Method for improving stability of polypeptide containing multiple cysteines in solid-phase synthesis

Abstract

The invention discloses a method for improving the stability of a polypeptide containing multiple cysteines in solid-phase synthesis, and mainly solves the technical problem of poor stability of the polypeptide caused by easy oxidation, degradation, elimination, denaturation and inactivation of the polypeptide. Comprising the following steps: 1) purifying polypeptides containing multiple cysteines synthesized by a solid phase through liquid chromatography to obtain a preparation solution containing trifluoroacetate; 2) enabling the prepared liquid to pass through a deep filter filled with hydrophilic silicon dioxide aerogel balls as a filter material, and adsorbing and removing metal ions and trace peroxides contained in the prepared liquid; 3) treating the prepared solution with the weakly alkaline cation exchange resin, wherein the pH value is 6.3-6.5; the preparation method comprises the following steps of 1, preparing a sodium trifluoroacetate solution, 2, preparing the sodium trifluoroacetate solution, 4, removing sodium trifluoroacetate in the prepared solution by using a tangential flow filtration method and applying low-pressure driving to obtain a weakly acidic salt-free prepared solution, 5, carrying out freezing and vacuum drying to obtain a solid powder final product, 6, carrying out vacuum packaging and freeze storage, and 7, carrying out purity stability according with scientific research after storage.

Description

Technical Field

[0001] The 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 a molecular formula of C3H7NO2S and a chemical name of (S)-2-amino-3-mercaptopropionic acid. Cysteine ​​is usually present as a white crystalline powder and is odorless. It is easily soluble in water but insoluble in alcohol and ether. Cysteine ​​is an unstable compound that is prone to redox reactions. It is easily oxidized in air and water, and is also easily oxidized to cystine in acidic or alkaline solutions. It is stable at room temperature and pressure, and relatively stable in weakly acidic solutions.

[0003] The stability of peptides containing multiple cysteines synthesized by solid phase has always been poor due to the presence of multiple cysteines. From the entire production process from synthesis to purification, the peptide products will contain metal ions and trace peroxides in the water environment. The presence of metal ions will cause β elimination of the peptide, which will then degrade and make the peptide unstable. The presence of trace peroxides will not only contaminate the product but also cause the peptide to oxidize and cause poor stability. The crude product synthesized by solid phase is cut by a strong acid reagent, and the liquid phase is used to prepare a strong acid mobile phase system. The pure peptide prepared by the liquid phase contains mostly trifluoroacetate in the form of salt. This strong acidic environment causes the thiol groups of the peptides containing multiple cysteines to exchange with each other, forming an incorrect disulfide bond, and the peptide is oxidized and has poor stability. Peptides containing multiple cysteines are also prone to β elimination and degradation in an alkaline environment. In addition, as the storage form, time, temperature, light, air and other conditions change, the peptide will also undergo oxidation, degradation, denaturation, inactivation and other phenomena, all of which will cause the poor stability of the peptides containing multiple cysteines. At this point, it becomes particularly important to ensure the stability of such polypeptides.

[0004] Polypeptides containing multiple cysteines are widely available in the field of polypeptide scientific research, and there are many varieties. As the proportion of cysteine ​​in the polypeptide increases, the stability of the polypeptide becomes worse. Currently, the processing technologies for improving the stability of such polypeptides are mostly vacuum drying and dehydration, vacuum packaging to isolate the air, and low-temperature storage to reduce the influence of temperature and light to ensure the stability of such polypeptides. Alternatively, the polypeptide is desalted to produce a salt-free polypeptide pure product to ensure that the polypeptide is in an acid-free and alkali-free environment to improve the stability of the polypeptide. However, vacuum drying, vacuum packaging and low-temperature storage are only short-term palliative measures, and still do not play a substantial role in the long-term preservation of such polypeptides. Simple desalting technology methods will reduce the solubility of such polypeptides. In order to study the re-dissolution in the future, it is very easy to aggregate or precipitate due to low solubility, causing polypeptide denaturation or even inactivation. The present invention proposes a method for improving the stability of polypeptides containing multiple cysteine, which is easy to operate and fast, and multiple method theories are comprehensively applied, which can not only obtain polypeptide products with higher activity, but also improve the stability of such polypeptides, and ensure that such products can be more stable for subsequent scientific research. Summary of the invention

