Chromatographic filler for purification of tetanus immunoglobulin or its fragments

Abstract

The present invention relates to a chromatographic filler for purifying tetanus immunoglobulin or a fragment thereof, a preparation method thereof, and a method for purifying tetanus immunoglobulin or a fragment thereof. The chromatographic filler for purifying tetanus immunoglobulin or a fragment thereof comprises: an affinity capture ligand selected from TeNT-Hc-C869A subunit antigen, and a chromatographic filler selected from any one of NHS-activated Sepharose 4FF, CNBr-activated Sepharose 4B, PabPur SulfoLink Beads 4FF, or epoxy-activated Seplife FF for protein binding / coupling. The chromatographic filler for purifying tetanus immunoglobulin or a fragment thereof provided by the present invention can improve the purity and titer of the purified tetanus immunoglobulin or a fragment thereof.

Description

Technical Field

[0001] The present invention relates to the technical field of antitoxins, and in particular to a chromatography filler for purifying tetanus immunoglobulin or a fragment thereof, a preparation method thereof, and a method for purifying tetanus immunoglobulin or a fragment thereof. Background Art

[0002] Tetanus is a serious infectious disease with a mortality rate as high as 20-40%. Any trauma can lead to tetanus. Tetanus antibodies are a rapid and effective short-term preventive and treatment for tetanus. Currently, two main products are marketed for the prevention and treatment of tetanus: human tetanus immunoglobulin (TIG) and tetanus antitoxin (TAT). Tetanus antitoxin (TAT) is an equine antiserum product that neutralizes the tetanus toxin produced by Clostridium tetani, alleviating symptoms and reducing mortality. The production process of TAT includes horse immunization, plasma collection, pepsin digestion, ammonium sulfate precipitation, ultrafiltration, desalination, concentration, and small protein removal. Human tetanus immunoglobulin (TIG) is a specific immunoglobulin preparation extracted from blood donor plasma. It does not require a skin test, but it carries the risk of infection, low production volume, and high cost.

[0003] The purity and specific activity of TAT often determine the intensity of its allergic reaction in humans. Because TAT contains a large amount of heterologous immunoglobulins, it is prone to allergic reactions after administration. In particular, for allergic patients, cumbersome desensitization procedures are required for emergency treatment, which increases patient waiting time and pain, and also increases the workload of medical staff. Compared with traditional TAT, monoclonal antibodies have higher purity and specificity, can more effectively neutralize tetanus toxin without causing allergic reactions. However, due to the complexity of research and development, production, and quality control, the manufacturing cost of monoclonal antibodies is high, and the market price is high, which limits their widespread application. If a highly effective immunogen can be introduced into the production of TAT to produce high-titer immune plasma, and a new and more effective purification method can be adopted to significantly reduce the heterologous protein content, while significantly improving the specific activity, reducing the proportion of patients with allergies and maintaining a low cost, it will have important clinical application significance.

[0004] Tetanus toxin is composed of a light chain (A subunit) and a heavy chain (B subunit). The C subunit (Hc) of the heavy chain specifically binds to receptors on nerve cell membranes, a key step in the toxin's pathogenesis. Current research focuses on the C subunit. Using monoclonal antibodies to specifically neutralize the C subunit can prevent the toxin from binding to nerve cell receptors, thereby preventing the toxin from entering nerve cells and preventing the onset of tetanus. Traditional equine tetanus antitoxin is a polyclonal antibody, of which only 5-10% is capable of binding to tetanus toxin. If antibodies with higher specificity for the C subunit could be effectively purified, they would not only effectively neutralize tetanus toxin but also remove other ineffective antibodies and contaminants, reducing the risk of allergic reactions and providing a new approach for the prevention and treatment of tetanus.

[0005] In the prior art, adding affinity chromatography as a more efficient purification process is an improvement direction. For example, patent application number 202211378319 discloses: a method for purifying tetanus antitoxin, a chromatography filler, and tetanus antitoxin. The application uses a chromatography filler containing an affinity substance to adsorb tetanus antitoxin from a protein solution, and adjusts appropriate conditions to desorb the tetanus antitoxin to purify the product solution to obtain tetanus antitoxin, thereby achieving the enrichment of tetanus antibody titer;

[0006] Patent application number 202210460052 discloses a method for purifying tetanus immunoglobulin and its affinity chromatography filler. The method involves performing affinity chromatography on the product using a chromatography column; the column is packed with a chromatography filler coupled to an affinity substance that specifically binds to tetanus antibodies. The affinity chromatography filler comprises a cross-linked agarose matrix and a ligand, which includes finished tetanus toxoid, semi-finished products, and stock solutions.

