Chromatographic filler for purification of tetanus immunoglobulin or fragments thereof

Abstract

The invention relates to a chromatographic filler for purifying tetanus immune globulin or a fragment thereof, a preparation method of the chromatographic filler and a method for purifying the tetanus immune globulin or the fragment thereof. The invention relates to a chromatographic filler for purifying tetanus immune globulin or a fragment thereof, which comprises an affinity capture ligand selected from a TeNT-Hc-C869A subunit antigen, and any one of a chromatographic filler which is selected from a group consisting of NHS-activated Sepharose 4FF, CNBr-activated Sepharose 4B, PabPur SulfoLink Beads 4FF or epoxy-activated Seplife FF and is used for binding / coupling a protein. The invention further discloses a preparation method of the chromatographic filler for purifying the tetanus immune globulin or the fragment of the tetanus immune globulin or the fragment of the tetanus immune globulin or the fragment of the tetanus immune globulin or the fragment of the tetanus immune globulin or the fragment of the tetanus immune globulin. The chromatographic filler for purifying the tetanus immune globulin or the fragment thereof, provided by the invention, can improve the purity and the titer of the purified tetanus immune globulin or the 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 and a preparation method thereof, as well as a method for purifying tetanus immunoglobulin or a fragment thereof. Background Art

[0002] Tetanus is a serious infectious disease with a mortality rate of up to 20-40%. Any trauma may lead to the occurrence of tetanus. Tetanus antibody is a fast and effective short-term drug for the prevention and treatment of tetanus. There are currently two main products on the market for the prevention and treatment of tetanus: tetanus human immunoglobulin (TIG) and tetanus antitoxin (TAT). Among them, tetanus antitoxin (TAT) is a horse-derived antiserum product that can neutralize tetanus toxin produced by Clostridium tetani, relieve symptoms and reduce mortality. The production process of TAT includes horse immunization, plasma collection, pepsin digestion, ammonium sulfate precipitation, ultrafiltration, desalting, concentration and removal of small molecule proteins. Tetanus human immunoglobulin (TIG) is a specific immunoglobulin preparation extracted from the plasma of blood donors. No skin test is required when used, but there are problems such as infection risk, low yield and high cost.

[0003] The purity and specific activity of TAT often determine the intensity of its allergic reaction in humans. Since TAT contains a large amount of heterologous immunoglobulins, it is easy to cause allergic reactions after administration, especially for allergic patients, which need to adopt cumbersome desensitization methods for emergency treatment, which increases the waiting time and pain of patients and increases the workload of medical staff. Compared with traditional TAT, monoclonal antibodies have higher purity and specificity, can more effectively neutralize tetanus toxin and will not cause 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 the widespread application of monoclonal antibodies. If an efficient immunogen can be introduced into the production of TAT to produce high-titer immune plasma, and a new and more effective purification method is adopted, the heterologous protein content can be greatly reduced, while the specific activity can be greatly improved, the proportion of patients with allergies can be reduced and the cost can be kept low, 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) in the heavy chain can specifically bind to receptors on the nerve cell membrane, which is a key step in the pathogenesis of tetanus toxin. Current research focuses on the C subunit. By specifically neutralizing the C subunit with monoclonal antibodies, the toxin can be prevented from binding to nerve cell receptors, thereby preventing the toxin from entering nerve cells and preventing the occurrence of tetanus. Traditional equine tetanus antitoxin is a polyclonal antibody, of which only 5-10% can bind to tetanus toxin. If antibodies with higher specificity for the C subunit can be effectively purified, it can not only effectively neutralize tetanus toxin, but also remove other ineffective antibodies and impurities, reduce the risk of allergic reactions, and provide a new path for the prevention and treatment of tetanus.

[0005] In the prior art, adding affinity chromatography as a more effective purification process is an improvement direction. For example, the patent application number 202211378319 discloses: a method for purifying tetanus antitoxin, a chromatographic filler and a tetanus antitoxin. The application uses a chromatographic filler containing an affinity substance to adsorb the tetanus antitoxin in the protein solution, and adjusts the appropriate conditions to desorb the tetanus antitoxin, so as to purify the product solution, obtain the tetanus antitoxin, and achieve the enrichment of the tetanus antibody titer; Patent application number 202210460052 discloses: a method for purifying tetanus immunoglobulin and its affinity chromatography filler. The method for purifying tetanus immunoglobulin includes using a chromatography column to perform affinity chromatography on the product; the chromatography column is filled with a chromatography filler, and the chromatography filler is coupled with an affinity substance for specific binding to tetanus antibodies. The raw materials of the affinity chromatography filler include a cross-linked agarose-based medium and a ligand, and the ligand includes a tetanus toxoid finished product, a semi-finished product, and a stock solution.

