Synthesis method of adsorption resin for removing immune globulin

Abstract

The invention provides a synthesis method of adsorption resin for removing immune globulin, and relates to a high polymer material synthesis technology, polyethylene glycol (PEG) or sulfobetaine (SBMA) is grafted through RAFT polymerization, the thickness of a grafting layer is 50-150nm, the nonspecific adsorption capacity of albumin is less than 5ng / cm < 2 >, photosensitive groups (such as phenyl azide) and ultraviolet light (254nm) are introduced to trigger crosslinking, and the coating shedding rate is less than 0.5% / time. According to the present invention, the cyclic stability is improved by 2 times, the Protein A directional capture antibody is grafted on the surface of the acrylic resin grafted with the polyethylene glycol or sulfobetaine, the IgG adsorption capacity achieves 150 mg / g, the specificity is more than 95%, the polyethylene glycol (PEG) is grafted on the surface of the magnetic particle by using ATRP / RAFT controllable polymerization, the organic solvent use amount is reduced by more than 80%, and the product adsorption capacity achieves 150 mg / g.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of high polymer material synthesis, and particularly relates to a synthesis method for removing immunoglobulin adsorption resin. BACKGROUND

[0002] According to Chinese Patent No. CN114106231B, an adsorbent resin and a preparation method thereof are disclosed. The adsorbent resin is mainly obtained by chemical modification reaction of porous polystyrene-based microspheres with epoxy groups or halogenated groups and amine-containing compounds. The preparation method avoids the use of carcinogenic chloromethyl methyl ether during the preparation process, thereby solving the safety and environmental protection problems caused by the use of carcinogenic chloromethyl methyl ether in the traditional process. The new crosslinking agent system is adopted to realize the introduction of reaction functional groups (halogenated groups or epoxy groups) with amine substances and the post-crosslinking of the resin, so that the specific pore structure, specific surface area and ion exchange capacity of the adsorbent resin can be effectively controlled. The preparation and development of the new adsorbent resin help to expand the application of the adsorbent resin in important fields such as high-performance adsorbent, blood purification, catalysis and energy.

[0003] According to Chinese Patent No. CN118638253A, a cytokine adsorption resin material and a preparation method thereof are disclosed. The resin material is obtained by post-crosslinking reaction, chloroacetylation modification and amine group modification of styrene-divinylbenzene to have a cytokine adsorption function. The polyethyleneimine is used as an amination reagent, which has high endotoxin adsorption and can eliminate residual chlorine. The presence of long branches in the subsequent crosslinking reaction can also improve the strength of the crosslinking product, and the presence of crosslinking structure can improve the adsorption capacity of cytokines. The commonly used acetylation reagent and high-efficiency catalyst are selected, which is simple, efficient and low in cost. However, the product resin has high strength, the side chain amine group is stable, and it has high adsorption capacity for cytokines and endotoxins, which can reach more than 70%, and can realize efficient treatment of diseases such as sepsis.

[0004] The existing method for removing protein-bound toxins mainly uses traditional resin adsorption. The above patent documents and existing technologies have the following technical problems in use:

[0005] Problem one: low adsorption capacity. The specific surface area of traditional resin (such as styrene-divinylbenzene copolymer) is limited, and the density of functional groups is insufficient, resulting in that the IgG adsorption capacity is generally less than 50 mg / g.

[0006] Problem two: poor selectivity. Conventional ion exchange resin has non-specific adsorption to various plasma proteins (such as albumin and fibrinogen), and the efficiency of removing target immunoglobulin is less than 60%.

[0007] Problem three, poor ligand stability: covalently coupled Protein A or antibody is easy to fall off during the cleaning process, and the number of repeated use is less than 5 times;

[0008] Problem four, insufficient biocompatibility: residual unreacted monomers on the surface of the resin may cause blood clotting or complement activation. SUMMARY

[0009] Technical problems solved

[0010] In view of the deficiencies of the prior art, the present application provides a synthetic method for removing immunoglobulin adsorption resin, which is used to solve the above problems:

[0011] Technical scheme

[0012] In order to achieve the above purpose, the present application is implemented by the following technical scheme: a synthetic method for removing immunoglobulin adsorption resin, the preparation method comprising the following steps:

[0013] Sp1: preparation of porous resin matrix: raw material formula: monomer: hydroxyethyl methacrylate (HEMA and ethylene glycol dimethacrylate (EGDMA) molar ratio 6:1; porogen: cyclohexanol and toluene volume ratio 2:1, accounting for 40% of the total phase volume; initiator: azobisisobutyronitrile (AIBN) amount is 1.5% of the total mass of monomers; polymerization process: under nitrogen protection, pre-polymerization at 60℃ for 2 hours, curing at 75℃ for 8 hours, forming a multi-level pore structure with a pore size distribution of 50-500nm;

[0014] Sp2: surface-initiated ATRP grafting functional layer: reaction system: GMA monomer concentration 2mol / L, CuBr / PMDETA catalytic system, molar ratio 1:1.2; reaction condition: polymerization at 30℃ for 4 hours, generating an epoxy functional layer with a thickness of about 200nm;

[0015] Sp3: ligand directional immobilization: ligand selection: recombinant Protein A mutant (alkali-resistant enhanced type), coupled by epoxy ring-opening reaction; coupling buffer: pH 8.5 carbonate buffer containing 0.5M NaCl; reaction time: 24 hours, ligand loading capacity up to 12mg / g resin; double cross-linking reinforcement: using glutaraldehyde (0.1%) and carbodiimide (EDC / NHS) double cross-linking system to reduce ligand shedding;

[0016] Sp4: biologically inert modification: PEGylation treatment: immerse the resin in a PBS solution containing mPEG-SVA (molecular weight 5kDa), react at room temperature for 12 hours, and the surface contact angle is reduced to below 30°.

[0017] Preferably, the Sp1 also includes raw material selection and ratio optimization, which in detail includes a matrix resin system, the main monomer of which is bisphenol A type epoxy resin (DGEBA), providing high mechanical strength and chemical resistance; the crosslinking agent is divinylbenzene (DVB), with a molar ratio of 1:3 to the epoxy resin, forming a three-dimensional network structure; the initiator is azobisisobutyronitrile (AIBN), with a dosage of 1.2-1.5% of the total mass of the monomer.

[0018] Preferably, the raw material selection and ratio optimization in the Sp1 also in detail includes the design of the porogen system, which includes a two-component porogen: cyclohexanol (main porogen) and toluene (auxiliary porogen) in a volume ratio of 2:1, accounting for 40-45% of the total volume of the oil phase; lignin templating method: adding 5-40% lignin microspheres as sacrificial templates to regulate the pore size distribution (50-500 nm).

[0019] Preferably, the Sp2 also includes substrate pretreatment and initiator immobilization, which in detail includes substrate cleaning and activation, including a cleaning program: ultrasonic cleaning with ethanol and acetone for 30 minutes in turn to remove surface contaminants; plasma activation: oxygen plasma treatment (50W, 5 minutes) to increase the density of surface hydroxyl groups (by 50%-70%).

[0020] Preferably, the substrate pretreatment and initiator immobilization in the Sp2 also in detail includes initiator chemical modification silanization treatment: immersing the substrate in an ethanol solution containing 5% 3-aminopropyltriethoxysilane (APTES) and refluxing at 80°C for 6 hours to achieve an amino group density of 0.6mmol / g; bromination reaction: reacting with bromoisobutyryl bromide (BiBB) (dichloromethane solvent, reaction at 0°C for 12 hours) to introduce ATRP initiation sites (surface bromine atom density >1.2×10 -6 mol / cm 2 ).

[0021] Preferably, the Sp3 also includes substrate pretreatment and active site construction, which in detail includes surface activation, epoxy group activation: using 0.5M NaOH to treat the epoxy group-containing resin (such as GMA grafted layer) for 1 hour to generate hydroxyl groups, which then react with 1,4-butanediol epoxy propylate to introduce amino reaction sites.

[0022] Preferably, the Sp3 also in detail includes carboxyl activation: using EDC / NHS (molar ratio 1:1.5) to activate the carboxyl carrier (such as polyacrylic acid resin) in a pH 5.5 buffer to generate active ester bonds.

[0023] Preferably, the substrate pretreatment and active site construction in Sp3 further comprises biomolecular adaptation, which includes Protein A / G pre-fixation: Protein A is fixed on the surface of the substrate by epoxy-amino reaction (pH 8.5 carbonate buffer, 24 hours) to form Fc fragment binding sites.

