Preparation method of polypeptide vaccine

Abstract

The invention relates to the technical field of vaccine preparation methods, in particular to a polypeptide vaccine preparation method which comprises the following components in percentage by mass: 20-30% of a polypeptide antigen, 10-50% of a carrier, 1-10% of an adjuvant, 1-3% of a stabilizer and 1-2% of a preservative. The method comprises the following steps: step 1, antigen epitope preparation, step 2, epitope verification, step 3, polypeptide synthesis, step 4, polypeptide loading, step 5, efficiency evaluation, step 6, pretreatment, step 7, purification processing, step 8, ultrafiltration processing, step 9, identification, step 10, mixing processing and the like. A stabilizer and a preservative are added in the preparation process of the polypeptide vaccine, the stability and safety of the vaccine in the storage and use process can be kept, impurities can be efficiently removed through the steps of physical precipitation, HPLC purification, ultrafiltration processing and the like, the quality of the vaccine can be ensured, customized production can be conducted according to different pathogens and epitopes, and the method is suitable for popularization and application. The medicine is suitable for preventing and treating various diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine preparation methods, and in particular to a method for preparing a polypeptide vaccine. Background Art

[0002] Peptide vaccine refers to a vaccine prepared through chemical synthesis technology according to the amino acid sequence of a known or predicted antigen epitope in the pathogen antigen gene. Peptide vaccine is mainly used to prevent diseases caused by viral infections, such as influenza virus, herpes virus, etc. It can reduce viral infections to a certain extent and has certain benefits to the body.

[0003] During the preparation process of traditional peptide vaccines, it is impossible to efficiently and multiplex impurities, resulting in low purity of the peptides, affecting the quality of the vaccine. It is also impossible to fully guarantee the stability of the vaccine during storage and use, which can easily lead to vaccine failure. Traditional preparation steps may be cumbersome and time-consuming, resulting in low production efficiency and limited preparation flexibility. It cannot be customized for large-scale rapid production according to different pathogens and antigenic epitopes, further reducing the preparation efficiency of peptide vaccines.

[0004] Therefore, in order to address the problems that the above-mentioned traditional polypeptide vaccines cannot efficiently and multiplexly remove impurities during the preparation process, the preparation is cumbersome and time-consuming, and they cannot be customized for large-scale and rapid production according to different pathogens and antigenic epitopes, a polypeptide vaccine preparation method can be designed that can be mass-produced, is easy to prepare, and can adapt to a variety of polypeptide vaccines. Summary of the invention

[0005] In order to overcome the problems that traditional peptide vaccines cannot efficiently and multiplexly remove impurities, are relatively cumbersome and time-consuming to prepare, and cannot be customized for large-scale rapid production according to different pathogens and antigenic epitopes.

[0006] The technical scheme of the present invention is: a method for preparing a polypeptide vaccine, which comprises the following components by mass percentage: 20-30% polypeptide antigen, 10-50% carrier, 1-10% adjuvant, 1-3% stabilizer and 1-2% preservative, and the preparation steps are as follows;

[0007] Step 1: Antigenic epitope

[0008] Based on the pathogen antigen gene sequence, bioinformatics methods are used to predict and select effective antigen epitopes as candidate sequences for peptide vaccines. More advanced NeoPredPipe and MuPeXI bioinformatics tools and Deep Learning algorithms are introduced, combined with machine learning technology, to more accurately predict and screen antigen epitopes with high immunogenicity and specificity. At the same time, considering the variability and escape mechanism of pathogens, conservative and effective epitopes are selected as candidate sequences, thereby determining the effective ingredients of the vaccine and providing a basis for subsequent synthesis;

[0009] Step 2: Epitope Validation

[0010] After the antigen epitope is selected, an in vitro validation experiment is performed to synthesize candidate epitope peptides and test their ability to bind to specific antibodies or T cell receptors to evaluate their immunogenicity and specificity;

[0011] Step 3: Synthesize peptides

[0012] Start the peptide synthesizer, use solid phase peptide synthesis technology, gradually add amino acids to the resin, and synthesize peptides according to the designed sequence. The pH value in solid phase peptide synthesis is between 7.5 and 9 to ensure that the amino and carboxyl groups of the amino acids are in a proper dissociation state. The catalytic reaction temperature is between 60 and 80 ° C. The solvent can be N, N-dimethylformamide or dichloromethane;

[0013] Step 4: Peptide loading

[0014] Select recombinant virus particles, proteins or liposomes as appropriate carriers, with the carrier accounting for 10-50%. Use a vector expression system to load the polypeptide antigen into the carrier. You can also choose nanoparticles or virus-like particles (VLP) as new carrier materials, while considering the targeting and release characteristics of the carrier to achieve precise delivery and controlled release of polypeptide antigens.