[0006] However, both of the above patents lack detailed data on filler loading capacity, durability and specific activity after purification, 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.

[0007] In the existing technology, whether it is human tetanus immunoglobulin or equine tetanus antitoxin, the current production process cannot simultaneously meet the production requirements of high purity, high specific activity (titer), and low cost. Therefore, improving the purification method of tetanus antitoxin to increase its purity and specific activity, reduce costs and the risk of allergic reactions is an important direction for future research. Summary of the invention

[0008] 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.

[0009] In a first aspect, the present invention provides a chromatography filler for purifying tetanus immunoglobulin and its fragments, comprising: an affinity capture ligand selected from the TeNT-Hc-C869A subunit antigen; Any one of NHS-activated Sepharose 4FF, CNBr-activated Sepharose 4B, PabPur SulfoLinkBeads 4FF or epoxy-activated Seplife FF is selected as a chromatography medium for binding / coupling proteins; Wherein, the protein concentration of the affinity capture ligand is 3 to 10 mg / mL.

[0010] In one implementation, the affinity capture ligand is fixed on the chromatography filler by a coupling process, the coupling process uses NHS-activated Sepharose, CNBr-activated Sepharose, PabPur SulfoLink, epoxy-activated chromatography filler, and the ratio of the affinity capture ligand to the chromatography filler is 1:(2~4).

[0011] In a second aspect, the present invention further provides a method for preparing a chromatographic filler for 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: S11, filler pretreatment: providing the ligand, and washing it with a first coupling solution; S12, antigen coupling: providing the affinity capture ligand, replacing the affinity capture ligand into the second coupling solution to make its protein concentration 3-10 mg / ml, and stirring at a ratio of ligand volume: affinity capture ligand volume = 1:2; 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.

[0012] 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.

[0013] In one implementation, in S14, cleaning specifically includes: washing with a first cleaning solution and a second cleaning solution alternately for 3 times, each time washing with a cleaning solution of 3 times the volume of the filler; wherein the first cleaning solution is a mixed solution of 0.1mol / L Tris-HCl and 0.5mol / L NaCl, and its pH=8.0; the second cleaning solution is a mixed solution of 0.1mol / L acetate buffer and 0.5mol / L NaCl.

[0014] In one implementation, in S14, the storage specifically includes: washing with 20 mmol / L PBS solution before use or storing in 20% ethanol.

[0015] 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.

[0016] In one implementation, the following specific steps are included: S21, column loading: loading the prepared chromatography filler for purification of tetanus immunoglobulin and its fragments into a 5 mL pre-loaded empty column, and washing with purified water to obtain a pre-loaded column; S22, balancing: balancing the pre-packed column with a balancing buffer; S23, loading: the liquid containing tetanus immunoglobulin or its fragment is replaced with the equilibrium buffer and retained on the pre-loaded column; S24, washing: washing with equilibrium buffer after staying; 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 antitoxin or a fragment thereof.

[0017] In one implementation, the equilibrium 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, pH=2.5~4.0; The second elution buffer is 0.1 mol / L glycine, pH=2.0; The neutralizing solution is 1 mol / L disodium hydrogen phosphate, pH=8.0.

[0018] In one implementation, S23 specifically includes: taking a liquid containing tetanus immunoglobulin or a fragment thereof, replacing it with the equilibrium buffer or diluting it to a protein concentration of 10 mg / ml, and setting the loading speed so that the protein stays on the pre-loaded column for 5 minutes.

