A virus-removing membrane, its preparation method and application

Abstract

The present application relates to the technical field of biopharmaceutical separation and purification, and particularly relates to a virus-removing membrane, a preparation method and application thereof. The present application provides a virus-removing membrane, which comprises a base membrane and a functional layer grafted on the surface of the base membrane; the base membrane is an ethylene-vinyl alcohol copolymer porous membrane, a polyvinylidene fluoride porous membrane or a polyethersulfone porous membrane; the functional layer comprises an intermediate layer and a zwitterionic layer grafted on the surface of the base membrane in sequence; the intermediate layer is a tertiary amine type polyacrylate intermediate layer; and the zwitterionic layer is a polyacrylate carboxybetaine layer. The virus-removing membrane has high flux, high virus retention rate, high protein recovery rate and excellent anti-pollution capacity.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical separation and purification technology, and in particular to a virus-removing membrane, its preparation method, and its application. Background Technology

[0002] In downstream purification processes of biopharmaceuticals (such as IgG), virus removal is a critical step in ensuring the safety of the final product. Currently, commercially available virus-removing membranes mostly use polymeric materials such as polyethersulfone (PES) or polyvinylidene fluoride (PVDF), but these suffer from low flux, susceptibility to protein contamination, and insufficient anti-contamination properties. Furthermore, existing membrane materials are prone to clogging when processing high-concentration protein solutions over long periods, leading to significant flux decline and impacting production efficiency and cost.

[0003] To address these issues, researchers have attempted to improve the antifouling properties and virus removal efficiency of membranes through surface modification. For example, existing technologies disclose a method for preparing membrane materials with gradient pore structures via ultraviolet light grafting, which utilizes an asymmetric gradient pore size distribution to enhance membrane separation efficiency. However, this approach primarily focuses on improving separation efficiency through physical structural gradients, leaving room for improvement in the membrane material's antifouling properties, the selectivity of virus removal (i.e., maintaining a high target protein recovery rate while increasing virus rejection), and the overall performance optimization for specific applications (such as immune protein systems).

[0004] Therefore, developing a specialized virus-removing membrane that combines high throughput, high virus rejection rate, high protein recovery rate, and excellent anti-fouling ability is of great significance for reducing biopharmaceutical costs and ensuring product safety. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a virus-removing membrane, its preparation method and application, wherein the virus-removing membrane has the advantages of high throughput, high virus rejection rate, high protein recovery rate and excellent anti-contamination ability.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a virus-removing membrane, comprising a base membrane and a functional layer grafted onto the surface of the base membrane; The base membrane is an ethylene-vinyl alcohol copolymer porous membrane, a polyvinylidene fluoride porous membrane, or a polyethersulfone porous membrane; The functional layer includes an intermediate layer and a zwitterionic layer sequentially grafted onto the surface of the base film; The intermediate layer is a tertiary amine type polyacrylate intermediate layer; The zwitterionic layer is a polyacrylic acid carboxybetaine (PCBMA) layer.

[0007] Preferably, the pore size of the virus-removing membrane is asymmetrical; The pore size of the virus-removing membrane increases in a gradient from one side of the functional layer to the other side of the virus-removing membrane; The tertiary amine polyacrylate intermediate layer is a poly(dimethylaminoethyl methacrylate) layer, a poly(diethylaminoethyl methacrylate) layer, a poly(dimethylaminoethyl methacrylate) layer, or a poly(diisopropylaminoethyl methacrylate) layer.

[0008] Preferably, the base membrane is a flat sheet membrane or a hollow fiber membrane; The porosity of the base film is 50%~85%, and the average pore size on one side of the functional layer is 15~200nm.

[0009] Preferably, the grafting amount of tertiary amine polyacrylate in the intermediate layer is 1.0~2.0 mg / cm³. 2 The grafting chain length is 15~25.

[0010] The present invention also provides a method for preparing the virus-removing membrane described in the above technical solution, comprising the following steps: After immersing the base film in a photoinitiator solution, it is subjected to the first ultraviolet irradiation to obtain a base film with the photoinitiator fixed. After immersing the base film with the photoinitiator in a tertiary amine polyacrylate monomer solution, it is subjected to a second ultraviolet irradiation to obtain a tertiary amine polyacrylate modified base film. The tertiary amine-modified polyacrylate base membrane was impregnated in a methanol solution of 3-bromopropionic acid and then subjected to a quaternization reaction to obtain the virus-removing membrane.

[0011] Preferably, the concentration of the photoinitiator solution is 40~80 g / L; The photoinitiator in the photoinitiator solution is a benzophenone-based photoinitiator; The power of the first ultraviolet irradiation is 300~400W, and the time is 10~20min.

[0012] Preferably, the concentration of the tertiary amine polyacrylate monomer solution is 0.3~1.0 mol / L. The tertiary amine polyacrylate monomer in the tertiary amine polyacrylate monomer solution is dimethylaminoethyl methacrylate, diethylaminoethyl methacrylate, dimethylaminoethyl acrylate or diisopropylaminoethyl methacrylate. The solvent in the tertiary amine polyacrylate monomer solution is water or an alcohol-water mixture; The power of the second ultraviolet irradiation is 300~400W, and the light intensity density is 3.0~4.5mW / cm². 2 The time is 10-15 minutes.

