Preparation method and application of anti-human papilloma virus IgY antibody
By using a stepwise immunization process involving HPV L1 virus-like particles and Freund's adjuvant, and a multi-step chromatographic purification technique, high-titer, high-purity anti-HPV IgY antibodies were prepared. This solved the problems of unstable antibody titers and high impurity content in existing technologies, achieving broad-spectrum cross-protection and improved drug safety.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG ZHONGDA GENE TECHNOLOGY CO LTD
- Filing Date
- 2026-02-04
- Publication Date
- 2026-05-12
AI Technical Summary
In the existing technology, the immunogen design of anti-human papillomavirus IgY antibodies is difficult to induce broad-spectrum cross-protection, poultry immunization programs lack uniformity of response capability, and traditional extraction processes result in unstable antibody titers and high impurity content, affecting their safety and effectiveness in clinical applications.
A stepwise immunization process using HPV L1 virus-like particles and Freund's adjuvant, combined with multi-step chromatographic purification techniques including ultrafiltration, affinity chromatography, ion exchange, and size exclusion chromatography, was employed to prepare high-titer, high-purity anti-HPV IgY antibodies.
The induction of high-titer neutralizing antibodies with broad-spectrum cross-protection was achieved, antibody titer stability was improved, impurities were controlled at low levels, and the biological activity of the antibody and the safety of drug use were guaranteed.
Smart Images
Figure CN122011168A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of IgY antibody technology, specifically to a method for preparing and applying an anti-human papillomavirus IgY antibody. Background Technology
[0002] Human papillomavirus (HPV) infection is a major cause of various diseases, including cervical cancer and genital warts, with a persistently high global incidence rate, posing a serious threat to public health. Current prevention and treatment methods include prophylactic vaccines and topical physical / chemical therapies. However, existing prophylactic vaccines cannot cover all high-risk types and have limited effectiveness in already infected individuals. Topical medications such as interferon have limitations, including unstable efficacy, potential side effects, and high costs. In recent years, immunizing laying hens with specific antigens and extracting high-titer specific antibodies (IgY) from their egg yolks has emerged as a novel passive immunization strategy, showing potential in virus control due to its high safety, ease of large-scale production, and ability to target specific pathogens. However, the translation of this technology into HPV prevention and control still faces a series of challenges. Key bottlenecks include: First, in terms of immunogen design, while using L1 virus-like particles (VLPs) alone can induce type-specific antibodies, it is difficult to generate broad cross-protection covering multiple types. Existing technologies lack a multi-target immunogen design and screening system that can efficiently induce broad-spectrum, high-affinity neutralizing antibodies. Second, in terms of production processes, traditional avian immunization protocols are highly arbitrary and lack scientific screening and stepwise enhancement procedures based on animal immune response capabilities, resulting in large fluctuations in antibody titers and unstable yields. At the same time, traditional antibody extraction and purification processes (such as simple water extraction and salting out) are relatively crude, resulting in insufficient purity, specificity, and biological activity (such as neutralizing titer) of the obtained IgY, and high levels of impurities such as endotoxins, which seriously affect the safety and efficacy of subsequent formulation development and clinical applications.
[0003] Therefore, we have made improvements to this and proposed a method for preparing and applying an anti-human papillomavirus IgY antibody. Summary of the Invention
[0004] The purpose of this invention is to provide a method for preparing and applying an anti-human papillomavirus IgY antibody, so as to solve the problems mentioned in the background art.
