Preparation method of antitoxin serum containing F (ab ') 2 fragment

Abstract

The invention provides a preparation method of antitoxin serum containing an horse F (ab ') 2 fragment, and the specific steps are as follows: a recombinant streptococcus immunoglobulin G degrading enzyme is immobilized to obtain an immobilized enzyme chromatography column, the recombinant streptococcus immunoglobulin G degrading enzyme comprises a glutathione-S-transferase tag and a histidine tag; performing primary treatment on the immune plasma to obtain a pretreated antibody solution; the pretreatment antibody solution is added into an immobilized enzyme chromatography column for digestion, after digestion is completed, ultrafiltration, purification and solution preparation are sequentially conducted through an immobilized glutathione affinity column or a nickel affinity column, an antitoxin serum stock solution is obtained, and the antitoxin serum stock solution contains F (ab ') 2 fragments and does not contain Fc fragments, impure protein and small molecule impurities. Through the process steps, the Fc fragment and impure protein are effectively removed, and only the horse F (ab ') 2 fragment is reserved, so that the immunogenicity of antitoxin serum and the risk of causing anaphylactic reaction are remarkably reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for preparing antitoxin serum containing F(ab')2 fragments. Background Art

[0002] Antitoxin serum, also known as antitoxin or antiserum, is an immune plasma made by combining plasma (serum) produced by animal (mainly equine) or human immunity with corresponding toxins or toxoids, bacteria, viruses, etc. Immunoglobulin preparations made by digesting animal-derived plasma with porcine pepsin, ammonium sulfate precipitation and chromatography purification are widely used in clinical practice to treat poisoning symptoms caused by bites or stings from venomous animals. If the patient can be injected with antitoxin serum in time at the first time, the possibility of survival will be significantly increased, and precious time will be gained for subsequent rescue and treatment.

[0003] However, animal-derived antitoxin serum has two sides in the treatment process: on the one hand, it provides patients with specific antitoxin antibodies, which can effectively neutralize the corresponding exotoxins in the body and play a therapeutic and preventive role; on the other hand, because it is essentially a foreign protein and has antigenicity, it may stimulate the human body to produce antibodies against animal-derived serum proteins. After the patient is exposed to this foreign protein for the first time, if he receives a similar immune serum injection again, he may have a hypersensitivity reaction, that is, an allergic reaction, due to the production of antibodies. Summary of the invention

[0004] The technical solution of the present invention is to provide a method for preparing antitoxin serum containing F(ab')2 fragments, aiming to solve the problem that heterologous proteins and impurities introduced by animal-derived pepsin in the production process of antitoxin serum are difficult to completely remove, leading to allergies.

[0005] To solve the above technical problems, the present invention is implemented as follows: a method for preparing an antitoxin serum containing a F(ab')2 fragment, the steps comprising: S1, the recombinant Streptococcus immunoglobulin G degrading enzyme is immobilized to obtain an immobilized enzyme chromatography column, wherein the recombinant Streptococcus immunoglobulin G degrading enzyme comprises a glutathione-S-transferase tag and a histidine tag; S2, preliminarily treating the immune plasma to obtain a pretreated antibody solution; S3. Add the pretreated antibody solution to an immobilized enzyme chromatography column for digestion. After digestion, pass it through an immobilized glutathione affinity column or a nickel affinity column for ultrafiltration, purification and solution preparation to obtain an antitoxin serum stock solution, which includes the F(ab')2 fragment but does not include the Fc fragment, miscellaneous proteins and small molecule impurities.

[0006] In some embodiments of the present invention, step S1 comprises: S1.1, prepare coupling solution A and coupling solution B respectively; S1.2, placing the NHS activation medium in the coupling solution A for washing, wherein the volume ratio of the NHS activation medium to the coupling solution A is 1:15-30; S1.3, placing the recombinant Streptococcus immunoglobulin G degrading enzyme in the coupling solution B, and then adding the washed NHS activation medium to stir and mix to obtain a mixed solution, wherein the volume ratio of the NHS activation medium to the recombinant Streptococcus immunoglobulin G degrading enzyme is 1:1-2; S1.4, allowing the mixed solution to stand for sedimentation, removing the supernatant after the sedimentation is completed, adding a blocking solution to the precipitate of the mixed solution for a blocking operation to obtain an immobilized enzyme, wherein the blocking time is 2 to 4 hours and the blocking temperature is 20 to 30°C; S1.5, placing the immobilized enzyme alternately in cleaning solution A and cleaning solution B for alternate washing, wherein the volume ratio of the immobilized enzyme to the cleaning solution A is 1:2-5, and the volume ratio of the immobilized enzyme to the cleaning solution B is 1:2-5; S1.6. Place the immobilized enzyme after washing in a chromatography column and add a preservation solution to obtain an immobilized enzyme chromatography column.

[0007] In some embodiments of the present invention, in step S1.4, when the deposition temperature is 20-30°C, the deposition time is 3-6 hours, and when the deposition temperature is 0-10°C, the deposition time is 12-20 hours.

[0008] In some embodiments of the present invention, the coupling solution A comprises at least one of hydrochloric acid, acetic acid, phosphoric acid, and formic acid, and the concentration of the coupling solution A is 0.5-2 mM; The coupling solution B includes a buffer and an alkaline regulator, wherein the buffer includes at least one of sodium bicarbonate, disodium hydrogen phosphate, and triethanolamine, and the concentration of the buffer is 0.05-0.5M, and the alkaline regulator includes at least one of sodium hydroxide, potassium hydroxide, and sodium carbonate, and the concentration of the alkaline regulator is 0.1-1M, and the pH of the coupling solution is 8-9; The blocking solution includes at least one of tris(hydroxymethyl)aminomethane hydrochloride, sodium bicarbonate-sodium carbonate buffer, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, the concentration of the blocking solution is 0.05-0.5M, and the pH of the blocking solution is 8-9; The cleaning solution A comprises an alkaline pH buffer and an ionic strength regulator, wherein the alkaline pH buffer comprises at least one of tris(hydroxymethyl)aminomethane hydrochloride, sodium bicarbonate-sodium carbonate buffer, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, and the concentration of the alkaline pH buffer is 0.05-0.5M, and the ionic strength regulator comprises at least one of potassium chloride, sodium hydroxide, and potassium hydroxide, and the concentration of the ionic strength regulator is 0.1-1M, and the pH of the cleaning solution A is 8-9; The cleaning solution B comprises an acidic pH buffer and an ionic strength regulator, wherein the acidic pH buffer comprises at least one of an acetate buffer, a citrate buffer, and a phosphate buffer, and the concentration of the acidic pH buffer is 0.05-0.5 M, and the ionic strength regulator comprises at least one of potassium chloride, sodium hydroxide, and potassium hydroxide, and the concentration of the ionic strength regulator is 0.1-1 M, and the pH of the cleaning solution A is 3-4; The preservation solution includes a neutral buffer and an antibacterial agent, the neutral buffer includes at least one of phosphate buffered saline, 3-(N-morpholine)propanesulfonic acid buffer, and tris(hydroxymethyl)aminomethane hydrochloride, the concentration of the neutral buffer is 15-30 mM, the pH of the neutral buffer is 7-8, and the antibacterial agent includes at least one of isopropanol, ethanol, and benzalkonium chloride, and the concentration of the antibacterial agent is 10%-30%.

[0009] In some embodiments of the present invention, step S2 includes: S2.1. Filter and centrifuge the immune plasma in sequence to obtain antibody serum; S2.2, adding an acidic buffer to the antibody serum, stirring and mixing, adding a precipitating solution during the stirring process, and performing centrifugation and filtering operations to obtain an antibody filtrate, wherein the volume ratio of the antibody serum to the acidic buffer is 1:2-4; S2.3, adding a buffer to the antibody filtrate, and then dropping an alkaline solution to obtain an antibody buffer, wherein the pH value of the antibody buffer is 7-7.5, and the volume ratio of the buffer to the antibody filtrate is 1:8-15; S2.4, placing the antibody buffer in an ice bath, adding a protein precipitant and stirring, and allowing to stand for sedimentation after the stirring is completed to obtain an antibody sedimentation solution; S2.5, centrifuging the antibody deposition liquid to separate the clear liquid and the precipitate of the antibody deposition liquid, adding a stabilizing liquid to the precipitate of the antibody deposition liquid, and sequentially performing centrifugation, filtration, ultrafiltration, and concentration to obtain a pretreated antibody solution, wherein the volume ratio of the stabilizing liquid to the clear liquid of the antibody deposition liquid is 1:8~15.