[0013] Preferably, the mass ratio of the tertiary amine polyacrylate modified base film to the 3-bromopropionic acid in the methanol solution of 3-bromopropionic acid is 1:(0.08~0.12).

[0014] Preferably, the quaternization reaction is carried out at a temperature of 50-60°C for 8-24 hours.

[0015] The present invention also provides the application of the virus-removing membrane described in the above technical solution or the virus-removing membrane prepared by the preparation method described in the above technical solution in the purification process of biological products.

[0016] This invention provides a virus-removing membrane, comprising a base membrane and a functional layer grafted onto the surface of the base membrane; the base membrane is an ethylene-vinyl alcohol copolymer porous membrane, a polyvinylidene fluoride porous membrane, or a polyethersulfone porous membrane; the functional layer comprises an intermediate layer and a zwitterionic layer sequentially grafted onto the surface of the base membrane; the intermediate layer is a tertiary amine polyacrylate intermediate layer; the zwitterionic layer is a polyacrylic acid carboxybetaine layer. The polyacrylic acid carboxybetaine layer has a superhydrophilic and electrically neutral surface, which can greatly inhibit the non-specific adsorption of various proteins. The intermediate layer has a charge function, and the positive charge it provides can reduce the aggregation and adsorption of positively charged human immunoglobulin G (IgG) through electrostatic repulsion, thereby improving the permeability. Therefore, the virus removal membrane of the present invention achieves high efficiency in virus retention (LRV≥4.5) and high permeability (≥80%) for target IgG, breaking through the "trade-off" effect between flux and retention rate in traditional membrane technology. At the same time, the positive charge provided by the intermediate layer in the virus removal membrane can reduce the aggregation and adsorption of positively charged IgG through electrostatic repulsion. The zwitterionic layer obtained by further quaternization has a superhydrophilic and electrically neutral surface, which can greatly inhibit the non-specific adsorption of various proteins. According to the embodiments, the adsorption of protein by the zwitterionic layer is reduced by about 78% compared with the intermediate layer, and the flux attenuation is significantly improved when filtering high-concentration protein solutions.

[0017] Furthermore, the virus-removing membrane of this invention has an asymmetric pore structure; by grafting polymers along the membrane thickness direction, the pore size gradually increases from one functional layer to the other support layer, forming a "from sparse to dense" filtration channel. Adopting a "large-pore side feeding, dense side liquid exit" filtration mode, gradient interception of pollutants can be achieved, effectively mitigating membrane surface clogging and extending membrane lifespan. The present invention also provides a method for preparing the virus-removing membrane described in the above technical solution, comprising the following steps: immersing a base membrane in a photoinitiator solution and then subjecting it to a first ultraviolet irradiation to obtain a base membrane with a photoinitiator immobilized; immersing the base membrane with the photoinitiator immobilized in a tertiary amine polyacrylate monomer solution and then subjecting it to a second ultraviolet irradiation to obtain a tertiary amine polyacrylate modified base membrane; and immersing the tertiary amine polyacrylate modified base membrane in a methanol solution of 3-bromopropionic acid and then subjecting it to a quaternization reaction to obtain the virus-removing membrane. The preparation method described in this invention uses ultraviolet (UV) grafting as its core. First, a photoinitiator is fixed, and then tertiary amine polyacrylate monomers are grafted under UV light to form a gradient grafting that gradually decreases from the surface to the interior of the film. The main reason for this is that the pre-adsorbed photoinitiator is not uniformly distributed within the base film; rather, its concentration is high at the surface and gradually decreases towards the interior. Simultaneously, under UV irradiation, the light intensity decreases exponentially with penetration depth, allowing the tertiary amine polyacrylate monomers to graft only at the active sites, ultimately achieving a gradient grafting with a gradually decreasing grafting amount from the film surface to the interior. Furthermore, this preparation method requires no expensive equipment or complex post-processing, has mild reaction conditions, low energy consumption, and is easily scaled up to continuous production, demonstrating significant potential for industrial application. Attached Figure Description

[0018] Figure 1 SEM images of the poly(dimethylaminoethyl methacrylate) (PDMAEMA) modified base films described in Examples 1-3; Figure 2 SEM images of the virus-removing membranes described in Examples 3-5; Figure 3 Here is a SEM image of the EVAL base film described in Comparative Example 1; Figure 4 The static BSA adsorption results are shown for the poly(dimethylaminoethyl methacrylate) modified base membrane described in Example 3 and the virus-removing membrane described in Example 5. Figure 5 The flux decay curves of the poly(dimethylaminoethyl methacrylate) modified base membrane described in Example 3 and the virus-removing membrane described in Example 5 during the filtration of a 30 g / L IgG solution. Detailed Implementation

[0019] This invention provides a virus-removing membrane, comprising a base membrane and a functional layer grafted onto the surface of the base membrane; The base membrane is an ethylene-vinyl alcohol copolymer porous membrane, a polyvinylidene fluoride porous membrane, or a polyethersulfone porous membrane; The functional layer includes an intermediate layer and a zwitterionic layer sequentially grafted onto the surface of the base film; The intermediate layer is a tertiary amine type polyacrylate intermediate layer; The zwitterionic layer is a polyacrylic acid carboxylic betaine layer.

