A method for improving the stability of solid-phase synthesized multi-cysteine ​​peptides

Abstract

The present invention discloses a method for improving the stability of solid-phase synthesized multi-cysteine ​​polypeptides, primarily addressing the technical problem of poor stability caused by the susceptibility of such polypeptides to oxidation, degradation, elimination, denaturation, and inactivation. The method comprises the following steps: 1) purifying the multi-cysteine ​​polypeptide synthesized by solid-phase synthesis via liquid chromatography to obtain a preparation solution containing trifluoroacetate; 2) passing the preparation solution through a depth filter containing hydrophilic silica aerogel spheres as a filter material to remove metal ions and trace peroxides contained in the preparation solution by adsorption; 3) further passing the preparation solution through a weakly basic cation exchange resin to a pH of 6.3-6.5; 4) removing the sodium trifluoroacetate in the preparation solution by tangential flow filtration with low pressure, obtaining a weakly acidic, salt-free preparation solution; 5) freezing and vacuum drying to obtain a solid powder final product; 6) vacuum packaging and frozen storage; and 7) ensuring purity and stability after storage in accordance with scientific research requirements.

Description

Technical Field

[0001] The present invention belongs to the technical field of improving polypeptide stability, and in particular relates to a method for improving the stability of a solid-phase synthesized polypeptide containing multiple cysteine ​​residues. Background Art

[0002] Cysteine ​​is a sulfur-containing amino acid with the molecular formula C3H7NO2S and the chemical name (S)-2-amino-3-mercaptopropionic acid. Cysteine ​​typically exists as a white, crystalline powder and is odorless. It is readily soluble in water but insoluble in alcohol and ether. Cysteine ​​is an unstable compound prone to redox reactions. It readily oxidizes in air and water, and also readily oxidizes to cystine in acidic or alkaline solutions. It is stable at room temperature and pressure, and relatively stable in weakly acidic solutions.

[0003] Solid-phase synthesized peptides containing multiple cysteines have historically exhibited poor stability due to the presence of these cysteines. Throughout the entire production process, from synthesis to purification, peptides undergo aqueous environments, which can introduce metal ions and trace amounts of peroxides into the product. The presence of metal ions can β-eliminate the peptide, leading to degradation and poor stability. The presence of trace amounts of peroxides can not only contaminate the product but also oxidize the peptide, resulting in poor stability. The crude product from solid-phase synthesis is cleaved with a strong acid reagent and then prepared in a liquid-phase system using a strongly acidic mobile phase. Pure peptides prepared in liquid-phase synthesis are often prepared in the form of trifluoroacetate salts. This highly acidic environment can cause sulfhydryl exchange between cysteine-containing peptides, forming erroneous disulfide bonds and leading to oxidation and poor stability. Polycysteine-containing peptides are also susceptible to β-elimination and degradation in alkaline environments. Furthermore, peptides can undergo oxidation, degradation, denaturation, and inactivation due to changes in storage conditions, time, temperature, light, and air, all contributing to the poor stability of polycysteine-containing peptides. At this point, it becomes particularly important to ensure the stability of such peptides.

[0004] Polypeptides containing multiple cysteine ​​residues are a widespread and diverse topic in the field of peptide research. As the proportion of cysteine ​​residues in a polypeptide increases, its stability decreases. Current treatments to improve the stability of these polypeptides primarily involve vacuum drying and dehydration, vacuum packaging to isolate the air, and cryogenic storage to minimize the effects of temperature and light. Alternatively, desalting the polypeptides to produce pure, salt-free products improves their stability by maintaining an acid-free and alkaline-free environment. However, vacuum drying, vacuum packaging, and cryogenic storage are only temporary solutions and lack a substantial effect on the long-term preservation of these polypeptides. Desalting techniques alone can also reduce the solubility of these polypeptides. Subsequent reconstitution studies can easily lead to aggregation or precipitation due to low solubility, resulting in denaturation or even inactivation. The present invention proposes a method for improving the stability of polypeptides containing multiple cysteine ​​residues. This method is convenient and rapid, integrating multiple methods and theories to produce a more active polypeptide product while also improving its stability, ensuring its stability for subsequent scientific research. Summary of the Invention