[0010] In some embodiments of the present invention, in step S2.3, the buffer comprises at least one of phosphate buffered saline, 3-(N-morpholine) propanesulfonic acid buffer, and tris(hydroxymethyl)aminomethane hydrochloride, the concentration of the buffer is 0.05-0.5M, and the pH of the buffer is 7-8; The alkaline solution includes at least one of sodium hydroxide, potassium hydroxide, and sodium bicarbonate, and the concentration of the alkaline solution is 1-3M; In step S2.4, the protein precipitant includes at least one of ammonium sulfate, isopropanol, and polyethylene glycol, and the concentration of the protein precipitant is 250-300 g / L; In step S2.5, the stabilizing solution includes at least one of phosphate buffered saline, sodium bicarbonate buffer, and citric acid buffer, and the concentration of the stabilizing solution is 0.005-0.05M.

[0011] In some embodiments of the present invention, in step S2.2, the precipitation solution includes an acidic regulator and a precipitant, the acidic regulator includes at least one of acetic acid, hydrochloric acid, and phosphoric acid, and the precipitant includes at least one of ethanol, n-octanoic acid, and isopropanol; The step S2.2 comprises: S2.2.1, adding an acidic buffer to the antibody serum and stirring and mixing, wherein the acidic buffer comprises at least one of an acetate buffer, a citrate buffer, and a phosphate buffer, the concentration of the acidic buffer is 0.02-0.1 M, the pH of the acidic buffer is 4-5, and the volume ratio of the antibody serum to the acidic buffer is 1:2-4; S2.2.2. Maintain stirring and mixing operation, first add the acidic regulator during stirring, then dropwise add the precipitant, after the dropwise addition is completed, let it stand and settle, then perform centrifugation and filtration operations in sequence to obtain the antibody filtrate.

[0012] In some embodiments of the present invention, step S3 includes: S3.1, connecting the immobilized enzyme chromatography column to a fluid control device, first introducing the equilibrium solution, and then introducing the pre-treated antibody solution to perform enzymatic hydrolysis reaction, wherein the reaction time is 30 to 60 minutes; S3.2, passing the pretreated antibody solution after the enzymatic hydrolysis reaction into an immobilized glutathione affinity column or a nickel affinity column for chromatography reaction; S3.3, performing ultrafiltration cycle on the pretreated antibody solution after the chromatography reaction is completed to obtain an antibody ultrafiltrate; S3.4. Add an osmotic pressure regulator, a preservative and an acid-base regulator to the antibody ultrafiltrate in sequence, sterilize and filter to obtain an antitoxin serum stock solution, wherein the pH of the antitoxin serum stock solution is 6-7.

[0013] In some embodiments of the present invention, in step S3, the equilibrium solution includes at least one of water for injection, deionized water, pure water, and glycerol; The osmotic pressure regulator includes at least one of sodium chloride, potassium chloride and sodium sulfate, and the concentration of the osmotic pressure regulator is 8-9 g / L; The preservative includes at least one of benzalkonium chloride, m-cresol, and sodium benzoate, and the concentration of the preservative is 2-3 g / L; The acid-base regulator includes at least one of dilute hydrochloric acid, dilute sodium hydroxide, sodium bicarbonate, and acetic acid.

[0014] The present invention provides a method for preparing an antitoxin serum containing a F(ab')2 fragment, which has the following advantages: The present invention uses an immobilized recombinant Streptococcus immunoglobulin G degrading enzyme (IdeS) to specifically cleave IgG antibodies in immune plasma. The IdeS enzyme is fixed to the chromatography column by chemical bonding so that it can be reused in multiple reactions, ensuring the catalytic stability of the immobilized enzyme column. When the pretreated antibody solution flows through the immobilized enzyme column, the IdeS enzyme specifically cleaves the heavy chain portion of IgG to generate the required F(ab')2 fragment. This process not only ensures the efficient generation of the F(ab')2 fragment, but also avoids excessive digestion of the Fc fragment and unremoved miscellaneous proteins and small molecule impurities, thereby simplifying the subsequent purification steps.

[0015] In addition, the designed IdeS enzyme carries a double tag of glutathione-S-transferase (GST) and histidine (His), which is convenient for fixation on the affinity column, easy to operate, and easy to remove the residual IdeS enzyme that falls off the column after the hydrolysis reaction. After digestion, the mixture can be further purified by GST or Ni affinity column to ensure that any detached enzyme is completely removed. This method not only simplifies the operation process and reduces the risk of residual impurities, but also significantly improves the purity and safety of F(ab')2 products, and effectively reduces the risk of allergic reactions caused by heterologous proteins and their fragments in antitoxin serum.

[0016] This immobilized dual-labeled enzyme solution reduces production costs and improves efficiency, and is an ideal solution for efficient and safe IgG preparation of F(ab')2. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is an SDS-PAGE gel electrophoresis diagram of an immobilized enzyme in one embodiment of the present invention; Figure 2 This is the SDS-PAGE gel electrophoresis diagram of the antitoxin serum stock solution in one embodiment of the present invention. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. 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.

[0019] The present invention provides a method for preparing an antitoxin serum containing a F(ab')2 fragment, the steps comprising: S1, the recombinant Streptococcus immunoglobulin G degrading enzyme was immobilized to obtain an immobilized enzyme chromatography column, wherein the recombinant Streptococcus immunoglobulin G degrading enzyme includes a glutathione-S-transferase tag and a histidine tag; S2, preliminarily treating the immune plasma to obtain a pretreated antibody solution; S3. Add the pretreated antibody solution to the immobilized enzyme chromatography column for digestion. After the digestion is completed, pass it through an immobilized glutathione affinity column or a nickel affinity column for ultrafiltration, purification and solution preparation to obtain an antitoxin serum stock solution. The antitoxin serum stock solution includes the F(ab')2 fragment but does not include the Fc fragment, miscellaneous proteins and small molecule impurities.

[0020] The present invention utilizes recombinant streptococcal immunoglobulin G degrading enzyme (IdeS) to specifically cleave IgG antibodies in immune plasma. The recombinant IdeS enzyme is fixed to the medium of the chromatography column by chemical bonding, so that it can be reused in multiple reactions, ensuring the catalytic stability of the enzyme chromatography column. When the pretreated antibody solution flows through the immobilized enzyme chromatography column, the IdeS enzyme can specifically cleave the heavy chain part of IgG to generate the required F(ab')2 fragment. After the traditional animal serum is digested with gastric enzymes, a large amount of undigested Fc fragments, impurities and incompletely digested Fc fragments and impurities may remain in the solution. These components have a large molecular weight distribution range and are not easy to completely remove. Their residues in the final antitoxin preparation may trigger an immune response. In contrast, through the specific action of the IdeS enzyme, the efficient generation of F(ab')2 fragments is achieved, while avoiding excessive digestion of Fc and other impurities, which increases the difficulty of the downstream purification process. In addition, the present invention designs a recombinant IdeS enzyme with a double tag of glutathione-S-transferase (GST) and histidine (His), which is fixed on an affinity column to make the use and removal of the enzyme easier. After the digestion is completed, the mixture can be further treated with a GST or Ni affinity column to ensure that any detached enzyme is completely removed, thereby greatly improving the purity and safety of the F(ab')2 product. After the recombinant IdeS enzyme removal step is completed, the product is ultrafiltration technology to remove Fc, small molecule impurities and undigested foreign proteins. After further purification, it is formulated into an antitoxin serum stock solution. The method of the present invention can efficiently remove Fc fragments and foreign proteins, retaining only F(ab')2 fragments, ensuring the high purity and safety of the final antitoxin serum preparation, while optimizing the activity of the antitoxin serum and significantly reducing its immunogenicity and allergic risks. This method not only simplifies the operating procedures and reduces the risk of residual impurities, but also reduces the contamination of animal-derived foreign proteins that may be introduced in traditional processes. By adopting the scheme of immobilized double-labeled IdeS enzymes, the production cost is significantly reduced and the efficiency is improved. It is an ideal solution for the efficient and safe preparation of F(ab')2 from IgG.

