Fiber fabric virus removing membrane with high virus interception rate and its preparation process

Abstract

The application relates to a high-virus-removal-rate fiber virus-removal membrane, comprising a porous body, the porous body having non-directional winding channels, the two sides of the porous body being respectively a first outer surface and a second outer surface, the porous body comprising a pre-filter layer and a separation layer, the separation layer being located on the side of the pre-filter layer close to the second outer surface, and the pre-filter layer and the separation layer being connected by a continuous fiber; the SEM-measured average pore diameter of the pre-filter layer is larger than that of the separation layer; the thickness of the separation layer is 5-60 mu m, and the SEM-measured average pore diameter of the separation layer is 25-85 nm; for PP7 phages, the top washing LRV of the virus-removal membrane is not less than 4, and the ratio of the top washing LRV of the virus-removal membrane to the initial LRV is not less than 0.7. The application further discloses a preparation process of the virus-removal membrane. The fiber virus-removal membrane has a high protein yield, and has a high virus-removal effect before and after top washing, so that the virus pollution risk can be reduced, and the economic benefit can be improved.

Description

Technical Field

[0001] This application relates to the field of membrane separation technology, and in particular to a cellulose virus removal membrane with high virus rejection rate and its preparation process. Background Technology

[0002] Membrane separation technology refers to the technology of separating, classifying, enriching, and purifying binary or multi-component mixtures of fluids using external forces (such as pressure) or chemical potential differences. Due to its advantages such as high separation efficiency, low energy consumption, no need for external chemical reagents, and ability to separate systems that conventional methods cannot (such as azeotropic systems), membrane separation technology is widely used in various industries. Particularly in the biological and pharmaceutical fields, the characteristic of membrane separation technology not easily causing denaturation of active substances makes it widely used in the production processes of various biological agents.

[0003] This is because the introduction of various viruses is unavoidable during the production of biopharmaceuticals, and both domestic and international regulations have clear requirements regarding the viral safety of biopharmaceuticals. In fact, a report on viral safety assessment test results must be attached when submitting a drug application, and the content of this report directly affects the review outcome. Therefore, for all types of biopharmaceutical companies, virus removal and / or virus inactivation steps in the biopharmaceutical production process are indispensable.

[0004] Various biological agents have seen rapid development due to their ability to prevent, treat, and diagnose various infectious diseases, immune diseases, and other diseases that are difficult to prevent or treat using conventional methods. Biological agents are generally processed from microorganisms (bacteria, rickettsiae, viruses, etc.) and their metabolites, effective antigenic components, animal toxins, human or animal blood or tissues, etc. In addition to general production requirements, the production of vaccines and broad-spectrum biological agents has a series of unique characteristics, such as the need for culturing microorganisms, viruses, and live cells, followed by further processing of the obtained biological materials, including purification, cleaning, passivation, extraction, freezing, and lyophilization.

[0005] Chinese invention patent application CN113842792A discloses an asymmetric PES filter membrane for virus removal. This PES filter membrane comprises a main body, including a pre-filtration layer and a separation layer for virus retention. The other side of the pre-filtration layer and the other side of the separation layer are transitioned by continuous fibers. This PES membrane has a typical bilayer structure (a large-pore pre-filtration layer and a small-pore separation layer) and exhibits good virus retention (LRV > 4). However, the poor hydrophilicity of the PES material itself often results in a low protein yield.

[0006] For example, Chinese Patent CN1759924B discloses a multilayer composite ultrafiltration membrane. This composite ultrafiltration membrane includes at least one first porous membrane layer having a first surface and an equivalent second surface, and at least one second porous membrane layer having an equivalent first surface and a second surface. The first and second layers are superimposed and have a porosity transition region from the equivalent first surface of the second layer to the equivalent second surface of the first layer. At least one of the layers is an asymmetric ultrafiltration membrane. While this multilayer membrane structure provides good filtration for small-sized viruses, the transition region of the composite membrane exhibits a rapid pore size change. While this rapid pore size change can effectively trap viruses, it also traps particles of a wider range, leading to a decrease in flux and lifespan. Furthermore, as a polyethersulfone (PES) membrane, the poor hydrophilicity of PES results in a high adsorption rate for viruses, leading to a decrease in protein yield.

[0007] The two types of filter membranes mentioned above have a high protein retention effect due to the use of PES raw material with poor hydrophilicity as the membrane forming material, resulting in a low protein yield. For various biological and pharmaceutical companies, an excessively low protein yield is a fatal flaw.

[0008] Japanese invention patent JP1984204911A discloses that regenerated cellulose membranes (RC membranes) have good clearance ability for HIV (approximately 100 nm), and due to the good hydrophilic properties of cellulose, their adsorption of active substances (proteins) is low, often resulting in good protein yields. However, their clearance ability for viruses with a size of 20–100 nm, such as hepatitis B virus (approximately 42 nm), nAnB virus (30–60 nm), and mouse parvovirus (approximately 20 nm), is poor (LRV < 4), therefore, they can no longer meet the current stringent requirements for virus clearance.

[0009] Furthermore, Chinese invention patent application CN105980038A discloses a virus-removing membrane comprising cellulose, having a first surface for supplying a protein-containing solution and a second surface for discharging permeate that has passed through the membrane. It achieves a logarithmic removal rate of over 4 (LRV > 4) for porcine parvovirus (approximately 18–26 nm), and due to its cellulose composition, it exhibits good hydrophilicity and low protein adsorption, often resulting in a high protein yield. However, this membrane is prepared using the cuprammonium process, which causes significant pollution, incurs high environmental remediation costs, and requires the use of highly irritating and odorous ammonia gas (ammonia water) during production, potentially causing health problems for operators.

[0010] The high price of proteins drives biotechnology and pharmaceutical companies to continuously pursue higher protein yields in the virus removal stage. This has made cellulose-based virus removal membranes, with their high protein yield, a promising future development direction. However, even with cellulose-based virus removal membranes offering high protein yields, a top-washing operation is still necessary in both laboratory and production processes. The purpose of this operation is to wash out the proteins trapped by the virus removal membrane, obtaining a top-wash solution for further protein recovery to improve yield. However, top-washing often requires a process stoppage. During this stoppage, external pressure needs to be removed to release the proteins from the membrane's tortuous pathways, allowing for better protein washing and recovery. However, after the pressure is removed, trapped viruses also release from the membrane's tortuous pores, making them easily washable and resulting in a high virus titer in the top-wash solution.

[0011] For cellulose-based filter membranes, the phenomenon of virus elution during top washing is more severe. This may be because, although cellulose raw materials have good hydrophilicity, cellulose is relatively soft and has poor mechanical strength. Cellulose-based virus-removing membranes made from cellulose raw materials as film-forming materials are prone to significant deformation under pressure. During the process residence time, as the external pressure is removed, the cellulose-based virus-removing membrane that was deformed under external pressure will recover its shape. During the recovery process, there is a complex change in the pore structure of the virus-removing membrane. The greater the deformation of the virus-removing membrane, the more likely it is to release viruses trapped in the complex pore structure during the recovery process, which will then be washed out into the top washing solution.

[0012] Based on the above issues, how to ensure that the cellulosic virus removal membrane has a high protein yield while maintaining a high virus retention efficiency before and after top washing, and reducing the possibility of virus being washed out during the top washing operation, is an urgent problem to be solved. Summary of the Invention

[0013] In view of the shortcomings of the prior art, the purpose of this application is to provide a cellulose virus-removing membrane with high virus rejection rate and its preparation process. The cellulose virus-removing membrane has a high protein yield and a high virus rejection effect before and after top washing, which can not only reduce the risk of virus contamination, but also improve economic benefits.

[0014] In a first aspect, this application provides a cellulose virus-removing membrane with a high virus rejection rate, employing the following technical solution:

[0015] A cellulose virus-removing membrane with high virus rejection rate comprises a porous body having non-directional tortuous pathways within the porous body. One side surface of the porous body is a first outer surface, and the other side surface of the porous body is a second outer surface.

[0016] The porous body includes a pre-filter layer and a separation layer for trapping viruses. One side of the pre-filter layer is a first outer surface, and the separation layer is located on the side of the pre-filter layer near the second outer surface. The pre-filter layer and the separation layer are connected by continuous fibers.

[0017] The average pore size measured by SEM of the pre-filter layer is greater than the average pore size measured by SEM of the separation layer.

[0018] The thickness of the separation layer is 5–60 μm, and the average pore size of the separation layer measured by SEM is 25–85 nm.

[0019] For PP7 bacteriophage, the top wash LRV of the devirulent membrane is not less than 4, and the ratio of the top wash LRV of the devirulent membrane to the initial LRV is not less than 0.7.

[0020] By adopting the above technical solution, the virus-removing membrane in this application has a continuously varying pore structure in the thickness direction. The porous main body near the first outer surface has larger pore sizes, while the porous main body on the side of the pre-filtration layer near the second outer surface has smaller pore sizes. Therefore, the virus-removing membrane in this application has an anisotropic pore structure in the thickness direction, belonging to an asymmetric structure. The porous main body with a larger average pore size serves as the pre-filtration layer, which can filter out larger particles in the feed liquid, reducing the possibility of large particles clogging the separation layer and giving the virus-removing membrane a high flux and contaminant holding capacity. The porous main body with a smaller average pore size serves as the separation layer, which can retain the viruses to be filtered, greatly reducing the virus titer in the filtrate and lowering the viral risk of biological agents.