[0015] Step 5: Efficiency Assessment

[0016] After the carrier is loaded with the polypeptide, the binding efficiency is evaluated. The loading amount and binding stability of the polypeptide on the carrier are determined by ELISA detection instrument or Western Blot system detection method. In the antigen epitope verification experiment, the binding ability of the candidate epitope polypeptide with the specific antibody or T cell receptor should reach the predetermined threshold. According to the evaluation results, the ratio and binding conditions of the carrier and polypeptide are adjusted so that the binding efficiency of the carrier and polypeptide should reach more than 90%;

[0017] Step 6: Preprocessing

[0018] The synthesized polypeptide is subjected to a rough extraction operation by physical precipitation to remove unnecessary macromolecules and other impurities;

[0019] Step 7: Purification and processing

[0020] Inject the filtered and degassed mobile phase into the liquid reservoir, rinse the metal filter with the mobile phase, and then immerse the filter in the mobile phase in the liquid reservoir, start the pump, run it for five minutes to eliminate bubbles in the system, run the mobile phase at a fixed rate until the baseline is stable, set the sample operation parameters, inject the solution into the injection valve, start the injection command, and the solution enters the chromatographic column with the mobile phase for separation. Monitor various readings through the software, stop running when the detector detects all peaks of the solution, and gradually change the concentration of the organic solvent in the mobile phase to achieve effective separation of polypeptides and impurities. You can also use more efficient chromatographic separation technology such as affinity chromatography or ion exchange chromatography, combined with mass spectrometry technology for detection, to achieve efficient separation and accurate detection of polypeptides and impurities. The above is a continuous flow production technology, fully automatic mechanized production;

[0021] Step 8: Ultrafiltration Processing

[0022] Carefully collect the purified product of the target peptide from the HPLC chromatogram to ensure that there are no impurities or contaminants mixed in, and use ultrafiltration equipment to further remove small molecule impurities in the solution or adjust the molecular weight distribution of the peptide solution;

[0023] Step 9: Identification

[0024] Use mass spectrometry, nuclear magnetic resonance and other technologies to use mass spectrometers and NMR instruments to identify and verify the structure of the purified peptides to ensure that they meet the design requirements, thereby ensuring the structural correctness and biological activity of the peptides;

[0025] Step 10: Mixing

[0026] The above raw materials are conveyed into a mixer, and 1-10% of adjuvant, 1-3% of stabilizer and 1-2% of preservative are added to improve the immunogenicity and stability of the vaccine;

[0027] Step 11: Preparation

[0028] The prepared polypeptide vaccine is made into a preparation suitable for injection. If it needs to be prepared as a liquid vaccine, a filling machine can be used for filling and packaging. If it needs to be prepared as a freeze-dried powder vaccine, a freeze dryer is used for preparation. It can also be prepared in the form of a microneedle patch or oral capsule to adapt to different vaccination routes and patient needs;

[0029] Step 12: Quality Control

[0030] A series of quality control tests are conducted on the prepared polypeptide vaccine using microbial limit testers, stability test chambers, UV spectrophotometers, etc. to ensure that the quality of the vaccine meets relevant standards and regulations and to ensure the safety and effectiveness of the vaccine.

[0031] Preferably, stabilizers and preservatives are added during the preparation of the polypeptide vaccine, which helps to maintain the stability and safety of the vaccine during storage and use. Through physical precipitation, HPLC purification, ultrafiltration processing and other steps, impurities can be efficiently removed, the purity of the polypeptide can be improved, and the quality of the vaccine can be ensured. At the same time, a suitable vector is selected, and a vector expression system is used to load the polypeptide antigen into the vector, which improves the immunogenicity and stability of the polypeptide. The preparation of the entire polypeptide vaccine mainly relies on chemical synthesis, which makes the production process relatively simple and easy to scale, which can meet the needs of large-scale production, and also makes the polypeptide synthesis process more automated and efficient. It can also be customized according to different pathogens and antigenic epitopes, which is suitable for the prevention and treatment of various diseases, greatly improving the preparation speed of polypeptide vaccines.

[0032] Preferably, the adjuvant can be one or more of interferon, interleukin, tumor necrosis factor, granulocyte-macrophage colony stimulating factor, cytosine-phosphate-guanosine oligodeoxynucleotide, double-stranded RNA, monophosphoryl lipid A, and a complex adjuvant containing aluminum salt and MPL, CpGODN and TLR ligand.

[0033] Preferably, the stabilizer can be one or more of Tween series, poloxamer, ascorbic acid, glutathione and ethylenediaminetetraacetic acid to prepare the liquid vaccine.

[0034] Preferably, the preservative may be one of thimerosal, phenol and 2-phenoxyethanol.

[0035] Preferably, the stabilizer can be one or more of sucrose, trehalose, glycine, arginine, phosphate buffer and Tris buffer to prepare the freeze-dried vaccine.

[0036] Preferably, in step 4, if the polypeptide solution contains small molecule impurities such as salts, low molecular weight organic matter, etc., a dialysis device can be used for preliminary purification.

[0037] Preferably, the mixer in step eight can be a vortex mixer or a magnetic stirrer, and a centrifuge can also be used to remove the generated precipitate.

[0038] Preferably, the rotation speed of the centrifuge ranges from 5000 to 2000 rpm, and the centrifugation time ranges from 8 to 15 minutes.