[0005] To achieve the above objectives, the present invention provides the following technical solution: Includes the following steps: S1. Preparation of immunogen: The human papillomavirus (HPV) antigen is subjected to a first emulsification treatment with Freund's complete adjuvant to obtain the primary immunogen; the HPV antigen is subjected to a second emulsification treatment with Freund's incomplete adjuvant to obtain the enhanced immunogen; S2, Stepwise Immunization: Primary immunization: The primary immunizing agent is injected into laying hens via multiple injection sites. The injection dose per hen is 0.8-1.2 ml, and the number of injection sites is 20-80, including subcutaneous tissue and muscle tissue. First booster immunization: On days 12-16 after the initial immunization, the booster immunizer is injected into the same laying hen via multiple injection sites. The injection dose per hen is 0.8-1.2 ml, and the number of injection sites is 15-60. Series of booster immunizations: After the first booster immunization, repeat the above booster immunization every 12-16 days, for a total of 2-4 times; Maintenance immunization: After completing a series of booster immunizations, lay hens should be given a booster immunization every 6-10 weeks using the aforementioned booster immunogen, and the maintenance immunization period should be no less than 24 weeks; S3. Collection and pretreatment of hyperimmune eggs: Starting from 18-25 days after the initial immunization, collect eggs laid by immunized hens, disinfect the surface, and take the yolk liquid for later use. S4. Preliminary extraction of antibodies: Mix the egg yolk liquid and diluent at a volume ratio of 1:4 to 1:15, stir evenly, adjust the pH of the mixture to 4.8-5.5 with acid, let it stand at 2-8℃ for 6-24 hours, and then centrifuge at 2-8℃ and 8000-15000×g for 10-30 minutes to collect the first supernatant; S5. Preliminary concentration and purification of antibodies: The first supernatant is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 30kDa to 300kDa to concentrate it 5-30 times, and then replaced with neutral buffer to obtain concentrated antibody solution. S6. Fine purification of the antibody: The concentrated antibody solution is purified sequentially by passing it through at least one chromatography column, wherein the chromatography column is selected from affinity chromatography column, ion exchange chromatography column and size exclusion chromatography column; S7. Antibody formulation: The finely purified antibody solution was filtered through 0.45μm and 0.22μm filter membranes for sterilization, then stabilizers and preservatives were added, and the pH was adjusted to 6.5-7.8.
[0006] As a preferred technical solution of this application, the HPV antigen in step S1 is selected from: HPV virus-like particles, recombinant expressed HPVL1 protein, recombinant expressed HPVE6 protein, recombinant expressed HPVE7 protein, or immunogenic fragments of proteins; the first emulsification treatment and the second emulsification treatment adopt the dual-syringe injection method or the high-speed shear emulsification method until a stable emulsion that does not diffuse when dropped into water is formed.
[0007] As a preferred technical solution of this application, step S2 further includes an immune monitoring step: before each immunization and 7-10 days after immunization, serum or egg yolk samples are collected from immunized chickens, and the titer of anti-HPV specific antibodies is determined by enzyme-linked immunosorbent assay (ELISA); the timing of subsequent booster immunizations is dynamically adjusted based on the antibody titer change curve; when the antibody titer increases to a plateau, the next booster immunization is implemented.
[0008] As a preferred technical solution of this application, the diluent in step S4 is deionized water, phosphate buffer, Tris-HCl buffer, or acetate-sodium acetate buffer; the acid solution is a 0.5-2.0N hydrochloric acid solution, acetic acid solution, or citric acid solution.
[0009] As a preferred technical solution of this application, the neutral buffer in step S5 is a phosphate buffer or Tris-HCl buffer with a pH of 7.0-7.5 and a concentration of 10-100mM; the ultrafiltration process is carried out at 2-8℃ and adopts tangential flow filtration mode.
[0010] As a preferred technical solution of this application, the fine purification in step S6 adopts one of the following three chromatographic pathways: Path 1: The concentrated antibody solution flows sequentially through an affinity chromatography column and a size exclusion chromatography column; Path 2: The concentrated antibody solution flows sequentially through an ion exchange chromatography column and a size exclusion chromatography column; Path 3: The concentrated antibody solution flows sequentially through an affinity chromatography column, an ion exchange chromatography column, and a size exclusion chromatography column.
[0011] As a preferred technical solution of this application, when the affinity chromatography column is used, its ligand is recombinant protein G, recombinant protein L or egg yolk immunoglobulin specific ligand; affinity elution is performed using glycine-hydrochloric acid buffer at pH 2.5-3.5, and the elution is immediately neutralized with Tris buffer at pH 8.0-9.0.
[0012] As a preferred technical solution of this application, when the ion exchange chromatography column is used, it is an anion exchange chromatography column and the medium is a quaternary ammonium salt type; the elution is carried out using a buffer solution containing 0-1.0M sodium chloride at pH 7.0-8.5 for linear or stepwise gradient elution.
[0013] As a preferred technical solution of this application, when the size exclusion chromatography column is used, its separation range is 10-1000kDa; the eluent is a phosphate or Tris buffer with pH 6.5-7.5 containing 100-200mM sodium chloride, and the flow rate is 0.5-1.5ml / min.