[0019] 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; the purity and potency of the purified tetanus antitoxin can be improved; the column purification load of the chromatographic filler for purifying tetanus immunoglobulin and its fragments is high, the stability is good, and the specificity is good; the preparation process of the chromatographic filler for purifying tetanus immunoglobulin and its fragments is simple, the cost is low, linear amplification can be achieved, the quality is easy to control, and the production requirements of high-purity, high-specific activity and low-cost tetanus antitoxin are met at the same time, and the application prospect is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is an SDS-PAGE diagram of horse plasma before and after purification by the tetanus antitoxin purification method provided by the present invention; Figure 2 This is a diagram showing the results of SDS-PAGE detection of human immunoglobulin before and after purification by the tetanus antitoxin purification method provided by the present invention; Figure 3 This is a western blot test result diagram of horse tetanus immunoglobulin (F(ab')2) purification performed by the tetanus antitoxin purification method provided by the present invention; The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with 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 description of the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" described below means 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 representation of the above terms is not necessarily for the same embodiment or example. Moreover, the technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0022] The present invention provides a chromatographic filler for purifying tetanus immunoglobulin and its fragments, comprising: an affinity capture ligand selected from the TeNT-Hc-C869A subunit antigen; Any one of NHS-activated Sepharose 4FF, CNBr-activated Sepharose 4B, PabPur SulfoLinkBeads 4FF or epoxy-activated Seplife FF is selected as a chromatography medium for binding / coupling proteins; Wherein, the protein concentration of the affinity capture ligand is 3 to 10 mg / mL.

[0023] 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 genetic engineering derivatives, including single-chain variable fragments (scFv), diabodies / tribodies, nanobodies, bispecific / trispecific antibodies (Bispecific and TrispecificAntibodies), minibodies (Minibodies), fusion proteins and PEGylated fragments.

[0024] Specifically, the affinity capture ligand is fixed on the chromatography filler through a coupling process, the coupling process uses NHS-activated Sepharose, CNBr-activated Sepharose, PabPur SulfoLink, and epoxy-activated chromatography filler, and the ratio of the affinity capture ligand to the chromatography filler is 1:(2~4).

[0025] Among them, NHS chemical coupling is to activate Sepharose with NHS (N-hydroxysuccinimide), react the amine group in the protein with the carboxyl group activated by NHS to form a covalent bond. In the buffer solution, the amino group of the protein reacts with the NHS group to form a stable covalent coupling; CNBr chemical coupling is to activate Sepharose with cyanogen bromide (CNBr). After CNBr activation, an active carbonyl group is formed. The amino group in the protein reacts with the active site. Under alkaline conditions, the protein is covalently bound to the activated Sepharose; Epoxy chemical coupling is to activate Sepharose through epoxy groups (Epoxy), so that the amino or thiol group of the protein reacts with the epoxy group to form a covalent bond; SulfoLink chemical coupling is to use SulfoLink resin, which contains an activated thiol reaction site, and the thiol group in the protein forms a thioether bond with the activated group on the SulfoLink resin, which can be used for thiol-modified proteins. The above coupling methods can covalently bind proteins to Sepharose under specific conditions.

[0026] By using specific affinity capture ligands (TeNT-Hc-C869A subtoxin antigen) and ligands (such as NHS-activated Sepharose 4FF, etc.), the chromatography medium can specifically bind to tetanus immunoglobulin and its fragments, improve purification efficiency and reduce impurity content.

[0027] The present invention also provides a method for preparing a filler for purifying tetanus immunoglobulin and its fragments by chromatography, which is used to prepare the above-mentioned chromatographic filler for purifying tetanus immunoglobulin and its fragments, and comprises the following specific steps: S11, filler pretreatment: providing the ligand, and washing it with a first coupling solution; S12, antigen coupling: providing the affinity capture ligand, replacing the affinity capture ligand into the second coupling solution to make its protein concentration 3-10 mg / ml, and stirring at a ratio of ligand volume: affinity capture ligand volume = 1:2; 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.

[0028] Specifically, in S11, the first coupling solution is precooled 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 1 mol / L HCl solution.

[0029] 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.

[0030] In S13, the blocking solution is a 0.1 mol / L Tris-HCl mixed solution, and its pH=8.5.

[0031] In S14, washing specifically includes: washing with a first cleaning solution and a second cleaning solution alternately for 3 times, each time washing with a cleaning solution of 3 times the volume of the filler; storage specifically includes: washing with a 20mmol / L PBS solution and then using or storing in 20% ethanol. The first cleaning solution is a mixed solution of 0.1mol / L Tris-HCl and 0.5mol / L NaCl, and its pH is 8.0; the second cleaning solution is a mixed solution of 0.1mol / L acetate buffer and 0.5mol / L NaCl; the pH of the PBS solution is 7.4.