[0039] Beneficial effects of the present invention:

[0040] 1. Stabilizers and preservatives are added during the preparation of the peptide vaccine, which helps to maintain the stability and safety of the vaccine during storage and use. Physical precipitation, HPLC purification, ultrafiltration processing and other steps can effectively remove impurities, improve the purity of the peptide, and ensure the quality of the vaccine. At the same time, a suitable vector is selected and a vector expression system is used to load the peptide antigen into the vector, which improves the immunogenicity and stability of the peptide. The adjuvant can be CpG Adjuvants with immunomodulatory functions such as ODN and TLR ligands are used to enhance the immunogenicity and stability of the vaccine, while new carrier materials such as nanoparticles or virus-like particles (VLPs) are used, which have better biocompatibility and immunostimulatory ability to achieve precise delivery and controlled release of polypeptide antigens. The preparation of the entire polypeptide vaccine mainly relies on chemical synthesis, which makes the production process relatively simple and easy to scale, and can meet the needs of large-scale production. It also makes the polypeptide synthesis process more automated and efficient, and can also be customized according to different pathogens and antigenic epitopes. It is suitable for the prevention and treatment of various diseases, greatly improving the preparation speed of polypeptide vaccines. Compared with traditional polypeptide vaccines, impurities cannot be removed efficiently and multiple times during the preparation process, and the preparation is cumbersome and time-consuming. It cannot be customized and mass-produced according to different pathogens and antigenic epitopes. This polypeptide vaccine can be mass-produced rapidly. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Shown is a flow chart of the method for preparing the polypeptide vaccine of the present invention. DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0043] See also Figure 1 , the present invention provides an embodiment: a method for preparing a polypeptide vaccine, which comprises the following components, by mass percentage: 20-30% polypeptide antigen, 10-50% carrier, 1-10% adjuvant, 1-3% stabilizer and 1-2% preservative, and the preparation steps are as follows;

[0044] Step 1: Antigenic epitope

[0045] Based on the pathogen antigen gene sequence, bioinformatics methods are used to predict and select effective antigen epitopes as candidate sequences for peptide vaccines. More advanced NeoPredPipe and MuPeXI bioinformatics tools and Deep Learning algorithms are introduced, combined with machine learning technology, to more accurately predict and screen antigen epitopes with high immunogenicity and specificity. At the same time, considering the variability and escape mechanism of pathogens, conservative and effective epitopes are selected as candidate sequences, thereby determining the effective ingredients of the vaccine and providing a basis for subsequent synthesis;

[0046] Step 2: Epitope Validation

[0047] After the antigen epitope is selected, an in vitro validation experiment is performed to synthesize candidate epitope peptides and test their ability to bind to specific antibodies or T cell receptors to evaluate their immunogenicity and specificity;

[0048] Step 3: Synthesize peptides

[0049] Start the peptide synthesizer, use solid phase peptide synthesis technology, gradually add amino acids to the resin, and synthesize peptides according to the designed sequence. The pH value in solid phase peptide synthesis is between 7.5 and 9 to ensure that the amino and carboxyl groups of the amino acids are in a proper dissociation state. The catalytic reaction temperature is between 60 and 80 ° C. The solvent can be N, N-dimethylformamide or dichloromethane;

[0050] Step 4: Peptide loading

[0051] Select recombinant virus particles, proteins or liposomes as appropriate carriers, with the carrier accounting for 10-50%. Use a vector expression system to load the polypeptide antigen into the carrier. You can also choose new carrier materials such as nanoparticles or virus-like particles (VLPs), while considering the targeting and release characteristics of the carrier to achieve precise delivery and controlled release of polypeptide antigens.

[0052] Step 5: Efficiency Assessment

[0053] After the carrier is loaded with the polypeptide, the binding efficiency is evaluated. The loading amount and binding stability of the polypeptide on the carrier are determined by ELISA detection instrument or Western Blot system detection method. In the antigen epitope verification experiment, the binding ability of the candidate epitope polypeptide with the specific antibody or T cell receptor should reach the predetermined threshold. According to the evaluation results, the ratio and binding conditions of the carrier and polypeptide are adjusted so that the binding efficiency of the carrier and polypeptide should reach more than 90%;

[0054] Step 6: Preprocessing

[0055] The synthesized polypeptide is subjected to a rough extraction operation by physical precipitation to remove unnecessary macromolecules and other impurities;

[0056] Step 7: Purification and processing

[0057] Inject the filtered and degassed mobile phase into the liquid reservoir, rinse the metal filter with the mobile phase, and then immerse the filter in the mobile phase in the liquid reservoir, start the pump, run it for five minutes to eliminate bubbles in the system, run the mobile phase at a fixed rate until the baseline is stable, set the sample operation parameters, inject the solution into the injection valve, start the injection command, and the solution enters the chromatographic column with the mobile phase for separation. Monitor various readings through the software, stop running when the detector detects all peaks of the solution, and gradually change the concentration of the organic solvent in the mobile phase to achieve effective separation of polypeptides and impurities. You can also use more efficient chromatographic separation technology such as affinity chromatography or ion exchange chromatography, combined with mass spectrometry technology for detection, to achieve efficient separation and accurate detection of polypeptides and impurities. The above is a continuous flow production technology, fully automatic mechanized production;

[0058] Step 8: Ultrafiltration Processing

[0059] Carefully collect the purified product of the target peptide from the HPLC chromatogram to ensure that there are no impurities or contaminants mixed in, and use ultrafiltration equipment to further remove small molecule impurities in the solution or adjust the molecular weight distribution of the peptide solution;

[0060] Step 9: Identification

[0061] Use mass spectrometry, nuclear magnetic resonance and other technologies to use mass spectrometers and NMR instruments to identify and verify the structure of the purified peptides to ensure that they meet the design requirements, thereby ensuring the structural correctness and biological activity of the peptides;

[0062] Step 10: Mixing

[0063] The above raw materials are conveyed into a mixer, and 1-10% of adjuvant, 1-3% of stabilizer and 1-2% of preservative are added to improve the immunogenicity and stability of the vaccine;

[0064] Step 11: Preparation

[0065] The prepared polypeptide vaccine is made into a preparation suitable for injection. If it needs to be prepared as a liquid vaccine, a filling machine can be used for filling and packaging. If it needs to be prepared as a freeze-dried powder vaccine, a freeze dryer is used for preparation. It can also be prepared in the form of a microneedle patch or oral capsule to adapt to different vaccination routes and patient needs;

[0066] Step 12: Quality Control

[0067] A series of quality control tests are conducted on the prepared polypeptide vaccine using microbial limit testers, stability test chambers, UV spectrophotometers, etc. to ensure that the quality of the vaccine meets relevant standards and regulations and to ensure the safety and effectiveness of the vaccine.