[0020] In this invention, the base membrane is an ethylene-vinyl alcohol copolymer (EVAL), a polyvinylidene fluoride porous membrane, or a polyethersulfone porous membrane; the base membrane is preferably a flat sheet membrane or a hollow fiber membrane; the porosity of the base membrane is preferably 50%~85%, more preferably 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%; the average pore size on one side of the functional layer is preferably 15~200 nm, more preferably 15 nm, 20 nm, 50 nm, 100 nm, 150 nm, or 200 nm.

[0021] In this invention, the intermediate layer is a tertiary amine polyacrylate intermediate layer, preferably a poly(dimethylaminoethyl methacrylate) layer, a poly(diethylaminoethyl methacrylate) layer, a poly(dimethylaminoethyl methacrylate) layer, or a poly(diisopropylaminoethyl methacrylate) layer; the grafting amount of the tertiary amine polyacrylate material in the intermediate layer is preferably 1.0~2.0 mg / cm³. 2 More preferably 1.0 mg / cm³ 2 1.2 mg / cm 2 1.4 mg / cm 2 1.6 mg / cm 2 1.8 mg / cm 2 Or 2.0 mg / cm 2 The graft chain length is preferably 15 to 25, more preferably 15, 20 or 25.

[0022] In this invention, controlling the grafting amount and graft chain length of the tertiary amine polyacrylate intermediate layer can ensure the uniformity of hydrophilic modification of the pores without causing pore blockage.

[0023] In this invention, the zwitterionic layer is electrically neutral under physiological pH conditions.

[0024] In this invention, the pore size of the virus-removing membrane is preferably an asymmetric structure; the pore size of the virus-removing membrane preferably increases in a gradient from one side of the functional layer to the other side of the virus-removing membrane. In this invention, the aforementioned asymmetric gradient pore structure constitutes a synergistic structure of a physical barrier for virus retention and an anti-contamination barrier of the zwitterionic layer.

[0025] In this invention, the pure water flux of the virus-removing membrane at an operating pressure of 0.1 MPa is 50~600 L·m. -2 ·h -1 ·bar -1 The logarithmic removal rate (LRV) for MS2 phage is not less than 4.5; the permeability of 30 g / L human immunoglobulin G (IgG) solution is not less than 80%.

[0026] The present invention also provides a method for preparing the virus-removing membrane described in the above technical solution, comprising the following steps: After immersing the base film in a photoinitiator solution, it is subjected to the first ultraviolet irradiation to obtain a base film with the photoinitiator fixed. After immersing the base film with the photoinitiator in a tertiary amine polyacrylate monomer solution, it is subjected to a second ultraviolet irradiation to obtain a tertiary amine polyacrylate modified base film. The tertiary amine-modified polyacrylate base membrane was impregnated in a methanol solution of 3-bromopropionic acid and then subjected to a quaternization reaction to obtain the virus-removing membrane.

[0027] In this invention, unless otherwise specified, all raw materials used in the preparation are commercially available products well known to those skilled in the art.

[0028] In this invention, the base film is immersed in a photoinitiator solution and then subjected to first ultraviolet irradiation to obtain a base film with the photoinitiator fixed.

[0029] In this invention, the base film is preferably obtained by preparation; the preparation method of the base film preferably includes the following steps: The polymer, pore-forming agent, and organic solvent are mixed to obtain the casting solution; After the casting solution is applied to the substrate surface, it is cured to obtain the nascent film. The nascent membrane was subjected to solvent displacement and purification processes in sequence to obtain the base membrane.

[0030] This invention mixes a polymer, a pore-forming agent, and an organic solvent to obtain a casting solution.

[0031] In this invention, the polymer is preferably an ethylene-vinyl alcohol copolymer, polyvinylidene fluoride, or polyethersulfone. In this invention, the ethylene-vinyl alcohol copolymer preferably contains 40% to 50% by mass, more preferably 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% by mass. In an embodiment of this invention, the ethylene-vinyl alcohol copolymer may contain 44% by mass.

[0032] In this invention, the polymer content in the casting solution is preferably 15% to 25% by mass, more preferably 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25%.

[0033] In this invention, the pore-forming agent is preferably n-octanol; the organic solvent is preferably dimethyl sulfoxide (DMSO). In this invention, the mass percentage concentration of the pore-forming agent in the casting solution is preferably 10% to 20%, more preferably 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.

[0034] In this invention, the mixing process is preferably carried out by mixing the polymer with an organic solvent and then adding a pore-forming agent.

[0035] After the mixing is completed, the present invention preferably includes sequential stirring and degassing, wherein the stirring temperature is preferably 110°C and the stirring time is preferably 10 hours. The degassing is preferably carried out under vacuum conditions, wherein the degassing temperature is preferably 80°C and the degassing time is preferably 12 hours.