[0005] The purpose of the present invention is to improve the stability of a solid-phase synthesized polypeptide containing multiple cysteine ​​residues, mainly to solve the technical problems that such polypeptides have poor stability over a long period of time, and that a simple desalting method will reduce the solubility of such polypeptides, easily causing 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: A method for improving the stability of a solid phase synthesized polypeptide containing multiple cysteine ​​residues comprises the following steps: 1) The multi-cysteine ​​peptide synthesized from solid phase is purified by liquid chromatography to obtain a preparation solution containing trifluoroacetate. At this time, the preparation solution is highly acidic and the peptide has poor stability; 2) The prepared liquid is passed through a deep filter filled with hydrophilic silica aerogel balls as filter material to adsorb and remove metal ions and trace peroxides contained in the prepared liquid; 3) The preparation solution after removing metal ions and trace peroxides is quickly passed through a 30-mesh weakly alkaline (NaOH) cation exchange resin. When the exchange solution is preliminarily verified to be weakly acidic by colorimetric method, the rapid exchange is stopped and the weakly acidic polypeptide preparation solution is collected for standby use; 4) The weakly acidic peptide preparation solution is then slowly passed through a 20-mesh weakly alkaline (NaOH) cation exchange resin, and the pH value of the preparation solution after 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 exchange contains sodium trifluoroacetate organic salt; 5) The polypeptide preparation liquid containing sodium trifluoroacetate organic salt obtained after the secondary exchange is filtered by tangential flow filtration, using ether as the sodium trifluoroacetate organic salt removal agent, and applying a liquid phase pump to drive the filtration speed at low pressure, and passing through a filter column filled with hydrophobic silica aerogel balls to remove the sodium trifluoroacetate salt in the preparation liquid to obtain a weakly acidic salt-free preparation liquid; 6) Freezing and vacuum drying the prepared solution to obtain a solid powder final product; 7) Vacuum packing and frozen storage of the solid powder final product; 8) After storage, the peptide product is tested again and the purity of the peptide meets the purity for scientific research.

[0007] The preferred solution is: the adsorption filtration speed in step 2 is 10-12 ml / min.

[0008] The preferred solution is: the particle size of the weakly alkaline (NaOH) cation exchange resin in step 3 is 30 mesh.

[0009] The preferred solution is: the rapid exchange rate in step 3 is 30-33 ml / min.

[0010] The preferred solution is: the particle size of the weakly alkaline (NaOH) cation exchange resin in step 4 is 20 mesh.

[0011] The preferred solution is: the slow exchange rate in step 4 is 18-20 ml / min.

[0012] A preferred solution is: in step 5, the low-pressure driven filtration speed is 8-10 ml / min.

[0013] The beneficial effect of the present invention is that in order to improve the stability of polypeptides containing multiple cysteine ​​residues, the technical solution proposed by the present invention can effectively store such polypeptides for a long time and guarantee the activity of the polypeptides to the maximum extent. First, the metal ions and trace peroxides in the water environment are removed by adsorption. The hydrophilic silica aerogel spheres have high porosity, high specific surface area and good chemical stability. The filtration mechanism is achieved by adsorption, and this step belongs to a physical process. The operation is simple and it is easier to ensure the stability of the polypeptide. Then, ion exchange is carried out with weakly alkaline (NaOH) cation exchange resin. First, a large-particle resin is used for rapid pretreatment to rapidly reduce the acidity of the preparation liquid. Then, fine exchange is carried out with a small-particle resin to control the pH value of the preparation liquid after the exchange to 6.3-6.5, so that the polypeptide product is in a weakly acidic environment, reducing the easy oxidation of the product while ensuring that the polypeptide product still has a certain solubility, thereby once again improving the stability of the polypeptide. After weakly alkaline (NaOH) cation exchange, sodium trifluoroacetate organic salt is produced. The characteristics of cysteine ​​being insoluble in ether and sodium trifluoroacetate being slightly soluble in ether are utilized, and the tangential flow filtration method is adopted to drive slowly at low pressure through the filter column filled with hydrophobic silica aerogel balls. The hydrophobic silica aerogel balls have the characteristics of high strength and high crack resistance, which can not only withstand the pressure drive applied by the liquid phase pump, but also achieve the purpose of removing sodium trifluoroacetate organic salt. Combined with vacuum drying, vacuum packaging, low temperature storage and other technologies, the stability of such polypeptides is improved to achieve the purpose of long-term storage of polypeptides. The hydrophilic silica aerogel balls, hydrophobic silica aerogel balls, and weakly alkaline (NaOH) cation exchange resins introduced in the present invention, the adsorption method and tangential flow filtration method adopted are all to ensure that such polypeptides are processed faster, and the existence environment after treatment is better and more stable. In addition, these chemical materials are easy to obtain and easy to replace, and can cover various research-type polypeptides containing multiple cysteine ​​with high samples, which ensures the stability of the product for subsequent scientific research and improves the accuracy of the research results. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the liquid chromatogram of the product of Example 1.