[0068] Example 1

[0069] The present invention provides an embodiment: a method for preparing a polypeptide vaccine, comprising the following components, by mass percentage: 20% of a polypeptide antigen, 10% of a carrier, 1% of an adjuvant, 1% of a stabilizer and 1% of a preservative, wherein the adjuvant may be one or more of interferon, interleukin, tumor necrosis factor, granulocyte-macrophage colony stimulating factor, cytosine-phosphate-guanosine oligodeoxynucleotide, double-stranded RNA, monophosphoryl lipid A, a composite adjuvant comprising aluminum salt and MPL, CpG ODN and TLR ligand, the stabilizer may be one or more of Tween series, poloxamer, ascorbic acid, glutathione and ethylenediaminetetraacetic acid to prepare a liquid vaccine, the preservative may be one of thimerosal, phenol and 2-phenoxyethanol, the stabilizer may be one or more of sucrose, trehalose, glycine, arginine, phosphate buffer and Tris buffer to prepare a freeze-dried powder vaccine, and the preparation steps are as follows;

[0070] Step 1: Antigenic epitope

[0071] Based on the pathogen antigen gene sequence, bioinformatics methods are used to predict and select effective antigen epitopes as candidate sequences for peptide vaccines. More advanced NeoPredPipe and MuPeXI bioinformatics tools and Deep Learning algorithms are introduced, combined with machine learning technology, to more accurately predict and screen antigen epitopes with high immunogenicity and specificity. At the same time, considering the variability and escape mechanism of pathogens, conservative and effective epitopes are selected as candidate sequences, thereby determining the effective ingredients of the vaccine and providing a basis for subsequent synthesis;

[0072] Step 2: Epitope Validation

[0073] After the antigen epitope is selected, an in vitro validation experiment is performed to synthesize candidate epitope peptides and test their ability to bind to specific antibodies or T cell receptors to evaluate their immunogenicity and specificity;

[0074] Step 3: Synthesize peptides

[0075] Start the peptide synthesizer, use solid phase peptide synthesis technology, gradually add amino acids to the resin, and synthesize peptides according to the designed sequence. The pH value in solid phase peptide synthesis is between 7.5 and 9 to ensure that the amino and carboxyl groups of the amino acids are in a proper dissociation state. The catalytic reaction temperature is between 60 and 80 ° C. The solvent can be N, N-dimethylformamide or dichloromethane;

[0076] Step 4: Peptide loading

[0077] Select recombinant virus particles, proteins or liposomes as appropriate carriers, with the carrier accounting for 10-50%. Use a vector expression system to load the polypeptide antigen into the carrier. You can also choose new carrier materials such as nanoparticles or virus-like particles (VLPs), while considering the targeting and release characteristics of the carrier to achieve precise delivery and controlled release of polypeptide antigens.

[0078] Step 5: Efficiency Assessment

[0079] After the carrier is loaded with the polypeptide, the binding efficiency is evaluated. The loading amount and binding stability of the polypeptide on the carrier are determined by ELISA detection instrument or Western Blot system detection method. In the antigen epitope verification experiment, the binding ability of the candidate epitope polypeptide with the specific antibody or T cell receptor should reach the predetermined threshold. According to the evaluation results, the ratio and binding conditions of the carrier and polypeptide are adjusted so that the binding efficiency of the carrier and polypeptide should reach more than 90%;

[0080] Step 6: Preprocessing

[0081] The synthesized polypeptides are subjected to a crude extraction operation by physical precipitation to remove unnecessary macromolecules and other impurities. If the polypeptide solution contains small molecule impurities such as salts and low molecular weight organic matter, a dialysis device can be used for preliminary purification;

[0082] Step 7: Purification and processing

[0083] Inject the filtered and degassed mobile phase into the liquid reservoir, rinse the metal filter with the mobile phase, and then immerse the filter in the mobile phase in the liquid reservoir, start the pump, run it for five minutes to eliminate bubbles in the system, run the mobile phase at a fixed rate until the baseline is stable, set the sample operation parameters, inject the solution into the injection valve, start the injection command, and the solution enters the chromatographic column with the mobile phase for separation. Monitor various readings through the software, stop running when the detector detects all peaks of the solution, and gradually change the concentration of the organic solvent in the mobile phase to achieve effective separation of polypeptides and impurities. You can also use more efficient chromatographic separation technology such as affinity chromatography or ion exchange chromatography, combined with mass spectrometry technology for detection, to achieve efficient separation and accurate detection of polypeptides and impurities. The above is a continuous flow production technology, fully automatic mechanized production;