[0021] The specific steps of the recombinant streptococcal immunoglobulin G degrading enzyme with a glutathione-S-transferase tag and a histidine tag are as follows: Prepare GST tag sequence and His tag sequence. The GST tag sequence can be located at the N-terminus (i.e., the start end of the enzyme) or the C-terminus (i.e., the end of the enzyme). The specific location is determined according to the structure and functional requirements of the degradation enzyme. The His tag sequence can add 6 or more histidine residues (such as 6xHis) at the other end of the GST tag or the other end of the enzyme. For example, the tag combination can be: GST tag-degradation enzyme-His tag or His tag-degradation enzyme-GST tag.

[0022] Design primers for PCR amplification of gene fragments containing GST and His tags. The primer sequence contains the GST tag, the gene sequence of the degradation enzyme, and the His tag sequence. Suitable restriction sites are introduced at appropriate sites to facilitate subsequent cloning operations.

[0023] To construct a recombinant gene with a tag, the GST and His tag sequences are connected to the degradation enzyme gene fragment using PCR technology, which can be completed by fusion PCR to splice the tag gene and the degradation enzyme gene together. The equipment used in PCR technology is well known to those skilled in the art and will not be described in detail here.

[0024] The synthesized tagged degradation enzyme gene is inserted into an appropriate expression vector, such as pET, pGEX, etc., with a promoter and terminator to ensure efficient expression in the host. The inserted fragment and the vector can be connected by T4 DNA ligase to form a complete recombinant vector.

[0025] The constructed recombinant expression vector is transformed into a host cell (such as E. coli), and a resistant strain is selected for culture and amplification. Under appropriate induction conditions (such as IPTG induction), the degradation enzyme with GST and His tags is expressed in the host, and the protein with GST tag is purified using a GST affinity column, and then the protein with His tag is further purified through a nickel affinity column.

[0026] The culture, amplification, affinity column and other experimental equipment and usage methods used in the present invention are well known to those skilled in the art and will not be described in detail here.

[0027] In one embodiment, step S1 includes: S1.1, prepare coupling solution A and coupling solution B respectively; The coupling solution A includes at least one of hydrochloric acid, acetic acid, phosphoric acid, and formic acid, and the concentration of the coupling solution A is 0.5-2 mM, for example, 1 mM or 1.5 mM. The main function of these acidic substances is to clean and activate the NHS activation medium in the subsequent reaction, remove the unreacted NHS groups on the surface, and adjust its surface chemical environment to provide a stable chemical environment for the subsequent enzyme coupling.

[0028] The coupling solution B includes a buffer and an alkaline regulator, wherein the buffer includes at least one of sodium bicarbonate, disodium hydrogen phosphate, and triethanolamine, and the concentration of the buffer is 0.05-0.5 M, for example, 0.1 M or 0.2 M. The alkaline regulator includes at least one of sodium hydroxide, potassium hydroxide, and sodium carbonate, and the concentration of the alkaline regulator is 0.1-1 M, for example, 0.2 M or 0.3 M. The pH of the coupling solution is 8-9, for example, 8.3 or 8.5.

[0029] The addition of buffer can maintain the pH of the solution in the range of 8-9, which is crucial for the covalent coupling reaction between the NHS group and the enzyme. Appropriate pH conditions can ensure the stability of the reaction and avoid enzyme inactivation or hydrolysis of the NHS group on the surface of the medium. Alkaline regulators can increase the pH of the solution to a weak alkaline state, activate the amino group in the enzyme molecule, make it easier to react with the NHS group, thereby forming a stable amide bond and realizing the covalent immobilization of the enzyme.

[0030] S1.2, placing the NHS activation medium in the coupling solution A for washing, the washing time can be 30 minutes, 40 minutes, etc. Wherein, the volume ratio of the NHS activation medium to the coupling solution A is 1:15-30, for example, 1:15, 1:20.

[0031] NHS activated medium is an activated solid phase carrier material with NHS groups (N-hydroxysuccinimide ester groups) on its surface. This group can react with primary amine groups (such as amino groups on lysine residues) in molecules such as proteins to form covalent bonds. This property makes NHS activated medium very suitable for the immobilization of proteins, enzymes, antibodies, etc. in the field of biotechnology.

[0032] S1.3, placing the recombinant Streptococcus immunoglobulin G degrading enzyme in coupling solution B, so that the concentration of the recombinant Streptococcus immunoglobulin G degrading enzyme is 5-10 mg / ml, for example, 6 mg / ml, 8 mg / ml, etc. Then adding the washed NHS activation medium and stirring and mixing to obtain a mixed solution, wherein the volume ratio of the NHS activation medium to the recombinant Streptococcus immunoglobulin G degrading enzyme is 1:1-2, for example, 1:1, 1:1.5.

[0033] Coupling solution B provides a stable weak alkaline environment (pH 8-9), in which the primary amine groups in the recombinant Streptococcus immunoglobulin G degrading enzyme are in an active state and easily react with the NHS groups on the surface of the NHS activated medium. In this way, a stable amide bond can be formed on the surface of the medium to achieve covalent immobilization of the enzyme, thereby improving the coupling efficiency of the enzyme.

[0034] By placing the recombinant Streptococcal immunoglobulin G degrading enzyme in coupling solution B and then adding the washed NHS activated medium for stirring and mixing, sufficient contact and uniform distribution between the enzyme and the medium can be ensured. This operation ensures that the NHS group can fully combine with the active group of the enzyme through stirring and setting the volume ratio, thereby improving the efficiency of the coupling reaction.

[0035] Setting the volume ratio of NHS activation medium to enzyme at 1:1~2 can ensure that the concentration of enzyme in the entire coupling reaction is moderate, thereby avoiding excessive or insufficient enzyme consumption. If the concentration of the enzyme is too high, it may cause interaction or aggregation between enzymes, reducing the coupling efficiency; if the concentration of the enzyme is too low, it may cause a large number of unreacted NHS groups on the surface of the NHS activation medium, thereby wasting the activation medium. A reasonable volume ratio can maximize the use of enzymes and activation medium, ensure the efficient conduct of the coupling reaction, and reduce resource waste and production costs.

[0036] By using the buffer and alkaline regulator in the coupling solution B, a stable weak alkaline environment can be provided to prevent drastic fluctuations in pH, thereby protecting the structural stability of the enzyme and avoiding enzyme inactivation. Coupling under such conditions can maximize the activity of the enzyme and extend the service life of the immobilized enzyme.

[0037] S1.4, allowing the mixed solution to stand for sedimentation, removing the supernatant after sedimentation, adding a blocking solution to the precipitate of the mixed solution for blocking operation to obtain an immobilized enzyme, wherein the blocking time is 2 to 4 hours, for example, 2 hours or 3 hours, and the blocking temperature is 20 to 30°C, for example, 20°C or 25°C.

[0038] The blocking solution includes at least one of tris(hydroxymethyl)aminomethane hydrochloride, sodium bicarbonate-sodium carbonate buffer, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer. The concentration of the blocking solution is 0.05-0.5M, such as 0.1M or 0.2M. The pH of the blocking solution is 8-9, such as 8.3 or 8.5.

[0039] During the preparation of immobilized enzymes, NHS groups are used to form covalent bonds with primary amine groups of enzymes. However, after coupling, some unreacted NHS groups may still remain on the surface of the activated medium. If these residual NHS groups are not blocked, they may react nonspecifically with other molecules or proteins in subsequent use, resulting in the failure of the immobilized enzyme or reducing its specificity. The main technical effect of the blocking operation is to inactivate the residual NHS groups by reacting with the buffer in the blocking solution, thereby reducing nonspecific binding.