[0024] Preferably, the substrate pretreatment and active site construction in Sp3 further comprises glycosyl directed modification: after the aldehyde group is generated by periodate oxidation (0.1M NaIO4, 4℃ dark reaction for 2 hours) on the antibody Fc end glycosyl, it is coupled with the carrier hydrazine group.

[0025] Preferably, Sp4 further comprises surface inertization strategy and material selection, which is PEG grafting, zwitterionic polymer, diamond-like coating and albumin pre-coating.

[0026] The synthesis method optimizes process parameters through machine learning algorithm, establishes a model based on historical data (synthesis efficiency, adsorption advantage and clinical applicability), improves mass transfer efficiency with hierarchical porous matrix, realizes precise and controllable growth of functional layer with ATRP technology, reduces ligand shedding rate to 0.8% per time through double crosslinking, which is better than the average level (3-5% per time) in the industry, and the hemolysis rate and thrombosis risk are significantly reduced after PEG modification, so the method can be directly used for blood perfusion.

[0027] Advantages

[0028] The present application provides a synthesis method for removing immunoglobulin adsorption resin.

[0029] Advantages:

[0030] 1. The present application adopts precise and controllable grafting, which includes free radical concentration regulation, directional grafting mechanism, epoxy functional layer construction, anti-pollution surface modification and light-responsive grafting steps. First, reversible chain transfer agent (such as RAFT reagent) or dormant species (such as ATRP initiation system) is introduced, the free radical concentration is reduced through dynamic balance, the chain termination reaction is inhibited, the polymer chain growth with narrow molecular weight distribution (PDI<1.2) is realized, the initiation sites (such as bromo isobutyryl bromide modified amino substrate) are fixed on the surface through surface-initiated polymerization (SI-CRP) technology, it is ensured that the polymer brush only grows in the preset area, the grafting density error is less than 10%, glycidyl methacrylate (GMA) is used as monomer, epoxy-modified polymer brush is formed through ATRP grafting, the epoxy group density is 1.0-1.5 mmol / g, which is used for directional fixation of antibodies, polyethylene glycol (PEG) or sulfobetaine (SBMA) is grafted through RAFT polymerization, the grafting layer thickness is 50-150 nm, and the non-specific adsorption amount of albumin is less than 5 ng / cm2 , the introduction of photosensitive groups (such as phenyl azide), ultraviolet light (254nm) triggered cross-linking, coating shedding rate <0.5% / time, cycle stability is improved 2 times, acrylic resin grafted with grafted polyethylene glycol or sulfobetaine is resurfaced to graft Protein A to capture antibodies, IgG adsorption capacity reaches 150mg / g, and specificity >95%;

[0031] 2、The present application adopts a directional fixation strategy, which is divided into active group introduction, affinity ligand loading, Fc fragment preferential binding, covalent cross-linking reinforcement, inert molecule shielding and surface charge optimization. Reactive groups such as epoxy groups (GMA monomers) and amino groups (APTES silanization) are grafted on the surface of the resin by ATRP or RAFT polymerization, the density control precision reaches ±0.1mmol / g, Protein A is covalently fixed by epoxy ring-opening reaction (pH 8.5 carbonate buffer), the loading capacity reaches 5-10mg / g, the uniformity error of the binding site is <10%, antibody solution (1-5mg / mL) is incubated with Protein A modified carrier (4℃, 12 hours), the Fc fragment specific binding rate is >95%, the Fab region is freely exposed, stable covalent bonds are formed by thiol-alkene reaction or photo-induced cross-linking (254nm ultraviolet light), the ligand shedding rate is <0.5% / time, the efficiency remains >95% after 10 cycles, unreacted sites are blocked by polyethylene glycol (PEG) or zwitterionic polymer (such as SBMA), and the non-specific adsorption amount of albumin is reduced to <5ng / cm 2 The surface Zeta potential is adjusted to ±5mV by sulfonic acid group or carboxyl modification to reduce electrostatic adsorption interference (non-target protein adsorption is reduced by 80%);