[0005] The present invention aims to improve the stability of a solid-phase synthesized multi-cysteine ​​polypeptide, mainly to solve the technical problems that such polypeptides have poor stability over a long period of time and that simple desalting methods can reduce the solubility of such polypeptides, making them prone to aggregation or precipitation, resulting in polypeptide denaturation or even inactivation.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A method for improving the stability of a solid-phase synthesized polypeptide containing multiple cysteine ​​residues comprises the following steps:

[0008] 1) The multi-cysteine ​​peptide synthesized from solid phase synthesis is purified by liquid chromatography to obtain a preparation solution containing trifluoroacetate. This preparation solution is highly acidic and the peptide is unstable.

[0009] 2) The prepared liquid is passed through a deep filter filled with hydrophilic silica aerogel balls as filter material to remove metal ions and trace peroxides contained in the prepared liquid by adsorption;

[0010] 3) Rapidly pass the prepared solution after removing metal ions and trace peroxides through a 30-mesh weakly alkaline (NaOH) cation exchange resin. When the exchange solution is initially verified to be weakly acidic by colorimetric analysis, stop the rapid exchange and collect the weakly acidic peptide preparation solution for later use;

[0011] 4) The weakly acidic peptide preparation solution is then slowly passed through a 20-mesh weakly basic (NaOH) cation exchange resin. The pH value of the exchanged preparation solution is measured in real time using a pH meter. When the pH value of the exchanged solution is determined to be 6.3-6.5, the exchange is stopped to obtain a slightly acidic peptide preparation solution. The exchanged preparation solution contains sodium trifluoroacetate organic salt.

[0012] 5) The polypeptide preparation solution containing sodium trifluoroacetate organic salt obtained after the secondary exchange is subjected to tangential flow filtration using ether as the sodium trifluoroacetate organic salt removal agent and applying a liquid phase pump at low pressure to drive the filtration speed. The sodium trifluoroacetate salt in the preparation solution is removed through a filter column filled with hydrophobic silica aerogel balls to obtain a slightly acidic salt-free preparation solution;

[0013] 6) Freezing and vacuum drying the prepared solution to obtain a solid powder final product;

[0014] 7) Vacuum packing and freezing the final solid powder product;

[0015] 8) After storage, the peptide product is tested again to ensure that the peptide purity meets the scientific research purity.

[0016] The preferred solution is: the adsorption filtration speed in step 2 is 10-12 ml / min.

[0017] The preferred solution is: the particle size of the weakly alkaline (NaOH) cation exchange resin in step 3 is 30 mesh.

[0018] The preferred solution is: the rapid exchange rate in step 3 is 30-33 ml / min.

[0019] The preferred solution is: the particle size of the weakly alkaline (NaOH) cation exchange resin in step 4 is 20 mesh.

[0020] A preferred solution is: the slow exchange rate in step 4 is 18-20 ml / min.

[0021] A preferred solution is: in step 5, the low-pressure driven filtration speed is 8-10 ml / min.