[0014] As a preferred technical solution of this application, in step S7, the stabilizer is selected from one or more of trehalose, sucrose, mannitol, glycerol and human serum albumin, and its mass-volume concentration in the final antibody solution is 0.5%-10%; the preservative is selected from one or more of thimerosal, phenol, m-cresol and chlorhexidine gluconate, and its mass-volume concentration in the final antibody solution is 0.01%-0.5%.
[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. By adopting the "HPV L1 virus-like particle (VLP)" immunogen strategy, the cross-protective potential of the high immunogenicity of L1 VLP is fully utilized. Combined with a highly efficient adjuvant system, it effectively induces poultry to produce high-titer neutralizing antibodies with broad cross-reactivity. This overcomes the shortcomings of single antigen targeting limited types and narrow protection range, and provides a more comprehensive antibody resource for dealing with multi-type HPV infection.
[0016] 2. Pre-immune screening ensured the uniformity of animal matrix response; the stepwise immunization procedure effectively maintained a consistently high and stable antibody titer; combined with multi-step chromatographic purification techniques such as polyethylene glycol / sodium alginate selective precipitation, affinity chromatography, and ion exchange, the purity, monomer ratio, and in vitro neutralization titer of anti-HPV specific IgY were significantly improved, while key impurities such as endotoxins were controlled at extremely low levels, comprehensively ensuring the product's bioactivity, stability, and drug safety. Attached Figure Description
[0017] Figure 1 This is a schematic diagram of the overall structure of the present invention. Detailed Implementation
[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0019] This invention provides a technical solution: such as Figure 1 The preparation method and application of an anti-human papillomavirus IgY antibody shown herein include the following steps: S1. Preparation of immunogen: The human papillomavirus (HPV) antigen is subjected to a first emulsification treatment with Freund's complete adjuvant to obtain the primary immunogen; the HPV antigen is subjected to a second emulsification treatment with Freund's incomplete adjuvant to obtain the enhanced immunogen; S2, Stepwise Immunization: Primary immunization: The primary immunizing agent is injected into laying hens via multiple injection sites. The injection dose per hen is 0.8-1.2 ml, and the number of injection sites is 20-80, including subcutaneous tissue and muscle tissue. First booster immunization: On days 12-16 after the initial immunization, the booster immunizer is injected into the same laying hen via multiple injection sites. The injection dose per hen is 0.8-1.2 ml, and the number of injection sites is 15-60. Series of booster immunizations: After the first booster immunization, repeat the above booster immunization every 12-16 days, for a total of 2-4 times; Maintenance immunization: After completing a series of booster immunizations, lay hens should be given a booster immunization every 6-10 weeks using the aforementioned booster immunogen, and the maintenance immunization period should be no less than 24 weeks; S3. Collection and pretreatment of hyperimmune eggs: Starting from 18-25 days after the initial immunization, collect eggs laid by immunized hens, disinfect the surface, and take the yolk liquid for later use. S4. Preliminary extraction of antibodies: Mix the egg yolk liquid and diluent at a volume ratio of 1:4 to 1:15, stir evenly, adjust the pH of the mixture to 4.8-5.5 with acid, let it stand at 2-8℃ for 6-24 hours, and then centrifuge at 2-8℃ and 8000-15000×g for 10-30 minutes to collect the first supernatant; S5. Preliminary concentration and purification of antibodies: The first supernatant is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 30kDa to 300kDa to concentrate it 5-30 times, and then replaced with neutral buffer to obtain concentrated antibody solution. S6. Fine purification of the antibody: The concentrated antibody solution is purified sequentially by passing it through at least one chromatography column, wherein the chromatography column is selected from affinity chromatography column, ion exchange chromatography column and size exclusion chromatography column; S7. Antibody formulation: The finely purified antibody solution was filtered through 0.45μm and 0.22μm filter membranes for sterilization, then stabilizers and preservatives were added, and the pH was adjusted to 6.5-7.8.
[0020] Furthermore, in step S1, the HPV antigen is selected from: HPV virus-like particles, recombinant expressed HPVL1 protein, recombinant expressed HPVE6 protein, recombinant expressed HPVE7 protein, or immunogenic fragments of proteins; the first emulsification treatment and the second emulsification treatment are performed using a dual-syringe do-push injection method or a high-speed shear emulsification method until a stable emulsion that does not diffuse when dropped into water is formed.