[0032] 3, The application adopts green process, which is divided into supercritical CO2 system, biological thickening agent, metal catalyst replacement, closed cycle process, in-situ polymerization modification and porous resin green synthesis. The supercritical CO2 (critical temperature 31.1 DEG C, pressure 7.38 MPa) is used to replace the traditional organic solvent, the RAFT polymerization of the fluorine-containing monomer on the surface of ramie fiber is realized, the residual amount of the solvent is less than 0.1 ppm, the reaction efficiency is increased by 30%, the sodium alginate is used to replace the synthetic thickening agent, the stable polyacrylic acid free radical polymerization system is formed, the gelation occurrence rate is reduced from 30% to less than 5%, the waste liquid discharge is reduced by 70%, the enzyme catalysis or the photo-initiation system (such as phenyl azide ultraviolet activation) is used to replace the metal catalyst such as CuBr / PMDETA, the Cu residual in the resin is less than 1 ppm, and the medical grade material standard is met. In the lincomycin resin synthesis, the steam generator is used to recover the porogen (dichloroethane), the recovery rate is 95%, the comprehensive energy consumption is reduced by 40%, the ATRP / RAFT controlled polymerization is used to graft polyethylene glycol (PEG) on the surface of the magnetic particles, the amount of the organic solvent used is reduced by more than 80%, and the product adsorption capacity is 150 mg / g. BRIEF DESCRIPTION OF DRAWINGS

[0033] Fig. 1 The preparation method flow chart of the application is shown in the figure.

[0034] Fig. 2 The process control step chart of the preparation method of the application is shown in the figure. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the application. Embodiment one:

[0037] As shown in the figure, a synthetic method for removing an immunoglobulin adsorption resin, the preparation method comprises the following steps: Figs. 1-2 Sp1: porous resin matrix preparation: raw material formula: monomer: hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethacrylate (EGDMA) molar ratio 6:1; porogen: cyclohexanol and toluene volume ratio 2:1, accounting for 40% of the total phase volume; initiator: azobisisobutyronitrile (AIBN) amount is 1.5% of the total mass of the monomer; polymerization process: under the protection of nitrogen, pre-polymerization at 60 DEG C for 2 hours, curing at 75 DEG C for 8 hours, forming a multi-level pore structure with a pore size distribution of 50-500 nm;

[0038]

[0039] ​Sp2: Surface-initiated ATRP grafting functional layer: Reaction system: GMA monomer concentration 2 mol / L, CuBr / PMDETA catalytic system, molar ratio 1:1.2; Reaction conditions: polymerization at 30°C for 4 hours, generating an epoxy functional layer with a thickness of about 200 nm;

[0040] Sp3: Ligand directed immobilization: Ligand selection: recombinant Protein A mutant (alkaline resistance enhanced), coupled by epoxy ring-opening reaction; Coupling buffer: pH 8.5 carbonate buffer containing 0.5M NaCl; Reaction time: 24 hours, ligand loading up to 12mg / g resin; Double cross-linking reinforcement: using glutaraldehyde (0.1%) and carbodiimide (EDC / NHS) double cross-linking system to reduce ligand shedding;

[0041] Sp4: Biologically inert modification: PEGylation treatment: immerse the resin in a PBS solution containing mPEG-SVA (molecular weight 5kDa), react at room temperature for 12 hours, the surface contact angle is reduced to below 30°.

[0042] The synthesis method optimizes process parameters through machine learning algorithms, establishes a model based on historical data (synthesis efficiency, adsorption advantage and clinical applicability), improves mass transfer efficiency with hierarchical porous matrix, realizes precise and controllable growth of functional layer with ATRP technology, reduces ligand shedding rate to 0.8% per time with double cross-linking, which is better than the industry average level (3-5% per time), and significantly reduces the risk of hemolysis and thrombosis after PEG modification, which can be directly used for blood perfusion. Specific embodiment two:

[0044] As shown in the above specific embodiments, the following contents are further disclosed: Figs. 1-2

[0045] To ensure the realization of high efficiency, stability, clinical safety and batch stability of the immunoglobulin clearance adsorption resin, through five core steps of raw material quality control, polymerization process control, product purification and residual detection, batch consistency and stability test, the synthesis method precisely regulates the density and distribution of functional groups on the resin surface through controllable radical polymerization (CRP) technology, combined with immunoglobulin directed immobilization strategy, to realize high specificity adsorption. Its quality control system covers the whole process of raw material screening, reaction process monitoring, product characterization and performance verification, the specific method is as follows:

[0046] Sp1: Raw material quality control

[0047] Purity of initiator and monomer:

[0048] ​The purity of the initiator (e.g. bromoisobutyryl bromide) is determined by high performance liquid chromatography (HPLC) to be ≥ 99.5%, and the content of the polymerization inhibitor in the monomer (glycidyl methacrylate) is < 10 ppm, to ensure the stability of the polymerization kinetics.