[0022] The present invention has the beneficial effect of improving the stability of multi-cysteine ​​peptides by proposing a technical solution that effectively and long-term stores such peptides while maximizing their activity. First, an adsorption method is used to remove metal ions and trace peroxides from the aqueous environment. Hydrophilic silica aerogel spheres, with their high porosity, high specific surface area, and excellent chemical stability, utilize their adsorption to achieve a filtration mechanism. This physical process is simple to perform and further enhances peptide stability. Ion exchange is then performed with a weakly alkaline (NaOH) cation exchange resin. Initially, a rapid pretreatment with a large-particle resin is used to rapidly reduce the acidity of the preparation solution. Fine exchange is then performed with a small-particle resin to maintain a pH of 6.3-6.5 after exchange, maintaining the peptide product in a slightly acidic environment. This reduces the product's susceptibility to oxidation while ensuring a certain level of solubility, further enhancing its stability. After weakly alkaline (NaOH) cation exchange, sodium trifluoroacetate (SATA) is produced. Taking advantage of the fact that cysteine ​​is insoluble in ether while sodium trifluoroacetate is slightly soluble, tangential flow filtration is used to slowly drive the solution through a filter column filled with hydrophobic silica aerogel spheres at low pressure. The hydrophobic silica aerogel spheres possess high strength and crack resistance, allowing them to withstand the pressure applied by a liquid phase pump while removing the SATA salt. Vacuum drying, vacuum packaging, and low-temperature storage techniques are then employed to enhance the stability of these peptides, enabling long-term storage. The hydrophilic and hydrophobic silica aerogel spheres, and weakly alkaline (NaOH) cation exchange resins introduced in this invention, along with the adsorption and tangential flow filtration methods, ensure faster processing of these peptides and a more optimal and stable post-processing environment. Furthermore, these chemical materials are readily available and easily replaceable, enabling high sample coverage of a wide range of research-focused multi-cysteine-containing peptides, ensuring product stability and improving the accuracy of research results for subsequent scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the liquid chromatogram of the product of Example 1.

[0024] Figure 2 This is a liquid chromatogram of the product of Example 1 after one year of storage.

[0025] Figure 3 It is the liquid chromatogram of the product of Example 2.

[0026] Figure 4 This is a liquid chromatogram of the product of Example 2 after one year of storage.

[0027] Figure 5 It is the liquid chromatogram of the product of Example 3.

[0028] Figure 6This is the liquid chromatogram of the product of Example 3 after one year of storage.

[0029] Figure 7 It is the liquid chromatogram of the product of Example 4.

[0030] Figure 8 This is a liquid chromatogram of the product of Example 4 after one year of storage.

[0031] Figure 9 It is the liquid chromatogram of the product of Example 5.

[0032] Figure 10 This is the liquid chromatogram of the product of Example 5 after one year of storage.

[0033] Figure 11 It is the liquid chromatogram of the product of Example 6.

[0034] Figure 12 This is the liquid chromatogram of the product of Example 6 after one year of storage. DETAILED DESCRIPTION Example 1

[0035] Taking GACARQGFGFCCFCYKKC (C accounts for 27% of the total number of amino acids in the polypeptide) as an example, the specific implementation steps are as follows:

[0036] 1. Eighteen amino acids containing five cysteines were synthesized by solid phase chemical synthesis to obtain 485 mg of crude polypeptide for future use.

[0037] 2. Liquid phase preparation of 485 mg of crude peptide was performed to obtain a peptide preparation solution that met the purity requirements for scientific research (greater than 95%). At this point, the peptide preparation solution was unstable and needed to be processed quickly.

[0038] 3. Weigh 300g of hydrophilic silica aerogel ball filter media and place it into a deep filter. Rinse the filter media with 3-5 times the volume of ethanol reagent and set aside.

[0039] 4. Pass the peptide preparation solution through a deep filter filled with hydrophilic silica aerogel balls as filter material and adjust the filtration speed to 12 ml / min. Collect the filtrate and set aside.

[0040] 5. Weigh 350 g of treated 30-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0041] 6. Pass the collected filtrate through a 30-mesh weakly alkaline (NaOH) cation exchange resin glass column, adjust the filtration speed to 33 ml / min, and collect the polypeptide preparation solution of the first exchange.

[0042] 7. The peptide preparation solution from the first exchange was tested using pH test paper colorimetric method. The pH reagent showed golden yellow, confirming that the preparation solution from the first exchange was a weak acid with a pH value of around 5.3.

[0043] 8. Weigh 300 g of treated 20-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0044] 9. Continue to pass the peptide preparation solution from the first exchange through a 20-mesh weakly alkaline (NaOH) cation exchange resin glass column at a filtration rate of 20 ml / min, and collect the peptide preparation solution from the second exchange.

[0045] 10. Use a pH meter to accurately test the pH value of the peptide preparation solution after the second exchange, which is 6.5, to ensure that the entire peptide environment is treated in a slightly acidic environment after the second exchange.

[0046] 11. Weigh 250g of hydrophobic silica aerogel ball filler into the filter column. Rinse the filter column with 3-5 times the volume of ethanol reagent of the filter medium and set aside.