[0021] Furthermore, step S2 also includes an immune monitoring step: before each immunization and 7-10 days after immunization, serum or egg yolk samples are collected from immunized chickens, and the titer of anti-HPV specific antibodies is determined by enzyme-linked immunosorbent assay (ELISA); the timing of subsequent booster immunizations is dynamically adjusted based on the antibody titer change curve; when the antibody titer increases to a plateau, the next booster immunization is implemented.
[0022] Furthermore, in step S4, the diluent is deionized water, phosphate buffer, Tris-HCl buffer, or acetate-sodium acetate buffer; the acid is a 0.5-2.0N hydrochloric acid solution, acetic acid solution, or citric acid solution.
[0023] Furthermore, in step S5, the neutral buffer is a phosphate buffer or Tris-HCl buffer with a pH of 7.0-7.5 and a concentration of 10-100 mM; the ultrafiltration process is carried out at 2-8°C using a tangential flow filtration mode.
[0024] Furthermore, the fine purification in step S6 employs one of the following three chromatographic pathways: Path 1: The concentrated antibody solution flows sequentially through an affinity chromatography column and a size exclusion chromatography column; Path 2: The concentrated antibody solution flows sequentially through an ion exchange chromatography column and a size exclusion chromatography column; Path 3: The concentrated antibody solution flows sequentially through an affinity chromatography column, an ion exchange chromatography column, and a size exclusion chromatography column.
[0025] Furthermore, when using the affinity chromatography column, its ligand is recombinant protein G, recombinant protein L, or egg yolk immunoglobulin-specific ligand; affinity elution is performed using glycine-hydrochloric acid buffer at pH 2.5-3.5, followed immediately by neutralization with Tris buffer at pH 8.0-9.0.
[0026] Furthermore, when the ion exchange chromatography column is used, it is an anion exchange chromatography column, and the medium is a quaternary ammonium salt type; elution is performed using a buffer solution containing 0-1.0M sodium chloride at pH 7.0-8.5 for linear or stepwise gradient elution.
[0027] Furthermore, when using the size exclusion chromatography column, the separation range is 10-1000 kDa; the eluent is a phosphate or Tris buffer with pH 6.5-7.5 containing 100-200 mM sodium chloride, and the flow rate is 0.5-1.5 ml / min.
[0028] Furthermore, in step S7, the stabilizer is selected from one or more of trehalose, sucrose, mannitol, glycerol, and human serum albumin, and its mass-volume concentration in the final antibody solution is 0.5%-10%; the preservative is selected from one or more of thimerosal, phenol, m-cresol, and chlorhexidine gluconate, and its mass-volume concentration in the final antibody solution is 0.01%-0.5%.
[0029] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.
[0030] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A method for preparing and applying an anti-human papillomavirus IgY antibody, characterized in that, Includes the following steps: S1. Preparation of immunogen: The human papillomavirus antigen is subjected to a first emulsification treatment with Freund's complete adjuvant to obtain the primary immunogen; the HPV antigen is subjected to a second emulsification treatment with Freund's incomplete adjuvant to obtain the enhanced immunogen. S2, Stepwise Immunization: Primary immunization: The primary immunizing agent is injected into laying hens via multiple injection sites. The injection dose per hen is 0.8-1.2 ml, and the number of injection sites is 20-80, including subcutaneous tissue and muscle tissue. First booster immunization: On days 12-16 after the initial immunization, the booster immunizer is injected into the same laying hen via multiple injection sites. The injection dose per hen is 0.8-1.2 ml, and the number of injection sites is 15-60. Series of booster immunizations: After the first booster immunization, repeat the above booster immunization every 12-16 days, for a total of 2-4 times; Maintenance immunization: After completing a series of booster immunizations, lay hens should be given a booster immunization every 6-10 weeks using the aforementioned booster immunogen, and the maintenance immunization period should be no less than 24 weeks; S3. Collection and pretreatment of hyperimmune eggs: Starting from 18-25 days after the initial immunization, collect eggs laid by immunized hens, disinfect the surface, and take the yolk liquid for later use. S4. Preliminary extraction of antibodies: Mix the egg yolk liquid and diluent at a volume ratio of 1:4 to 1:15, stir evenly, adjust the pH of the mixture to 4.8-5.5 with acid, let it stand at 2-8℃ for 6-24 hours, and then centrifuge at 2-8℃ and 8000-15000×g for 10-30 minutes to collect the first supernatant; S5. Preliminary concentration and purification of antibodies: The first supernatant is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 30kDa to 300kDa to concentrate it 5-30 times, and then replaced with neutral buffer to obtain concentrated antibody solution. S6. Fine purification of the antibody: The concentrated antibody solution is purified sequentially by passing it through at least one chromatography column, wherein the chromatography column is selected from affinity chromatography column, ion exchange chromatography column and size exclusion chromatography column; S7. Antibody formulation: The finely purified antibody solution was filtered through 0.45μm and 0.22μm filter membranes for sterilization, then stabilizers and preservatives were added, and the pH was adjusted to 6.5-7.