[0007] However, both of the above patents lack detailed data on filler loading capacity, durability and post-purification potency and activity, and it is not clear whether the subtoxins used are wild-type or mutants, making it difficult to evaluate their effectiveness in reducing costs and improving product quality.

[0008] Current production processes for either human tetanus immunoglobulin or equine tetanus antitoxin cannot simultaneously meet the requirements of high purity, high specific activity (potency), and low cost. Therefore, improving tetanus antitoxin purification methods to increase purity and specific activity, while reducing costs and the risk of allergic reactions, is an important research direction for the future. Summary of the Invention

[0009] In the prior art, human tetanus immunoglobulin and equine tetanus antitoxin are either monoclonal antibodies or fragments thereof, and the current production processes cannot simultaneously meet the production requirements of high purity, high specific activity and low cost. Therefore, the present invention provides a chromatographic filler for purifying tetanus immunoglobulin and its fragments and a preparation method thereof, as well as a method for purifying tetanus immunoglobulin and its fragments to solve the above problems.

[0010] In a first aspect, the present invention provides a chromatography filler for purifying tetanus immunoglobulin and fragments thereof, comprising:

[0011] an affinity capture ligand selected from the TeNT-Hc-C869A subunit antigen;

[0012] Any one of NHS-activated Sepharose 4FF, CNBr-activated Sepharose 4B, PabPur SulfoLinkBeads 4FF, or epoxy-activated Seplife FF for protein binding / coupling;

[0013] Wherein, the protein concentration of the affinity capture ligand is 3 to 10 mg / mL.

[0014] In one implementation, the affinity capture ligand is immobilized on the chromatography filler through a coupling process using NHS-activated Sepharose, CNBr-activated Sepharose, PabPur SulfoLink, or epoxy-activated chromatography filler, and the ratio of the affinity capture ligand to the chromatography filler is 1:(2-4).

[0015] In a second aspect, the present invention further provides a method for preparing a chromatography filler for tetanus immunoglobulin and its fragments, which is used to prepare the above-mentioned chromatography filler for purification of tetanus immunoglobulin and its fragments, comprising the following specific steps:

[0016] S11, filler pretreatment: providing the ligand and washing it with a first coupling solution;

[0017] S12, Antigen Coupling: Provide the affinity capture ligand, replace the affinity capture ligand into the second coupling solution to a protein concentration of 3-10 mg / ml, and stir at a ratio of ligand volume: affinity capture ligand volume = 1:2;

[0018] S13, blocking: remove the supernatant after coupling and add blocking solution, and block at room temperature for 4 hours;

[0019] S14. Cleaning and storage: washing with cleaning solution and then storing with preservation solution.

[0020] In one implementation, the first coupling solution is a 1 mol / L HCl solution, the second coupling solution is a mixed solution of 0.11 mol / L NaHCO3 and 0.5 mol / L NaCl, and the pH of the second coupling solution is 8.3; the blocking solution is a 0.1 mol / L Tris-HCl mixed solution, and its pH is 8.5.

[0021] In one implementation, in S14, the cleaning specifically includes: washing three times alternately with a first cleaning solution and a second cleaning solution, each time washing with a cleaning solution of three times the volume of the filler; wherein the first cleaning solution is a mixed solution of 0.1 mol / L Tris-HCl and 0.5 mol / L NaCl, and its pH = 8.0; the second cleaning solution is a mixed solution of 0.1 mol / L acetate buffer and 0.5 mol / L NaCl.

[0022] In one implementation, in S14, the storage specifically includes: washing with 20 mmol / L PBS solution before use or storing in 20% ethanol.

[0023] In a third aspect, the present invention also provides a method for purifying tetanus antitoxin, which comprises loading a liquid containing anti-tetanus immunoglobulin and its fragments onto the above-mentioned chromatographic filler for chromatography, or loading it onto a filler prepared by the above-mentioned method for preparing a chromatographic filler for purifying tetanus immunoglobulin and its fragments for chromatography.