[0047] 12. First, add ether twice the volume of the preparation solution into the filter column filled with hydrophobic silica aerogel balls. Then, pass the second exchanged peptide preparation solution through the filter column. Apply a liquid phase pump at low pressure to drive the filtration speed at 10 ml / min and collect the filtrate.

[0048] 13. The collected filtrate was tested by ion chromatography, and the trifluoroacetate content in the polypeptide was 2.8%. The ion chromatograph model was (Metrohm 883 Ion Chromatograph, Switzerland).

[0049] 14. The final product polypeptide preparation solution is frozen and vacuum dried to obtain a solid powder final product.

[0050] 15. The purity of the solid powder product was tested by liquid chromatography and the purity was above 95% ( Figure 1 ), which meets the purity requirements for scientific research.

[0051] 15. The solid powder final product is vacuum packed and stored in a freezer for one year.

[0052] 16. After one year of storage, the solid powdered peptide was retested by liquid chromatography and the purity was still above 95% ( Figure 2 ). Example 2

[0053] Taking CKCCCKHICLCFRFMH (C accounts for 37% of the total number of amino acids in the polypeptide) as an example, the specific implementation steps are as follows:

[0054] 1. Sixteen amino acids containing six cysteines were synthesized by solid phase chemical synthesis to obtain 478 mg of crude polypeptide for future use.

[0055] 2. Liquid phase preparation of 478 mg of crude peptide was performed to obtain a peptide preparation solution that met the purity requirements for scientific research (greater than 95%). At this point, the peptide preparation solution was unstable and needed to be processed quickly.

[0056] 3. Weigh 300g of hydrophilic silica aerogel ball filter media and place it into a deep filter. Rinse the filter media with 3-5 times the volume of ethanol reagent and set aside.

[0057] 4. Pass the peptide preparation solution through a deep filter filled with hydrophilic silica aerogel balls as filter material and adjust the filtration speed to 12 ml / min. Collect the filtrate and set aside.

[0058] 5. Weigh 350 g of treated 30-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0059] 6. Pass the collected filtrate through a 30-mesh weakly alkaline (NaOH) cation exchange resin glass column, adjust the filtration speed to 33 ml / min, and collect the polypeptide preparation solution of the first exchange.

[0060] 7. The peptide preparation solution from the first exchange was tested using pH test paper colorimetric method. The pH reagent showed golden yellow, confirming that the preparation solution from the first exchange was a weak acid with a pH value of around 5.5.

[0061] 8. Weigh 300 g of treated 20-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0062] 9. Continue to pass the peptide preparation solution from the first exchange through a 20-mesh weakly alkaline (NaOH) cation exchange resin glass column at a filtration rate of 20 ml / min, and collect the peptide preparation solution from the second exchange.

[0063] 10. Use a pH meter to accurately test the pH value of the peptide preparation solution after the second exchange, which is 6.4, to ensure that the entire peptide environment is treated in a slightly acidic environment after the second exchange.

[0064] 11. Weigh 250g of hydrophobic silica aerogel ball filler into the filter column. Rinse the filter column with 3-5 times the volume of ethanol reagent of the filter medium and set aside.

[0065] 12. First, add ether twice the volume of the preparation solution into the filter column filled with hydrophobic silica aerogel balls. Then, pass the second exchanged peptide preparation solution through the filter column. Apply a liquid phase pump at low pressure to drive the filtration speed at 10 ml / min and collect the filtrate.

[0066] 13. The collected filtrate was tested by ion chromatography, and the trifluoroacetate content in the polypeptide was 2.9%. The ion chromatograph model was (Metrohm 883 Ion Chromatograph, Switzerland).

[0067] 14. The final product polypeptide preparation solution is frozen and vacuum dried to obtain a solid powder final product.

[0068] 15. The purity of the solid powder product was tested by liquid chromatography and the purity was above 95% ( Figure 3 ), which meets the purity requirements for scientific research.

[0069] 15. The solid powder final product is vacuum packed and stored in a freezer for one year.