[0021] However, it is generally believed that if a virus-removing membrane has a better virus retention effect, it often also means a higher retention rate of other materials in the feed solution (such as proteins) and a decrease in flux. Therefore, a high virus retention rate and a high protein yield are often contradictory. For example, to obtain a higher virus retention rate, the average pore size measured by SEM in the separation layer can be reduced or the thickness of the separation layer can be increased. This creates a tortuous pathway with a smaller average pore size measured by SEM in the separation layer, resulting in efficient virus retention. This tortuous pathway with a smaller average pore size measured by SEM not only has a better virus retention effect, but also, combined with the greater adsorption force generated by the larger specific surface area, a higher protein retention effect. Therefore, an improved virus retention effect often also means an improved protein retention effect, leading to a decrease in protein yield.

[0022] However, the inventors of this application have discovered that when the thickness of the separation layer in the cellulose virus removal membrane is 5–60 μm and the average pore size measured by SEM is 25–85 nm, the cellulose virus removal membrane not only has a good filtration effect on small-sized viruses (such as the typical model virus PP7 bacteriophage), but also has a high protein yield.

[0023] This is likely because typical PES and PVDF membranes require a thin, small-pore separation layer for virus filtration to ensure high virus rejection and protein yield. If the separation layer is too thick (regardless of whether the average pore size measured by SEM increases), the increased thickness of the tortuous pathway structure with strong rejection and adsorption properties can cause the less hydrophilic PES and PVDF membranes to adsorb or retain large amounts of protein, leading to a significant decrease in protein yield. Increasing the average pore size measured by SEM of the separation layer, while improving protein yield, often reduces virus rejection. Cellulose virus-removing membranes, on the other hand, have better hydrophilicity (cellulose membranes are generally considered the least likely to adsorb proteins among organic membranes). Even with a thicker separation layer and a larger average pore size measured by SEM (lower specific surface area), they exhibit lower protein adsorption, resulting in a higher protein yield. Meanwhile, the thicker separation layer, combined with an appropriately increased average pore size measured by SEM, ensures good retention of viruses of similar size and pore structure through a longer, more tortuous pathway in the thickness direction. Conversely, it reduces retention of proteins significantly smaller than their pore size, thus achieving high protein yield while maintaining high virus retention. In other words, contrary to the common belief that high virus retention and high protein yield are mutually exclusive, by using cellulose-based raw materials, increasing the separation layer thickness (5–60 μm), and appropriately increasing the average pore size measured by SEM (25–85 nm), the cellulose virus-removing membrane not only achieves high virus retention but also high protein yield. Furthermore, this cellulose virus-removing membrane also exhibits high flux, resulting in high filtration efficiency and improved production efficiency.

[0024] In addition, due to the high price of various active protein substances, improving the protein yield in each process is of great significance. In the virus filtration process, using a cellulose-based virus-removing membrane with good hydrophilicity to filter viruses from the feed solution can reduce the adsorption of proteins by the virus-removing membrane, thereby increasing the protein yield while ensuring a high virus rejection rate and achieving greater economic benefits. Based on this, whether in the laboratory validation stage (to avoid wasting protein or to better reflect the actual production process) or in the actual production process of biopharmaceutical companies, a top-washing operation is generally required after using a membrane filter (or filter membrane) for virus filtration. This is because, even when using a cellulose-based filter membrane with good hydrophilicity, some protein will still be retained in the filter membrane during the virus filtration process. The high price of protein dictates that even if the amount of protein retained by the filter membrane is small, a top-washing operation is necessary to remove the protein from the filter membrane.

[0025] Taking the laboratory validation stage as an example, the top wash operation method generally involves removing the external pressure and allowing the actual production process to remain in a pressure-free state for a period of time (e.g., 5 to 15 minutes) during continuous filtration when the flow rate of the membrane filter (or filter membrane) decreases to 75% or only a small amount of feed solution remains. This allows the protein to be released from the pore structure. Subsequently, a buffer solution (which can be adjusted according to the actual feed solution system) is added to elute the released protein from the filter membrane to obtain the top wash solution. The top wash solution is then post-processed to recover the protein, thereby improving the protein yield.

[0026] In this application, to obtain a higher virus rejection rate (initial LRV) and a high protein yield, the average pore size of the separation layer, as measured by SEM, was appropriately increased. It is generally believed that during the top wash operation, when external pressure is removed and the process is paused, not only are proteins released from the tortuous pathways of the porous matrix, but viruses with stronger motility are also more easily released from the pore structure. This phenomenon is more pronounced in cellulosic devirulent membranes with poor mechanical strength. Due to the increased average pore size of the separation layer as measured by SEM, viruses can more easily migrate through the tortuous pathways to the lower end of the devirulent membrane. These released and moved viruses to the lower end of the membrane are easily eluted into the top wash solution; therefore, the virus titer in the top wash solution is often much higher than the virus titer in the filtrate during continuous filtration. Correspondingly, for cellulosic devirulent membranes, the top wash LRV often decreases significantly compared to the initial LRV.

[0027] However, the inventors of this application unexpectedly discovered that for cellulosic virus removal membranes, when the thickness of the separation layer is 5-60 μm and the average pore size of the separation layer measured by SEM is 25-85 nm, the virus removal membrane not only achieves good virus filtration effect during continuous filtration, but also achieves a logarithmic removal rate of over 5 for PP7 phage (i.e., initial LRV > 5); and after the top wash operation, its logarithmic removal rate for PP7 phage remains high (i.e., top wash LRV). Compared with the initial LRV during continuous filtration, the top wash LRV retention rate can reach over 80%, and the top wash LRV can still be maintained at no less than 4. This is a completely new technical route and improvement direction, and the effect is also unexpected.

[0028] This may be because, for cellulosic virus-removing membranes, appropriately increasing the average pore size of the separation layer (as measured by SEM) while simultaneously increasing the separation layer thickness can not only reduce the impact of increased separation layer thickness on the membrane flux, allowing it to maintain a high flux, but also significantly improve the mechanical properties of the membrane due to its denser three-dimensional network structure and excellent self-supporting properties. This greatly reduces the deformation of the membrane under pressure, thus naturally reducing the possibility of a large release of viruses when the membrane recovers its shape. Furthermore, even with increased average pore size, the separation layer's relatively small pore size, combined with its long, tortuous pathways, still maintains good virus filtration. However, even with increased average pore size, the significant difference between the protein in the feed solution and the pore size of the separation layer makes it difficult to retain small proteins, even with the tortuous pathways. In other words, increasing the separation layer thickness and its average pore size (as measured by SEM) has different effects on the retention efficiency of viruses and proteins.

[0029] It is important to note that for cellulosic virus removal membranes, increasing the thickness of the separation layer without increasing the average pore size measured by SEM may improve the virus filtration efficiency of the membrane. However, a thicker separation layer with smaller pores may prevent proteins from being washed out during the top wash, reducing protein yield. Furthermore, a thicker separation layer with smaller pores has a significant impact on the flux of the virus removal membrane, potentially causing a decrease in the flux.

[0030] If only the average pore size measured by SEM of the separation layer is increased without increasing the thickness of the separation layer, although the flux of the virus removal membrane is larger and proteins are easily washed out during top washing, the separation layer with low thickness and small pore size often has poor virus retention effect and cannot ensure that the virus removal membrane has the required virus filtration effect. In addition, although the three-dimensional network structure formed by the separation layer is relatively dense, its support effect is very limited due to its low thickness, which makes the virus removal membrane prone to large deformation when under pressure. Once the deformation of the virus removal membrane is too large, it may lead to a decrease in membrane flux and load, and may even lead to tearing or damage of the membrane.

[0031] Therefore, increasing the thickness of the separation layer and increasing the average pore size measured by SEM must be done simultaneously to ensure that the separation layer has a high virus rejection rate, a low protein rejection rate, and minimal deformation when the virus removal membrane is compressed. This reduces the possibility of releasing excessive virus due to the large recovery deformation of the virus removal membrane during the top wash process, thereby ensuring that the virus removal membrane still has a high logarithmic removal rate (i.e., top wash LRV) during top wash.

[0032] In addition, it is generally believed that a high LRV during top washing indicates that the virus-removing membrane still has a good retention effect on viruses during top washing, which also often means that the virus-removing membrane has a good retention effect on proteins during top washing. However, the inventors of this application unexpectedly discovered that although the virus's top washing LRV is high during top washing, the protein elution rate is not low. This may be because, as mentioned above, viruses and proteins have different sensitivities to changes in the separation layer structure. Appropriately increasing the separation layer thickness and the average pore size measured by SEM can improve the virus retention rate during top washing, but a larger average pore size measured by SEM means that even if the separation layer thickness is increased, the increase in protein retention rate is not significant. Therefore, even if the virus's top washing LRV is not less than 4 during top washing, a small amount of protein retained by the virus-removing membrane can still be eluted.