[0024] In one implementation, the following specific steps are included:

[0025] S21, column packing: loading the prepared chromatography filler for purification of tetanus immunoglobulin and its fragments into a 5 mL prepacked empty column, and washing with purified water to obtain a prepacked column;

[0026] S22, equilibration: equilibrate the prepacked column with an equilibration buffer;

[0027] S23, sample loading: the liquid containing tetanus immunoglobulin or its fragment is replaced with the equilibration buffer and retained on the prepacked column;

[0028] S24, washing: washing with equilibration buffer after the dwell time;

[0029] S25, first elution: eluting with a first elution buffer, collecting the eluate and neutralizing it with a neutralizing solution;

[0030] S26. Second elution: Elution is performed with a second elution buffer, the eluate is collected and neutralized with a neutralizing solution to obtain purified tetanus antitoxin or a fragment thereof.

[0031] In one implementation, the equilibration buffer is a mixed solution of 0.5 mol / L NaCl and 20 mmol / L Na2HPO4, with a pH of 7.0;

[0032] The first elution buffer is 0.1 mol / L glycine, pH=2.5~4.0;

[0033] The second elution buffer is 0.1 mol / L glycine, pH=2.0;

[0034] The neutralizing solution is 1 mol / L disodium hydrogen phosphate, pH=8.0.

[0035] In one implementation, S23 specifically includes: taking a liquid containing tetanus immunoglobulin or a fragment thereof, replacing it with the equilibration buffer or diluting it to a protein concentration of 10 mg / ml, and setting the loading speed so that the protein stays on the prepacked column for 5 minutes.

[0036] Beneficial effects: The chromatographic filler for purifying tetanus immunoglobulin and its fragments provided by the present invention uses subtoxin TeNT-Hc-C869A as an affinity capture ligand to prepare a tetanus immunoglobulin and its fragment affinity chromatography column, which can be used to purify horse plasma, enzyme-digested horse tetanus immunoglobulin (F(ab')2) and human tetanus toxin immunoglobulin; it can improve the purity and potency of the purified tetanus antitoxin; the column purification load of the chromatographic filler for purifying tetanus immunoglobulin and its fragments is high, stable and specific; the preparation process of the chromatographic filler for purifying tetanus immunoglobulin and its fragments is simple, low in cost, can achieve linear amplification, and is easy to control quality, while meeting the production requirements of high-purity, high specific activity and low-cost tetanus antitoxin, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is an SDS-PAGE image of horse plasma before and after purification of tetanus antitoxin by the purification method provided by the present invention;

[0038] Figure 2 This is a diagram showing the SDS-PAGE test results before and after purification of human immunoglobulin using the tetanus antitoxin purification method provided by the present invention;

[0039] Figure 3 This is a graph showing the results of western blot detection of horse tetanus immunoglobulin (F(ab')2) purified by the tetanus antitoxin purification method provided by the present invention;

[0040] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the descriptions of the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" described below mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0042] The present invention provides a chromatography filler for purifying tetanus immunoglobulin and its fragments, comprising:

[0043] an affinity capture ligand selected from the TeNT-Hc-C869A subunit antigen;

[0044] Any one of NHS-activated Sepharose 4FF, CNBr-activated Sepharose 4B, PabPur SulfoLinkBeads 4FF, or epoxy-activated Seplife FF for protein binding / coupling;

[0045] Wherein, the protein concentration of the affinity capture ligand is 3 to 10 mg / mL.

[0046] Among them, NHS-activated Sepharose 4FF is a filler activated by NHS (N-hydroxysuccinimide), CNBr-activated Sepharose 4B is an agarose filler activated by cyanogen bromide (CNBr); PabPur SulfoLinkBeads 4FF are SulfoLink activated microbeads, and Epoxy Activated Seplife FF is an epoxy resin activated filler. The tetanus immunoglobulin and its fragments include tetanus immunoglobulin and its natural fragments (Fab, F(ab')2) and recombinant genetically engineered derivatives, including single-chain variable fragments (scFv), diabodies / tribodies, nanobodies, bispecific / trispecific antibodies (bispecific and trispecific antibodies), minibodies, fusion proteins, and PEGylated fragments.

[0047] Specifically, the affinity capture ligand is fixed on the chromatography filler through a coupling process, and the coupling process uses NHS-activated Sepharose, CNBr-activated Sepharose, PabPur SulfoLink, and epoxy-activated chromatography filler. The ratio of the affinity capture ligand to the chromatography filler is 1:(2~4).