[0084] Step 8: Ultrafiltration Processing

[0085] Carefully collect the purified product of the target peptide from the HPLC chromatogram to ensure that there are no impurities or contaminants mixed in, and use ultrafiltration equipment to further remove small molecule impurities in the solution or adjust the molecular weight distribution of the peptide solution;

[0086] Step 9: Identification

[0087] Use mass spectrometry, nuclear magnetic resonance and other technologies to use mass spectrometers and NMR instruments to identify and verify the structure of the purified peptides to ensure that they meet the design requirements, thereby ensuring the structural correctness and biological activity of the peptides;

[0088] Step 10: Mixing

[0089] The above raw materials are conveyed to a mixer, and 1% of an adjuvant, 1% of a stabilizer and 1% of a preservative are added to improve the immunogenicity and stability of the vaccine. The mixer can be a vortex mixer or a magnetic stirrer, and a centrifuge can also be used to remove the generated precipitate. The speed range of the centrifuge is between 5000-2000 rpm, and the centrifugation time is between 8-15 minutes;

[0090] Step 11: Preparation

[0091] The prepared polypeptide vaccine is made into a preparation suitable for injection. If it needs to be prepared as a liquid vaccine, a filling machine can be used for filling and packaging. If it needs to be prepared as a freeze-dried powder vaccine, a freeze dryer is used for preparation. It can also be prepared in the form of a microneedle patch or oral capsule to adapt to different vaccination routes and patient needs;

[0092] Step 12: Quality Control

[0093] A series of quality control tests are conducted on the prepared polypeptide vaccine using microbial limit testers, stability test chambers, UV spectrophotometers, etc. to ensure that the quality of the vaccine meets relevant standards and regulations and to ensure the safety and effectiveness of the vaccine.

[0094] Example 2

[0095] The present invention provides an embodiment: a method for preparing a polypeptide vaccine, comprising the following components, by mass percentage: 25% of a polypeptide antigen, 30% of a carrier, 5% of an adjuvant, 2% of a stabilizer and 1.5% of a preservative, wherein the adjuvant may be one or more of interferon, interleukin, tumor necrosis factor, granulocyte-macrophage colony stimulating factor, cytosine-phosphate-guanosine oligodeoxynucleotide, double-stranded RNA, monophosphoryl lipid A, a composite adjuvant comprising aluminum salt and MPL, CpG ODN and TLR ligand, the stabilizer may be one or more of Tween series, poloxamer, ascorbic acid, glutathione and ethylenediaminetetraacetic acid to prepare a liquid vaccine, the preservative may be one of thimerosal, phenol and 2-phenoxyethanol, the stabilizer may be one or more of sucrose, trehalose, glycine, arginine, phosphate buffer and Tris buffer to prepare a freeze-dried powder vaccine, and the preparation steps are as follows;

[0096] Step 1: Antigenic epitope

[0097] Based on the pathogen antigen gene sequence, bioinformatics methods are used to predict and select effective antigen epitopes as candidate sequences for peptide vaccines. More advanced NeoPredPipe and MuPeXI bioinformatics tools and Deep Learning algorithms are introduced, combined with machine learning technology, to more accurately predict and screen antigen epitopes with high immunogenicity and specificity. At the same time, considering the variability and escape mechanism of pathogens, conservative and effective epitopes are selected as candidate sequences, thereby determining the effective ingredients of the vaccine and providing a basis for subsequent synthesis;

[0098] Step 2: Epitope Validation

[0099] After the antigen epitope is selected, an in vitro validation experiment is performed to synthesize candidate epitope peptides and test their ability to bind to specific antibodies or T cell receptors to evaluate their immunogenicity and specificity;

[0100] Step 3: Synthesize peptides

[0101] Start the peptide synthesizer, use solid phase peptide synthesis technology, gradually add amino acids to the resin, and synthesize peptides according to the designed sequence. The pH value in solid phase peptide synthesis is between 7.5 and 9 to ensure that the amino and carboxyl groups of the amino acids are in a proper dissociation state. The catalytic reaction temperature is between 60 and 80 ° C. The solvent can be N, N-dimethylformamide or dichloromethane;

[0102] Step 4: Peptide loading

[0103] Select recombinant virus particles, proteins or liposomes as appropriate carriers, with the carrier accounting for 10-50%. Use a vector expression system to load the polypeptide antigen into the carrier. You can also choose new carrier materials such as nanoparticles or virus-like particles (VLPs), while considering the targeting and release characteristics of the carrier to achieve precise delivery and controlled release of polypeptide antigens.

[0104] Step 5: Efficiency Assessment

[0105] After the carrier is loaded with the polypeptide, the binding efficiency is evaluated. The loading amount and binding stability of the polypeptide on the carrier are determined by ELISA detection instrument or Western Blot system detection method. In the antigen epitope verification experiment, the binding ability of the candidate epitope polypeptide with the specific antibody or T cell receptor should reach the predetermined threshold. According to the evaluation results, the ratio and binding conditions of the carrier and polypeptide are adjusted so that the binding efficiency of the carrier and polypeptide should reach more than 90%;

[0106] Step 6: Preprocessing

[0107] The synthesized polypeptides are subjected to a crude extraction operation by physical precipitation to remove unnecessary macromolecules and other impurities. If the polypeptide solution contains small molecule impurities such as salts and low molecular weight organic matter, a dialysis device can be used for preliminary purification;