[0040] The composition of the blocking solution includes tris-HCl, sodium bicarbonate-sodium carbonate buffer and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES). These buffers have good pH buffering capacity and can maintain the pH stability of the immobilized enzyme during the blocking operation. pH stability is crucial for enzyme activity, so in the blocking operation, a suitable weak alkaline environment (pH 8~9) helps maintain the activity of the enzyme and prevents its inactivation during the reaction process.

[0041] The sealing operation can make the surface of the immobilized enzyme more uniform and flat, reducing the nonspecific adsorption and reaction that may occur in the subsequent reaction process.

[0042] The concentration range of the blocking solution is 0.05~0.5M. This setting can ensure that the buffer has sufficient buffering capacity and reaction efficiency during the blocking process, while avoiding side effects (such as enzyme denaturation or inactivation) caused by excessive concentration. The specific concentration (such as 0.1M or 0.2M) can be flexibly selected according to experimental requirements to achieve the best blocking effect.

[0043] The blocking operation is carried out under mild conditions of 20-30°C, which can effectively protect the activity of the enzyme and avoid protein denaturation caused by high temperature. The blocking time is set long enough to allow the buffer to fully react with the unreacted NHS groups, making the blocking process complete and efficient.

[0044] During the static deposition operation, when the deposition temperature is 20-30°C, the deposition time is 3-6 hours, for example, 4 hours or 5 hours; when the deposition temperature is 0-10°C, the deposition time is 12-20 hours, for example, 13 hours or 15 hours.

[0045] The matching of temperature and time can ensure the stability and uniformity of the deposition process. Higher temperatures can speed up the deposition process, so a shorter deposition time is sufficient for the deposition reaction to reach equilibrium. At lower temperatures, the reaction rate is slower and a longer deposition time is required to ensure sufficient and uniform deposition.

[0046] During the preparation of immobilized enzymes, excessively high temperatures may lead to loss of enzyme activity. By controlling the deposition temperature within the range of 20-30°C or lower, 0-10°C, the effect of high temperature on the enzyme can be reduced, thereby protecting the activity of the enzyme. Extending the deposition time at a lower temperature can ensure that the deposition process is fully carried out while lowering the temperature, achieving the goal of both ensuring enzyme activity and completing deposition.

[0047] At different deposition temperatures, the solution viscosity, molecular movement rate and diffusion rate will be different. By properly selecting the deposition temperature and time, the influence of these physical and chemical factors can be controlled so that the deposition reaction can proceed evenly and stably. The setting of temperature and time helps to avoid the unevenness and variation of the deposited layer thickness caused by too fast or too slow deposition rate.

[0048] Appropriate low temperature and long deposition time can reduce unnecessary nonspecific adsorption during the deposition process and avoid defects caused by too fast deposition. Low temperature conditions can effectively inhibit the diffusion and nonspecific binding of impurity molecules, thereby improving the purity and uniformity of the deposited layer.

[0049] S1.5, placing the immobilized enzyme alternately in the cleaning solution A and the cleaning solution B for alternate washing, the alternating times may be 3 to 6 times, for example, 3 times or 5 times. The volume ratio of the immobilized enzyme to the cleaning solution A is 1:2 to 5, for example, 1:3 or 1:4, and the volume ratio of the immobilized enzyme to the cleaning solution B is 1:2 to 5, for example, 1:3 or 1:4.

[0050] The cleaning solution A includes an alkaline pH buffer and an ionic strength regulator, the alkaline pH buffer includes at least one of tris(hydroxymethyl)aminomethane hydrochloride, sodium bicarbonate-sodium carbonate buffer, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, the concentration of the alkaline pH buffer is 0.05~0.5M, for example, it can be 0.1M or 0.2M, the ionic strength regulator includes at least one of potassium chloride, sodium hydroxide, and potassium hydroxide, the concentration of the ionic strength regulator is 0.1~1M, for example, it can be 0.2M or 0.3M, and the pH of the cleaning solution A is 8~9, for example, it can be 8.3 or 8.5.

[0051] The cleaning solution B includes an acidic pH buffer and an ionic strength regulator, the acidic pH buffer includes at least one of acetate buffer, citrate buffer, and phosphate buffer, the concentration of the acidic pH buffer is 0.05~0.5M, for example, it can be 0.1M or 0.2M, the ionic strength regulator includes at least one of potassium chloride, sodium hydroxide, and potassium hydroxide, the concentration of the ionic strength regulator is 0.1~1M, for example, it can be 0.2M or 0.3M, and the pH of the cleaning solution A is 3~4, for example, it can be 3.3 or 3.5.

[0052] During the preparation of immobilized enzymes, some unbound impurities or other proteins may be adsorbed on the surface. By alternately using cleaning solution A (alkaline) and cleaning solution B (acidic), the changes in molecular charge and solubility under different pH conditions can be used to effectively remove nonspecific adsorbed substances remaining on the surface of the immobilized enzyme and ensure the purity of the enzyme.

[0053] Cleaning solution A provides a weak alkaline environment (pH 8-9), which can help remove nonspecific binders formed under acidic conditions. Cleaning solution B provides an acidic environment (pH 3-4) that can remove impurities that are stable under alkaline conditions. This acid-base alternating cleaning can effectively reduce nonspecific adsorption on the surface of the immobilized enzyme and prevent residual chemicals from affecting the activity of the immobilized enzyme. This alternating cleaning method can completely remove residual substances on the surface without destroying the structure of the immobilized enzyme.

[0054] Both cleaning solution A and cleaning solution B include ionic strength regulators (such as potassium chloride, sodium hydroxide, potassium hydroxide, etc.), which can adjust the ionic strength of the solution to avoid the adverse effects of too low or too high ionic strength on the activity of the immobilized enzyme. Reasonable ionic strength can maintain the stability of the immobilized enzyme and prevent the denaturation or inactivation of the protein.

[0055] The volume ratio of the cleaning solution to the immobilized enzyme is 1:2~5. This volume ratio design can ensure that the amount of cleaning solution is sufficient to cover the immobilized enzyme and provide sufficient cleaning effect. In the process of alternating cleaning, a reasonable volume ratio can ensure that each round of cleaning operation can act evenly and comprehensively on the surface of the immobilized enzyme.

[0056] S1.6. Place the washed immobilized enzyme in a chromatography column and add a preservation solution to obtain an immobilized enzyme chromatography column.

[0057] The preservation solution includes a neutral buffer and an antibacterial agent, wherein the neutral buffer includes at least one of phosphate buffered saline, 3-(N-morpholine) propanesulfonic acid buffer, and tris(hydroxymethyl)aminomethane hydrochloride, wherein the concentration of the neutral buffer is 15-30 mM, for example, 20 mM or 25 mM, and the pH of the neutral buffer is 7-8, for example, 7.3 or 7.5. The antibacterial agent includes at least one of isopropanol, ethanol, and benzalkonium chloride, and the concentration of the antibacterial agent is 10%-30%, for example, 15% or 20%.

[0058] The neutral buffer in the preservation solution can provide a stable pH condition, allowing the immobilized enzyme to maintain its natural conformation and catalytic activity in a neutral environment. This pH range is the optimal pH range for most enzymes and can effectively prevent enzyme inactivation or denaturation caused by pH fluctuations.

[0059] The appropriate concentration of neutral buffer can provide sufficient buffering capacity to prevent pH fluctuations caused by external factors (such as temperature or slight contamination) during storage. By maintaining a neutral environment, adverse effects on the structure and function of the immobilized enzyme can be avoided, thereby improving the stability of the enzyme.

[0060] The antibacterial agents added to the preservation solution can effectively inhibit and kill microorganisms, preventing microbial contamination during long-term storage. The concentration of these antibacterial agents is set at 10%~30%, which can provide sufficient antibacterial activity without damaging the immobilized enzyme. In particular, benzalkonium chloride, as a broad-spectrum antibacterial agent, can effectively prevent the growth of microorganisms such as bacteria and mold during the storage process.

[0061] By adding appropriate neutral buffers and antibacterial agents, the preservation solution can provide a stable, sterile storage environment for the immobilized enzyme, preventing enzyme activity decline or contamination problems caused by improper storage. This optimized storage condition helps to extend the service life of the immobilized enzyme, thereby improving the stability and economy of the process.