[0033] Because the viral titer in the wash solution after top washing is low, the possibility of the wash solution contaminating the filtrate is low. In addition, the difficulty of subsequent treatment of the wash solution is greatly reduced and the efficiency of treatment is greatly improved, which is of great significance to various biological and pharmaceutical companies.

[0034] It is understood that the "initial LRV" in this application refers to the logarithmic removal rate of the virus in the filtrate obtained during the continuous filtration process of the virus removal membrane; while the "top wash LRV" in this application refers to the logarithmic removal rate of the virus in the top wash solution obtained after the continuous filtration of the virus removal membrane is completed and the virus removal membrane is further subjected to a top wash operation.

[0035] PP7 bacteriophage is a typical virus. For example, the TR41 document issued by the Pharmaceutical Research and Development Authority (PDA) uses PP7 bacteriophage as a model virus for small-sized viruses to evaluate the virus retention capacity of filter membranes or membrane filters. Therefore, using PP7 bacteriophage as a model virus to compare the virus retention capacity of filter membranes during continuous filtration and during top washing is a reliable and acceptable method for evaluating the virus removal efficiency of filter membranes before and after top washing.

[0036] The term "non-directional tortuous pathway" in this application refers to a porous body with irregularly oriented groove structures and / or discretely distributed pore structures, and these non-directional tortuous pathways are interconnected, so that the feed liquid can penetrate the filter membrane through the interconnected pathways. Viruses and large particles in the feed liquid are trapped in the non-directional tortuous pathways inside the filter membrane or the porous body, thereby achieving the effect of filtering out viruses.

[0037] In this application, "continuous fiber transition" means that all fibers in the porous body along the membrane thickness direction are integrally formed and interconnected as a whole. No additional adhesives or other substances are needed to bond the fibers together. Unless torn or peeled by external force, the three-dimensional network of fibers will not separate from each other. Simultaneously, the continuously transitioning three-dimensional network of fibers is also interconnected with the first and second outer surfaces.

[0038] The parameters such as average pore size, layer thickness, and average fiber diameter in this application can all be calculated by morphological characterization of the membrane structure using scanning electron microscopy, followed by measurement using computer software (such as Matlab, NIS-Elements, etc.) or manually. Significantly smaller or larger dimensions are not considered during measurement. It should be noted that porosity can also be calculated using computer software (such as Matlab, NIS-Elements, etc.) or measured by gravimetric methods. Regarding the testing of average pore size, in addition to measurement and analysis of SEM images, the average pore size of each layer can be directly analyzed using an average pore size distribution analyzer, or the average pore size can be tested using the bubble pressure method. The above measurement methods for each parameter are only preliminary; it is understood that those skilled in the art can obtain these parameters using other measurement methods.

[0039] In addition, it must be noted that since cellulose exhibits significant shrinkage during drying, all morphological parameters of cellulose filter membranes are measured by taking SEM images under wet or freeze-drying conditions.

[0040] Optionally, the side of the separation layer away from the pre-filter layer is the bottom surface of the separation layer. The virus-removing membrane containing 20nm colloidal gold is top-washed. After top-washing, the distance between the peak part of the virus-removing membrane containing 20nm colloidal gold and the bottom surface of the separation layer is D1, where D1 is 0.2 to 5μm.

[0041] By adopting the above technical solution, it is difficult to observe the retention position of PP7 bacteriophage in the deviruation membrane (unless fluorescent genes are introduced into PP7 bacteriophage through gene editing, but the observation effect is generally not good), and therefore it is difficult to intuitively show and evaluate the retention behavior of the deviruation membrane for PP7 bacteriophage in the thickness direction.

[0042] To more intuitively demonstrate the retention position of 20nm viruses in the virus removal membrane after continuous filtration or top washing, the virus removal membrane of this application was used to capture 20nm colloidal gold.

[0043] Because the separation layer of the cellulose virus-removing membrane in this application has a larger pore size, it is generally believed that a large-pore separation layer is more likely to cause leakage of 20 nm colloidal gold during top washing. However, the inventors of this application unexpectedly discovered that when the distance between the peak capture site of 20 nm colloidal gold and the bottom surface of the separation layer after top washing is 0.2–5 μm, it not only ensures that the proteins trapped by the virus-removing membrane can be eluted into the washing solution during top washing, but also ensures that a large amount of virus is still trapped in the virus-removing membrane during top washing, thus maintaining a high LRV during top washing. This has important theoretical guiding significance for determining the top washing effect of the virus-removing membrane.

[0044] This may be because the amount of 20nm colloidal gold retained in the separation layer initially increases and then gradually decreases. In other words, the amount of 20nm colloidal gold captured along the thickness of the separation layer follows a curve similar to a normal distribution. Although the separation layer at the peak captures the largest amount of 20nm colloidal gold, some 20nm colloidal gold is still captured below the peak. Therefore, to reduce the risk of 20nm colloidal gold leakage during continuous filtration and to minimize the possibility of virus elution during top washing, it is necessary to control the distance between the peak capture area of ​​the 20nm colloidal gold and the bottom surface of the separation layer.

[0045] When D1 is too large, it means that after top washing, the distance between the part that plays a major role in retaining 20nm colloidal gold and the bottom surface of the separation layer is large. Although the top washing LRV is high, there is too much of the part of the separation layer that does not play a role in retention. This part of the separation layer will have a significant impact on the flux of the virus removal membrane, affecting the efficiency of continuous filtration and top washing operations. When D1 is too small, since the 20nm colloidal gold has a distribution pattern similar to a normal distribution in the thickness direction within a certain range, the distance between the colloidal gold at the lower end of the peak capture part of 20nm colloidal gold and the lower end is too small, which can easily lead to leakage.

[0046] It is understood that the "peak capture location" in this application refers to the location where 20nm colloidal gold is captured by a virus-removing membrane, and the darkest area is determined by optical microscopy after cutting a slice from the virus-removing membrane containing 20nm colloidal gold; or the location of the peak after testing with an EDS spectrometer is the capture peak location. Since the spectral shift peak is positively correlated with the amount of 20nm colloidal gold, the maximum peak of the spectrum is the peak capture location of 20nm colloidal gold. The determination of the distribution of colloidal gold in the porous membrane can be performed with reference to the test method in Chinese Patent CN105980038B - Virus-removing Membrane.

[0047] Optionally, in the cross-section of the virus-removing membrane, the distance between the peak region of the 20nm colloidal gold capture and the bottom surface of the separation layer is D0, where D0 is 0.5–5μm.

[0048] By adopting the above technical solution, the size of D0 mainly affects the initial LRV and flux of the virus removal membrane, which may be due to...

[0049] If D0 is too large, it indicates that a large amount of 20nm colloidal gold is trapped at the upper end of the separation layer. Virus removal membranes with this structure often have higher initial LRV and top-wash LRV. However, since the 20nm colloidal gold is essentially captured at the upper end of the separation layer, the lower end of the separation layer has little retention effect. If this non-retention portion of the separation layer constitutes too large a proportion, it will not only have little effect on further improving the initial LRV and top-wash LRV, but will also significantly impact the flux of the virus removal membrane. Lower flux has an excessive impact on the efficiency of continuous filtration and top-washing operations of the virus removal membrane, leading to a decrease in overall production efficiency. Furthermore, with a large amount of virus trapped at the upper end of the separation layer, viruses and viruses and fibers easily form bridging structures, resulting in protein retention. Therefore, if D0 is too large, it may also lead to a decrease in protein yield.

[0050] If D0 is too small, it means that a large amount of 20nm colloidal gold is trapped at the lower end of the separation layer. This type of virus-removing membrane not only has a low initial LRV, but also the LRV often drops significantly after the top wash because the 20nm colloidal gold is likely to move to the lower end. This often leads to an increased risk of virus leakage from the virus-removing membrane.

[0051] When D0 is 0.5–5 μm, 20 nm colloidal gold, distributed in a near-normal pattern, can be retained relatively completely in the virus-removing membrane. Since the peak of the 20 nm colloidal gold capture point is not far from the bottom surface of the separation layer, the lower portion of the separation layer, which has almost no retention effect and thus significantly impacts flux, constitutes a small proportion. This allows for a high throughput while maintaining a high initial LRV. Furthermore, because the bridging effect between the virus and the separation layer fibers is relatively close to the bottom surface of the separation layer, these bridging effects are less likely to cause excessive protein retention, whether during continuous filtration or top washing. This results in a high protein yield for the virus-removing membrane.

[0052] Optionally, D1 is less than D0, and the difference between D0 and D1 is 0.1 to 3 μm.

[0053] By adopting the above technical solution, the inventors of this application discovered that the peak capture site of 20nm colloidal gold shifts downward in the direction of the virus-free membrane thickness after top washing (based on the flow direction of the feed liquid, the first outer surface of the virus-free membrane is considered the upper end, and the second outer surface is considered the lower end; the downward shift refers to the movement towards the second outer surface). This means that even inactive colloidal gold will be released from the virus-free porous structure after the external pressure is removed for a certain period of time. Therefore, the peak capture site of 20nm colloidal gold will also shift downward after the top washing operation, which greatly increases the risk of leakage of 20nm colloidal gold (i.e., approximately 20nm virus).