[0036] After obtaining the casting solution, the present invention applies the casting solution to the substrate surface and then cures it to obtain a primary film.

[0037] The present invention does not impose any special limitations on the substrate; any clean substrate known to those skilled in the art can be used.

[0038] In this invention, the coating method is preferably casting coating; the thickness of the liquid film obtained after coating is preferably 230μm~270μm, more preferably 230μm, 240μm, 250μm, 260μm or 270μm. In an embodiment of this invention, the thickness of the liquid film obtained after coating can be 250μm.

[0039] In this invention, the curing method is preferably a coagulation bath; the temperature of the coagulation bath is preferably 40°C. In this invention, the solution used in the coagulation bath is preferably pure water or an aqueous solution of N,N-dimethylacetamide.

[0040] In this invention, the coagulation bath process is preferably to quickly immerse the coated liquid film in the solution used in the coagulation bath until the film automatically detaches from the substrate and completes the phase transformation, and then remove it to obtain the nascent film.

[0041] After obtaining the primary membrane, the present invention performs solvent replacement and purification treatments on the primary membrane in sequence to obtain the base membrane.

[0042] In this invention, the solvent replacement process is preferably performed by immersing the nascent membrane in an organic solvent and deionized water in sequence; the organic solvent is preferably anhydrous ethanol, the immersion time in the organic solvent is preferably 48 hours, and the immersion time in the deionized water is preferably 12 hours.

[0043] The present invention does not impose any special limitations on the purification process. It can be carried out using a process well known to those skilled in the art, and the residual solvent and porogen in the pore structure of the base membrane can be completely removed.

[0044] In this invention, the above preparation method is also suitable for the preparation of hollow fiber membranes as the base membrane.

[0045] In this invention, the photoinitiator in the photoinitiator solution is preferably a benzophenone-based photoinitiator, and the solvent is preferably acetone.

[0046] In this invention, the concentration of the photoinitiator in the photoinitiator solution is preferably 40-80 g / L, more preferably 40 g / L, 50 g / L, 60 g / L, 70 g / L or 80 g / L; in an embodiment of this invention, the concentration of the photoinitiator in the photoinitiator solution can be 60 g / L.

[0047] In this invention, the impregnation is preferably carried out under light-protected conditions, and the impregnation time is preferably 1 to 5 hours, more preferably 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours. In an embodiment of this invention, the impregnation time can be 3 hours.

[0048] After the impregnation is completed, the present invention preferably includes using filter paper to absorb excess solution from the surface, placing it in a closed quartz reactor filled with nitrogen, with the functional layer side of the membrane as the light-receiving surface (i.e. the side that contacts the coagulation bath during the phase transformation process).

[0049] Before the first ultraviolet irradiation, it is preferable to allow the mixture to stand in a protective atmosphere, preferably a nitrogen atmosphere, for a period of 20 minutes. In this invention, the purpose of the standing period is to remove oxygen that inhibits polymerization.

[0050] In this invention, the power of the first ultraviolet irradiation is preferably 300~400W (surface light intensity calibration is 4.0~4.2mW / cm). 2 The power of the first ultraviolet irradiation can be 300W, 320W, 340W, 360W, 380W, or 400W; the time is preferably 10-20 minutes, more preferably 10 minutes, 12 minutes, 14 minutes, 16 minutes, 18 minutes, or 20 minutes. In an embodiment of the present invention, the power of the first ultraviolet irradiation can be 400W, and the time can be 15 minutes.

[0051] In this invention, the function of the first ultraviolet irradiation is to fix the photoinitiator to hydrogen on the surface of the base film.

[0052] After obtaining the base film with the photoinitiator fixed, the present invention impregnates the base film with the photoinitiator fixed in a tertiary amine polyacrylate monomer solution and then subjectes it to a second ultraviolet irradiation to obtain a tertiary amine polyacrylate modified base film.

[0053] In this invention, the concentration of the tertiary amine polyacrylate monomer solution is preferably 0.3~1.0 mol / L, more preferably 0.3 mol / L, 0.31 mol / L, 0.32 mol / L, 0.33 mol / L, 0.34 mol / L, 0.35 mol / L, 0.36 mol / L, 0.37 mol / L, 0.5 mol / L, 0.7 mol / L or 0.90 mol / L.

[0054] In this invention, the tertiary amine polyacrylate monomer in the tertiary amine polyacrylate monomer solution is preferably dimethylaminoethyl methacrylate, diethylaminoethyl methacrylate, dimethylaminoethyl acrylate, or diisopropylaminoethyl methacrylate.

[0055] In this invention, the solvent in the tertiary amine polyacrylate monomer solution is preferably water or an alcohol-water mixture, wherein the volume ratio of alcohol to water in the alcohol-water mixture is preferably 1:(1~4), more preferably 1:1, 1:2, 1:3 or 1:4; the alcohol is preferably methanol, ethanol, isopropanol or n-butanol, more preferably ethanol or isopropanol. In embodiments of this invention, the concentration of the tertiary amine polyacrylate monomer solution can be 0.35 mol / L, and the solvent can be ethanol.