[0062] The components in the preservation solution help to reduce the nonspecific adsorption of the immobilized enzyme on the surface during storage. In particular, the use of phosphate buffer or MOPS buffer containing an appropriate ion concentration can effectively control the ion strength and avoid nonspecific adsorption of proteins or other impurities, thereby maintaining the purity of the immobilized enzyme.

[0063] Step S1 also includes: S1.7, take the pre-packed column in the immobilized enzyme chromatography column, and react according to the preset reaction system. The preset reaction system is shown in Table 1.

[0064] Table 1: Components concentration IgG 1mg Tris-HCl (pH 7.5) 50mM IdeS 100μl After the reaction is completed, the activity of the enzyme is detected by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The reaction system sets specific pH and buffer conditions to ensure the specific cleavage of IgG by IdeS enzyme. Through SDS-PAGE analysis, it can be detected whether IgG is specifically cleaved into F(ab')2 fragments and Fc fragments by IdeS enzyme, thereby verifying the activity of the immobilized enzyme.

[0065] The results are as follows Figure 1 As shown, the prepared immobilized enzyme chromatography column can be recycled 30 times without a significant decrease in activity, showing good reusability.

[0066] Figure 1 Including a single lane on the left and multiple lanes on the right, a standard marker and intact IgG (lane 1) are shown on the left, providing a control for intact IgG, and the SDS-PAGE on the right shows the IgG fragments cut with IdeS immobilized enzyme at different cycle times. From left to right in the figure are marked as: Lane M: marker, used to mark protein molecular weight, Lane 1: the result of the first reaction using IdeS immobilized enzyme, Lane 2: the result of the 4th cycle, Lane 3: the result of the 7th cycle, Lane 4: the result of the 10th cycle, Lane 5: the result of the 13th cycle, Lane 6: the result of the 16th cycle, Lane 7: the result of the 19th cycle, Lane 8: the result of the 22nd cycle, Lane 9: the result of the 25th cycle, Lane 10: the result of the 30th cycle.

[0067] In lane 1 (first use), it can be clearly seen that IgG is cut by the IdeS immobilized enzyme to form characteristic fragments, showing two significant bands of approximately 50 kDa and 25 kDa, representing the Fc fragment and the F(ab')2 fragment. As the number of cycles increases (from lanes 1 to 10), the intensity of the 25 kDa and 50 kDa bands in each lane remains basically the same, indicating that the cutting effect of the IdeS immobilized enzyme has not changed significantly with the increase in the number of uses.

[0068] From lane 1 to lane 10 (i.e., from the first use to the 30th cycle), the presence and intensity of the 25 kDa and 50 kDa bands in each lane did not change significantly, indicating that the activity of the immobilized IdeS enzyme was still stably maintained after multiple uses. No obvious decrease in band intensity or the appearance of new bands was observed, indicating that the specificity and cleavage efficiency of the enzyme were well maintained.

[0069] Compared with the bands in the intact IgG on the left (lane 1), no similar bands were seen in each lane on the right, indicating that the intact IgG was successfully cleaved with almost no residue. This further verifies the efficient cleavage ability of the IdeS enzyme.

[0070] In one embodiment, step S2 includes: S2.1. Filter and centrifuge the immune plasma in sequence to obtain antibody serum. The centrifugation temperature can be 4°C, 8°C, etc., the centrifugation speed can be 10000~15000r / min, for example, 12000r / min, 13000r / min, etc., and the centrifugation time can be 10~20min, for example, 15min, 18min, etc. The immune plasma can be extracted from animals or humans after immunizing them using toxins / toxoids or vaccines as antigens.

[0071] The filtration operation can effectively remove large particle impurities, cell debris and other insoluble substances in immune plasma. This operation ensures the smooth progress of subsequent process treatment and avoids clogging or interference of further processing and purification of antibodies due to particles.

[0072] The main purpose of centrifugation is to obtain a purer supernatant (antibody supernatant) by settling suspended particles, precipitated proteins and other high-molecular impurities in plasma through high-speed rotation. Centrifugation can significantly reduce the content of non-antibody components in plasma and improve the efficiency of subsequent antibody extraction and purification.

[0073] The combined treatment of filtration and centrifugation can significantly improve the purity of antibody serum and remove impurities such as cell debris and precipitated proteins in immune plasma. This purification process provides high-quality raw materials for subsequent antibody extraction and processing.

[0074] During the filtration and centrifugation process, appropriate temperature and conditions can be selected to reduce the impact and damage to the antibody and ensure that the biological activity and function of the antibody are maintained. This is of great significance for the subsequent digestion and fragmentation of the antibody.

[0075] S2.2, adding an acidic buffer to the antibody supernatant, stirring and mixing, adding a precipitation solution during the stirring process, and performing centrifugation and filtering operations to obtain an antibody filtrate, wherein the volume ratio of the antibody supernatant to the acidic buffer is 1:2-4, for example, 1:2, 1:3, etc. The precipitation solution includes an acidic regulator and a precipitant, the acidic regulator includes at least one of acetic acid, hydrochloric acid, and phosphoric acid, and the precipitant includes at least one of ethanol, n-octanoic acid, and isopropanol.

[0076] Specifically, step S2.2 includes: S2.2.1. Add an acidic buffer to the antibody serum and stir to mix, wherein the acidic buffer includes at least one of an acetate buffer, a citrate buffer, and a phosphate buffer, and the concentration of the acidic buffer is 0.02-0.1 M, for example, 0.03 M, 0.05 M, etc. The pH of the acidic buffer is 4-5, for example, 4.3, 4.5, etc. The volume ratio of the antibody serum to the acidic buffer is 1:2-4, for example, 1:3, 1:4, etc.

[0077] By adding an acidic buffer to the antibody serum, the pH of the solution can be precisely adjusted to 4-5. This pH range is the region where most antibodies exist stably in solution, and it is also an ideal acidic environment for precipitation of impurities and proteins. The ionic strength and pH adjustment of the acidic buffer can significantly affect the solubility of the antibody, optimize the precipitation conditions of the antibody, keep the target antibody dissolved, and promote the precipitation of impurities or other non-target components.

[0078] S2.2.2, maintain stirring and mixing operation, first add acidic regulator during stirring, then drop precipitant, let stand for sedimentation after drop addition, centrifuge and filter in sequence to obtain antibody filtrate. Centrifuge temperature can be 4°C, 8°C, etc., centrifuge speed can be 10000~15000r / min, for example, 12000r / min, 13000r / min, etc., centrifuge time can be 2~10min, for example, 5min, 8min, etc.

[0079] After adding an acidic buffer to the antibody supernatant, the pH of the solution can be further accurately lowered by further adding an acidic regulator. This operation is intended to enhance the precipitation efficiency of foreign proteins and impurities and ensure the purity of the antibody filtrate. The choice of acidic regulator can adjust the acid strength of the solution as needed to promote the precipitation process.

[0080] The addition of precipitants can significantly reduce the dielectric constant of the solution and promote the selective precipitation of proteins and lipids. In particular, n-octanoic acid and isopropanol have a strong effect on precipitating lipids and impurities, and can effectively remove non-target components in the antibody serum. This operation ensures uniformity and completeness of the precipitation process by gradually adding acidic regulators and precipitants, combined with stirring and static sedimentation. Finally, pure antibody filtrate can be obtained by centrifugation and filtration.

[0081] During the operation, the pH range of the solution is accurately controlled by selecting appropriate acid buffers and acid regulators to ensure the stability of the antibody and avoid antibody denaturation or loss of activity caused by low pH or instability of the solution medium.

[0082] Setting the volume ratio of antibody serum to acidic buffer at 1:2~4 can ensure that the pH and ionic strength of the solution can be fully adjusted under dilution conditions. This volume ratio selection helps to improve the precipitation efficiency of impurities and reduce the impact on antibodies.

[0083] S2.3, adding a buffer to the antibody filtrate, and then dropping an alkaline solution to obtain an antibody buffer, wherein the pH value of the antibody buffer is 7 to 7.5, for example, 7.3, 7.4, etc. The volume ratio of the buffer to the antibody filtrate is 1:8 to 15, for example, 1:9, 1:10, etc.