[0054] It is generally believed that the smaller the distance the peak capture site of 20nm colloidal gold moves downward before and after the top wash (i.e., the difference between D0 and D1), the better. This indicates that less 20nm colloidal gold is washed away during the top wash, thus resulting in a smaller decrease in LRV during the top wash.

[0055] However, the inventors of this application have discovered that a smaller difference between D0 and D1 is not always better. While a smaller difference between D0 and D1 often means a higher retention rate of the top wash LRV compared to the initial LRV, it also often indicates that the pore size at the lower end of the separation layer is too small or that the average pore size at the lower end of the separation layer decreases rapidly along the thickness direction of the membrane (other conditions that can affect the downward movement of 20nm colloidal gold may also produce this result). Regardless of the reason, this can hinder the downward movement of 20nm colloidal gold, which often also affects the downward movement of the feed solution and protein, thus affecting throughput and protein yield. When the difference between D0 and D1 is too small, although the top wash LRV is larger, it has a greater impact on throughput and protein yield. Therefore, the difference between D0 and D1 is not necessarily better the smaller it is. When the difference between D0 and D1 is 0.1 to 3 μm, it can not only ensure a high protein yield and high LRV of the head wash, but also ensure a high flux of the virus removal membrane, which is quite unexpected.

[0056] Optionally, the average pore size change gradient of the separation layer measured by SEM is 0–3 nm / μm; for PP7 phage, the ratio of the top wash LRV of the virus-removing membrane to the initial LRV is 0.85–0.95.

[0057] By adopting the above technical solution, when the average SEM pore size of the separation layer remains basically constant from top to bottom, the separation layer is considered to have a basically symmetrical structure. A basically symmetrical separation layer does not have a layer structure with small pore sizes. When the average SEM pore size of the separation layer gradually decreases from top to bottom, the separation layer is considered to have an asymmetrical structure. An asymmetrical separation layer has a smaller average SEM pore size closer to the bottom, thus having large-pore and small-pore regions. For example, if the average SEM pore size of the separation layer gradually decreases from 40 nm to 20 nm, the separation layer has a large-pore layer structure with an average SEM pore size of approximately 40 nm and a small-pore layer structure with an average SEM pore size of 20 nm. In this case, the average SEM pore size of the separation layer is approximately 30 nm, which is an asymmetrical structure. However, if the average SEM pore size of the separation layer is uniformly distributed at 30 nm, it is considered to have no small-pore layer structure and is a symmetrical structure.

[0058] Based on this, the inventors of this application discovered that even for separation layers with essentially the same average pore size measured by SEM, a larger gradient in the average pore size measured by SEM often indicates a lower flux of the virus removal membrane and a decrease in the top wash LRV; conversely, a smaller gradient in the average pore size measured by SEM often results in a higher flux of the virus removal membrane. This may be because the relatively dense structure of the separation layer makes it the main region affecting the virus removal flux. Asymmetric separation layers, due to their small pore size regions, exert a strong resistance on the feed solution, leading to a decrease in the virus removal membrane flux. Conversely, symmetrical separation layers, due to their near isotropic nature, experience relatively uniform resistance to the feed solution, with no obvious high-resistance regions, resulting in a relatively higher flux of the virus removal membrane. Furthermore, the non-uniformity of the mechanical properties of asymmetric separation layers significantly reduces their self-supporting capacity, making them more prone to deformation under pressure. This, in turn, makes them more likely to release viruses during the top wash process, leading to a decrease in the top wash LRV.

[0059] The inventors of this application have also discovered that, with an appropriate increase in the thickness and average pore size of the separation layer (thickness of 5–60 μm and average pore size of 25–85 nm), when the separation layer has a symmetrical structure or the gradient of the average pore size is small (i.e., the gradient of the average pore size of the separation layer is 0–3 nm / μm), not only is the flux of the virus removal membrane also less affected, but a higher top-wash LRV (the ratio of the top-wash LRV of the virus removal membrane to the initial LRV is 0.85–0.95) can also be obtained. This results in a virus removal membrane with high throughput, high protein yield, high initial LRV, and high top-wash LRV. High protein yield, high virus rejection rate, and high throughput are generally considered to be mutually exclusive, so this effect is quite unexpected.

[0060] In addition, it is generally believed that the higher the top wash LRV, the better, in order to further reduce the risk of virus leakage during top washing. However, the inventors of this application have found that as the top wash LRV gradually increases, the difficulty of increasing the top wash LRV increases sharply. A small increase in the top wash LRV often comes at the cost of a significant decrease in throughput and a decrease in protein yield. This will not only reduce production efficiency but also reduce economic benefits. Therefore, the top wash LRV is not necessarily better the higher it is.

[0061] It is understandable that the average pore size variation gradient refers to the magnitude of the change in the average pore size of the separation layer in the thickness direction from the top to the bottom. The larger the average pore size variation gradient, the faster the average pore size of the separation layer changes in the thickness direction.

[0062] Optionally, the porosity of the separation layer is 6-30%, and the thickness variation rate of the separation layer is not higher than 10%.

[0063] By adopting the above technical solutions, the porosity and thickness of the separation layer have a significant impact on the virus filtration efficiency and flux of the virus removal membrane. Although excessively high porosity or excessively low thickness results in higher flux, both the initial LRV and the top wash LRV are often low, leading to a higher risk of virus leakage. Conversely, excessively low porosity or excessively high thickness, while resulting in better virus filtration, often leads to lower flux, lower protein yield, and lower filtration efficiency, making it unsuitable for actual production.

[0064] In addition, the inventors of this application discovered that even with virus-removing membranes where the porosity and thickness of the separation layer are essentially the same, the top-wash LRV may still be low, a rather unexpected result. Through comparison, the inventors found that when the thickness variation rate of the separation layer exceeds 10%, even if the average thickness and porosity of the separation layer are roughly the same, the top-wash LRV may still fluctuate significantly. This may be because, when the feed solution flows along the thickness direction of the virus-removing membrane, the average pore size of the separation layer is smaller, resulting in greater resistance, making it the main area affecting feed solution flow. If the separation layer thickness is smaller in certain areas, the resistance to the feed solution is also smaller, allowing the feed solution to easily generate a larger flux in these thinner areas. Viruses can easily penetrate these weak points, leading to a decrease in the top-wash LRV.

[0065] Understandably, the rate of change of the separation layer thickness is the ratio of the difference between the maximum and minimum thickness to the average thickness of the separation layer. The greater the rate of change of thickness, the more uneven the thickness of the separation layer.

[0066] Optionally, the flux of the virus removal membrane is greater than 35 L·h. -1 ·m -2 @30psi; the protein yield of the viral membrane is not less than 98%.

[0067] By adopting the above technical solution, the virus removal membrane not only has a good virus filtration effect, but also has a high throughput and a high protein yield.

[0068] Optionally, the first outer surface includes a plurality of elongated and interconnected first fibers, the average diameter of which, as measured by SEM, is 70–650 nm, and adjacent and interconnected first fibers surround each other to form a first hole.

[0069] By employing the above-mentioned technical solutions, the pre-filter layer and the large pore size of the first outer surface can achieve a good contaminant holding effect, but their mechanical properties are often poor. Under external pressure, the pore structure is prone to collapse, which not only reduces the contaminant holding capacity but also affects the flux of the virus removal membrane. This phenomenon is even more pronounced and has a greater impact on the already soft cellulose-based virus removal membrane.

[0070] During the use of virus-removing membranes, they are subjected to significant pressure from the feed solution (e.g., 30 psi, and even up to 50 psi when verifying membrane integrity). When the average diameter of the first fiber, measured by SEM, is 70–650 nm, it not only forms a pore structure with a suitable pore size but also provides excellent support for these pores, greatly reducing the possibility of pore collapse under high pressure. This is crucial for cellulose-based virus-removing membranes. The first outer surface, as the part directly in contact with the feed solution and directly bearing pressure, is susceptible to deformation due to excessive feed pressure, which could lead to a decrease in flux and load capacity. The first fiber with a specific average diameter measured by SEM on its first outer surface, combined with a thicker separation layer structure, provides mutual support and reinforcement, resulting in strong self-support for the virus-removing membrane and significantly reducing deformation under high pressure. Unlike conventional cellulosic virus removal membranes (such as the cellulosic filter membrane in CN105980038A), which can often only be used at pressures of 15 psi or even lower, the virus removal membrane of this application can be used for a long time at a pressure of 30 psi, which is a very significant and commercially valuable improvement.

[0071] Optionally, the separation layer includes a second outer surface having second pores, the average pore diameter of the second pores measured by SEM is 15-35 nm, the pore area ratio of the second outer surface is 2-15%, and the fibers of the separation layer are long strips with an average fiber diameter of 20-50 nm measured by SEM.

