rate of 0.8°C / min, and crystallized at this temperature for 3.5 h, during which time the system was intermittently stirred at a speed of 50 r / min (stirring for 40 min and stopping for 60 min); then the system was cooled to 3°C at a rate of 0.3°C / min, and crystallization was continued for 7 h to obtain a crystalline suspension. 4. Separation and purification: The crystallized suspension was centrifuged at 3000 r / min for 18 min to obtain crude crystals; the crude crystals were washed three times with a mixture of a mixed solvent (water and ethanol, volume ratio 3:1) and a water mixture (volume ratio 1:1.5), and centrifuged at 3000 r / min for 15 min after each wash; the crude crystals were then washed twice with anhydrous ethanol, and centrifuged after each wash; finally, the washed crystals were placed in a vacuum freeze dryer and dried at -35℃ and a vacuum degree of 5 Pa for 10 h to obtain the Arg-Arg hydrochloride product.

[0022] The Arg-Arg hydrochloride product obtained in this embodiment has a purity of 99.7%, and no peptide degradation products were detected by high-performance liquid chromatography (HPLC). (See [link to relevant documentation]). Figure 1 The product has good water solubility (solubility ≥50g / 100mL water at 25℃). Example 2

[0023] The polypeptide raw material processed in this embodiment is Ly-Arg-Arg (lysine-arg-arg), and the specific salt conversion method is as follows: 1. Raw material pretreatment: Mix 50g Ly-Arg-Arg peptide raw material with 500g mixed solvent (water to acetone volume ratio 4:1), stir at 250r / min at 25℃ for 20min to dissolve, and obtain raw material solution; filter the raw material solution with a 0.20μm microfiltration membrane to remove mechanical impurities and aggregates; 2. Staged pH adjustment: Slowly add a 0.8 mol / L phosphoric acid solution (containing 0.3% sodium bisulfite antioxidant by mass) dropwise to the filtered raw material solution at a dropping rate of 1.5 mL / min; in the first stage, adjust the pH to 8.0 and stir at 25℃ for 20 min; in the second stage, adjust the pH to 6.0 and stir at 22℃ for 20 min; in the third stage, adjust the pH to 4.0 and stir at 18℃ for 20 min. 3. Gradient crystallization: The adjusted system is placed in a crystallization tank, and the temperature is first reduced to 12℃ at a rate of 1℃ / min, and crystallized at this temperature for 3 hours, during which the system is intermittently stirred at a speed of 80 r / min (stirring for 40 min and stopping for 60 min); then the temperature is reduced to 5℃ at a rate of 0.5℃ / min, and crystallization continues for 6 hours to obtain a crystalline suspension. 4. Separation and purification: The crystallized suspension was centrifuged at 4000 r / min for 15 min to obtain crude crystals; the crude crystals were washed twice with a mixture of a mixed solvent (water and acetone volume ratio 3:1) and a mixture of water (volume ratio 1:1.5), and centrifuged after each wash; the crude crystals were then washed once with anhydrous acetone, and centrifuged after washing; finally, the washed crystals were placed in a vacuum freeze dryer and dried at -30℃ and a vacuum degree of 10 Pa for 8 h to obtain the Ly-Arg-Arg phosphate product.

[0024] Testing showed that the Ly-Arg-Arg phosphate product obtained in this example had a purity of 99.66% and contained no peptide degradation products. (See [link to relevant documentation]). Figure 2 Solubility at 25℃ is ≥45g / 100mL water. Example 3