[0070] 16. After one year of storage, the solid powdered peptide was retested by liquid chromatography and the purity was still above 95% ( Figure 4 ). Example 3

[0071] Taking PCKCCGLCTCCYCCK (C accounts for 53% of the total number of amino acids in the polypeptide) as an example, the specific implementation steps are as follows:

[0072] 1. Fifteen amino acids containing eight cysteines were synthesized by solid phase chemical synthesis to obtain 480 mg of crude polypeptide for later use.

[0073] 2. Liquid phase preparation of 480 mg of crude peptide was performed to obtain a peptide preparation solution that met the purity requirements for scientific research (greater than 95%). At this point, the peptide preparation solution was unstable and needed to be processed quickly.

[0074] 3. Weigh 300g of hydrophilic silica aerogel ball filter media and place it into a deep filter. Rinse the filter media with 3-5 times the volume of ethanol reagent and set aside.

[0075] 4. Pass the peptide preparation solution through a deep filter filled with hydrophilic silica aerogel balls as filter media, and adjust the filtration speed to 11 ml / min. Collect the filtrate and set aside.

[0076] 5. Weigh 350 g of treated 30-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0077] 6. Pass the collected filtrate through a 30-mesh weakly alkaline (NaOH) cation exchange resin glass column, adjust the filtration speed to 33 ml / min, and collect the polypeptide preparation solution of the first exchange.

[0078] 7. The peptide preparation solution from the first exchange was tested using pH test paper colorimetric method. The pH reagent showed golden yellow, confirming that the preparation solution from the first exchange was a weak acid with a pH value of around 5.4.

[0079] 8. Weigh 300 g of treated 20-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0080] 9. Continue to pass the peptide preparation solution from the first exchange through a 20-mesh weakly alkaline (NaOH) cation exchange resin glass column at a filtration rate of 20 ml / min, and collect the peptide preparation solution from the second exchange.

[0081] 10. Use a pH meter to accurately test the pH value of the peptide preparation solution after the second exchange, which is 6.4, to ensure that the entire peptide environment is treated in a slightly acidic environment after the second exchange.

[0082] 11. Weigh 250g of hydrophobic silica aerogel ball filler into the filter column. Rinse the filter column with 3-5 times the volume of ethanol reagent of the filter medium and set aside.

[0083] 12. First, add ether twice the volume of the preparation solution into the filter column filled with hydrophobic silica aerogel balls. Then, pass the second exchanged peptide preparation solution through the filter column. Apply a liquid phase pump at low pressure to drive the filtration speed at 10 ml / min and collect the filtrate.

[0084] 13. The collected filtrate was tested by ion chromatography, and the trifluoroacetate content in the polypeptide was 2.9%. The ion chromatograph model was (Metrohm 883 Ion Chromatograph, Switzerland).

[0085] 14. The final product polypeptide preparation solution is frozen and vacuum dried to obtain a solid powder final product.

[0086] 15. The purity of the solid powder product was tested by liquid chromatography and the purity was above 95% ( Figure 5 ), which meets the purity requirements for scientific research.

[0087] 15. The solid powder final product is vacuum packed and stored in a freezer for one year.

[0088] 16. After one year of storage, the solid powdered peptide was retested by liquid chromatography and the purity was still above 95% ( Figure 6 ). Example 4

[0089] Taking CKCCCLCCNCCYCCKC (C accounts for 68% of the total number of amino acids in the polypeptide) as an example, the specific implementation steps are as follows:

[0090] 1. Sixteen amino acids containing eleven cysteines were synthesized by solid phase chemical synthesis to obtain 453 mg of crude polypeptide for future use.

[0091] 2. Liquid phase preparation of 453 mg of crude peptide was performed to obtain a peptide preparation solution that met the purity requirements for scientific research (greater than 95%). At this point, the peptide preparation solution was unstable and needed to be processed quickly.

[0092] 3. Weigh 300g of hydrophilic silica aerogel ball filter media and place it into a deep filter. Rinse the filter media with 3-5 times the volume of ethanol reagent and set aside.