[0056] The present invention does not impose any special limitations on the impregnation process; any process well known to those skilled in the art can be used. In an embodiment of the present invention, the impregnation time can be 20 minutes.

[0057] Before the second ultraviolet irradiation, a vacuum degassing process is preferably performed, wherein the vacuum degree of the vacuum degassing process is preferably -0.09 MPa and the time is preferably 30 min. In this invention, the second ultraviolet irradiation is preferably carried out in a protective atmosphere, preferably a nitrogen atmosphere; the power of the second ultraviolet irradiation is preferably 300-400W, more preferably 300W, 320W, 340W, 360W, 380W, or 400W; the time is preferably 3-15 minutes, more preferably 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, or 15 minutes. In an embodiment of this invention, the power of the second ultraviolet irradiation can be 400W (light intensity density set to 3.0-4.5 mW / cm²). 2 The time can be 15 minutes.

[0058] In this invention, controlling the second ultraviolet irradiation time within the above-mentioned range can effectively control the thickness of the tertiary amine polyacrylate modified layer in the tertiary amine polyacrylate modified base film.

[0059] During the second ultraviolet irradiation process, it is preferable to change the water every 8 hours to thoroughly remove homopolymers and unreacted monomers.

[0060] After obtaining the tertiary amine polyacrylate modified base film, the present invention impregnates the tertiary amine polyacrylate modified base film in a methanol solution of 3-bromopropionic acid and then performs a quaternization reaction to obtain the virus-removing membrane.

[0061] In this invention, the concentration of 3-bromopropionic acid in the methanol solution of 3-bromopropionic acid is preferably 1.0~2.0 g / L, more preferably 1.0 g / L, 1.2 g / L, 1.4 g / L, 1.6 g / L, 1.8 g / L, or 2.0 g / L; the solvent is preferably anhydrous methanol. In embodiments of this invention, the concentration of 3-bromopropionic acid in the methanol solution of 3-bromopropionic acid can be 1.0 g / L or 2.0 g / L.

[0062] In this invention, the preferred mass ratio of the tertiary amine-type polyacrylate-modified base film to the 3-bromopropionic acid in the methanol solution of 3-bromopropionic acid is 1:(0.08~0.12), more preferably 1:0.08, 1:0.09, 1:0.1, 1:0.11, or 1:0.12. In an embodiment of this invention, the mass ratio of the tertiary amine-type polyacrylate-modified base film to the 3-bromopropionic acid in the methanol solution of 3-bromopropionic acid can be 1:0.1.

[0063] In this invention, the temperature of the quaternization reaction is preferably 50-60°C, more preferably 50°C, 52°C, 54°C, 56°C, 58°C, or 60°C; the time is preferably 8-24 hours, more preferably 8 hours, 10 hours, 15 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In an embodiment of this invention, the temperature of the quaternization reaction can be 60°C, and the time can be 20 hours or 24 hours.

[0064] After the quaternization reaction is completed, the present invention preferably further includes washing with methanol and deionized water sequentially to remove unreacted substances. The present invention does not impose any special limitations on the washing process; any process well known to those skilled in the art can be used.

[0065] The present invention also provides the application of the virus-removing membrane described in the above technical solution or the virus-removing membrane prepared by the preparation method described in the above technical solution in the purification process of biological products.

[0066] In this invention, the support layer (large pore side) of the virus removal membrane is preferably the feed side, and the functional layer side (small pore side) is preferably the permeation side.