[0015] Figure 2 This is the liquid chromatogram of the product of Example 1 after being stored for one year.

[0016] Figure 3 It is the liquid chromatogram of the product of Example 2.

[0017] Figure 4 This is the liquid chromatogram of the product of Example 2 after being stored for one year.

[0018] Figure 5 It is the liquid chromatogram of the product of Example 3.

[0019] Figure 6This is the liquid chromatogram of the product of Example 3 after being stored for one year.

[0020] Figure 7 It is the liquid chromatogram of the product of Example 4.

[0021] Figure 8 This is the liquid chromatogram of the product of Example 4 after being stored for one year.

[0022] Fig. 9 It is the liquid chromatogram of the product of Example 5.

[0023] Fig.10 This is the liquid chromatogram of the product of Example 5 after being stored for one year.

[0024] Fig.11 It is the liquid chromatogram of the product of Example 6.

[0025] Fig.12 This is the liquid chromatogram of the product of Example 6 after being stored for one year. DETAILED DESCRIPTION Example 1

[0026] 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: 1. Eighteen amino acids containing five cysteines were synthesized by solid phase chemical synthesis to obtain 485 mg of crude polypeptide for later use.

[0027] 2. Liquid phase preparation was performed on 485 mg of crude peptide to obtain a peptide preparation solution that met the purity requirements for scientific research (greater than 95%). At this time, the peptide preparation solution had poor stability and needed to be processed quickly.

[0028] 3. Weigh 300g of hydrophilic silica aerogel ball filter material and put it into the deep filter. Rinse the filter material with 3-5 times the volume of ethanol reagent and set aside.

[0029] 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 for later use.

[0030] 5. Weigh 350 g of treated 30-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0031] 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 first exchanged polypeptide preparation solution.

[0032] 7. The first exchanged peptide preparation solution was tested using the pH test paper colorimetric method. The pH reagent showed golden yellow, confirming that the first exchanged preparation solution was a weak acid with a pH value of about 5.3.

[0033] 8. Weigh 300 g of treated 20-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0034] 9. Continue to pass the peptide preparation solution of the first exchange through a 20-mesh weakly alkaline (NaOH) cation exchange resin glass column, adjust the filtration speed to 20 ml / min, and collect the peptide preparation solution of the second exchange.

[0035] 10. Use a pH meter to accurately detect the pH value of the second exchange peptide preparation solution, which is 6.5, to ensure that the entire peptide environment is treated in a weakly acidic environment after the second exchange.

[0036] 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, and set aside.

[0037] 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 to 10 ml / min, and collect the filtrate.

[0038] 13. The content of trifluoroacetate in the polypeptide was detected by ion chromatography using the collected filtrate, and the result was 2.8%. The ion chromatography instrument model was (Metrohm 883 ion chromatograph, Switzerland).

[0039] 14. The final product polypeptide preparation solution is frozen and vacuum dried to obtain a solid powder final product.

[0040] 15. The purity of the solid powder final product was tested by liquid chromatography, and the purity reached more than 95% ( Figure 1 ), which meets the purity requirements for scientific research.

[0041] 15. The solid powder final product is vacuum packed and frozen for storage for one year.

[0042] 16. After one year of storage, the solid powder peptide was retested by liquid chromatography and the purity was still above 95% ( Figure 2 ). Example 2

[0043] Taking CKCCCKHICLCFRFMH (C accounts for 37% of the total number of polypeptide amino acids) as an example, the specific implementation steps are as follows: 1. Sixteen amino acids containing six cysteines were synthesized by solid phase chemical synthesis to obtain 478 mg of crude polypeptide for later use.

[0044] 2. Liquid phase preparation was performed on 478 mg of crude peptide to obtain a peptide preparation solution that met the purity requirements for scientific research (greater than 95%). At this time, the peptide preparation solution had poor stability and needed to be processed quickly.

[0045] 3. Weigh 300g of hydrophilic silica aerogel ball filter material and put it into the deep filter. Rinse the filter material with 3-5 times the volume of ethanol reagent and set aside.

[0046] 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 for later use.