[0032] This example provides multiple examples for testing, wherein the detection methods used in this example are all pharmacopoeia methods, which may be slightly modified. Examples 1-4 The specific process selection of Examples 1-4 is shown in Table 1.

[0033] Table 1. Screening of different filler types in Examples 1-4 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 The affinity capture ligand used in Examples 1 to 4 is TeNT-Hc-C869A subtoxin antigen. From the table above, it can be seen that NHS-activated Sepharose 4FF is the most preferred filler. Although CNBr-activated Sepharose 4B also has a relatively high loading capacity, due to the highly toxic CNBr group, there will be great regulatory risks in actual use. Therefore, the preferred ligand is NHS-activated Sepharose 4FF.

[0034] The present invention also provides a method for purifying tetanus antitoxin, which comprises the following specific steps: S21, column loading: loading the prepared chromatography filler for purification of tetanus immunoglobulin and its fragments into a 5 mL pre-loaded empty column, and washing with purified water to obtain a pre-loaded column; S22, balancing: balancing the pre-packed column with a balancing buffer; S23, loading: taking tetanus antitoxin from horse or tetanus immunoglobulin from human, replacing it with the equilibrium buffer, and retaining it on the preloaded column; S24, washing: washing with equilibrium buffer after staying; 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 antitoxin.

[0035] Specifically, the equilibrium buffer is a mixed solution of 0.5 mol / L NaCl and 20 mmol / L Na2HPO4, and its pH=7.0; The first elution buffer is 0.1 mol / L glycine, pH=2.5~4.0; The second elution buffer is 0.1 mol / L glycine, pH=2.0; The neutralizing solution is 1 mol / L disodium hydrogen phosphate, pH=8.0.

[0036] In S21, 5CV (column volume) of purified water was used for washing; In S22, the equilibrium volume is 5CV; S23 specifically includes: taking tetanus antitoxin from horse or tetanus immunoglobulin from human and replacing or diluting it with the equilibrium buffer to a protein concentration of 10 mg / ml, and setting the loading speed so that the protein stays on the pre-loaded column for 5 minutes; In S24, washing was performed with 10 CV volume of equilibration buffer; In S25, elution is performed with 10CV volume of the first elution buffer and neutralization is performed with 1 / 10 volume of the neutralization solution; 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.

[0037] After the second elution, 5CV column volume can be flushed with 0.1 mol / L sodium hydroxide.

[0038] Embodiment 5-8 The chromatographic filler for purifying tetanus immunoglobulin and its fragments in Example 1 is provided for purifying tetanus antitoxin. The specific process selections of Examples 1-4 are shown in Table 2.

[0039] Table 2. Purification results under different elution conditions Example 5 Example 6 Example 7 Example 8 First elution condition pH4.0 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 300IU / mg 320IU / mg 340IU / mg 350 U / mg According to the results in Table 2, 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.

[0040] The chromatographic filler prepared in Example 1 for purification of tetanus immunoglobulin and its fragments was provided, and stability test and purification test of the filler were performed. Specifically, the test results are shown in Tables 3 and 4.

[0041] Table 3. Packing durability test data Table 4: Filler storage temperature stability test data table 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 of the purification process are not significantly reduced. After 5 cycles without alkali washing, the loading capacity is retained by more than 80%, and the alkali washing is performed once after 5 cycles. After 5 cycles, the loading capacity is retained by more than 80%, and the protein loading and purity are not significantly reduced. In addition, the protein loading and purity of 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.

[0042] The present invention also performs purification tests on antibodies from different sources, and the purification results are shown in Table 5.

[0043] Table 5: Purification results of antibodies from different sources Table 6. Filler preparation process enlargement test results Combined with reference Figure 1-Figure 3 , Table 5 and Table 6, Figure 1 The reduced SDS-PAGE diagram of the 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 is shown in FIG. 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 3The invention provides a method for purifying tetanus antitoxin to produce horse tetanus immunoglobulin (F(ab' )2 ) Purification western blot test result diagram. Among them, Western blot detection is an antigen shedding test. There is no antigen shedding during flow-through, washing, and elution. According to the control concentration, the antigen concentration in flow-through, washing, and elution is less than 0.01%; According to the results in Table 6, the purity of the eluate detected by SEC-HPLC and SDS-PAGE is more than 95%, and the specific activity of the titer detection is 10-15 times higher than that of the original solution.