[0067] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0068] Example 1 Ethylene-vinyl alcohol copolymer (EVAL, 44% ethylene content) was dissolved in dimethyl sulfoxide (DMSO), and a pore-forming agent (n-octanol) was added. The mixture was mechanically stirred at 110°C for 10 hours until completely dissolved, and then allowed to stand in a vacuum oven at 80°C for 12 hours to remove bubbles, thus obtaining a casting solution (the mass concentration of ethylene-vinyl alcohol copolymer in the casting solution was 20%, and the mass percentage concentration of n-octanol in the casting solution was 15%). The casting solution was poured onto a clean glass plate and coated using a precision doctor blade to obtain a 250 μm liquid film. The film was then quickly immersed in a 40°C pure water coagulation bath. After the film automatically detached from the glass plate and completed the phase transformation, it was removed to obtain the nascent film. The nascent membrane was immersed in anhydrous ethanol for 48 hours to extract the residual solvent and pore-forming agent, and then soaked in deionized water for 12 hours to replace the ethanol, to obtain the EVAL base membrane (denoted as M1). The EVAL-based membrane was placed in an acetone solution of 60 g / L benzophenone and allowed to adsorb in the dark for 3 hours. The membrane was then removed, excess solution was blotted off with filter paper, and the membrane was placed in a sealed quartz reactor filled with nitrogen and allowed to stand for 20 minutes. Subsequently, the functional layer side of the membrane (i.e., the side in contact with the coagulation bath during phase transformation) was used as the light-receiving surface and exposed to light through a 400W high-pressure mercury lamp (surface light intensity calibrated to 4.0~4.2 mW / cm²). 2 Irradiation for 15 minutes under the irradiation condition caused BP to abstract hydrogen and fix it on the membrane surface, resulting in a base film with photoinitiator fixed on it. The base film immobilized with the photoinitiator was rapidly immersed in a 0.35 mol / L solution of dimethylaminoethyl methacrylate (DMAEMA) monomer for 20 min, during which vacuum degassing (-0.09 MPa) was performed for 30 min to remove air from the pores. Then, a second ultraviolet irradiation was performed for 15 min under a nitrogen protective atmosphere (power 400 W, light intensity set to 4.1 mW / cm²). 2 During this process, the water was changed every 8 hours to thoroughly remove the homopolymer and unreacted monomers, resulting in a poly(dimethylaminoethyl methacrylate) modified base film (denoted as M1-g-PDMA, with a grafting amount of 1.4 mg / cm).2 (Graft chain length is 15). The M1-g-PDMA (5cm×5cm) was completely immersed in 40mL of methanol solution containing 40mg of 3-bromopropionic acid (i.e., the concentration of 3-bromopropionic acid was 1.0g / L, and the mass ratio of the poly(dimethylaminoethyl methacrylate) modified base membrane to the 3-bromopropionic acid in the methanol solution was 1:0.1). The reaction was carried out in a constant temperature oil bath at 60℃ with magnetic stirring for 24h. After the reaction was completed, the membrane was removed, washed three times with anhydrous methanol and n-hexane, and then soaked in deionized water overnight. After vacuum drying, the virus-free membrane (denoted as M1-0424) was obtained.

[0069] Example 2 Referring to Example 1, the difference is that, in preparing the nascent membrane, a liquid membrane with a thickness of 250 μm was rapidly immersed in an aqueous solution containing 10 vol% N,N-dimethylacetamide at 40 °C for coagulation; the final EVAL-based membrane (denoted as M2) was obtained. A poly(dimethylaminoethyl methacrylate) modified base film (denoted as M2-g-PDMA, with a grafting amount of 1.6 mg / cm²) was prepared according to the preparation method in Example 1. 2 (The grafting chain is 20). The virus-free membrane (denoted as M2-0424) was prepared according to the preparation method of Example 1.

[0070] Example 3 Referring to Example 1, the difference is that, in preparing the nascent membrane, a liquid membrane with a thickness of 250 μm was rapidly immersed in an aqueous solution containing 30 vol% N,N-dimethylacetamide at 25°C for coagulation; the final EVAL-based membrane (denoted as M3) was obtained. A poly(dimethylaminoethyl methacrylate) modified base film (denoted as M3-g-PDMA, with a grafting amount of 1.8 mg / cm³) was prepared according to the preparation method in Example 1. 2 (Graft chain length is 25). The virus-free membrane (denoted as M3-0424) was prepared according to the preparation method of Example 1.

[0071] Example 4 Referring to Example 3, the difference is that the M3-g-PDMA (5cm×5cm in size) was completely immersed in 40mL of methanol solution containing 80mg of 3-bromopropionic acid (i.e., the concentration of 3-bromopropionic acid was 2.0g / L), and reacted in a constant temperature oil bath at 60℃ under magnetic stirring for 20h. After the reaction was completed, the membrane was taken out, washed three times with anhydrous methanol and n-hexane in sequence, soaked in deionized water overnight, and vacuum dried to obtain the virus-free membrane (denoted as M3-0820).

[0072] Example 5 Referring to Example 3, the difference is that the M3-g-PDMA (5cm×5cm in size) was completely immersed in 40mL of methanol solution containing 80mg of 3-bromopropionic acid (i.e., the concentration of 3-bromopropionic acid was 2.0g / L), and reacted in a constant temperature oil bath at 60℃ under magnetic stirring for 24h. After the reaction was completed, the membrane was taken out, washed three times with anhydrous methanol and n-hexane in sequence, soaked in deionized water overnight, and vacuum dried to obtain the virus-free membrane (denoted as M3-0824).

[0073] Comparative Example 1 The EVAL base film (denoted as M3) was prepared according to Example 3.

[0074] Comparative Example 2 The EVAL-based film prepared according to Example 3; Prepare a mixed grafting solution containing 15 vol% DMAEMA monomer and 1.0 wt% benzophenone photoinitiator (solvent is tert-butanol). The EVAL base film is immersed in the mixed grafting solution for 30 minutes to allow the monomer and initiator to diffuse evenly into the depth of the membrane pores. The film is then removed and sandwiched between two pieces of quartz glass. After being irradiated under a 400W UV lamp for 15 minutes, it is cleaned to obtain a uniformly grafted EVAL film. (This method will cause the membrane pores, especially the pores of the support layer, to be filled and blocked by the polymer, and will not be able to form a functionalized gradient concentrated in the skin layer.)