[0048] Among them, NHS chemical conjugation uses NHS (N-hydroxysuccinimide) to activate Sepharose, allowing amine groups in proteins to react with NHS-activated carboxyl groups to form covalent bonds. In a buffer solution, the amino groups of the protein react with the NHS groups to form a stable covalent bond. CNBr chemical conjugation uses cyanogen bromide (CNBr) to activate Sepharose. After CNBr activation, the activated carbonyl groups are formed. The amino groups in the protein react with these active sites, and under alkaline conditions, the protein covalently binds to the activated Sepharose. Epoxy chemical conjugation uses epoxy groups to activate Sepharose, allowing amino or sulfhydryl groups in the protein to react with epoxy groups to form covalent bonds. SulfoLink chemical conjugation uses SulfoLink resin, which contains activated sulfhydryl reaction sites. The sulfhydryl groups in the protein form thioether bonds with the activated groups on the SulfoLink resin and can be used for sulfhydryl-modified proteins. All of the above conjugation methods can covalently bind proteins to Sepharose under specific conditions.

[0049] By using a specific affinity capture ligand (TeNT-Hc-C869A subtoxin antigen) and ligand (such as NHS-activated Sepharose 4FF, etc.), this chromatography medium can specifically bind to tetanus immunoglobulin and its fragments, improving purification efficiency and reducing impurity content.

[0050] The present invention also provides a method for preparing a filler for chromatographic purification of tetanus immunoglobulin and its fragments, which is used to prepare the above-mentioned chromatographic filler for purification of tetanus immunoglobulin and its fragments, comprising the following specific steps:

[0051] S11, filler pretreatment: providing the ligand and washing it with a first coupling solution;

[0052] S12, Antigen Coupling: Provide the affinity capture ligand, replace the affinity capture ligand into the second coupling solution to a protein concentration of 3-10 mg / ml, and stir at a ratio of ligand volume: affinity capture ligand volume = 1:2;

[0053] S13, blocking: remove the supernatant after coupling and add blocking solution, and block at room temperature for 4 hours;

[0054] S14. Cleaning and storage: washing with cleaning solution and then storing with preservation solution.

[0055] Specifically, in S11, the first coupling solution is pre-cooled at 4°C and then washed for 30 minutes, and during the washing, 1 ml of the ligand is washed with about 20 ml of the first coupling solution; the first coupling solution is a 1 mol / L HCl solution.

[0056] In S12, the affinity capture ligand is the self-prepared TeNT-Hc-C869A subtoxin antigen; the second coupling solution is a mixed solution of 0.11 mol / L NaHCO3 and 0.5 mol / L NaCl, and the pH of the second coupling solution is 8.3.

[0057] In S13, the blocking solution is a 0.1 mol / L Tris-HCl mixed solution with a pH of 8.5.

[0058] In S14, washing specifically includes: alternating washing with a first cleaning solution and a second cleaning solution three times, each washing step using three times the volume of the filler; and storage specifically includes: washing with a 20 mmol / L PBS solution before use or storage in 20% ethanol. The first cleaning solution is a mixture of 0.1 mol / L Tris-HCl and 0.5 mol / L NaCl, with a pH of 8.0; the second cleaning solution is a mixture of 0.1 mol / L acetate buffer and 0.5 mol / L NaCl; and the pH of the PBS solution is 7.4.

[0059] This embodiment provides multiple examples for detection, wherein the detection methods used in this embodiment are all pharmacopoeial methods, which may be slightly modified

[0060] Examples 1-4

[0061] The specific process selections for Examples 1-4 are shown in Table 1.

[0062] Table 1. Screening of different filler types in Examples 1-4

[0063] Example 1 Example 2 Example 3 Example 4 Affinity capture ligand protein concentration 6mg / ml 6mg / ml 6mg / ml mg / ml Ligand type NHS-activated Sepharose 4FF CNBr-activated Sepharose 4B PabPur SulfoLink Beads 4FF Epoxy Activated Seplife FF Medium loading (protein / medium volume) 34mg / ml 30mg / ml 15mg / ml 15mg / ml

[0064] The affinity capture ligand used in Examples 1-4 was the TeNT-Hc-C869A subtoxin antigen. As shown in the table above, NHS-activated Sepharose 4FF is the most preferred medium. Although CNBr-activated Sepharose 4B also has a relatively high binding capacity, the CNBr group is highly toxic, posing significant regulatory risks in practical use. Therefore, NHS-activated Sepharose 4FF is the preferred ligand.