[0049] Resin matrix screening

[0050] Polystyrene microspheres with uniform pore size distribution (10-50 nm) are selected, with a specific surface area > 500 m 2 / g, and a surface amino density error of ≤ ± 0.05 mmol / g, and the uniformity of surface functionalization is verified by X-ray photoelectron spectroscopy (XPS).

[0051] Sp2: Polymerization process control

[0052] Reaction kinetics monitoring

[0053] Temperature and time control: the prepolymerization temperature is controlled at 60-80°C to avoid local overheating leading to uneven crosslinking density, and the time is adjusted according to the monomer conversion rate (which can be monitored in real time by viscosity or by sampling for detection of the conversion rate).

[0054] Temperature and oxygen content control: multiple washing with acetone or ethanol to remove unreacted monomers and catalyst residues (the residual amount of metal ions is ≤ 10 ppm detected by ICP-MS).

[0055] Grafting density calibration: the epoxy group density is determined by the thiol fluorescent probe method (Ellman reagent) to be 1.0-1.5 mmol / g, and the grafting layer thickness error is < 10 nm (verified by atomic force microscopy).

[0056] Sp3: Product purification and residual detection

[0057] Unreacted monomer removal

[0058] Supercritical CO2 extraction technology is used to remove residual monomers, and the solvent residual amount is < 0.1 ppm (detected by GC-MS).

[0059] Metal catalyst residue control

[0060] The Cu residue is < 1 ppm detected by ICP-MS, meeting the biological compatibility standards for medical materials

[0061] Verification of the effectiveness of the blocking agent

[0062] Unreacted sites are blocked using PEG-2000, and the amount of non-specific adsorption is < 5 ng / cm 2 (bovine serum albumin adsorption experiment).

[0063] Sp4: Verification of functional groups and adsorption performance

[0064] Standardized process parameters

[0065] Establish the standard operation procedure (SOP) of reaction temperature, time, and feed ratio, and record the deviation of process parameters (such as temperature fluctuation ≤±1℃) of each batch.

[0066] Statistical process control (SPC)

[0067] Monitor the batch-to-batch fluctuation of key indicators (such as specific surface area, crosslinking degree) through control charts (such as X-bar chart, R chart), and ensure that Cpk (process capability index) ≥1.33.

[0068] Sample retention and traceability

[0069] Retain samples of each batch and periodically retest performance (such as adsorption capacity every 3 months), and establish a quality traceability file.

[0070] Sp5: batch consistency and stability test

[0071] Repeated use stability:

[0072] After 10 cycles of adsorption-elution, the adsorption capacity decreases by <5%, and there is no functional layer shedding (SEM observation of surface integrity);

[0073] Long-term storage stability:

[0074] After 12 months of storage at 4℃, the antibody binding activity retention rate is >90%, and there is no microbial contamination (sterility test);

[0075] Environmental adaptability:

[0076] Within the pH range of 2-10, the swelling rate of the resin is <5%, and the mechanical strength retention rate is >95% (compression modulus test).

[0077] The above quality control methods introduce a miniature optical spectrum sensor for real-time feedback of the polymerization reaction process through intelligent monitoring, reduce human error, and upgrade the green process by replacing acid-base treatment with enzyme-catalyzed deprotection, reducing the COD value of waste liquid by >50%. Multi-dimensional characterization combined with time-of-flight secondary ion mass spectrometry (ToF-SIMS) and cryo-EM resolves the uniformity of the three-dimensional structure of the functional layer. This quality control system ensures the efficiency, stability, and clinical safety of immunoglobulin adsorption resin through strict process parameter control and multi-dimensional performance verification. Specific embodiment three:

[0079] As Figs. 1-2 shown, according to the content in the above specific embodiments, the following content is further disclosed:

[0080] To further verify the effects of the preparation method and quality control method in specific embodiment one and specific embodiment two, the following experiments are designed for verification:

[0081] The experimental purpose is to verify the feasibility and stability of the preparation method in the technical scheme, evaluate the effectiveness of the quality control method in the technical scheme, compare the differences in key performance indicators between the technical scheme and the existing technical scheme, and prove its superiority.