[0075] Test case Figure 1 The images shown are SEM images of the poly(dimethylaminoethyl methacrylate) modified base films described in Examples 1-3 (where a is M1-g-PDMA, b is M2-g-PDMA, and c is M3-g-PDMA). Figure 1 It can be seen that the cross-section of the poly(dimethylaminoethyl methacrylate) modified base film described in Examples 1-3 has a sponge-like porous structure, and the pore volume gradually decreases as PDMAEMA is filled. Figure 2 The images shown are SEM images of the virus-removing membranes described in Examples 3-5 (where a is M3-0424, b is M3-0820, and c is M3-0824). Figure 2 It can be seen that, compared with the M3-g-ODMA membrane, the cross-sectional structure of the virus-removing membrane described in Examples 3-5 shows that the volume of the finger-shaped cavity in the membrane cross-section is significantly reduced. It is speculated that the high temperature of 60°C during the reaction process causes the polymer chains to cross-link after quaternization, resulting in cross-linking on the membrane surface and in the pores, which further reduces the pore size of the membrane. Figure 3 The image shown is a SEM image of the EVAL base film described in Comparative Example 1. Figure 3It can be seen that the cross-section of the EVAL base film described in Comparative Example 1 exhibits a porous sponge structure. SEM images of the poly(dimethylaminoethyl methacrylate) modified base membranes described in Examples 1-3, the virus-removing membranes described in Examples 3-5, the EVAL base membrane described in Comparative Example 1, and the uniformly grafted EVAL membrane described in Comparative Example 2 were used to test the permeation and separation performance. The test procedure was as follows: The pure water flux of the membrane samples was tested using an Amicon® stirred ultrafiltration cup. Before the test, the membrane was fully wetted and pre-pressurized at 0.1 MPa for 30 min until the flux stabilized. The test temperature was controlled at 25℃ and the operating pressure at 0.1 MPa. After the system stabilized, the permeate volume was collected periodically. The pure water flux was calculated by combining the effective filtration area of ​​the membrane with the test time. Each group of samples was measured in parallel three times and the average value was taken. A 30 g / L concentration of IgG-PBS buffer was prepared and cross-flow filtered at 0.1 MPa and 25℃. After the system stabilized, the stock solution and permeate were collected separately. The absorbance was measured at 280 nm using a UV spectrophotometer. The concentration was converted by combining the standard curve, and the IgG permeability was calculated. Each group of samples was tested in parallel three times. MS2 bacteriophage was used as a simulated virus, and 4.0 × 10⁻⁶ PCR broth was prepared. 6 MS2 bacterial suspension with a concentration of PFU / mL was filtered through an ultrafiltration cup at room temperature and 0.1 MPa pressure, and the feed solution and membrane permeate were collected separately. The number of bacteriophage colonies in both groups of samples was counted using the bilayer plate method, and the removal efficiency of the membrane for MS2 was calculated based on the change in colony concentration. Each experiment was repeated three times to ensure accurate and reliable results. Colloidal gold particle solution (particle concentration 6.54 × 10⁻⁶) was prepared using 0.1 mmol / L PBS buffer. 11 The stock solution was prepared using NPS / mL. Under stable conditions, the membrane samples were filtered through a colloidal gold solution, and the filtrate was collected. The absorbance (Abs) at 521 nm was measured using a UV-Vis spectrophotometer, and the colloidal gold concentration in the permeate was calculated. The membrane's retention performance for colloidal gold was then calculated. Each group of samples was tested in triplicate, and the average value was taken. The test results are shown in Table 1. Table 1. SEM images of the poly(dimethylaminoethyl methacrylate) modified base membranes described in Examples 1-3, the virus-removing membranes described in Examples 3-5, and the permeation and separation performance of the EVAL base membrane described in Comparative Example 1 and the uniformly grafted EVAL membrane described in Comparative Example 2.