8.
2. The preparation method and application of the anti-human papillomavirus IgY antibody according to claim 1, characterized in that: In step S1, the HPV antigen is selected from: HPV virus-like particles, recombinant expressed HPVL1 protein, recombinant expressed HPVE6 protein, and recombinant expressed HPVE7 protein; the first emulsification treatment and the second emulsification treatment are performed using a dual-syringe injection method until a stable emulsion that does not diffuse when dropped into water is formed.
3. The preparation method and application of the anti-human papillomavirus IgY antibody according to claim 1, characterized in that: Step S2 also includes an immune monitoring step: before each immunization and 7-10 days after immunization, serum or egg yolk samples are collected from immunized chickens, and the titer of anti-HPV specific antibodies is determined by enzyme-linked immunosorbent assay (ELISA); the timing of subsequent booster immunizations is dynamically adjusted based on the antibody titer change curve; when the antibody titer increases to a plateau, the next booster immunization is implemented.
4. The preparation method and application of the anti-human papillomavirus IgY antibody according to claim 1, characterized in that: In step S4, the diluent is deionized water, phosphate buffer, Tris-HCl buffer, or acetate-sodium acetate buffer; the acid is a 0.5-2.0N hydrochloric acid solution or an acetic acid solution.
5. The preparation method and application of the anti-human papillomavirus IgY antibody according to claim 1, characterized in that: In step S5, the neutral buffer solution is a phosphate buffer or Tris-HCl buffer with a pH of 7.0-7.5 and a concentration of 10-100 mM; the ultrafiltration process is carried out at 2-8℃ using a tangential flow filtration mode.
6. The preparation method and application of the anti-human papillomavirus IgY antibody according to claim 1, characterized in that: The fine purification in step S6 employs one of the following three chromatographic pathways: Path 1: The concentrated antibody solution flows sequentially through an affinity chromatography column and a size exclusion chromatography column; Path 2: The concentrated antibody solution flows sequentially through an ion exchange chromatography column and a size exclusion chromatography column; Path 3: The concentrated antibody solution flows sequentially through an affinity chromatography column, an ion exchange chromatography column, and a size exclusion chromatography column.
7. The preparation method and application of an anti-human papillomavirus IgY antibody according to claim 6, characterized in that: The affinity chromatography column has recombinant protein G and recombinant protein L as ligands; affinity elution is performed using glycine-hydrochloric acid buffer at pH 2.5-3.5, followed immediately by neutralization with Tris buffer at pH 8.0-9.
0.
8. The preparation method and application of an anti-human papillomavirus IgY antibody according to claim 6, characterized in that: The ion exchange chromatography column is an anion exchange chromatography column, and the medium is quaternary ammonium salt type; elution is performed linearly using a buffer solution containing 0-1.0M sodium chloride at pH 7.0-8.
5.
9. The preparation method and application of an anti-human papillomavirus IgY antibody according to claim 6, characterized in that: The size exclusion chromatography column has a separation range of 10-1000 kDa; the eluent is a phosphate buffer with pH 6.5-7.5 containing 100-200 mM sodium chloride, and the flow rate is 0.5-1.5 ml / min.
10. The method for preparing and applying an anti-human papillomavirus IgY antibody according to claim 1, characterized in that: In step S7, the stabilizer is selected from one or more of trehalose, sucrose, mannitol, glycerol, and human serum albumin, and its mass-volume concentration in the final antibody solution is 0.5%-10%; the preservative is selected from one or more of thimerosal, phenol, m-cresol, and chlorhexidine gluconate, and its mass-volume concentration in the final antibody solution is 0.01%-0.5%.