[0072] By adopting the above technical solution, the pre-filter layer includes a first outer surface and the separation layer includes a second outer surface. Therefore, the virus removal membrane is a typical two-layer structure (a pre-filter layer with a larger average pore size measured by SEM and a separation layer with a smaller average pore size measured by SEM). The pre-filter layer is used to filter larger particles, thereby increasing the dirt holding capacity and service life of the virus removal membrane. The separation layer plays a major role in retaining viruses, reducing the risk of virus leakage.

[0073] The lower SEM average pore size and lower pore area ratio of the second outer surface ensure that the virus-removing membrane not only has a high initial LRV but also a high top-wash LRV. The SEM average pore size of the separation layer is 25–85 nm, while that of the second outer surface is 15–35 nm. This means that the SEM average pore size of the second outer surface is generally not larger than that of the separation layer. During top washing, the portion of the separation layer near the second outer surface can effectively trap viruses, thereby increasing the top-wash LRV and reducing the risk of virus leakage.

[0074] It is generally believed that the separation layer with smaller pore size is the main factor affecting the flux of the virus removal membrane. The feed solution needs to bypass the fiber structure when passing through the separation layer; therefore, the smaller the average fiber diameter measured by SEM in the separation layer, the less resistance it poses to the feed solution, and the higher the flux of the virus removal membrane. However, the inventors of this application have discovered that a smaller average fiber diameter measured by SEM in the separation layer is not necessarily better. This may be because when the average fiber diameter measured by SEM in the separation layer is too small, the self-supporting capacity of the three-dimensional network structure formed by the separation layer is weak, making it easily compressed under the pressure of the feed solution. The pore structure of the separation layer is also compressed, and the smaller pore size after compression actually leads to a decrease in flux. Furthermore, when the average fiber diameter measured by SEM in the separation layer is too small, even with a cellulose-based raw material with good hydrophilicity, the excessively large specific surface area will result in a higher protein adsorption rate, and the protein yield will decrease accordingly. Therefore, when the average fiber diameter of the separation layer measured by SEM is 20-50 nm, it can ensure that the three-dimensional network structure of the separation layer has good self-support and relatively low resistance to the feed liquid. The two work together to ensure that the virus removal membrane has high flux and virus filtration effect.

[0075] Optionally, the thickness ratio of the separation layer to the porous body is 8-25%, and the porosity of the virus removal membrane is 20%-60%.

[0076] By employing the above technical solutions, cellulosic virus removal membranes, due to their excellent hydrophilicity, can still achieve high protein yields even with a thick separation layer. However, for thicker virus removal membranes, the feed solution travels a longer, tortuous path through the membrane thickness. While viruses are more easily trapped in this long, winding path, the flux often decreases, and the protein adsorption rate is often higher, leading to a lower protein yield. Therefore, when the porous body is thick, it is necessary not only to appropriately reduce the proportion of the small-pore separation layer but also to appropriately increase the overall porosity of the virus removal membrane. This will improve the flux and protein yield of the virus removal membrane while ensuring high initial LRV and top wash LRV.

[0077] The inventors of this application have discovered that when the thickness of the porous body is large (since the thickness of the separation layer is relatively low, the thickness of the porous body is correspondingly large), the thickness of the separation layer can be appropriately reduced to about 8% to 25%, and the porosity of the virus-removing membrane can be appropriately increased to 20% to 60%. At this time, even if the thickness of the virus-removing membrane is large, it is possible to obtain high throughput and protein yield while ensuring high initial LRV and top wash LRV.

[0078] Optionally, the average aperture of the first hole on the first outer surface measured by SEM is 400-5000 nm, the ratio of the average aperture of the first hole and the second hole measured by SEM is 15-200, and the area ratio of the hole on the first outer surface is 8-45%.

[0079] By adopting the above technical solution, when the thickness of the porous body is large, the average pore size of the first pore measured by SEM is 400-5000 nm, and the pore area ratio of the first outer surface is 8-45%. These two factors work together to ensure that the first outer surface of the virus-removing membrane can effectively guide the feed liquid into and through the pore structure of the first outer surface, thus entering the membrane interior and achieving higher flux. Furthermore, as the first outer surface directly bearing pressure, although it forms mutual support with the separation layer, it must itself possess sufficient pressure resistance. The inventors of this application have discovered that for the first fiber with an average diameter of 70-650 nm measured by SEM, and the average pore size of the first pore measured by SEM is 400-5000 nm, the first fiber provides good support for the first pore, ensuring that the virus-removing membrane can be used for a long time under a pressure of 30 psi.

[0080] When the average pore size and pore area ratio of the first pore measured by SEM are too low, the flux of the virus removal membrane is often low and the filtration efficiency is low due to the large thickness of the porous body. When the average pore size and pore area ratio of the first pore measured by SEM are too high, although the feed liquid can more easily enter the membrane through the pores on the first outer surface, the pre-filtration layer is less effective at pre-filtering large particles, which can easily cause blockage of the separation layer and reduce its service life. In addition, if the average pore size and pore area ratio of the first pore measured by SEM are further increased, it may even lead to a significant decrease in flux and loading capacity. This is because an excessively high average pore size and pore area ratio of the first pore will inevitably lead to insufficient support of the first fiber, resulting in a decrease in the mechanical properties of the membrane structure near the first outer surface. Under the pressure of the feed liquid, the membrane structure near the first outer surface is likely to collapse under pressure, leading to a decrease in flux.

[0081] In addition, the ratio of the average pore size measured by SEM of the first and second pores to some extent characterizes the degree of pore size variation in the thickness direction of the porous substrate. A larger ratio generally indicates a greater degree of pore size variation. Due to the greater thickness of the porous substrate, viruses are more easily trapped in long, tortuous pathways, and the resistance to the feed solution is already greater. Therefore, if the ratio of the average pore size measured by SEM of the first and second pores is too large, it often leads to a significant decrease in the flux of the virus removal membrane; conversely, if the ratio is too small, it greatly increases the risk of virus leakage. A ratio of 15 to 200 for the average pore size measured by SEM of the first and second pores can achieve a high flux while ensuring a low risk of virus leakage.

[0082] Optionally, the SEM measured average pore size of the pre-filter layer is 160–600 nm, the porosity of the pre-filter layer is 35%–75%, the SEM measured average pore size of the pre-filter layer gradually decreases from the side near the first outer surface to the side near the second outer surface, and the pore size of the pre-filter layer near the first outer surface decreases at a greater rate than the pore size of the pre-filter layer near the second outer surface.

[0083] By adopting the above technical solution, the pre-filtration layer has a significant impact on the service life and flux of the virus removal membrane. However, due to the larger thickness of the porous substrate, the flux is often lower. When the SEM-measured average pore size of the pre-filtration layer is 160–600 nm and the porosity is 35%–75%, the pre-filtration layer has good contaminant holding capacity, thus improving the service life of the virus removal membrane. Larger SEM-measured average pore size and porosity result in less resistance to the feed liquid and less impact on flux. Furthermore, as the most pressure-bearing part of the membrane thickness, the pre-filtration layer must have good pressure resistance and deformation resistance. If the SEM-measured average pore size and porosity of the pre-filtration layer are too large, even if the first outer surface and separation layer provide good reinforcement and self-support, the pre-filtration layer is still likely to deform under high-pressure feed liquid, leading to a decrease in the flux and loading capacity of the virus removal membrane.

[0084] In addition, the SEM measurement of the average pore size of the pre-filter layer shows a rapid change rate followed by a slower rate, which allows large particles to be trapped by the pre-filter layer more quickly, reducing the impact of large particles on the separation layer. Furthermore, since there is no abrupt change in the average pore size between the pre-filter and the separation layer, proteins are less likely to be trapped in the abrupt change in the average pore size. Therefore, even with a relatively thick viral membrane, a high protein yield can still be obtained.

[0085] Optionally, the thickness ratio of the separation layer to the porous body is 40-95%, and the porosity of the virus removal membrane is 15-50%.

[0086] By adopting the above technical solution, when the thickness of the porous substrate is small (due to the high proportion of the separation layer thickness, the thickness of the porous substrate is correspondingly small), the flow path of the feed liquid across the membrane thickness is short and the resistance is low. Although the virus removal membrane often has a high flux under low resistance, the short-distance tortuous channels often result in poor virus retention. A small porous substrate thickness can easily lead to a decrease in initial LRV and top wash LRV, increasing the risk of virus leakage. Therefore, when the thickness of the porous substrate is small, it is necessary to increase the proportion of the separation layer and reduce the overall porosity of the virus removal membrane to improve the virus filtration capacity of the virus removal membrane. This allows the thinner virus removal membrane to maintain high initial LRV and top wash LRV while achieving high flux and high protein yield.

[0087] The inventors of this application have discovered that when the separation layer accounts for 40-95% and the porosity of the virus removal membrane needs to be low, to about 15-50%, it is possible to ensure that the virus removal membrane has a high initial LRV and top wash LRV, and a high flux of the virus removal membrane.

[0088] Optionally, the average aperture of the first hole measured by SEM is 300–4500 nm, the ratio of the average aperture of the first hole and the second hole measured by SEM is 10–150, and the aperture area ratio of the first outer surface is 5–40%.