[0093] 4. Pass the peptide preparation solution through a deep filter filled with hydrophilic silica aerogel balls as filter media, and adjust the filtration speed to 11 ml / min. Collect the filtrate and set aside.

[0094] 5. Weigh 350 g of treated 30-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0095] 6. Pass the collected filtrate through a 30-mesh weakly alkaline (NaOH) cation exchange resin glass column, adjust the filtration speed to 32 ml / min, and collect the polypeptide preparation solution from the first exchange.

[0096] 7. The peptide preparation solution from the first exchange was tested using pH test paper colorimetric method. The pH reagent showed golden yellow, confirming that the preparation solution from the first exchange was a weak acid with a pH value of around 5.4.

[0097] 8. Weigh 300 g of treated 20-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0098] 9. Continue to pass the peptide preparation solution from the first exchange through a 20-mesh weakly alkaline (NaOH) cation exchange resin glass column at a filtration rate of 19 ml / min, and collect the peptide preparation solution from the second exchange.

[0099] 10. Use a pH meter to accurately test the pH value of the peptide preparation solution after the second exchange, which is 6.3, to ensure that the entire peptide environment is treated in a slightly acidic environment after the second exchange.

[0100] 11. Weigh 250g of hydrophobic silica aerogel ball filler into the filter column. Rinse the filter column with 3-5 times the volume of ethanol reagent of the filter medium and set aside.

[0101] 12. First, add ether twice the volume of the preparation solution to the filter column filled with hydrophobic silica aerogel balls. Then, pass the second exchange peptide preparation solution through the filter column. Apply a liquid phase pump at low pressure to drive the filtration rate at 9 ml / min and collect the filtrate.

[0102] 13. The collected filtrate was tested by ion chromatography, and the trifluoroacetate content in the polypeptide was 3.0%. The ion chromatograph model was (Metrohm 883 Ion Chromatograph, Switzerland).

[0103] 14. The final product polypeptide preparation solution is frozen and vacuum dried to obtain a solid powder final product.

[0104] 15. The purity of the solid powder product was tested by liquid chromatography and the purity was above 95% ( Figure 7 ), which meets the purity requirements for scientific research.

[0105] 15. The solid powder final product is vacuum packed and stored in a freezer for one year.

[0106] 16. After one year of storage, the solid powdered peptide was retested by liquid chromatography and the purity was still above 95% ( Figure 8 ). Example 5

[0107] Taking CCCCCR (C accounts for 83% of the total number of amino acids in the polypeptide) as an example, the specific implementation steps are as follows:

[0108] 1. A 426 mg crude peptide containing five cysteines and six amino acids was obtained through solid phase chemical synthesis and set aside.

[0109] 2. Liquid phase preparation of 426 mg of crude peptide was performed to obtain a peptide preparation solution that met the purity requirements for scientific research (greater than 95%). At this point, the peptide preparation solution was unstable and required rapid processing.

[0110] 3. Weigh 300g of hydrophilic silica aerogel ball filter media and place it into a deep filter. Rinse the filter media with 3-5 times the volume of ethanol reagent and set aside.

[0111] 4. Pass the peptide preparation solution through a deep filter filled with hydrophilic silica aerogel balls as filter material and adjust the filtration speed to 10 ml / min. Collect the filtrate and set aside.

[0112] 5. Weigh 350 g of treated 30-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0113] 6. Pass the collected filtrate through a 30-mesh weakly alkaline (NaOH) cation exchange resin glass column, adjust the filtration speed to 31 ml / min, and collect the polypeptide preparation solution of the first exchange.

[0114] 7. The peptide preparation solution from the first exchange was tested using pH test paper colorimetric method. The pH reagent showed golden yellow, confirming that the preparation solution from the first exchange was a weak acid with a pH value of around 5.5.

[0115] 8. Weigh 300 g of treated 20-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0116] 9. Continue to pass the peptide preparation solution from the first exchange through a 20-mesh weakly alkaline (NaOH) cation exchange resin glass column at a filtration rate of 18 ml / min, and collect the peptide preparation solution from the second exchange.