[0044] In addition, the chromatographic filler provided by the present invention can purify antibodies from various sources, including tetanus immunoglobulin plasma from humans and animals, tetanus immunoglobulin-rich liquid after preliminary treatment, and natural fragments of tetanus immunoglobulin (such as Fab, F(ab')2) and recombinant genetic engineering derivatives. These derivatives include single-chain variable fragments (scFv), diabodies / tribodies, nanobodies, bispecific and trispecific antibodies, minibodies, fusion proteins, and PEGylated fragments.

[0045] Tylenol Biopharm's TNM002 tetanus toxin monoclonal antibody, which is currently on the market, has shown excellent ability in neutralizing tetanus toxin. Preclinical studies have shown that 1 mg of TNM002 monoclonal antibody (IgG) has a neutralizing potency of up to 250 IU of tetanus immunoglobulin, 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). Although monoclonal antibodies have higher potency, traditional tetanus immunoglobulins and their fragments, after purification by affinity chromatography, provide high-purity alternative products with similar neutralizing activity at a lower cost, which is more cost-effective.

[0046] 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. The affinity chromatography column is suitable for purifying immune horse plasma containing tetanus immunoglobulin, enzyme-digested horse tetanus immunoglobulin F(ab')2 fragments, and human tetanus toxin immunoglobulin. The purity of the purified tetanus antitoxin can be increased to more than 95%, and the titer is increased by about 5.5 times and 30 times, which greatly improves the purity and titer of the purified tetanus antitoxin. In addition, the affinity chromatography column is also suitable for purifying human or animal 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, etc.

[0047] The chromatographic filler used for purifying tetanus immunoglobulin and its fragments has high column purification capacity, good stability and high specificity. Its preparation process is simple, cost-effective, can achieve linear amplification, and is easy to control quality. It meets the requirements of high-purity, high-specific activity and low-cost tetanus antitoxin production and has broad application prospects.

[0048] 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 specification 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: an affinity capture ligand selected from the TeNT-Hc-C869A subunit antigen; Any one of NHS-activated Sepharose 4FF, CNBr-activated Sepharose 4B, PabPur SulfoLink Beads4FF or epoxy-activated Seplife FF is selected as a chromatography medium for binding / coupling proteins; Wherein, the protein concentration of the affinity capture ligand is 3 to 10 mg / mL.

2. The chromatographic filler for purifying tetanus immunoglobulin and its fragments according to claim 1, characterized in that: 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).

3. A method for preparing a chromatographic filler for purifying tetanus immunoglobulin and its fragments, characterized in that: The method for preparing the chromatographic 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 ligand, and washing it with a first coupling solution; S12, antigen coupling: providing the affinity capture ligand, replacing the affinity capture ligand into the second coupling solution to make its protein concentration 3-10 mg / ml, and stirring at a ratio of ligand volume: affinity capture ligand volume = 1:2; 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 chromatographic 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 / L Tris-HCl mixed solution, and its pH is 8.

5.

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

6. The method for preparing a chromatographic 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 and its fragments, 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 the 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 and its fragments according to claim 7, characterized in that: The specific steps include: S21, column loading: loading the prepared chromatography filler for purification of tetanus immunoglobulin and its fragments into a 5 mL pre-loaded empty column, and washing with purified water to obtain a pre-loaded column; S22, balancing: balancing the pre-packed column with a balancing buffer; S23, loading: taking a liquid containing tetanus immunoglobulin or a fragment thereof, replacing it with the equilibrium buffer, and retaining it on the pre-loaded column; S24, washing: washing with equilibrium buffer after staying; 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 antitoxin or a fragment thereof.

9. The method for purifying tetanus immunoglobulin and fragments thereof according to claim 8, characterized in that: The equilibrium 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, pH=2.5~4.0; The second elution buffer is 0.1 mol / L glycine, pH=2.0; The neutralizing solution is 1 mol / L disodium hydrogen phosphate, pH=8.

0.

10. The method for purifying tetanus immunoglobulin and fragments 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 equilibrium buffer or diluting it to a protein concentration of 10 mg / ml, and setting the loading speed so that the protein stays on the pre-loaded column for 5 minutes.

Images