[0047] 5. Weigh 350 g of treated 30-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0048] 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 first exchanged polypeptide preparation solution.

[0049] 7. The first exchanged peptide preparation solution was tested using the pH test paper colorimetric method. The pH reagent showed golden yellow, confirming that the first exchanged preparation solution was a weak acid with a pH value of about 5.5.

[0050] 8. Weigh 300 g of treated 20-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0051] 9. Continue to pass the peptide preparation solution of the first exchange through a 20-mesh weakly alkaline (NaOH) cation exchange resin glass column, adjust the filtration speed to 20 ml / min, and collect the peptide preparation solution of the second exchange.

[0052] 10. Use a pH meter to accurately detect the pH value of the second exchange peptide preparation solution, which is 6.4, to ensure that the entire peptide environment is treated in a weakly acidic environment after the second exchange.

[0053] 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, and set aside.

[0054] 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 to 10 ml / min, and collect the filtrate.

[0055] 13. The content of trifluoroacetate in the polypeptide was detected by ion chromatography using the collected filtrate, and the result was 2.9%. The ion chromatograph model was (Metrohm 883 ion chromatograph, Switzerland).

[0056] 14. The final product polypeptide preparation solution is frozen and vacuum dried to obtain a solid powder final product.

[0057] 15. The purity of the solid powder final product was tested by liquid chromatography, and the purity reached more than 95% ( Figure 3 ), which meets the purity requirements for scientific research.

[0058] 15. The solid powder final product is vacuum packed and frozen for storage for one year.

[0059] 16. After one year of storage, the solid powder peptide was retested by liquid chromatography and the purity was still above 95% ( Figure 4 ). Example 3

[0060] Taking PCKCCGLCTCCYCCK (C accounts for 53% of the total number of polypeptide amino acids) as an example, the specific implementation steps are as follows: 1. Fifteen amino acids containing eight cysteines were synthesized by solid phase chemical synthesis to obtain 480 mg of crude polypeptide for later use.

[0061] 2. Perform liquid phase preparation on 480 mg of crude peptide to obtain a peptide preparation solution that meets the purity of scientific research (greater than 95%). At this time, the peptide preparation solution has poor stability and needs to be processed quickly.

[0062] 3. Weigh 300g of hydrophilic silica aerogel ball filter material and put it into the deep filter. Rinse the filter material with 3-5 times the volume of ethanol reagent and set aside.

[0063] 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 11 ml / min. Collect the filtrate for later use.

[0064] 5. Weigh 350 g of treated 30-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0065] 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 first exchanged polypeptide preparation solution.

[0066] 7. The first exchanged peptide preparation solution was tested using the pH test paper colorimetric method. The pH reagent showed golden yellow, confirming that the first exchanged preparation solution was a weak acid with a pH value of about 5.4.

[0067] 8. Weigh 300 g of treated 20-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0068] 9. Continue to pass the peptide preparation solution of the first exchange through a 20-mesh weakly alkaline (NaOH) cation exchange resin glass column, adjust the filtration speed to 20 ml / min, and collect the peptide preparation solution of the second exchange.

[0069] 10. Use a pH meter to accurately detect the pH value of the second exchange peptide preparation solution, which is 6.4, to ensure that the entire peptide environment is treated in a weakly acidic environment after the second exchange.

[0070] 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, and set aside.

[0071] 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 to 10 ml / min, and collect the filtrate.

[0072] 13. The content of trifluoroacetate in the polypeptide was detected by ion chromatography using the collected filtrate, and the result was 2.9%. The ion chromatograph model was (Metrohm 883 ion chromatograph, Switzerland).

[0073] 14. The final product polypeptide preparation solution is frozen and vacuum dried to obtain a solid powder final product.

[0074] 15. The purity of the solid powder final product was tested by liquid chromatography, and the purity reached more than 95% ( Figure 5 ), which meets the purity requirements for scientific research.

[0075] 15. The solid powder final product is vacuum packed and frozen for storage for one year.

[0076] 16. After one year of storage, the solid powder peptide was retested by liquid chromatography and the purity was still above 95% ( Figure 6 ). Example 4

[0077] Taking CKCCCLCCNCCYCCKC (C accounts for 68% of the total number of polypeptide amino acids) as an example, the specific implementation steps are as follows: 1. 11 cysteines and 16 amino acids were synthesized by solid phase chemical synthesis to obtain 453 mg of crude polypeptide for later use.