[0065] The present invention also provides a method for purifying tetanus antitoxin, which comprises the following specific steps:

[0066] S21, column packing: loading the prepared chromatography filler for purification of tetanus immunoglobulin and its fragments into a 5 mL prepacked empty column, and washing with purified water to obtain a prepacked column;

[0067] S22, equilibration: equilibrate the prepacked column with an equilibration buffer;

[0068] S23, sample loading: equine tetanus antitoxin or human tetanus immunoglobulin is replaced with the equilibration buffer and retained on the pre-packed column;

[0069] S24, washing: washing with equilibration buffer after the dwell time;

[0070] S25, first elution: eluting with a first elution buffer, collecting the eluate and neutralizing it with a neutralizing solution;

[0071] S26. Second elution: Elute with a second elution buffer, collect the eluate and neutralize it with a neutralizing solution to obtain purified tetanus antitoxin.

[0072] Specifically, the equilibration buffer is a mixed solution of 0.5 mol / L NaCl and 20 mmol / L Na2HPO4, with a pH of 7.0;

[0073] The first elution buffer is 0.1 mol / L glycine, pH=2.5~4.0;

[0074] The second elution buffer is 0.1 mol / L glycine, pH=2.0;

[0075] The neutralizing solution is 1 mol / L disodium hydrogen phosphate, pH=8.0.

[0076] In S21, 5CV (column volume) of purified water was used for washing;

[0077] In S22, the equilibrium volume is 5CV;

[0078] S23 specifically includes: taking tetanus antitoxin from horse or tetanus immunoglobulin from human, replacing it with the equilibration buffer or diluting it to a protein concentration of 10 mg / ml, and setting the loading speed so that the protein stays on the prepacked column for 5 minutes;

[0079] In S24, wash with 10 CV volume of equilibration buffer;

[0080] In S25, elution was performed with 10CV volume of the first elution buffer and neutralization was performed with 1 / 10 volume of the neutralization solution;

[0081] In S26, elution is performed with a 10CV volume of the second elution buffer, and neutralization is performed with a 1 / 10 volume of the neutralization solution.

[0082] After the second elution, 5CV column volume can be flushed with 0.1 mol / L sodium hydroxide.

[0083] Examples 5-8

[0084] The chromatographic filler for purifying tetanus immunoglobulin and its fragments in Example 1 was provided for purifying tetanus antitoxin. The specific process selections of Examples 1-4 are shown in Table 2.

[0085] Table 2. Purification results under different elution conditions

[0086] Example 5 Example 6 Example 7 Example 8 First elution conditions pH 4.0 pH 3.5 pH 3.0 pH 2.5 First elution protein yield 0.25% 1.38% 2.69% 4.44% Second elution conditions pH 2.0 pH 2.0 pH 2.0 pH 2.0 Second elution protein yield 5.75% 4.62% 3.31% 1.56% Second elution titer 300 IU / mg 320 IU / mg 340 IU / mg 350 U / mg

[0087] The results in Table 2 show that as the pH of the first elution decreases, the titer ratio (specific activity) of the eluted protein becomes higher and higher. Preferably, the pH of the first elution is 2.5 and the pH of the second elution is 2.0.

[0088] The chromatographic filler prepared in Example 1 for purification of tetanus immunoglobulin and its fragments was provided, and stability testing and purification testing of the filler were performed. Specifically, the test results are shown in Tables 3 and 4.

[0089] Table 3. Filler durability test data

[0090]

[0091] Table 4: Filler storage temperature stability test data

[0092]

[0093] Combining the results of Table 3 and Table 4, it can be seen that the filler is stored at 4°C-25°C for 1 month, and the protein loading and purity during the purification process are not significantly reduced. After recycling for 5 times without alkali washing, the loading capacity remains more than 80%. According to the recycling method of 5 times and alkali washing once, the loading capacity remains more than 80% after 5 uses, and the protein loading and purity are not significantly reduced. In addition, the protein loading and purity during the purification process are not significantly reduced under the conditions of 4°C and 25°C. The chromatographic filler for purification of tetanus immunoglobulin and its fragments provided by the present invention has good stability.

[0094] The present invention also performs purification tests on antibodies from different sources, and the purification results are shown in Table 5.