[0025] The polypeptide raw material processed in this embodiment is Arg-Gly-Ap-Arg (arginine-glycine-aspartic acid-arginine), and the specific salt conversion method is as follows: 1. Raw material pretreatment: 50g of Arg-Gly-Ap-Arg peptide raw material was mixed with 250g of mixed solvent (water to acetonitrile volume ratio 2:1) and stirred at 150r / min at 18℃ for 30min to dissolve and obtain raw material solution; because the raw material contained a small amount of hydrophobic impurities, it was first centrifuged at 1800r / min for 10min for pretreatment, and then filtered with a 0.1μm microfiltration membrane to remove impurities and aggregates; 2. Staged pH adjustment: Slowly add a 0.2 mol / L formic acid solution (containing 0.1% vitamin C antioxidant by mass) dropwise to the filtered raw material solution at a dropping rate of 0.5 mL / min; in the first stage, adjust the pH to 7.5 and stir at 20℃ for 30 min; in the second stage, adjust the pH to 5.5 and stir at 18℃ for 30 min; in the third stage, adjust the pH to 3.5 and stir at 15℃ for 30 min. 3. Gradient crystallization: The adjusted system is placed in a crystallization tank, and the temperature is first reduced to 8℃ at a rate of 0.5℃ / min, and crystallized at this temperature for 4 hours, during which the system is intermittently stirred at a speed of 30 r / min (stirring for 40 min and stopping for 60 min); then the temperature is reduced to 2℃ at a rate of 0.2℃ / min, and crystallization continues for 8 hours to obtain a crystalline suspension; 4. Separation and purification: The crystallized suspension was centrifuged at 2000 r / min for 20 min to obtain crude crystals; the crude crystals were washed three times with a mixture of a mixed solvent (water and acetonitrile volume ratio 2:1) and a water mixture (volume ratio 1:2), and centrifuged after each wash; the crude crystals were then washed twice with anhydrous acetonitrile, and centrifuged after each wash; finally, the washed crystals were placed in a vacuum freeze dryer and dried at -40℃ and a vacuum degree of 5 Pa for 12 h to obtain the Arg-Gly-Ap-Arg formate product.

[0026] Testing showed that the Arg-Gly-Ap-Arg formate product obtained in this example had a purity of 99.66% and contained no peptide degradation products. (See [link to relevant documentation]). Figure 3 Solubility at 25℃ is ≥55g / 100mL water. Comparative Example

[0027] This comparative example uses the traditional one-step acid-adjusting crystallization method to treat the same mixed raw materials as in Example 1. The specific method is as follows: 1. Raw material pretreatment: Same as step 1 in Example 1; 2. One-step acid adjustment: Add 1 mol / L hydrochloric acid solution dropwise to the raw material solution to directly adjust the pH to 4.3, and stir for 20 min; 3. Rapid crystallization: The system was directly cooled to 0℃ and crystallized for 6 hours; 4. Separation and purification: Same as step 4 in Example 1.

[0028] The purity of the product obtained in this comparative example was found to be 97.55% upon testing. (See attached image) Figure 4 The value was significantly lower than that of Example 1.

[0029] The above examples and comparative examples demonstrate that the various arginine salt conversion methods of the present invention can effectively improve the purity and yield of the product, and have significant advantages.

[0030] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for salt transfer containing multiple arginine polypeptides, characterized in that, Includes the following steps: 1) Raw material pretreatment: Mix multiple arginine peptide raw materials with a mixed solvent at a mass ratio of 1:5-1:

10. The mixed solvent is a mixture of water and a polar organic solvent at a volume ratio of 2:1-4:

1. Stir and dissolve the mixture at 150-250 r / min for 20-30 min at 15-30℃ to obtain a raw material solution. Filter the raw material solution using a 0.1-0.22 μm microfiltration membrane to remove mechanical impurities and macromolecular aggregates. 2) Segmented pH adjustment: Slowly add an acid solution with a concentration of 0.2-1 mol / L to the filtered raw material solution at a dropping rate of 0.5-2 mL / min, adjusting the pH value of the system in three stages: the first stage adjusts the pH to 7.5-8.0, and then stirs at a constant temperature of 20-25℃ for 20-30 min; the second stage adjusts the pH to 5.5-6.0, and then stirs at a constant temperature of 18-22℃ for 20-30 min; the third stage adjusts the pH to 3.5-4.0, and then stirs at a constant temperature of 15-18℃ for 20-30 min. 3) Gradient crystallization: After adjustment, place the system in a crystallization tank, first reduce the temperature to 8-12℃ at a rate of 0.5-1℃ / min, and maintain the temperature for crystallization for 3-4 hours. During this period, stir intermittently at a speed of 30-80 r / min for 40 minutes and stop for 60 minutes. Then reduce the temperature to 2-5℃ at a rate of 0.2-0.5℃ / min and continue crystallization for 6-8 hours to obtain a crystalline suspension. 4) Separation and purification: Centrifuge the crystallized suspension at 2000-4000 r / min for 15-20 min to obtain crude crystals; first wash the crude crystals 2-3 times with a mixture of mixed solvent and water at a volume ratio of 1:2-1:1, then wash 1-2 times with anhydrous polar organic solvent, centrifuging after each wash; finally place the washed crystals in a vacuum freeze dryer and dry them at -40 to -30℃ and a vacuum degree ≤10Pa for 8-12 h to obtain a product containing multiple arginine polypeptide salts; The arginine-containing polypeptide is one of Arg-Arg, Ly-Arg-Arg, and Arg-Gly-Ap-Arg; the polar organic solvent is one of ethanol, acetone, and acetonitrile; the acid solution is one of hydrochloric acid, phosphoric acid, and formic acid, and an antioxidant of 0.1%-0.3% by mass is added to the acid solution, wherein the antioxidant is vitamin C or sodium bisulfite.

2. The method for salt transfer of a plurality of arginine polypeptides according to claim 1, characterized in that, In step 1, the mass ratio of the multiple arginine peptide raw materials to the mixed solvent is 1:6-1:8, the volume ratio of water to polar organic solvent in the mixed solvent is 3:1, the stirring temperature is 18-25℃, and the stirring time is 25min.

3. The method for salt transfer containing multiple arginine polypeptides according to claim 1, characterized in that, In step 2, the concentration of the acid solution is 0.5-0.8 mol / L, the dropping rate is 1 mL / min, and the amount of antioxidant added is 0.2% by mass. The pH values ​​adjusted in three stages are 7.8, 5.8, and 3.9, and the corresponding constant temperature stirring temperatures are 22℃, 20℃, and 16℃, respectively.

4. The method for salt transfer containing multiple arginine polypeptides according to claim 1, characterized in that, In step 3, the specific process of gradient cooling is as follows: first, the temperature is reduced to 10℃ at a rate of 0.8℃ / min, and the crystallization is maintained at this temperature for 3.5h, with an intermittent stirring speed of 50r / min; then, the temperature is reduced to 3℃ at a rate of 0.3℃ / min, and the crystallization continues for 7h.

5. The method for salt transfer containing multiple arginine polypeptides according to claim 1, characterized in that, In step 4, the centrifugation speed is 3000 r / min and the centrifugation time is 18 min; during washing, the volume ratio of the mixed solvent to water is 1:1.5, the washing is performed 3 times, and the washing is performed 2 times with anhydrous polar organic solvent.

6. The method for salt transfer of a plurality of arginine polypeptides according to claim 1, characterized in that, In step 4, the vacuum freeze-drying temperature is -35℃, the vacuum degree is 5Pa, and the drying time is 10h.

7. The method for salt transfer of a plurality of arginine polypeptides according to claim 1, characterized in that, The acid solution is hydrochloric acid, and the antioxidant is vitamin C.

8. The method for salt transfer of a plurality of arginine polypeptides according to claim 1, characterized in that, In step 1, if the arginine peptide raw materials contain hydrophobic impurities, after stirring and dissolving, they should be pretreated by centrifugation at a speed of 1500-2000 r / min for 8-10 min, and then filtered through a microfiltration membrane.

Images