[0078] 2. Liquid phase preparation was performed on 453 mg of crude peptide to obtain a peptide preparation solution that met the purity requirements for scientific research (greater than 95%). At this time, the peptide preparation solution had poor stability and needed to be processed quickly.

[0079] 3. Weigh 300g of hydrophilic silica aerogel ball filter material and put it into the deep filter. Rinse the filter material with 3-5 times the volume of ethanol reagent and set aside.

[0080] 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 11 ml / min. Collect the filtrate for later use.

[0081] 5. Weigh 350 g of treated 30-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0082] 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 first exchanged polypeptide preparation solution.

[0083] 7. The first exchanged peptide preparation solution was tested using the pH test paper colorimetric method. The pH reagent showed golden yellow, confirming that the first exchanged preparation solution was a weak acid with a pH value of about 5.4.

[0084] 8. Weigh 300 g of treated 20-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0085] 9. Continue to pass the peptide preparation solution of the first exchange through a 20-mesh weakly alkaline (NaOH) cation exchange resin glass column, adjust the filtration speed to 19 ml / min, and collect the peptide preparation solution of the second exchange.

[0086] 10. Use a pH meter to accurately detect the pH value of the second exchange peptide preparation solution, which is 6.3, to ensure that the entire peptide environment is treated in a weakly acidic environment after the second exchange.

[0087] 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, and set aside.

[0088] 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 to 9 ml / min, and collect the filtrate.

[0089] 13. The content of trifluoroacetate in the polypeptide was detected by ion chromatography using the collected filtrate, and the result was 3.0%. The ion chromatograph model was (Metrohm 883 ion chromatograph, Switzerland).

[0090] 14. The final product polypeptide preparation solution is frozen and vacuum dried to obtain a solid powder final product.

[0091] 15. The purity of the solid powder final product was tested by liquid chromatography, and the purity reached more than 95% ( Figure 7 ), which meets the purity requirements for scientific research.

[0092] 15. The solid powder final product is vacuum packed and frozen for storage for one year.

[0093] 16. After one year of storage, the solid powder peptide was retested by liquid chromatography and the purity was still above 95% ( Figure 8 ). Example 5

[0094] Taking CCCCCR (C accounts for 83% of the total number of polypeptide amino acids) as an example, the specific implementation steps are as follows: 1. A 426 mg crude peptide containing five cysteines and six amino acids was obtained through solid phase chemical synthesis and set aside.

[0095] 2. Liquid phase preparation was performed on 426 mg of crude peptide to obtain a peptide preparation solution that met the purity requirements for scientific research (greater than 95%). At this time, the peptide preparation solution had poor stability and needed to be processed quickly.

[0096] 3. Weigh 300g of hydrophilic silica aerogel ball filter material and put it into the deep filter. Rinse the filter material with 3-5 times the volume of ethanol reagent and set aside.

[0097] 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 for later use.

[0098] 5. Weigh 350 g of treated 30-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0099] 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 first exchanged polypeptide preparation solution.

[0100] 7. The first exchanged peptide preparation solution was tested using the pH test paper colorimetric method. The pH reagent showed golden yellow, confirming that the first exchanged preparation solution was a weak acid with a pH value of about 5.5.

[0101] 8. Weigh 300 g of treated 20-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0102] 9. Continue to pass the peptide preparation solution of the first exchange through a 20-mesh weakly alkaline (NaOH) cation exchange resin glass column, adjust the filtration speed to 18 ml / min, and collect the peptide preparation solution of the second exchange.

[0103] 10. Use a pH meter to accurately detect the pH value of the second exchange peptide preparation solution, which is 6.3, to ensure that the entire peptide environment is treated in a weakly acidic environment after the second exchange.

[0104] 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, and set aside.

[0105] 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 to 8 ml / min, and collect the filtrate.

[0106] 13. The content of trifluoroacetate in the polypeptide was detected by ion chromatography using the collected filtrate, and the result was 3.0%. The ion chromatograph model was (Metrohm 883 ion chromatograph, Switzerland).

[0107] 14. The final product polypeptide preparation solution is frozen and vacuum dried to obtain a solid powder final product.

[0108] 15. The purity of the solid powder final product was tested by liquid chromatography, and the purity reached more than 95% ( Fig. 9 ), which meets the purity requirements for scientific research.

[0109] 15. The solid powder final product is vacuum packed and frozen for storage for one year.