[0082] The experimental data are recorded, and the parameters of the preparation of the present application and the existing technology are detected. All key parameters and detection results are recorded in the table, and the average value of three parallel experiments is taken. As shown in Table 1 below:

[0083] Table 1 Preparation method parameters

[0084]

[0085] Specific embodiment three:

[0087] As Figs. 1-2 indicated above, according to the contents in the above specific embodiments, the following contents are further disclosed:

[0088] In order to further verify the effects of the preparation method and the quality control method in the above specific embodiment one and specific embodiment two, the following experiments are designed for verification:

[0089] The experimental purpose is to verify the feasibility and stability of the preparation method in the technical scheme, evaluate the effectiveness of the quality control method in the technical scheme, compare the differences in key performance indicators between the technical scheme and the existing technical scheme, and prove its superiority.

[0090] The experimental data are recorded, and the parameters of the preparation of the present application and the existing technology are detected. All key parameters and detection results are recorded in the table, and the average value of three parallel experiments is taken. This time is shown in Table 2 below:

[0091]

[0092]

[0093] Table 2 Preparation method parameters

[0094] Specific embodiment three:

[0096] As Figs. 1-2 indicated above, according to the contents in the above specific embodiments, the following contents are further disclosed:

[0097] In order to further verify the effects of the preparation method and the quality control method in the above specific embodiment one and specific embodiment two, the following experiments are designed for verification:

[0098] The experimental purposes are to verify the feasibility and stability of the preparation method in the technical scheme, to evaluate the effectiveness of the quality control method in the technical scheme, to compare the differences in key performance indicators between the technical scheme and the existing technical scheme, and to prove its superiority.

[0099] Experimental data records, parameter detection of the preparation of the present application and the existing technology, all key parameters and detection results are recorded in the table, the average value of three parallel experiments, this time is shown in Table 3 as follows:

[0100] The experimental analysis is as follows:

[0101]

[0102] Table 3 Preparation method parameters

[0103]

[0104] Structural design advantages: based on the integration of high polymer materials, biological engineering and advanced manufacturing technology, modern structural design shows multi-dimensional synergistic advantages in the fields of adsorption separation, biological sensing and drug delivery, etc. Its core features are reflected in the following aspects:

[0105] Multi-level pore synergistic effect gradient pore size regulation: through the construction of 10-50nm main pore and 1-3nm micropore composite structure by ultrahigh crosslinking technology, the specific surface area is >800m 2 / g, realizing the synergy of fast diffusion of macromolecules (I(gG, 150kDa) and precise capture of small molecules (drug metabolites); surface topology optimization: using RAFT polymerization to modify polystyrene microspheres, forming a branched graft layer (thickness <20nm), the uniformity error of antibody binding sites is <5%, non-specific adsorption is reduced by 80%, mechanical-chemical stability balance crosslinking network is strengthened: using divinylbenzene crosslinking agent (crosslinking degree >60%) to construct a three-dimensional network, the compressive strength is >50MPa, the swelling rate is <5% (pH 2-12 environment); environmental adaptability design:

[0106] Introducing polydopamine coating (thickness 50-100nm), resisting high temperature sterilization (121℃ / 30min) and organic solvent washing (ethanol, acetone), the performance attenuation is <3% after 10 times of circulation, light curing 3D printing: digital light processing (DLP) technology to form microfluidic chips, the channel precision reaches 10μm, realizing the dynamic adsorption capacity of IgG to be improved to 180mg / g; green porosity technology: using supercritical CO2 instead of porogen (dichloroethane) to prepare porous resin, the VOCs emission is reduced by 90%, the pore connectivity is >95%.