[0108] Step 7: Purification and processing

[0109] Inject the filtered and degassed mobile phase into the liquid reservoir, rinse the metal filter with the mobile phase, and then immerse the filter in the mobile phase in the liquid reservoir, start the pump, run it for five minutes to eliminate bubbles in the system, run the mobile phase at a fixed rate until the baseline is stable, set the sample operation parameters, inject the solution into the injection valve, start the injection command, and the solution enters the chromatographic column with the mobile phase for separation. Monitor various readings through the software, stop running when the detector detects all peaks of the solution, and gradually change the concentration of the organic solvent in the mobile phase to achieve effective separation of polypeptides and impurities. You can also use more efficient chromatographic separation technology such as affinity chromatography or ion exchange chromatography, combined with mass spectrometry technology for detection, to achieve efficient separation and accurate detection of polypeptides and impurities. The above is a continuous flow production technology, fully automatic mechanized production;

[0110] Step 8: Ultrafiltration Processing

[0111] Carefully collect the purified product of the target peptide from the HPLC chromatogram to ensure that there are no impurities or contaminants mixed in, and use ultrafiltration equipment to further remove small molecule impurities in the solution or adjust the molecular weight distribution of the peptide solution;

[0112] Step 9: Identification

[0113] Use mass spectrometry, nuclear magnetic resonance and other technologies to use mass spectrometers and NMR instruments to identify and verify the structure of the purified peptides to ensure that they meet the design requirements, thereby ensuring the structural correctness and biological activity of the peptides;

[0114] Step 10: Mixing

[0115] The above raw materials are conveyed to a mixer, and 5% of adjuvant, 2% of stabilizer and 1.5% of preservative are added to improve the immunogenicity and stability of the vaccine. The mixer can be a vortex mixer or a magnetic stirrer, and a centrifuge can also be used to remove the generated precipitate. The speed range of the centrifuge is between 5000-2000rpm, and the centrifugation time is between 8-15 minutes;

[0116] Step 11: Preparation

[0117] The prepared polypeptide vaccine is made into a preparation suitable for injection. If it needs to be prepared as a liquid vaccine, a filling machine can be used for filling and packaging. If it needs to be prepared as a freeze-dried powder vaccine, a freeze dryer is used for preparation. It can also be prepared in the form of a microneedle patch or oral capsule to adapt to different vaccination routes and patient needs;

[0118] Step 12: Quality Control

[0119] A series of quality control tests are conducted on the prepared polypeptide vaccine using microbial limit testers, stability test chambers, UV spectrophotometers, etc. to ensure that the quality of the vaccine meets relevant standards and regulations and to ensure the safety and effectiveness of the vaccine.

[0120] Example 3

[0121] The present invention provides an embodiment: a method for preparing a polypeptide vaccine, comprising the following components, by mass percentage: 30% of a polypeptide antigen, 50% of a carrier, 10% of an adjuvant, 3% of a stabilizer and 2% of a preservative, wherein the adjuvant may be one or more of interferon, interleukin, tumor necrosis factor, granulocyte-macrophage colony stimulating factor, cytosine-phosphate-guanosine oligodeoxynucleotide, double-stranded RNA, monophosphoryl lipid A, a composite adjuvant comprising aluminum salt and MPL, CpG ODN and TLR ligand, the stabilizer may be one or more of Tween series, poloxamer, ascorbic acid, glutathione and ethylenediaminetetraacetic acid to prepare a liquid vaccine, the preservative may be one of thimerosal, phenol and 2-phenoxyethanol, the stabilizer may be one or more of sucrose, trehalose, glycine, arginine, phosphate buffer and Tris buffer to prepare a freeze-dried powder vaccine, and the preparation steps are as follows;

[0122] Step 1 Epitope

[0123] Based on the pathogen antigen gene sequence, bioinformatics methods are used to predict and select effective antigen epitopes as candidate sequences for peptide vaccines. More advanced NeoPredPipe and MuPeXI bioinformatics tools and Deep Learning algorithms are introduced, combined with machine learning technology, to more accurately predict and screen antigen epitopes with high immunogenicity and specificity. At the same time, considering the variability and escape mechanism of pathogens, conservative and effective epitopes are selected as candidate sequences, thereby determining the effective ingredients of the vaccine and providing a basis for subsequent synthesis;

[0124] Step 2: Epitope Validation

[0125] After the antigen epitope is selected, an in vitro validation experiment is performed to synthesize candidate epitope peptides and test their ability to bind to specific antibodies or T cell receptors to evaluate their immunogenicity and specificity;

[0126] Step 3: Synthesize peptides

[0127] Start the peptide synthesizer, use solid phase peptide synthesis technology, gradually add amino acids to the resin, and synthesize peptides according to the designed sequence. The pH value in solid phase peptide synthesis is between 7.5 and 9 to ensure that the amino and carboxyl groups of the amino acids are in a proper dissociation state. The catalytic reaction temperature is between 60 and 80 ° C. The solvent can be N, N-dimethylformamide or dichloromethane;

[0128] Step 4: Peptide loading

[0129] Select recombinant virus particles, proteins or liposomes as appropriate carriers, with the carrier accounting for 10-50%. Use a vector expression system to load the polypeptide antigen into the carrier. You can also choose new carrier materials such as nanoparticles or virus-like particles (VLPs), while considering the targeting and release characteristics of the carrier to achieve precise delivery and controlled release of polypeptide antigens.