[0110] 16. After one year of storage, the solid powder peptide was retested by liquid chromatography and the purity was still above 95% ( Fig.10 ). Example 6

[0111] Taking CCCC (C accounts for 100% of the total number of polypeptide amino acids) as an example, the specific implementation steps are as follows: 1. A peptide containing four cysteines was synthesized by solid phase chemical synthesis to obtain 402 mg of crude peptide for later use.

[0112] 2. Liquid phase preparation was performed on 402 mg of crude peptide to obtain a peptide preparation solution that met the purity requirements for scientific research (greater than 95%). At this time, the peptide preparation solution had poor stability and needed to be processed quickly.

[0113] 3. Weigh 300g of hydrophilic silica aerogel ball filter material and put it into the deep filter. Rinse the filter material with 3-5 times the volume of ethanol reagent and set aside.

[0114] 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 for later use.

[0115] 5. Weigh 350 g of treated 30-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0116] 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 first exchanged polypeptide preparation solution.

[0117] 7. The first exchanged peptide preparation solution was tested using the pH test paper colorimetric method. The pH reagent showed golden yellow, confirming that the first exchanged preparation solution was a weak acid with a pH value of about 5.5.

[0118] 8. Weigh 300 g of treated 20-mesh weakly alkaline (NaOH) cation exchange resin and place it in a glass column.

[0119] 9. Continue to pass the peptide preparation solution of the first exchange through a 20-mesh weakly alkaline (NaOH) cation exchange resin glass column, adjust the filtration speed to 18 ml / min, and collect the peptide preparation solution of the second exchange.

[0120] 10. Use a pH meter to accurately detect the pH value of the second exchange peptide preparation solution, which is 6.5, to ensure that the entire peptide environment is treated in a weakly acidic environment after the second exchange.

[0121] 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, and set aside.

[0122] 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 polypeptide preparation solution through the filter column, apply a liquid phase pump at low pressure to drive the filtration speed to 8 ml / min, and collect the filtrate.

[0123] 13. The content of trifluoroacetate in the polypeptide was detected by ion chromatography using the collected filtrate, and the result was 2.9%. The ion chromatography instrument model was (Metrohm 883 ion chromatograph, Switzerland).

[0124] 14. The final product polypeptide preparation solution is frozen and vacuum dried to obtain a solid powder final product.

[0125] 15. The purity of the solid powder final product was tested by liquid chromatography, and the purity reached more than 95% ( Fig.11 ), which meets the purity requirements for scientific research.

[0126] 15. The solid powder final product is vacuum packed and frozen for storage for one year.

[0127] 16. After one year of storage, the solid powder peptide was retested by liquid chromatography and the purity was still above 95% ( Fig.12 ).

[0128] 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 worse the polypeptide stability, and the longer the polypeptide storage time, the more significant the method of the present invention is. The specific implementation case uses a small amount of research as a sample. The amount of each reagent and material involved can be adjusted in the same proportion according to the research requirements, which is also applicable to this patent and should also belong to 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) Purify the multi-cysteine ​​peptide synthesized from solid phase by liquid chromatography to obtain a preparation solution containing trifluoroacetate; 2) The prepared liquid is passed through a deep filter filled with hydrophilic silica aerogel balls as filter material to adsorb and remove metal ions and trace peroxides contained in the prepared liquid; 3) The preparation solution from which metal ions and trace amounts of peroxides have been removed is rapidly passed through a weakly alkaline cation exchange resin. When the exchange solution is initially verified to be weakly acidic by a colorimetric method, the rapid exchange is stopped and the weakly acidic polypeptide preparation solution is collected for later use; 4) The weakly acidic polypeptide preparation solution is continued to slowly pass through the weakly alkaline cation exchange resin, and the pH value of the preparation solution after the slow exchange is measured in real time with a pH meter, and the pH value of the exchange solution is determined to be 6.3-6.5, and the exchange is stopped to obtain a slightly weakly acidic polypeptide preparation solution. The preparation solution after the exchange contains sodium trifluoroacetate organic salt; 5) The polypeptide preparation liquid containing sodium trifluoroacetate organic salt obtained after the secondary exchange is filtered by 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, and passing through a filter column filled with hydrophobic silica aerogel balls to remove the sodium trifluoroacetate organic salt in the preparation liquid to obtain a weakly acidic salt-free preparation liquid; 6) Freezing and vacuum drying the prepared solution to obtain a solid powder final product; 7) Vacuum packing and frozen storage of the solid powder final product; 8) The peptide product was tested again after storage, and the purity of the peptide was consistent with the purity of scientific research.

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

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