[0095] Table 5: Purification results of antibodies from different sources

[0096]

[0097] Table 6. Filler preparation process scale-up test results

[0098]

[0099] See also Figure 1-Figure 3 , Table 5 and Table 6, Figure 1 This is a reduced SDS-PAGE image of plasma containing tetanus immunoglobulin from horse origin before and after purification by the purification method of tetanus immunoglobulin and its fragments provided by the present invention. Figure 2 This is a diagram showing the results of reduced SDS-PAGE detection of human tetanus immunoglobulin before and after purification by the tetanus antitoxin purification method provided by the present invention. Figure 3 The invention provides a method for purifying tetanus antitoxin to produce equine tetanus immunoglobulin (F(ab' )2 ) Purification western blot test results. Western blot analysis was performed to detect antigen shedding. No antigen shedding was observed during flow-through, wash, or elution. Based on control concentrations, the antigen concentrations during flow-through, wash, and elution were calculated to be less than 0.01%. Table 6 shows that the eluate was above 95% pure by SEC-HPLC and SDS-PAGE, and the specific activity of the eluate was 10-15 times higher than that of the original solution.

[0100] Furthermore, the chromatographic filler provided by the present invention is capable of purifying antibodies from a variety of sources, including tetanus immunoglobulin plasma from humans and animals, pre-treated tetanus immunoglobulin-rich solutions, and natural tetanus immunoglobulin fragments (such as Fab and F(ab')2) and recombinantly engineered derivatives. These derivatives include single-chain variable fragments (scFv), diabodies / tribodies, nanobodies, bispecific and trispecific antibodies, minibodies, fusion proteins, and PEGylated fragments.

[0101] Tylenol Biopharm's TNM002 tetanus toxin monoclonal antibody, currently on the market, demonstrates excellent neutralization potential against tetanus toxin. Preclinical studies have shown that 1 mg of TNM002 monoclonal antibody (IgG) has a neutralizing potency of 250 IU of tetanus immunoglobulin (IU), with a specific activity of approximately 250 IU / mg. This level is close to the specific activity of equine tetanus antitoxin (F(ab')2) purified by affinity chromatography (200-300 IU / mg). Despite the higher potency of monoclonal antibodies, traditional tetanus immunoglobulins and their fragments, purified by affinity chromatography, offer high-purity alternatives with similar neutralizing activity at a lower cost, offering a more cost-effective approach.

[0102] In summary, the present invention uses the subtoxin TeNT-Hc-C869A as an affinity capture ligand to prepare an affinity chromatography column for tetanus antibodies and fragments thereof. This affinity chromatography column is suitable for purifying immune horse plasma containing tetanus immunoglobulin, enzymatically digested equine tetanus immunoglobulin F(ab')2 fragments, and human tetanus toxin immunoglobulin. The purity of the purified tetanus antitoxin can be increased to over 95%, and the potency is increased by approximately 5.5-fold and 30-fold, significantly improving the purity and potency of the purified tetanus antitoxin. In addition, this affinity chromatography column is also suitable for purifying human or animal-derived immune plasma containing tetanus immunoglobulin, natural fragments of tetanus immunoglobulin (Fab, F(ab')2), and recombinant genetic engineering derivatives, including single-chain variable fragments (scFv), diabodies / tribodies, nanobodies, bispecific and trispecific antibodies, minibodies, fusion proteins, and PEGylated fragments.

[0103] This chromatographic filler for purifying tetanus immunoglobulin and its fragments has high column purification capacity, good stability, and high specificity. Its preparation process is simple, cost-effective, and allows for linear scale-up. Its quality is easily controlled, meeting the requirements for high-purity, high-specific activity, and low-cost production of tetanus antitoxin, and it holds broad application prospects.

[0104] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the present invention description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A chromatography filler for purifying tetanus immunoglobulin and its fragments, characterized in that: It includes: Affinity capture ligand and activated chromatography filler; the affinity capture ligand is TeNT-Hc-C869A tetanus toxin antigen; the activated chromatography filler is any one of NHS-activated Sepharose 4FF, CNBr-activated Sepharose 4B, PabPur SulfoLink Beads4FF or epoxy-activated Seplife FF, used for coupling the affinity capture ligand; Wherein, the protein concentration of the affinity capture ligand in the coupling binding solution is 3-10 mg / mL.