[0084] The buffer includes at least one of phosphate buffered saline, 3-(N-morpholine) propanesulfonic acid buffer, and tris(hydroxymethyl)aminomethane hydrochloride, and the concentration of the buffer is 0.05-0.5 M, such as 0.1 M, 0.2 M, etc. The pH of the buffer is 7-8, such as 7.3, 7.5, etc.

[0085] The alkaline solution includes at least one of sodium hydroxide, potassium hydroxide, and sodium bicarbonate. The concentration of the alkaline solution is 1-3M, for example, 1M, 2M, etc.

[0086] Adding a buffer to the antibody filtrate can provide a stable pH environment for the entire solution, and the pH of the antibody buffer can be set in the range of 7 to 7.5. This pH range is the ideal area for many antibodies to exist stably in solution, and can prevent the antibody from being inactivated or denatured under acidic or alkaline conditions.

[0087] The addition of buffer not only helps to adjust the pH of the solution, but also can further stabilize the conformation of the antibody by controlling the ionic strength and chemical environment. Using the appropriate buffer concentration can provide sufficient buffering capacity to prevent the rapid change of pH when adding alkaline solution, thereby effectively protecting the activity of the antibody.

[0088] The purpose of adding alkaline solution is to accurately adjust the pH of the antibody filtrate to the target range of 7 to 7.5. This fine adjustment helps to obtain consistent product quality during the production process and avoid antibody denaturation problems caused by too high or too low pH.

[0089] By mixing the buffer with the antibody filtrate in an appropriate volume ratio, the buffer environment can be gradually adjusted to reduce the ion shock and electrolyte imbalance caused by the rapid addition of alkaline solution. Reasonable selection and proportion control of the buffer can help maintain the electrolyte balance in the solution and avoid affecting the structure and function of the antibody.

[0090] S2.4, ice bathing the antibody buffer, adding a protein precipitant and stirring, and then standing and settling after stirring to obtain an antibody deposition solution. The protein precipitant includes at least one of ammonium sulfate, isopropanol, and polyethylene glycol, and the concentration of the protein precipitant is 250-300 g / L, for example, 277 g / L, 280 g / L, etc. The specific equipment for ice bath operation is well known to those skilled in the art and will not be described in detail here.

[0091] Under the low temperature conditions of the ice bath, the thermal motion of the protein slows down, the interaction between molecules is enhanced, and the solubility of the protein is reduced, thereby promoting precipitation. Low temperature treatment can also protect the structure and activity of the antibody and prevent antibody denaturation caused by high temperature. The role of the protein precipitant is to aggregate and precipitate the protein by changing the physical and chemical properties of the solution, which can effectively reduce the interaction between protein molecules and water molecules and promote the precipitation of the protein from the solution. Thereby, non-target proteins and impurities are selectively precipitated, while the target antibody is concentrated.

[0092] Low temperature ice bath treatment combined with appropriate precipitants can effectively achieve protein precipitation and separation without inactivating antibodies. By controlling the temperature and adding precipitants, the structure and function of the antibody can be protected throughout the operation.

[0093] By precipitating and separating non-target proteins and impurities in this step, the burden of handling the antibody solution in the subsequent purification process can be significantly reduced, thereby improving the efficiency of the entire production process. At the same time, the antibody solution after precipitation treatment has a higher concentration and is more suitable for subsequent filtration and refining operations.

[0094] S2.5, centrifuging the antibody deposit solution to separate the clear solution and the precipitate of the antibody deposit solution, adding a stabilizing solution to the precipitate of the antibody deposit solution, centrifuging, filtering, ultrafiltration, and concentration in sequence to obtain a pretreated antibody solution, wherein the volume ratio of the preservative solution to the clear solution of the antibody deposit solution is 1:8-15, for example, 1:9, 1:10, etc. The stabilizing solution includes at least one of a phosphate buffered saline solution, a sodium bicarbonate buffer solution, and a citric acid buffer solution, and the concentration of the stabilizing solution is 0.005-0.05M, for example, 0.01M, 0.03M, etc. The centrifugal speed can be 10000-15000r / min, for example, 12000r / min, 13000r / min, etc., and the centrifugal time can be 10-30min, for example, 15min, 18min, etc.

[0095] The function of the stabilizer is to provide a stable pH and ionic strength for the antibody, preventing the antibody from denaturing or inactivating due to pH changes or uneven ionic strength during centrifugation and filtration. By adding a low concentration of stabilizer to the precipitate, the effect of high ion concentration on the antibody structure can be avoided, while providing sufficient buffering capacity to maintain the stability of the antibody solution.

[0096] The stabilizer can improve the solubility of the antibody and prevent the antibody from re-precipitating by adjusting the pH and ion environment of the solution. Especially during operations such as centrifugation and filtration, the stabilizer can effectively reduce the aggregation and precipitation of antibody molecules, thereby improving the quality and purity of the final antibody pretreatment solution.

[0097] During the antibody precipitation process, adding a stabilizing solution to the precipitate can prevent the antibody molecules from being lost due to precipitation or adsorption. The stabilizing solution can provide an appropriate ionic environment to reduce the risk of antibodies adhering to the membrane surface or the inner wall of the container during ultrafiltration and concentration, thereby improving the recovery rate of the antibody.

[0098] The components in the stabilizing solution, such as phosphate buffer and sodium bicarbonate buffer, can provide the antibody with chemical conditions similar to the in vivo environment, thereby maintaining the biological activity of the antibody. After multiple centrifugation, filtration and ultrafiltration operations, the stabilizing solution can play a protective role, allowing the antibody to maintain its activity during long-term storage and multiple uses.

[0099] In one embodiment, step S3 includes: S3.1, connect the immobilized enzyme chromatography column to the fluid control device, first pass the equilibrium solution, and then pass the pre-treated antibody solution to perform enzymatic hydrolysis reaction, wherein the reaction time is 30 to 60 minutes, for example, 35 minutes, 40 minutes, etc. The equilibrium solution includes at least one of water for injection, deionized water, pure water, and glycerol. The fluid control device is used to control the flow rate of the solution. The fluid control device can be a plunger pump, a syringe pump, a gear pump, a diaphragm pump, a rotary vane pump, etc. The specific structure of the fluid control device is well known to those skilled in the art and will not be repeated here.

[0100] The operation of passing the equilibrium solution can adjust the chemical environment of the immobilized enzyme chromatography column to an appropriate state, ensuring that the activity of the enzyme is in the best state before contacting the antibody solution. The functions of the equilibrium solution include removing residual substances, adjusting pH and maintaining ionic strength, thereby providing a stable starting condition for subsequent enzymatic reactions.

[0101] The reaction time is set at 30-60 minutes. The main purpose is to ensure that the enzymatic reaction can be fully completed within the activity range of the immobilized enzyme, to ensure that the IgG in the pretreated antibody solution is effectively cleaved into the required antibody fragment (F(ab')2 fragment), and to avoid excessive degradation or side reactions caused by too long a reaction time.

[0102] By controlling the reaction time, the integrity of the antibody fragment can be maintained to the greatest extent possible, avoiding excessive cutting or enzyme inactivation. By connecting the fluid control device, the flow rate of the equilibrium solution and the antibody solution can be precisely controlled to ensure that the solution stays in the immobilized enzyme chromatography column long enough to achieve the best reaction efficiency. This precise control method can improve the overall efficiency of the enzymatic reaction while reducing the waste of enzymes and reactants.

[0103] During the process of introducing the equilibrium solution and carrying out the reaction, impurities and residual substances on the immobilized enzyme chromatography column can be removed, thereby extending the service life of the immobilized enzyme. Through reasonable reaction control and maintenance, the immobilized enzyme chromatography column can be used multiple times, reducing production costs.

[0104] S3.2. Pass the pretreated antibody solution after the enzymatic hydrolysis reaction into an immobilized glutathione affinity column or a nickel affinity column to perform a chromatography reaction.

[0105] By utilizing the specific binding ability of immobilized glutathione or nickel ions, target antibodies with specific tags (glutathione-S-transferase tag and histidine tag) can be efficiently separated. Through this selective adsorption, non-target proteins and impurities can be effectively removed in the unbinding or elution steps, thereby significantly improving the purity of the antibody.