[0089] By adopting the above technical solution, due to the relatively small thickness of the porous body, and given that the average pore size of the first pore (SEM measurement) is 300–4500 nm and the pore area ratio of the first outer surface is 5–40%, the first outer surface of the virus-removing membrane can effectively pre-filter large particles, reducing their impact on the separation layer and allowing the separation layer to better retain viruses. Furthermore, due to the minimal resistance to the feed liquid, it can penetrate the pore structure of the first outer surface and enter the membrane interior. Therefore, for a thin virus-removing membrane, with a high separation layer thickness and low overall porosity, the first fiber (SEM measurement average diameter 70–650 nm) can provide good support for the first pores (SEM measurement average pore size 300–4500 nm). Combined with the thicker, self-supporting decoupling strands of the separation layer, this significantly reduces the deformation of the virus-removing membrane under high pressure, ensuring that the membrane not only has excellent virus filtration performance but also high flux and load capacity.

[0090] When the average pore size and pore area ratio of the first outer surface measured by SEM are too low, although the thickness of the porous body is small, the flux of the virus removal membrane will still decrease significantly. This is because large particles in the feed solution can easily clog the first outer surface or the pre-filtration layer, and the concentrated large particles form a large number of bridging structures, greatly increasing the resistance of the feed solution. When the average pore size and pore area ratio of the first outer surface measured by SEM are too high, large particles can easily break through and accumulate at the lower end of the pre-filtration layer or even accumulate in the separation layer, thereby affecting the virus retention effect of the separation layer and leading to a decrease in service life.

[0091] In addition, the ratio of the average pore size measured by SEM of the first and second pores to some extent characterizes the degree of pore size variation in the thickness direction of the porous substrate. The larger the ratio, the greater the degree of pore size variation. Since the porous substrate has a small thickness and low porosity, the virus is less obstructed in short-distance tortuous pathways. In this case, if the ratio of the average pore size measured by SEM of the first and second pores is too large, the increase in flux is not significant, but it may lead to a decrease in the lifespan of the virus removal membrane and a decrease in the initial LRV and the top wash LRV. If the ratio of the average pore size measured by SEM of the first and second pores is too small, the first outer surface is easily blocked by large particles, and the lifespan of the virus removal membrane and the initial LRV and the top wash LRV may also decrease.

[0092] The SEM measurement average pore size ratio of the first and second pores is 10–150, which ensures high throughput of the low-thickness virus removal membrane while also providing high initial LRV, top wash LRV, and service life.

[0093] Optionally, the SEM measured average pore size of the pre-filter layer is 150–500 nm, the porosity of the pre-filter layer is 30–70%, the SEM measured average pore size of the pre-filter layer gradually decreases from the side near the first outer surface to the side near the second outer surface, and the pore size of the pre-filter layer near the first outer surface decreases at a greater rate than the pore size of the pre-filter layer near the second outer surface.

[0094] By adopting the above technical solution, the pre-filter layer has a significant impact on the service life and throughput of the virus removal membrane. Furthermore, due to the small thickness of the porous body, the virus filtration effect is often poor. More importantly, for thin virus removal membranes, the pre-filter layer accounts for a relatively small proportion. Therefore, large particles can easily pass through the pre-filter layer, affecting the separation layer and thus impacting the throughput and virus filtration effect of the virus removal membrane. The pore size at the upper end of the pre-filter layer (the side closest to the first outer surface) rapidly decreases, forming a pore size abrupt change zone. This abrupt change zone has little impact on the flow of proteins and small-sized viruses, but a significant impact on large particles. The synergistic effect of the significantly reduced pore size of the pre-filter layer compared to the first pore ensures that the upper end of the pre-filter layer has a good retention effect on large particles, reducing the possibility of large particles leaking from the pre-filter layer and affecting the separation layer. The pore size variation is small at the lower end of the pre-filter layer (closer to the second outer surface). The high porosity of the pre-filter layer (30-70%) can increase the dirt holding capacity of the pre-filter layer, ensuring that the pre-filter layer can hold a sufficient amount of large particles to increase the filter membrane's load.

[0095] Optionally, a protective layer is provided on the side of the separation layer away from the pre-filter layer, and the protective layer and the separation layer are transitioned by continuous fibers, the protective layer including a second outer surface;

[0096] The average pore size measured by SEM of the protective layer is greater than that of the separation layer but smaller than that of the pre-filter layer. The thickness of the porous body is 30–80 μm, and the ratio of the thickness of the protective layer to the thickness of the porous body is 5–20%.

[0097] While the above-mentioned technical solution achieves good virus filtration efficiency for the two-layer virus removal membrane, the separation layer, located on the membrane surface, is directly exposed and susceptible to mechanical damage. Further, introducing a protective layer outside the separation layer (with an average pore size measured by SEM) that is larger than the separation layer but smaller than the pre-filtration layer, and limiting the thickness of this protective layer, ensures effective protection of the separation layer. Furthermore, by limiting the thickness of the protective layer and its ratio to the thickness of the porous matrix, the possibility of mechanical damage to the separation layer, which plays a major role in virus removal, can be reduced, while ensuring a high flux of the virus removal membrane.

[0098] If the protective layer is too thin, although its impact on flux is small, its protection effect on the separation layer is poor; while if the protective layer is too thick, although its protection effect on the separation layer is good, its impact on flux is significant. With a porous substrate thickness of 30–80 μm, a protective layer with a thickness of 5–20% not only ensures good protection of the separation layer but also ensures a high flux of the virus removal membrane.

[0099] This is likely because, although the introduction of the protective layer increases the resistance of the feed liquid to the membrane thickness to some extent, the pre-filtration layer and the protective layer located on both sides of the separation layer can act as reinforcing ribs, thereby improving the mechanical properties of the virus-removing membrane and further reducing the possibility of flux reduction due to pore structure deformation under feed pressure. In other words, the flux of the virus-removing membrane is affected by both the increased resistance caused by the introduction of the protective layer and the reduction of pore structure compression deformation. Within a certain range, appropriately increasing the thickness of the protective layer may not even lead to a decrease in the flux of the virus-removing membrane, which is quite unexpected.

[0100] Optionally, the average aperture of the first hole measured by SEM is 300–4500 nm, the average aperture of the second hole measured by SEM is 100–500 nm, and the ratio of the average aperture of the first hole to the average aperture of the second hole measured by SEM is 2–25.

[0101] By adopting the above technical solution, the protective layer introduced on the outside of the separation layer affects the flux of the virus removal membrane to some extent. However, the inventors of this application have found that when the average pore size measured by SEM of the first pore is 300-4500 nm and the average pore size measured by SEM of the second pore is 100-500 nm, and the ratio of the two is 2-25, combined with the low thickness ratio of the protective layer, the virus removal membrane can still have a high flux while significantly reducing the mechanical damage resistance of the separation layer.

[0102] This may be because the average pore size measured by SEM of the first and second pores can characterize the membrane structure near the two surfaces of the virus membrane to a certain extent. When the average pore size measured by SEM of the first outer surface is too large, it not only leads to a poorer retention effect for large particles, increasing the possibility of large particles directly clogging the separation layer, but also may reduce the mechanical properties of the pre-filtration layer because the first fiber is insufficient to provide enough support. Under the pressure of the feed liquid, the pore structure may undergo compression deformation, resulting in a decrease in flux. When the average pore size measured by SEM of the first pore is too small, the feed liquid is subjected to excessive resistance from the solid part, which not only leads to a decrease in flux, but may also lead to a decrease in the dirt-holding capacity of the pre-filtration layer and a reduction in its service life. When the average pore size measured by SEM of the first pore is too large, external objects may still cause mechanical damage to the separation layer through the larger pore structure, and the reinforcing rib effect of the protective layer may also decrease. When the average pore size of the second pore is too small, although the protective layer has a better protection effect on the separation layer and can also play a better reinforcing rib role, the small pore size of the second pore has a greater impact on the flux and a greater impact on the efficiency of the entire filtration process.

[0103] The ratio of the average pore size of the first and second pores measured by SEM is 2 to 25, which ensures that the combination of the pore structures on the two surfaces of the virus removal membrane not only has a good virus retention effect and mechanical strength, but also a high throughput.

[0104] Optionally, the SEM measurement average pore size of the protective layer is 50–450 nm, and the porosity of the protective layer is 20–55%.

[0105] By adopting the above technical solution, with the protective layer thickness accounting for 5-25%, and when the average pore size of the protective layer is 50-450 nm and the porosity is 20-55%, not only is the protective layer's protective effect on the separation layer guaranteed, but it also ensures a high flux of the virus-removing membrane by reducing the compression deformation of the membrane under feed pressure. Furthermore, since virus removal filtration is generally dead-end filtration, the virus-removing membrane is often subjected to significant pressure perpendicular to the membrane, leading to a tendency for it to bulge downwards. This deformation easily causes compression of the pore structure near the upper end of the membrane, while stretching occurs near the lower end. The characteristics of cellulose fibers dictate that they are more prone to breakage under tension than under compression. Once the fibers inside the lower end of the virus-removing membrane break, the pore size will significantly increase, and viruses that should be trapped by the small pores are likely to leak out, potentially resulting in a substantial decrease in both the initial LRV and the top wash LRV. The protective layer introduced at the lower end of the separation layer can not only improve the separation layer's resistance to mechanical damage, but also reduce the possibility of initial LRV and top wash LRV decrease due to fiber breakage at the lower end of the virus removal membrane, thus reducing the possibility of virus leakage.