[0076] As shown in Table 1, the pure water flux of the membrane decreased significantly after the quaternization reaction. This phenomenon is attributed to two factors: First, the conversion from PDMAEMA to PCBMA introduced a bulky carboxybetaine side group, increasing the steric hindrance effect of the polymer chain within the pores, resulting in a slight reduction in the effective pore size; second, the zwitterionic groups have extremely strong hydration capabilities, forming a dense hydration layer on the membrane pore surface, increasing the resistance to fluid flow through the pores. Despite the decrease in pure water flux, the virus-removing membranes described in Examples 3-5 showed improved protein permeability when treating a 30 g / L IgG solution compared to the poly(dimethylaminoethyl methacrylate) modified base membranes described in Examples 1-3. This result fully demonstrates that the low protein affinity of the PCBMA zwitterionic layer effectively prevents IgG molecules from adhering to the membrane. Figure 4 The static BSA adsorption results of the poly(dimethylaminoethyl methacrylate) modified base membrane described in Example 3 and the virus-removing membrane described in Example 5 are obtained from... Figure 4 It can be seen that with the increase of adsorption time, the unit adsorption capacity of BSA for both groups of membranes showed a trend of first rapidly increasing and then gradually leveling off. The equilibrium adsorption capacity of the M3-g-PDMA membrane was as high as 49.25 mg / g. This is because the PDMAEMA layer is positively charged, and there is a strong electrostatic attraction between it and the negatively charged BSA protein, resulting in a large amount of protein being non-specifically adsorbed onto the membrane surface. After quaternization modification, the equilibrium adsorption capacity of the M3-0824 membrane was significantly reduced to 10.95 mg / g. Compared with the unmodified membrane, the BSA adsorption capacity of the M3-0824 membrane was reduced by about 4.5 times. This confirms that the zwitterionic layer of PCBMA constructs a dense hydration barrier on the membrane surface, endowing the membrane with excellent hydrophilicity and low protein affinity, ensuring high protein permeability during filtration. Figure 5 The flux decay curves of the poly(dimethylaminoethyl methacrylate) modified base membrane described in Example 3 and the virus-removing membrane described in Example 5 during the filtration of a 30 g / L IgG solution are shown below. Figure 5 It was found that when the IgG solution concentration was 30 g / L, the flux of the M3-g-PDMA membrane decreased by 70%, and that of the M3-0824 membrane decreased by approximately 60%. Compared to the M3-g-PDMA membrane, the flux decrease of the M3-0824 membrane was reduced by about 10%. Compared to before quaternization, the pure water flux of the membrane decreased significantly, but the permeability of IgG and the degree of flux decrease remained unchanged. This indicates that the low protein affinity of PCBMA allows IgG molecules to pass through the membrane pores smoothly and enhances the membrane's antifouling performance.

[0077] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A virus-removing membrane, characterized in that, Includes a base film and a functional layer grafted onto the surface of the base film; The base membrane is an ethylene-vinyl alcohol copolymer porous membrane, a polyvinylidene fluoride porous membrane, or a polyethersulfone porous membrane; The functional layer includes an intermediate layer and a zwitterionic layer sequentially grafted onto the surface of the base film; The intermediate layer is a tertiary amine type polyacrylate intermediate layer; The zwitterionic layer is a polyacrylic acid carboxylic betaine layer.

2. The virus-removing membrane as described in claim 1, characterized in that, The virus-removing membrane has an asymmetric pore structure. The pore size of the virus-removing membrane increases in a gradient from one side of the functional layer to the other side of the virus-removing membrane; The tertiary amine polyacrylate intermediate layer is a poly(dimethylaminoethyl methacrylate) layer, a poly(diethylaminoethyl methacrylate) layer, a poly(dimethylaminoethyl methacrylate) layer, or a poly(diisopropylaminoethyl methacrylate) layer.

3. The virus-removing membrane as described in claim 2, characterized in that, The base membrane is a flat sheet membrane or a hollow fiber membrane; The porosity of the base film is 50%~85%, and the average pore size on one side of the functional layer is 15~200nm.

4. The virus-removing membrane as described in claim 1, characterized in that, The grafting amount of tertiary amine polyacrylate in the intermediate layer is 1.0~2.0 mg / cm³. 2 The grafting chain length is 15~25.

5. The method for preparing the virus-removing membrane according to any one of claims 1 to 4, characterized in that, Includes the following steps: After immersing the base film in a photoinitiator solution, it is subjected to the first ultraviolet irradiation to obtain a base film with the photoinitiator fixed. After immersing the base film with the photoinitiator in a tertiary amine polyacrylate monomer solution, it is subjected to a second ultraviolet irradiation to obtain a tertiary amine polyacrylate modified base film. The tertiary amine-modified polyacrylate base membrane was impregnated in a methanol solution of 3-bromopropionic acid and then subjected to a quaternization reaction to obtain the virus-removing membrane.

6. The preparation method according to claim 5, characterized in that, The concentration of the photoinitiator solution is 40~80 g / L; The photoinitiator in the photoinitiator solution is a benzophenone-based photoinitiator; The power of the first ultraviolet irradiation is 300~400W, and the time is 10~20min.

7. The preparation method according to claim 5, characterized in that, The concentration of the tertiary amine polyacrylate monomer solution is 0.3~1.0 mol / L. The tertiary amine polyacrylate monomer in the tertiary amine polyacrylate monomer solution is dimethylaminoethyl methacrylate, diethylaminoethyl methacrylate, dimethylaminoethyl acrylate or diisopropylaminoethyl methacrylate. The solvent in the tertiary amine polyacrylate monomer solution is water or an alcohol-water mixture; The power of the second ultraviolet irradiation is 300~400W, and the light intensity density is 3.0~4.5mW / cm². 2 The time is 10-15 minutes.

8. The preparation method according to claim 5, characterized in that, The mass ratio of the tertiary amine polyacrylate modified base film to the 3-bromopropionic acid in the methanol solution of 3-bromopropionic acid is 1:(0.08~0.12).

9. The preparation method according to claim 5, characterized in that, The quaternization reaction is carried out at a temperature of 50-60℃ for 8-24 hours.

10. The application of the virus-removing membrane according to any one of claims 1 to 4 or the virus-removing membrane prepared by the preparation method according to any one of claims 5 to 9 in the purification process of biological products.

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