[0106] S3.3. After the chromatography reaction is completed, the pretreated antibody solution is subjected to an ultrafiltration cycle to obtain an antibody ultrafiltrate.

[0107] The pre-treated antibody solution is passed through an immobilized glutathione or column or a nickel affinity column to adsorb and remove the recombinant streptococcal immunoglobulin G degrading enzyme, ensuring that any detached recombinant streptococcal immunoglobulin G degrading enzyme is completely removed. Afterwards, the pre-treated antibody solution is subjected to an ultrafiltration cycle until the Fc fragment in the pre-treated antibody solution is completely removed to obtain an antibody ultrafiltrate. This ultrafiltration cycle step can also be replaced by a Protein A / Protein G or Protein A / G affinity column to remove Fc by affinity. The equipment and methods used for immobilized glutathione or nickel affinity columns, ProteinA, ProteinG, ProteinA / G affinity columns and ultrafiltration are well known to those skilled in the art and will not be repeated here.

[0108] Ultrafiltration technology can effectively remove small molecule impurities, unreacted enzyme fragments, salts and other low molecular weight substances in antibody solutions according to the pore size of the membrane. Through this selective filtration, the purity of the antibody solution can be significantly improved, providing high-quality antibody preparations for subsequent purification or application.

[0109] Ultrafiltration technology uses the principle of physical separation and performs filtration under the action of pressure difference. It usually has little effect on proteins (such as antibodies) and can effectively protect the activity of antibodies. By selecting the appropriate ultrafiltration membrane pore size and operating conditions, it is possible to remove impurities while maintaining the structure and function of the antibody.

[0110] Ultrafiltration cycles can continuously remove reaction byproducts and small molecule impurities, ensuring the stability and consistency of the antibody solution throughout the process. This approach can improve the overall efficiency of the process and reduce processing time and operation steps.

[0111] Compared with traditional precipitation or other purification methods, ultrafiltration is a physical separation technology that does not rely on chemical reagents, so it can reduce the use of organic solvents and salts, thereby reducing production costs and environmental burden.

[0112] S3.4, add osmotic pressure regulator, preservative and acid-base regulator to the antibody ultrafiltrate in sequence, and obtain antitoxin serum stock solution after sterilization and filtration, wherein the pH of the antitoxin serum stock solution is 6-7, for example, it can be 6.3 or 6.5. The osmotic pressure regulator includes at least one of sodium chloride, potassium chloride and sodium sulfate, and the concentration of the osmotic pressure regulator is 8-9 g / L, for example, it can be 8.3 g / L or 8.5 g / L; the preservative includes at least one of benzalkonium chloride, metacresol and sodium benzoate, and the concentration of the preservative is 2-3 g / L, for example, it can be 2.3 g / L or 2.5 g / L; the acid-base regulator includes at least one of dilute hydrochloric acid, dilute sodium hydroxide, sodium bicarbonate and acetic acid.

[0113] Osmotic pressure regulators are used to adjust the osmotic pressure of the antitoxin serum stock solution to a level that matches the osmotic pressure of human body fluids. This can prevent cell rupture or protein denaturation caused by osmotic pressure differences, thereby improving the biocompatibility and safety of the antitoxin. Setting the concentration of the osmotic pressure regulator at 8~9g / L can ensure that the osmotic pressure of the solution is within an appropriate range, thereby protecting the stability and activity of the antibody fragment.

[0114] The main function of preservatives is to inhibit and kill microorganisms to ensure the sterility and safety of the antitoxin serum during storage and use. By selecting a preservative with an appropriate concentration (2~3g / L), it is possible to ensure the antibacterial effect while avoiding adverse effects on antibodies or organisms.

[0115] Each preservative has its own unique antimicrobial spectrum and mechanism of action. Reasonable selection and combination of these preservatives can cover a wide range of microbial species and further improve the safety of antitoxins.

[0116] The pH of the antitoxin serum stock solution can be adjusted to a range of 6-7 by adding an acid-base regulator (such as dilute hydrochloric acid, dilute sodium hydroxide, sodium bicarbonate or acetic acid). This pH range is stable for many antibody fragments and proteins and can prevent antibody inactivation or structural changes caused by excessively high or low pH. Accurate pH adjustment is key to ensuring antibody activity and antitoxin function. By gradually adding an acid-base regulator, the pH of the solution can be finely controlled to avoid damage to the antibody due to rapid changes.

[0117] The sterilization filtration operation can effectively remove bacteria and microorganisms in the solution, further improving the safety of the antitoxin serum stock solution. This step, combined with the use of preservatives, can ensure the sterility of the antitoxin during storage and use.

[0118] Through osmotic pressure adjustment, preservative addition and pH control, these steps can enhance the stability of the antitoxin and avoid degradation or failure during storage and use. The final antitoxin serum solution has good physical and chemical properties and biocompatibility, and can effectively play its antitoxin role.

[0119] Step S3 also includes: S3.5. Take a certain volume of antitoxin serum stock solution sample and conduct stock solution testing.

[0120] The stock solution detection step is carried out by loading the sample according to the standard SDS-PAGE operation method, and the protein fragments in the stock solution are separated by electrophoresis so as to observe the characteristic bands and purity of the purified antitoxin (F(ab')2). The specific operation of the SDS-PAGE operation method is well known to those skilled in the art and will not be repeated here.

[0121] Figure 2 This is the electrophoresis image of the original solution detection. Figure 2 Including Marker (M) and antitoxin serum stock solution (lane 1), the molecular weight standard (Marker) of the protein is displayed in the lane of Marker (M), marking the common molecular weight position, and the 25kDa and 35kDa markers are shown in the figure. These markers are used to estimate the molecular weight of the electrophoresis band. Lane 1 shows the electrophoresis results of the prepared antitoxin serum stock solution. Two main bands can be clearly seen in this lane, with positions close to 25kDa and 35kDa, respectively, which are consistent with the target antibody fragment F(ab')2 fragment.

[0122] The two bands shown in lane 1 indicate that the antitoxin serum stock solution contains antibody fragments close to 25kDa and 35kDa, indicating that IgG was successfully cleaved into F(ab')2 fragments, and the Fc fragment and other impurity proteins were removed after the purification step. The results of SDS-PAGE were consistent with expectations, indicating that the preparation process achieved the expected effect.

[0123] From the electrophoresis bands in lane 1, the main bands are concentrated at the target molecular weight position, and no other obvious impurity protein bands are observed. This indicates that the purity of the prepared antitoxin serum stock solution is high and the process control is good.

[0124] The SDS-PAGE analysis results confirmed that the immobilized enzyme used in the preparation process was efficient and specific in cleaving IgG, and the quality of the antitoxin serum stock solution finally obtained met the expected requirements.

[0125] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an antitoxin serum containing a F(ab')2 fragment, characterized in that the steps include: S1. Immobilizing a recombinant Streptococcus immunoglobulin G degrading enzyme to obtain an immobilized enzyme chromatography column, wherein the recombinant Streptococcus immunoglobulin G degrading enzyme comprises a glutathione-S-transferase tag and a histidine tag; S2, preliminarily treating the immune plasma to obtain a pretreated antibody solution; S3. Add the pretreated antibody solution to an immobilized enzyme chromatography column for digestion. After digestion, pass it through an immobilized glutathione affinity column or a nickel affinity column for ultrafiltration, purification and solution preparation to obtain an antitoxin serum stock solution, which includes the F(ab')2 fragment but does not include the Fc fragment, miscellaneous proteins and small molecule impurities.

2. The method for preparing an antitoxin serum containing F(ab')2 fragment according to claim 1, characterized in that: The step S1 comprises: S1.1, prepare coupling solution A and coupling solution B respectively; S1.2, placing the NHS activation medium in the coupling solution A for washing, wherein the volume ratio of the NHS activation medium to the coupling solution A is 1:15-30; S1.3, placing the recombinant Streptococcus immunoglobulin G degrading enzyme in the coupling solution B, and then adding the washed NHS activation medium to stir and mix to obtain a mixed solution, wherein the volume ratio of the NHS activation medium to the recombinant Streptococcus immunoglobulin G degrading enzyme is 1:1-2; S1.4, allowing the mixed solution to stand for sedimentation, removing the supernatant after the sedimentation is completed, adding a blocking solution to the precipitate of the mixed solution for a blocking operation to obtain an immobilized enzyme, wherein the blocking time is 2 to 4 hours and the blocking temperature is 20 to 30°C; S1.5, placing the immobilized enzyme alternately in cleaning solution A and cleaning solution B for alternate washing, wherein the volume ratio of the immobilized enzyme to the cleaning solution A is 1:2-5, and the volume ratio of the immobilized enzyme to the cleaning solution B is 1:2-5; S1.