[0130] Step 5: Efficiency Assessment

[0131] After the carrier is loaded with the polypeptide, the binding efficiency is evaluated. The loading amount and binding stability of the polypeptide on the carrier are determined by ELISA detection instrument or Western Blot system detection method. In the antigen epitope verification experiment, the binding ability of the candidate epitope polypeptide with the specific antibody or T cell receptor should reach the predetermined threshold. According to the evaluation results, the ratio and binding conditions of the carrier and polypeptide are adjusted so that the binding efficiency of the carrier and polypeptide should reach more than 90%;

[0132] Step 6: Preprocessing

[0133] The synthesized polypeptides are subjected to a crude extraction operation by physical precipitation to remove unnecessary macromolecules and other impurities. If the polypeptide solution contains small molecule impurities such as salts and low molecular weight organic matter, a dialysis device can be used for preliminary purification;

[0134] Step 7: Purification and processing

[0135] Inject the filtered and degassed mobile phase into the liquid reservoir, rinse the metal filter with the mobile phase, and then immerse the filter in the mobile phase in the liquid reservoir, start the pump, run it for five minutes to eliminate bubbles in the system, run the mobile phase at a fixed rate until the baseline is stable, set the sample operation parameters, inject the solution into the injection valve, start the injection command, and the solution enters the chromatographic column with the mobile phase for separation. Monitor various readings through the software, stop running when the detector detects all peaks of the solution, and gradually change the concentration of the organic solvent in the mobile phase to achieve effective separation of polypeptides and impurities. You can also use more efficient chromatographic separation technology such as affinity chromatography or ion exchange chromatography, combined with mass spectrometry technology for detection, to achieve efficient separation and accurate detection of polypeptides and impurities. The above is a continuous flow production technology, fully automatic mechanized production;

[0136] Step 8: Ultrafiltration Processing

[0137] Carefully collect the purified product of the target peptide from the HPLC chromatogram to ensure that there are no impurities or contaminants mixed in, and use ultrafiltration equipment to further remove small molecule impurities in the solution or adjust the molecular weight distribution of the peptide solution;

[0138] Step 9: Identification

[0139] Use mass spectrometry, nuclear magnetic resonance and other technologies to use mass spectrometers and NMR instruments to identify and verify the structure of the purified peptides to ensure that they meet the design requirements, thereby ensuring the structural correctness and biological activity of the peptides;

[0140] Step 10: Mixing

[0141] The above raw materials are conveyed to a mixer, and 10% of adjuvant, 3% of stabilizer and 2% of preservative are added to improve the immunogenicity and stability of the vaccine. The mixer can be a vortex mixer or a magnetic stirrer, and a centrifuge can also be used to remove the generated precipitate. The speed range of the centrifuge is between 5000-2000rpm, and the centrifugation time is between 8-15 minutes;

[0142] Step 11: Preparation

[0143] The prepared polypeptide vaccine is made into a preparation suitable for injection. If it needs to be prepared as a liquid vaccine, a filling machine can be used for filling and packaging. If it needs to be prepared as a freeze-dried powder vaccine, a freeze dryer is used for preparation. It can also be prepared in the form of a microneedle patch or oral capsule to adapt to different vaccination routes and patient needs;

[0144] Step 12: Quality Control

[0145] A series of quality control tests are conducted on the prepared polypeptide vaccine using microbial limit testers, stability test chambers, UV spectrophotometers, etc. to ensure that the quality of the vaccine meets relevant standards and regulations and to ensure the safety and effectiveness of the vaccine.

[0146] Through the above steps, stabilizers and preservatives are added during the preparation of the polypeptide vaccine, which helps to maintain the stability and safety of the vaccine during storage and use. Through physical precipitation, HPLC purification, ultrafiltration processing and other steps, impurities can be efficiently removed and the purity of the polypeptide can be improved. It can be customized according to different pathogens and antigenic epitopes and is suitable for the prevention and treatment of various diseases. The preparation of the entire polypeptide vaccine mainly relies on chemical synthesis, which makes the production process relatively simple and easy to scale, and can meet the needs of large-scale production, so as to solve the problems that traditional polypeptide vaccines cannot efficiently and multiplexly remove impurities, and the preparation is relatively cumbersome and time-consuming, and cannot be customized according to different pathogens and antigenic epitopes for large-scale rapid production.

[0147] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of those skilled in the art without departing from the spirit of the present invention.