[0107] Function and efficiency improvement: Modern biomaterial technology has achieved breakthroughs in molecular recognition, specific adsorption, and process efficiency. The core optimization strategies and performance gains are as follows:

[0108] Enhanced molecular recognition: rProtein A / G magnetic beads are used to direct coupling of antibody Fc segments (binding constant Kd≈10 - 8 M), making the Fab antigen binding domain exposure rate >90%, and the antibody activity retention rate increased to more than 95%; biomimetic interface design: based on cell-like membrane phospholipid bilayer modification of porous resin surface, non-specific protein adsorption is reduced to <5ng / cm 2 , the target antibody capture efficiency in serum samples is increased by 40%; preparation process efficiency breakthrough controllable radical polymerization (CRP):

[0109] Through ATRP / RAFT technology to control polymer chain length (PDI≤1.2), antibody binding site density error <5%, batch adsorption capacity variation coefficient <3%, one-step purification process: combined with pre-fixed antibody magnetic beads and microfluidic chip, IgG purity in serum samples >90%, operation time reduced from 6 hours to 1.5 hours.

[0110] Clinical application potential: Immunoadsorption technology can specifically remove pathogenic factors and has shown significant clinical value in the treatment of various diseases. Its core application areas and technical advantages are as follows: rheumatoid arthritis treatment: gene recombinant protein A immunoadsorption column can remove abnormally elevated IgG in serum (2.0-2.5g IgG per adsorption cycle), treatment efficiency increased to more than 85%, especially suitable for severe patients with poor response to traditional drugs58; systemic lupus erythematosus: by directional adsorption of pathogenic IgG such as anti-dsDNA antibody, combined with immunosuppressive agents can reduce the disease activity index (SLEDAI) by more than 50%, significantly reducing the risk of organ damage58; myasthenia gravis: specific removal of acetylcholine receptor antibodies (AChR-Ab), patients' muscle strength score improvement rate >70% after treatment, ventilator dependence time shortened by 40%;

[0111] Tumor adjuvant therapy immune checkpoint molecule removal: ultra-high cross-linked resin immobilized polyphenol adsorbent can target removal of transforming growth factor-β (TGF-β) and PD-L1 in serum, reducing the risk of tumor immune escape, combined with immunotherapy can increase tumor remission rate by 30%25; chemotherapy efficiency and toxicity reduction: by adsorbing chemotherapy drug metabolites (such as oxaliplatin residual products), reducing liver and kidney damage, the incidence of grade III or higher toxicity reactions in patients after treatment is reduced to <15%.

[0112] The experiment proves that the technical scheme is superior to the prior art in preparation process, quality control and performance, and provides a reliable basis for the synthesis and development of the immunoglobulin adsorption resin.

[0113] It should be noted that the relational terms herein such as first and second and the like are used solely to distinguish one entity or action from another, without necessarily requiring or implying any such actual relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can also include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element preceded by "comprises a" does not, without more constraints, foreclose the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0114] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, alternatives, and variations can be made in the embodiments without departing from the spirit and scope of the present application as defined by the appended claims and their equivalents.

Claims

1. A synthetic method for cleaning an immunoglobulin adsorption resin, characterized by: The preparation method comprises the following steps: Sp1: porous resin matrix preparation: raw material formula: monomer: hydroxyethyl methacrylate (HEMA and ethylene glycol dimethacrylate (EGDMA) molar ratio 6:1; porogen: cyclohexanol and toluene volume ratio 2:1, accounting for 40% of the total phase volume; initiator: azobisisobutyronitrile (AIBN) amount is 1.5% of the total mass of monomers; polymerization process: under nitrogen protection, pre-polymerization at 60°C for 2 hours, curing at 75°C for 8 hours, forming a multi-level pore structure with a pore size distribution of 50-500nm; Sp2: surface-initiated ATRP grafting functional layer: reaction system: GMA monomer concentration 2mol / L, CuBr / PMDETA catalyst system, molar ratio 1:1.2; reaction conditions: polymerization at 30°C for 4 hours, generating an epoxy functional layer with a thickness of about 200nm; Sp3: ligand directed immobilization: ligand selection: recombinant Protein A mutant (alkali-resistant enhanced), coupled by epoxy ring-opening reaction; coupling buffer: pH 8.5 carbonate buffer containing 0.5M NaCl; reaction time: 24 hours, ligand loading capacity up to 12mg / g resin; double cross-linking reinforcement: using glutaraldehyde (0.1%) and carbodiimide (EDC / NHS) double cross-linking system to reduce ligand shedding; Sp4: biological inertness modification: PEGylation treatment: immerse the resin in a PBS solution containing mPEG-SVA (molecular weight 5kDa), react at room temperature for 12 hours, the surface contact angle is reduced to below 30°.