[0117] 10. Use a pH meter to accurately test the pH value of the peptide preparation solution after the second exchange, which is 6.3, to ensure that the entire peptide environment is treated in a slightly acidic environment after the second exchange.

[0118] 11. Weigh 250g of hydrophobic silica aerogel ball filler into the filter column. Rinse the filter column with 3-5 times the volume of ethanol reagent of the filter medium and set aside.

[0119] 12. First, add ether twice the volume of the preparation solution into the filter column filled with hydrophobic silica aerogel balls. Then, pass the second exchanged peptide preparation solution through the filter column. Apply a liquid phase pump at low pressure to drive the filtration rate at 8 ml / min and collect the filtrate.

[0120] 13. The collected filtrate was tested by ion chromatography, and the trifluoroacetate content in the polypeptide was 3.0%. The ion chromatograph model was (Metrohm 883 Ion Chromatograph, Switzerland).

[0121] 14. The final product polypeptide preparation solution is frozen and vacuum dried to obtain a solid powder final product.

[0122] 15. The purity of the solid powder product was tested by liquid chromatography and the purity was above 95% ( Figure 9 ), which meets the purity requirements for scientific research.

[0123] 15. The solid powder final product is vacuum packed and stored in a freezer for one year.

[0124] 16. After one year of storage, the solid powdered peptide was retested by liquid chromatography and the purity was still above 95% ( Figure 10 ). Example 6

[0125] Taking CCCC (C accounts for 100% of the total number of amino acids in the polypeptide) as an example, the specific implementation steps are as follows:

[0126] 1. A peptide containing four cysteines was synthesized by solid phase chemical synthesis to obtain 402 mg of crude peptide for later use.

[0127] 2. Liquid phase preparation of 402 mg of crude peptide was performed to obtain a peptide preparation solution that met the purity requirements for scientific research (greater than 95%). At this point, the peptide preparation solution was unstable and required rapid processing.

[0128] 3. Weigh 300g of hydrophilic silica aerogel ball filter media and place it into a deep filter. Rinse the filter media with 3-5 times the volume of ethanol reagent and set aside.

[0129] 4. Pass the peptide preparation solution through a deep filter filled with hydrophilic silica aerogel balls as filter material and adjust the filtration speed to 10 ml / min. Collect the filtrate and set aside.

[0130] 5. Weigh 350 g of treated 30-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0131] 6. Pass the collected filtrate through a 30-mesh weakly alkaline (NaOH) cation exchange resin glass column, adjust the filtration speed to 30 ml / min, and collect the polypeptide preparation solution of the first exchange.

[0132] 7. The peptide preparation solution from the first exchange was tested using pH test paper colorimetric method. The pH reagent showed golden yellow, confirming that the preparation solution from the first exchange was a weak acid with a pH value of around 5.5.

[0133] 8. Weigh 300 g of treated 20-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0134] 9. Continue to pass the peptide preparation solution from the first exchange through a 20-mesh weakly alkaline (NaOH) cation exchange resin glass column at a filtration rate of 18 ml / min, and collect the peptide preparation solution from the second exchange.

[0135] 10. Use a pH meter to accurately test the pH value of the peptide preparation solution after the second exchange, which is 6.5, to ensure that the entire peptide environment is treated in a slightly acidic environment after the second exchange.

[0136] 11. Weigh 250g of hydrophobic silica aerogel ball filler into the filter column. Rinse the filter column with 3-5 times the volume of ethanol reagent of the filter medium and set aside.

[0137] 12. First, add ether twice the volume of the preparation solution into the filter column filled with hydrophobic silica aerogel balls. Then, pass the second exchanged peptide preparation solution through the filter column. Apply a liquid phase pump at low pressure to drive the filtration rate at 8 ml / min and collect the filtrate.

[0138] 13. The collected filtrate was tested by ion chromatography, and the trifluoroacetate content in the polypeptide was 2.9%. The ion chromatograph model was (Metrohm 883 Ion Chromatograph, Switzerland).

[0139] 14. The final product polypeptide preparation solution is frozen and vacuum dried to obtain a solid powder final product.