[0106] Optionally, a porous support layer is provided on the side of the separation layer away from the pre-filter, and a leak-proof layer is provided on the side of the porous support layer away from the separation layer. The separation layer, the porous support layer and the leak-proof layer are connected by continuous fibers.

[0107] The average pore size measured by SEM of the porous support layer is greater than the average pore size measured by SEM of the separation layer and the leak-proof layer, but smaller than the average pore size measured by SEM of the pre-filter layer.

[0108] The average pore size of the porous support layer measured by SEM is 50–400 nm, and the average pore size of the anti-seepage layer measured by SEM is 25–35 nm.

[0109] By adopting the above technical solution, the four-layer structure is arranged sequentially along the direction of liquid flow in the membrane thickness direction, namely the pre-filtration layer with the largest pore size, the separation layer with the smaller pore size, the porous support layer with the medium pore size, and the anti-leakage layer with the smaller pore size.

[0110] The pre-filtration layer has a larger average pore size as measured by SEM, which can filter out large particles in the feed solution and reduce the possibility of large particles directly clogging the separation layer, thus reducing the lifespan of the virus removal membrane. The separation layer, with a smaller average pore size as measured by SEM, plays a major role in virus retention. After the feed solution, which has had large particles filtered out by the pre-filtration layer, passes through the separation layer, it is trapped or adsorbed on the surface or inside the separation layer. Smaller particles, such as proteins, can pass through the pore structure of the separation layer and move with the feed solution. Therefore, the virus concentration in the feed solution after passing through the separation layer is very low, while the protein concentration and purity are high. The feed solution after passing through the separation layer enters the porous support layer, which has a larger average pore size as measured by SEM compared to the separation layer. The porous support layer plays a good buffering role, reducing the deformation of the virus removal membrane under the pressure of the feed solution and improving its lifespan and filtration effect (especially for hollow fiber membranes, since the outer surfaces of the membrane fibers are bonded together with adhesive, the constantly deforming membrane fibers are easily desorbed from the adhesive, thus affecting the lifespan of the entire membrane filtration module).

[0111] As the feed solution flows through the porous support layer and then into the impermeable layer with smaller pores, this process, accompanied by a sudden decrease in pore size, creates a secondary retention effect. This effect traps the already low concentration of viruses in the feed solution, further enhancing the virus-removing membrane's ability to retain viruses. This secondary retention effect not only significantly improves the initial LRV of the virus-removing membrane but also greatly enhances its top-wash LRV.

[0112] In addition, when the average pore size of the porous support layer measured by SEM is 50–400 nm and the average pore size of the anti-permeability layer is 25–35 nm, the initial LRV and top wash LRV of the virus removal membrane are improved. This may be because the separation layer, porous support layer and anti-permeability layer cause the virus removal membrane to form a discontinuous trend of small pore size, medium pore size and small pore size in terms of membrane thickness. The movement state of the feed liquid, especially the virus in the feed liquid, becomes a discontinuous movement state. The discontinuous movement state of the virus, combined with the small average pore size measured by SEM of the anti-permeability layer, can ensure that the virus removal membrane has a high initial LRV and top wash LRV.

[0113] Furthermore, the unique dual-layer structure of the separation layer and the anti-leakage layer ensures that the virus-removing membrane maintains excellent virus retention even if defects occur during membrane fabrication. For example, even if defects develop in the pre-filtration layer and separation layer during membrane fabrication, the porous support layer combined with the anti-leakage layer can still achieve good retention (the probability of defects existing on both sides of a membrane simultaneously at the same location is very low). This significantly reduces the risk of virus leakage and improves the safety of the biological agent.

[0114] Optionally, the SEM average pore size of the pre-filter layer is 120–500 nm, the SEM average pore size of the separation layer is 25–35 nm, and the ratio of the SEM average pore size of the separation layer to the SEM average pore size of the leak-proof layer is 0.75–1.3.

[0115] By adopting the above technical solution, the pre-filter layer with an average pore size of 120–500 nm as measured by SEM can enable the virus removal membrane to have good dirt holding capacity, reduce the possibility of large particles clogging the separation layer, and improve the service life of the virus removal membrane. Furthermore, the good support of the first fiber, combined with the relatively small pore size of the pre-filter layer and the further introduced porous support layer, ensures that the virus removal membrane has minimal deformation under pressure, thereby reducing the decrease in flux and load capacity during use, and further ensuring that the virus removal membrane has good initial LRV and top-wash LRV.

[0116] Optionally, the thickness of the porous body is 25-100 μm, the ratio of the thickness of the separation layer to the thickness of the porous body is 5-25%, the ratio of the thickness of the porous support layer to the thickness of the porous body is 5-25%, and the ratio of the thickness of the leak-proof layer to the thickness of the porous body is 2-5%.

[0117] By adopting the above technical solutions, it is generally believed that although the dual retention layer structure of separation layer + anti-leakage layer can obtain better initial LRV and top wash LRV and reduce the risk of virus leakage, the virus removal membrane with such structure has a large resistance to the feed liquid, and therefore often has a low flux. The flux of the virus removal membrane has a very important impact on the efficiency of the entire filtration process.

[0118] In practice, when the thickness and average pore size of the separation layer are reasonable (the separation layer thickness accounts for 5-25%), the separation layer itself has a good virus filtration effect. Even during top washing, the amount of virus leaking from the separation layer is low. Therefore, the anti-leakage layer mainly plays an auxiliary role in retention. Considering that the thickness and average pore size of the anti-leakage layer do have a significant impact on the flux of the filter membrane, while ensuring high initial LRV and top washing LRV, it is necessary to condition the anti-leakage layer to ensure that the virus removal membrane has a high flux.

[0119] The inventors of this application have discovered that, in practice, by controlling conditions such as the average pore size and thickness of the porous support layer and the average pore size and thickness of the anti-leakage layer, even a virus-removing membrane with a double-layer structure can still achieve a high flux. This may be because, within a certain range, the initial LRV and top-wash LRV of the virus-removing membrane increase with the increase of the second separation layer; however, this improvement in virus filtration efficiency exhibits a diminishing marginal effect. As the thickness of the second separation layer increases, the virus filtration efficiency of the virus-removing membrane initially increases rapidly (rapid rise phase); as the thickness of the second separation layer further increases, although the virus filtration efficiency of the virus-removing membrane still improves, the rate of improvement slows significantly (slow rise phase and basically stable phase). Therefore, as long as the thickness of the second separation layer is in the rapid rise phase, the virus filtration efficiency of the virus-removing membrane can be significantly improved with a relatively thin anti-leakage layer, while minimizing the impact on the flux of the virus-removing membrane.

[0120] That is, when the impermeable layer accounts for 2 to 5% of the total membrane thickness, it can significantly improve the virus filtration effect of the virus removal membrane, while having little impact on the membrane flux. This makes it possible to obtain a dual-retention layer virus removal membrane with high virus filtration effect and high flux.

[0121] Secondly, this application provides a process for preparing a cellulose virus-removing membrane with high virus rejection rate, using the following technical solution:

[0122] A process for preparing a cellulose virus-removing membrane with high virus rejection rate includes the following steps:

[0123] S1. The casting solution is prepared and cast onto a carrier to form a liquid film; the casting solution comprises the following components by weight: 10-30 parts cellulose acetate; 20-50 parts good solvent; 3-6 parts non-solvent; 0.1-1 parts inorganic salt;

[0124] The good solvent is at least one selected from acetone, dioxane, dimethylacetamide, N-methylpyrrolidone, acetic acid, propionic acid, butyric acid, and valeric acid;

[0125] The non-solvent is water;

[0126] The cation of the inorganic salt is one or more of sodium, potassium, calcium, and magnesium, and the anion of the inorganic salt is one or more of sulfate, sulfite, or carbonate.

[0127] S2. Pre-phase separation: The first outer surface of the liquid film is pre-separated with a first pre-phase separation liquid to obtain a primary film; the first pre-phase separation liquid is a 60-90 wt% acetone aqueous solution, and the pre-phase separation time is 2-15 s.

[0128] S3. Solidification and phase separation: The nascent membrane is immersed in a coagulation bath for phase separation and solidification to obtain a film. The coagulation bath includes water and a penetrant. The concentration of the penetrant is 0.1-5 wt%. The penetrant is at least one of ethanol, n-propanol, isopropanol, n-butanol, n-pentanol, and sec-pentanol.