2. The synthetic method for cleaning the immunoglobulin adsorption resin according to claim 1, characterized by: The raw material selection and ratio optimization in Sp1 further comprises a matrix resin system, wherein the main monomer uses bisphenol A type epoxy resin (DGEBA) to provide high mechanical strength and chemical resistance; the cross-linking agent is divinylbenzene (DVB) with a molar ratio of 1:3 to the epoxy resin to form a three-dimensional network structure; and the initiator is azobisisobutyronitrile (AIBN) with a dosage of 1.2-1.5% of the total mass of monomers.

3. The synthetic method for cleaning the immunoglobulin adsorption resin according to claim 1, characterized by: The raw material selection and ratio optimization in Sp1 further comprises the design of the porogen system, which includes a two-component porogen: cyclohexanol (main porogen) and toluene (auxiliary porogen) with a volume ratio of 2:1, accounting for 40-45% of the total oil phase volume; and a lignin template method: adding 5-40% lignin microspheres as a sacrificial template to control the pore size distribution (50-500nm).

4. The synthetic method for cleaning the immunoglobulin adsorption resin according to claim 1, characterized by: The substrate pretreatment and initiator immobilization in Sp2 further comprises substrate cleaning and activation, which includes a cleaning program: sequentially ultrasonic cleaning with ethanol and acetone for 30 minutes to remove surface contaminants; plasma activation: oxygen plasma treatment (50W, 5 minutes) to increase the density of surface hydroxyl groups (by 50%-70%).

5. The synthetic method for cleaning the immunoglobulin adsorption resin according to claim 1, characterized by: The substrate pretreatment and initiator immobilization in Sp2 also include in detail initiator chemical modification silanization treatment: immersing the substrate in 5% 3- aminopropyltriethoxysilane (APTES) ethanol solution, refluxing at 80°C for 6 hours, and the amino group density reaches 0.6 mmol / g; bromination reaction: reacting with bromoisobutyryl bromide (BiBB) (dichloromethane solvent, reacting at 0°C for 12 hours), introducing ATRP initiation sites (the surface bromine atom density > 1.2 x 10 -6 mol / cm 2 ).

6. The synthetic method for cleaning the immunoglobulin adsorption resin according to claim 1, characterized by: The Sp3 also includes substrate pretreatment and active site construction, which further includes surface activation, epoxy activation: the epoxy-containing resin (e.g. GMA grafted layer) is treated with 0.5M NaOH for 1 hour to generate hydroxyl groups, which then react with 1,4-butanediol epoxy propyl oxide to introduce amino reactive sites.

7. The synthetic method for cleaning the immunoglobulin adsorption resin according to claim 1, characterized by: The Sp3 also includes carboxyl activation, which further includes activation of carboxyl carriers (e.g. polyacrylic acid resin) with EDC / NHS (molar ratio 1:1.5) in pH 5.5 buffer to generate active ester bonds.

8. The synthetic method for cleaning the immunoglobulin adsorption resin according to claim 1, characterized by: The Sp3 also includes biomolecule adaptation, which further includes Protein A / G pre-fixation: Protein A is fixed on the substrate surface (pH 8.5 carbonate buffer, 24 hours) through epoxy-amino reaction to form Fc fragment binding sites.

9. The synthetic method for cleaning the immunoglobulin adsorption resin according to claim 1, characterized by: The Sp3 also includes glycosyl directed modification, which further includes periodate oxidation of antibody Fc terminal glycosyl (0.1M NaIO4, 4℃ dark reaction for 2 hours) to generate aldehyde groups, which then couple with carrier hydrazine groups.

10. The synthetic method for cleaning an immunoglobulin adsorption resin according to claim 1, characterized by: The Sp4 also includes surface inertization strategies and material selection, which further includes PEG grafting, zwitterionic polymer, diamond-like coating, and albumin pre-coating.

Images