Claims

1. A method for preparing a polypeptide vaccine, It is characterized in that The composition comprises the following components by mass percentage: 20-30% of polypeptide antigen, 10-50% of carrier, 1-10% of adjuvant, 1-3% of stabilizer and 1-2% of preservative, and the preparation steps are as follows; Step 1: Antigenic epitope Based on the pathogen antigen gene sequence, bioinformatics methods are used to predict and select effective antigen epitopes as candidate sequences for peptide vaccines. More advanced NeoPredPipe and MuPeXI bioinformatics tools and Deep Learning algorithms are introduced, combined with machine learning technology, to more accurately predict and screen antigen epitopes with high immunogenicity and specificity. At the same time, considering the variability and escape mechanism of pathogens, conservative and effective epitopes are selected as candidate sequences, thereby determining the effective ingredients of the vaccine and providing a basis for subsequent synthesis; Step 2: Epitope Validation After the antigen epitope is selected, an in vitro validation experiment is conducted to synthesize candidate epitope peptides and test their ability to bind to specific antibodies or T cell receptors to evaluate their immunogenicity and specificity; Step 3: Synthesize peptides Start the peptide synthesizer, use solid phase peptide synthesis technology, gradually add amino acids to the resin, and synthesize peptides according to the designed sequence. The pH value in solid phase peptide synthesis is between 7.5 and 9 to ensure that the amino and carboxyl groups of the amino acids are in a proper dissociation state. The catalytic reaction temperature is between 60 and 80 ° C. The solvent can be N, N-dimethylformamide or dichloromethane; Step 4: Peptide loading Select recombinant virus particles, proteins or liposomes as appropriate carriers, with the carrier accounting for 10-50%. Use a vector expression system to load the polypeptide antigen into the carrier. You can also choose new carrier materials such as nanoparticles or virus-like particles (VLPs), while considering the targeting and release characteristics of the carrier to achieve precise delivery and controlled release of polypeptide antigens. Step 5: Efficiency Assessment After the carrier is loaded with the polypeptide, the binding efficiency is evaluated. The loading amount and binding stability of the polypeptide on the carrier are determined by ELISA detection instrument or Western Blot system detection method. In the antigen epitope verification experiment, the binding ability of the candidate epitope polypeptide with the specific antibody or T cell receptor should reach the predetermined threshold. According to the evaluation results, the ratio and binding conditions of the carrier and polypeptide are adjusted so that the binding efficiency of the carrier and polypeptide should reach more than 90%; Step 6: Preprocessing The synthesized polypeptide is subjected to a rough extraction operation by physical precipitation to remove unnecessary macromolecules and other impurities; Step 7: Purification and processing Inject the filtered and degassed mobile phase into the liquid reservoir, rinse the metal filter with the mobile phase, and then immerse the filter in the mobile phase in the liquid reservoir, start the pump, run it for five minutes to eliminate bubbles in the system, run the mobile phase at a fixed rate until the baseline is stable, set the sample operation parameters, inject the solution into the injection valve, start the injection command, and the solution enters the chromatographic column with the mobile phase for separation. Monitor various readings through the software, stop running when the detector detects all peaks of the solution, and gradually change the concentration of the organic solvent in the mobile phase to achieve effective separation of polypeptides and impurities. You can also use more efficient chromatographic separation technology such as affinity chromatography or ion exchange chromatography, combined with mass spectrometry technology for detection, to achieve efficient separation and accurate detection of polypeptides and impurities. The above is a continuous flow production technology, fully automatic mechanized production; Step 8: Ultrafiltration Processing Carefully collect the purified product of the target peptide from the HPLC chromatogram to ensure that there are no impurities or contaminants mixed in, and use ultrafiltration equipment to further remove small molecule impurities in the solution or adjust the molecular weight distribution of the peptide solution; Step 9: Identification Use mass spectrometry, nuclear magnetic resonance and other technologies to use mass spectrometers and NMR instruments to identify and verify the structure of the purified peptides to ensure that they meet the design requirements, thereby ensuring the structural correctness and biological activity of the peptides; Step 10: Mixing The above raw materials are conveyed into a mixer, and 1-10% of adjuvant, 1-3% of stabilizer and 1-2% of preservative are added to improve the immunogenicity and stability of the vaccine; Step 11: Preparation The prepared polypeptide vaccine is made into a preparation suitable for injection. If it needs to be prepared as a liquid vaccine, a filling machine can be used for filling and packaging. If it needs to be prepared as a freeze-dried powder vaccine, a freeze dryer is used for preparation. It can also be prepared in the form of a microneedle patch or oral capsule to adapt to different vaccination routes and patient needs; Step 12: Quality Control A series of quality control tests are conducted on the prepared polypeptide vaccine using microbial limit testers, stability test chambers, UV spectrophotometers, etc. to ensure that the quality of the vaccine meets relevant standards and regulations and to ensure the safety and effectiveness of the vaccine.

2. The method for preparing a polypeptide vaccine according to claim 1, Features: The adjuvant may be one or more of interferon, interleukin, tumor necrosis factor, granulocyte-macrophage colony stimulating factor, cytosine-phosphate-guanosine oligodeoxynucleotide, double-stranded RNA, monophosphoryl lipid A, composite adjuvant containing aluminum salt and MPL, CpG ODN and TLR ligand.

3. The method for preparing a polypeptide vaccine according to claim 1, Features: The stabilizer can be one or more of Tween series, poloxamer, ascorbic acid, glutathione and ethylenediaminetetraacetic acid to prepare the liquid vaccine.

4. The method for preparing a polypeptide vaccine according to claim 1, Features: The preservative may be one of thimerosal, phenol and 2-phenoxyethanol.

5. The method for preparing a polypeptide vaccine according to claim 1, Features: The stabilizer can be one or more of sucrose, trehalose, glycine, arginine, phosphate buffer and Tris buffer to prepare the freeze-dried vaccine.

6. The method for preparing a polypeptide vaccine according to claim 1, Features: In step 4, if the polypeptide solution contains small molecule impurities such as salts and low molecular weight organic matter, a dialysis device can be used for preliminary purification.

7. The method for preparing a polypeptide vaccine according to claim 1, Features: In step eight, the mixer may be a vortex mixer or a magnetic stirrer, and a centrifuge may also be used to remove the generated precipitate.

8. The method for preparing a polypeptide vaccine according to claim 6, Features: The speed range of the centrifuge is between 5000-2000rpm, and the centrifugation time is between 8-15 minutes.

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