6. Place the immobilized enzyme after washing in a chromatography column and add a preservation solution to obtain an immobilized enzyme chromatography column.

3. The method for preparing an antitoxin serum containing F(ab')2 fragment according to claim 2, characterized in that: In the step S1.4, when the deposition temperature is 20-30°C, the deposition time is 3-6 hours, and when the deposition temperature is 0-10°C, the deposition time is 12-20 hours.

4. The method for preparing an antitoxin serum containing F(ab')2 fragment according to claim 2, characterized in that: The coupling solution A includes at least one of hydrochloric acid, acetic acid, phosphoric acid, and formic acid, and the concentration of the coupling solution A is 0.5-2 mM; The coupling solution B includes a buffer and an alkaline regulator, wherein the buffer includes at least one of sodium bicarbonate, disodium hydrogen phosphate, and triethanolamine, and the concentration of the buffer is 0.05-0.5M, and the alkaline regulator includes at least one of sodium hydroxide, potassium hydroxide, and sodium carbonate, and the concentration of the alkaline regulator is 0.1-1M, and the pH of the coupling solution is 8-9; The blocking solution includes at least one of tris(hydroxymethyl)aminomethane hydrochloride, sodium bicarbonate-sodium carbonate buffer, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, the concentration of the blocking solution is 0.05-0.5M, and the pH of the blocking solution is 8-9; The cleaning solution A comprises an alkaline pH buffer and an ionic strength regulator, wherein the alkaline pH buffer comprises at least one of tris(hydroxymethyl)aminomethane hydrochloride, sodium bicarbonate-sodium carbonate buffer, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, and the concentration of the alkaline pH buffer is 0.05-0.5M, and the ionic strength regulator comprises at least one of potassium chloride, sodium hydroxide, and potassium hydroxide, and the concentration of the ionic strength regulator is 0.1-1M, and the pH of the cleaning solution A is 8-9; The cleaning solution B comprises an acidic pH buffer and an ionic strength regulator, wherein the acidic pH buffer comprises at least one of an acetate buffer, a citrate buffer, and a phosphate buffer, and the concentration of the acidic pH buffer is 0.05-0.5 M, and the ionic strength regulator comprises at least one of potassium chloride, sodium hydroxide, and potassium hydroxide, and the concentration of the ionic strength regulator is 0.1-1 M, and the pH of the cleaning solution A is 3-4; The preservation solution includes a neutral buffer and an antibacterial agent, the neutral buffer includes at least one of phosphate buffered saline, 3-(N-morpholine)propanesulfonic acid buffer, and tris(hydroxymethyl)aminomethane hydrochloride, the concentration of the neutral buffer is 15-30 mM, the pH of the neutral buffer is 7-8, and the antibacterial agent includes at least one of isopropanol, ethanol, and benzalkonium chloride, and the concentration of the antibacterial agent is 10%-30%.

5. The method for preparing an antitoxin serum containing F(ab')2 fragment according to claim 1, characterized in that: The step S2 comprises: S2.

1. Filter and centrifuge the immune plasma in sequence to obtain antibody serum; S2.2, adding an acidic buffer to the antibody serum, stirring and mixing, adding a precipitating solution during the stirring process, and performing centrifugation and filtering operations to obtain an antibody filtrate, wherein the volume ratio of the antibody serum to the acidic buffer is 1:2-4; S2.3, adding a buffer to the antibody filtrate, and then dropping an alkaline solution to obtain an antibody buffer, wherein the pH value of the antibody buffer is 7-7.5, and the volume ratio of the buffer to the antibody filtrate is 1:8-15; S2.4, placing the antibody buffer in an ice bath, adding a protein precipitant and stirring, and allowing to stand for sedimentation after the stirring is completed to obtain an antibody sedimentation solution; S2.5, centrifuging the antibody deposition liquid to separate the clear liquid and the precipitate of the antibody deposition liquid, adding a stabilizing liquid to the precipitate of the antibody deposition liquid, and sequentially performing centrifugation, filtration, ultrafiltration, and concentration to obtain a pretreated antibody solution, wherein the volume ratio of the preservation liquid to the clear liquid of the antibody deposition liquid is 1:8~15.

6. The method for preparing an antitoxin serum containing F(ab')2 fragment according to claim 5, characterized in that: In step S2.3, the buffer includes at least one of phosphate buffered saline, 3-(N-morpholine) propanesulfonic acid buffer, and tris(hydroxymethyl)aminomethane hydrochloride, the concentration of the buffer is 0.05-0.5M, and the pH of the buffer is 7-8; The alkaline solution includes at least one of sodium hydroxide, potassium hydroxide, and sodium bicarbonate, and the concentration of the alkaline solution is 1-3M; In step S2.4, the protein precipitant includes at least one of ammonium sulfate, isopropanol, and polyethylene glycol, and the concentration of the protein precipitant is 250-300 g / L; In step S2.5, the stabilizing solution includes at least one of phosphate buffered saline, sodium bicarbonate buffer, and citric acid buffer, and the concentration of the stabilizing solution is 0.005-0.05M.

7. The method for preparing an antitoxin serum containing F(ab')2 fragment according to claim 5, characterized in that: In the step S2.2, the precipitation solution includes an acidic regulator and a precipitant, the acidic regulator includes at least one of acetic acid, hydrochloric acid, and phosphoric acid, and the precipitant includes at least one of ethanol, n-octanoic acid, and isopropanol; The step S2.2 comprises: S2.2.1, adding an acidic buffer to the antibody serum and stirring and mixing, wherein the acidic buffer comprises at least one of an acetate buffer, a citrate buffer, and a phosphate buffer, the concentration of the acidic buffer is 0.02-0.1 M, the pH of the acidic buffer is 4-5, and the volume ratio of the antibody serum to the acidic buffer is 1:2-4; S2.2.

2. Maintain stirring and mixing operation, first add the acidic regulator during stirring, then dropwise add the precipitant, after the dropwise addition is completed, let it stand and settle, then perform centrifugation and filtration operations in sequence to obtain the antibody filtrate.

8. The method for preparing an antitoxin serum containing F(ab')2 fragment according to claim 1, characterized in that: The step S3 comprises: S3.1, connecting the immobilized enzyme chromatography column to a fluid control device, first introducing the equilibrium solution, and then introducing the pre-treated antibody solution to perform enzymatic hydrolysis reaction, wherein the reaction time is 30 to 60 minutes; S3.2, passing the pretreated antibody solution after the enzymatic hydrolysis reaction into an immobilized glutathione affinity column or a nickel affinity column for chromatography reaction; S3.3, performing ultrafiltration cycle on the pretreated antibody solution after the chromatography reaction is completed to obtain an antibody ultrafiltrate; S3.

4. Add an osmotic pressure regulator, a preservative and an acid-base regulator to the antibody ultrafiltrate in sequence, sterilize and filter to obtain an antitoxin serum stock solution, wherein the pH of the antitoxin serum stock solution is 6-7.

9. The method for preparing an antitoxin serum containing F(ab')2 fragment according to claim 8, characterized in that: In step S3, the equilibrium solution includes at least one of water for injection, deionized water, pure water, and glycerol; The osmotic pressure regulator includes at least one of sodium chloride, potassium chloride and sodium sulfate, and the concentration of the osmotic pressure regulator is 8-9 g / L; The preservative includes at least one of benzalkonium chloride, m-cresol, and sodium benzoate, and the concentration of the preservative is 2-3 g / L; The acid-base regulator includes at least one of dilute hydrochloric acid, dilute sodium hydroxide, sodium bicarbonate, and acetic acid.

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