2. The chromatography filler for purifying tetanus immunoglobulin and its fragments according to claim 1, characterized in that: The affinity capture ligand is immobilized on the activated chromatography filler through a coupling process, and the coupling process uses any one of NHS-activated Sepharose 4FF, CNBr-activated Sepharose 4B, PabPur SulfoLink Beads 4FF or epoxy-activated Seplife FF activated chromatography filler, and the volume ratio of the affinity capture ligand to the activated chromatography filler is 2:

1.

3. A method for preparing a chromatography filler for purifying tetanus immunoglobulin and its fragments, characterized in that: The method for preparing the chromatography filler for purifying tetanus immunoglobulin and its fragments according to any one of claims 1 to 2 comprises the following specific steps: S11, filler pretreatment: providing the activated chromatography filler and washing it with a first coupling solution; S12, Antigen Coupling: Provide the affinity capture ligand, replace the affinity capture ligand into the second coupling solution to a protein concentration of 3-10 mg / ml, and stir the affinity capture ligand and the activated chromatography medium in a volume ratio of 2:1; S13, blocking: remove the supernatant after coupling and add blocking solution, and block at room temperature for 4 hours; S14. Cleaning and storage: washing with cleaning solution and then storing with preservation solution.

4. The method for preparing a chromatography filler for purifying tetanus immunoglobulin and its fragments according to claim 3, characterized in that: The first coupling solution is a 1 mol / L HCl solution, the second coupling solution is a mixed solution of 0.11 mol / L NaHCO3 and 0.5 mol / L NaCl, and the pH of the second coupling solution is 8.3; the blocking solution is a 0.1 mol / LTris-HCl mixed solution, and its pH is 8.

5.

5. The method for preparing a chromatography filler for purifying tetanus immunoglobulin and its fragments according to claim 3, characterized in that: In S14, the cleaning specifically includes: washing three times alternately with a first cleaning solution and a second cleaning solution, each time washing with a cleaning solution of three times the volume of the filler; wherein the first cleaning solution is a mixed solution of 0.1 mol / L Tris-HCl and 0.5 mol / L NaCl, whose pH is 8.0; and the second cleaning solution is a mixed solution of 0.1 mol / L acetate buffer and 0.5 mol / L NaCl.

6. The method for preparing a chromatography filler for purifying tetanus immunoglobulin and its fragments according to claim 3, characterized in that: In S14, storage specifically includes: washing with 20 mmol / L PBS solution before use or storing in 20% ethanol.

7. A method for purifying tetanus immunoglobulin or a fragment thereof, characterized in that: The liquid containing tetanus immunoglobulin or its fragment is loaded onto the chromatography filler according to any one of claims 1 to 2 for chromatography, or loaded onto the filler prepared by the method for preparing a chromatography filler for purifying tetanus immunoglobulin and its fragments according to any one of claims 3 to 6 for chromatography.

8. The method for purifying tetanus immunoglobulin or a fragment thereof according to claim 7, characterized in that: The specific steps include: S21, column packing: loading the prepared chromatography filler for purification of tetanus immunoglobulin and its fragments into a 5 mL prepacked empty column, and washing with purified water to obtain a prepacked column; S22, equilibration: equilibrate the prepacked column with an equilibration buffer; S23, sample loading: taking a liquid containing tetanus immunoglobulin or a fragment thereof, replacing it with the equilibration buffer, and retaining it on the pre-packed column; S24, washing: washing with equilibration buffer after the dwell time; S25, first elution: eluting with a first elution buffer, collecting the eluate and neutralizing it with a neutralizing solution; S26. Second elution: Elution is performed with a second elution buffer, the eluate is collected and neutralized with a neutralizing solution to obtain purified tetanus immunoglobulin or a fragment thereof.

9. The method for purifying tetanus immunoglobulin or a fragment thereof according to claim 8, characterized in that: The equilibration buffer is a mixed solution of 0.5 mol / L NaCl and 20 mmol / L Na2HPO4, with a pH of 7.0; the first elution buffer is 0.1 mol / L glycine, with a pH of 2.5-4.0; the second elution buffer is 0.1 mol / L glycine, with a pH of 2.0; and the neutralization solution is 1 mol / L disodium hydrogen phosphate, with a pH of 8.

0.

10. The method for purifying tetanus immunoglobulin or a fragment thereof according to claim 8, characterized in that: S23 specifically includes: taking a liquid containing tetanus immunoglobulin or a fragment thereof, replacing it with the equilibration buffer or diluting it to a protein concentration of 10 mg / mL, and setting the loading speed so that the protein stays on the prepacked column for 5 minutes.

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