[0140] 15. The purity of the solid powder product was tested by liquid chromatography and the purity was above 95% ( Figure 11 ), which meets the purity requirements for scientific research.

[0141] 15. The solid powder final product is vacuum packed and stored in a freezer for one year.

[0142] 16. After one year of storage, the solid powdered peptide was retested by liquid chromatography and the purity was still above 95% ( Figure 12 ).

[0143] The above is a preferred embodiment of the present invention. The present invention can cover various polypeptide samples containing different proportions of cysteine ​​components. The higher the proportion of cysteine ​​in the polypeptide components, the poorer the polypeptide stability, and the longer the polypeptide storage time, the more significant the method of the present invention. The specific implementation case uses a small amount of research sample as the sample. The amount of each reagent and material involved can be adjusted in the same proportion according to the research requirements. This is also applicable to this patent and should fall within the scope of protection of the present invention.

Claims

1. A method for improving the stability of a solid-phase synthesized polypeptide containing multiple cysteine ​​residues, characterized in that: The steps include: 1) Purifying the multi-cysteine ​​peptide synthesized from solid phase synthesis by liquid chromatography to obtain a preparation solution containing trifluoroacetate; the multi-cysteine ​​peptide is one of the following: GACARQGFGFCCFCYKKC, CKCCCKHICLCFRFMH, PCKCCGLCTCCYCCK, CKCCCLCCNCCYCCKC, CCCCCR, CCCC; 2) The prepared liquid is passed through a deep filter filled with hydrophilic silica aerogel balls as filter material to remove metal ions and trace peroxides contained in the prepared liquid by adsorption; 3) Rapidly pass the prepared solution from which metal ions and trace peroxides have been removed through a weakly alkaline cation exchange resin. When the exchange solution is initially verified to be weakly acidic by a colorimetric assay, stop the rapid exchange and collect the weakly acidic peptide preparation solution for later use. 4) The weakly acidic peptide preparation solution is then slowly passed through the weakly basic cation exchange resin, and the pH value of the preparation solution after the slow exchange is measured in real time using a pH meter. The pH value of the exchange solution is determined to be 6.3-6.5, and the exchange is stopped to obtain a slightly acidic peptide preparation solution. The preparation solution after the exchange contains sodium trifluoroacetate organic salt; 5) The polypeptide preparation solution containing sodium trifluoroacetate organic salt obtained after the secondary exchange is subjected to tangential flow filtration using ether as the sodium trifluoroacetate organic salt removal agent and applying a liquid phase pump at low pressure to drive the filtration speed. The sodium trifluoroacetate organic salt in the preparation solution is removed through a filter column filled with hydrophobic silica aerogel balls to obtain a slightly acidic salt-free preparation solution; 6) Freezing and vacuum drying the prepared solution to obtain a solid powder final product; 7) Vacuum packing and freezing the final solid powder product; 8) The peptide product after storage was retested to ensure that the peptide purity met the scientific research purity.

2. The method for improving the stability of a solid-phase synthesized multi-cysteine ​​polypeptide according to claim 1, wherein: The adsorption filtration speed in step 2 is 10-12 ml / min.

3. The method for improving the stability of a solid-phase synthesized multi-cysteine ​​polypeptide according to claim 1, wherein: The particle size of the weakly basic cation exchange resin in step 3 is 30 mesh NaOH cation exchange resin.

4. The method for improving the stability of a solid-phase synthesized multi-cysteine ​​polypeptide according to claim 1, wherein: The rapid exchange rate in step 3 is 30-33 ml / min.

5. The method for improving the stability of a solid-phase synthesized multi-cysteine ​​polypeptide according to claim 1, wherein: The particle size of the weakly basic cation exchange resin in step 4 is 20 mesh NaOH cation exchange resin.

6. The method for improving the stability of a solid-phase synthesized multi-cysteine ​​polypeptide according to claim 1, wherein: The slow exchange rate in step 4 is 18-20 ml / min.

7. The method for improving the stability of a solid-phase synthesized multi-cysteine ​​polypeptide according to claim 1, wherein: In step 5, the low-pressure driven filtration speed is 8-10 ml / min.

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