[0129] S4. Post-treatment: The film is placed in a sodium hydroxide solution for hydrolysis, and after hydrolysis, it is washed to form a solid film; the solid film is further placed in a crosslinking agent for crosslinking treatment, and after the crosslinking treatment is completed and the film is washed, the finished film is obtained. The crosslinking agent is at least one of haloepoxide, dihaloalkanes and dihaloalcohols.

[0130] By adopting the above technical solution, using cellulose acetate as the film-forming raw material, and then hydrolyzing and regenerating the film to obtain a regenerated cellulose membrane, the entire production process has less environmental pollution, and the obtained virus-removing membrane has good hydrophilicity, thus having a low protein adsorption rate and achieving a high protein yield.

[0131] Specifically, this application involves adding non-solvents and inorganic salts to the casting solution because the inventors have discovered that the synergistic effect of adding non-solvents and inorganic salts to the casting solution can improve the stability during pre-phase separation and curing phase separation, regulate the phase separation rate, and improve the uniformity of the thickness of each layer of the virus-removing membrane and the dimensional stability of the pore structure of each layer. This is likely because, although inorganic salts can improve phase separation stability and regulate the phase separation rate, their solubility in good solvents is poor, and their dispersibility in casting solutions is also poor. The added inorganic solvent itself has the function of regulating the phase separation rate, and it can also promote the dissolution and uniform dispersion of inorganic salts. The synergistic effect of both improves the dimensional uniformity and stability of the pore structure of the virus-removing membrane.

[0132] However, it is important to note that the amount of non-solvent added needs to be strictly controlled. This is because, while non-solvents can synergistically improve phase separation stability with inorganic salts, increasing the amount of non-solvents will also increase the phase separation rate of the film-forming components. If too much non-solvent is added, it may lead to low overall porosity and low flux of the virus-removing membrane, rendering it unusable. Conversely, if too little non-solvent is added, its effect on promoting the solubility and dispersion of inorganic salts is weak, and its own effect on promoting the phase separation stability of the film-forming components is also weak, resulting in poor uniformity of the final virus-removing membrane.

[0133] In step S2, a first pre-phase separation liquid with a low water content is used to pre-separate the liquid film. The first outer surface of the liquid film (i.e., one side of the original support) separates phases at a relatively slow rate, thereby forming a pore structure with a larger pore size on the first outer surface. In step S3, the small molecule alcohol permeate added to the coagulation bath not only has a good effect on improving the phase separation stability, but also can adjust the surface tension of the coagulation bath and promote the penetration of the coagulation bath into the membrane interior, thereby forming a separation layer and other structures with good performance.

[0134] For cellulose-based raw materials, which are inherently soft, hydrolyzing cellulose acetate into regenerated cellulose can improve the hydrophilicity of the virus-removing membrane. However, this often means a decrease in the dimensional stability of the fiber structure and a decline in the mechanical properties of the virus-removing membrane. This is particularly detrimental to cellulose-based virus-removing membranes, which already have poor mechanical properties. Cross-linking treatment can connect multiple fiber polymer chains together, forming a microscopic three-dimensional network self-supporting structure. This significantly improves the mechanical properties and dimensional stability of the fiber structure in the virus-removing membrane, thereby enhancing its overall mechanical performance.

[0135] Optionally, in step S2, the second outer surface is further pre-separated with a second pre-separation liquid, wherein the second pre-separation liquid is a 40-60 wt% acetone aqueous solution, and the pre-separation time is 2-10 s.

[0136] By adopting the above technical solution, the water content of the second pre-separated liquid is higher than that of the first pre-separated liquid, resulting in a smaller pore size compared to the pore structure on the first outer surface. Under the action of the first and second pre-separated liquids, the first outer surface extends into the membrane thickness to form a pre-filtration layer, and the second outer surface extends into the membrane thickness to form a protective layer. The coagulation bath containing a permeate permeates into the membrane through the pore structure formed on the first and second outer surfaces. Rapid phase separation occurs inside the membrane under the action of the coagulation bath with a higher water content and larger quantity, thereby forming a separation layer structure with the smallest average pore size.

[0137] In order to form the desired second outer surface porosity structure and protective layer structure, it is necessary to control the water content in the second pre-phase separation liquid and the pre-phase separation time, thereby controlling the phase separation rate and phase separation time of the second outer surface, and forming a protective layer and second outer surface structure with appropriate thickness, porosity and pore size.

[0138] Optionally, in step S2, the second outer surface is further pre-separated with a third pre-separating liquid, wherein the third pre-separating liquid is a 20-40 wt% acetone aqueous solution, and the temperature of the third pre-separating liquid is 5-10°C lower than that of the liquid film, and the pre-separation time is 2-10 s.

[0139] By employing the above technical solution, a third pre-phase liquid with a higher water content than the first pre-phase liquid is used to pre-separate the second outer surface. The higher water content of the third pre-phase liquid allows for the formation of a pore structure with smaller pore size on the second outer surface. Under the synergistic effect of pre-phase separation and coagulation liquid over a certain period, a leak-proof layer with a smaller average pore size and thinner thickness can be formed. Due to the smaller average pore size of the leak-proof layer, even with the addition of a penetrant in the coagulation bath, it is still difficult for the low porosity and small average pore size of the leak-proof layer to pass through. Therefore, the region where the leak-proof layer extends into the membrane interior often has a slower phase separation rate, thus forming a porous support layer with an average pore size larger than that of the leak-proof layer. After the formation of the porous support layer with a larger pore size, the coagulation bath that infiltrates from the pre-filtration layer and the coagulation bath that infiltrates from the porous support layer work together to rapidly separate the phases inside the membrane, thereby forming a separation layer structure with a smaller average pore size.

[0140] To form both the impermeable layer and the porous support layer, it is necessary to control the water content in the third pre-separated liquid and the pre-separation time. This is because, to obtain an impermeable layer with small pore sizes and achieve good two-layer retention, the second outer surface needs a relatively fast phase separation rate. A common practice is to increase the water content in the third pre-separated liquid. However, the inventors of this application have discovered that simply adjusting the water content in the third pre-separated liquid is insufficient to obtain the desired impermeable layer structure. This may be because even subtle changes in the water content of the third pre-separated liquid have a significant impact on the membrane structure, especially for impermeable layers with small average pore sizes. In this case, by appropriately lowering the pre-separation temperature, the structure of the impermeable layer can be adjusted more finely. That is, for impermeable layers with specific pore structures, the water content in the third pre-separated liquid plays a major role, while lowering the pre-separation temperature helps to adjust the pore structure of the impermeable layer. The third pre-phase separation liquid and the pre-phase separation temperature work together to obtain a relatively stable anti-leakage layer with the required structure after a certain period of pre-phase separation and coagulation bath treatment, ensuring a good two-layer retention effect and enabling the virus removal membrane to have good initial LRV and top wash LRV.

[0141] In summary, this application includes at least one of the following beneficial technical effects:

[0142] 1. By selecting cellulose-based materials with good hydrophilicity as the film-forming material, and further combining a thicker separation layer with an appropriately increased average pore size measured by SEM, the virus-removing membrane is able to retain viruses of the same order of magnitude as the pore size of the separation layer, while exhibiting poor retention of proteins much smaller than the pore size of the separation layer. Therefore, a high protein yield and high virus retention rate can be obtained. In addition, the thicker separation layer combined with an appropriately increased average pore size measured by SEM enables the separation layer to form a denser and thicker three-dimensional network structure, giving the separation layer good self-supporting properties. This greatly reduces the possibility of deformation of the virus-removing membrane under pressure, and also reduces the large-scale release of viruses caused by the recovery deformation of the virus-removing membrane during top washing, thereby improving the top washing LRV and reducing the difficulty of protein recovery in the top washing solution.

[0143] 2. To better characterize the virus retention area in the virus-removing membrane, 20nm colloidal gold was captured using the virus-removing membrane. Due to the distribution of 20nm colloidal gold across the membrane thickness, a curve resembling a normal distribution is formed within a certain region. By limiting the distance between the peak absorption peak of 20nm colloidal gold during continuous filtration and the peak absorption peak of 20nm colloidal gold after top washing and the bottom surface of the separation layer, it is possible to reduce the adsorption of proteins by the virus-removing membrane and its impact on flux while ensuring that the virus-removing membrane has high initial LRV and high top washing LRV.

[0144] 3. By capturing 20nm colloidal gold using a virus-removing membrane, it was found that the peak capture site of 20nm colloidal gold in the virus-removing membrane tended to shift downwards before and after top washing. It is generally believed that the smaller the downward shift of the peak capture site, the better, in order to obtain a higher top-wash LRV. However, in the cellulose virus-removing membrane of this application, through the improvement of the separation layer structure, it has better mechanical properties and is less likely to release a large amount of virus due to recovery deformation. The smaller the downward shift of the peak capture site, the better its retention effect on released viruses, and the greater the impact on protein yield and throughput.

[0145] 4. By controlling the separation layer to have a small pore size variation gradient (or even remain basically constant), the uniformity of the resistance of the feed liquid in the separation layer can be improved, thereby increasing the flux of the virus removal membrane. In addition, the more uniform three-dimensional network structure in the separation layer can improve the self-supporting ability of the separation layer, thereby reducing the possibility of virus release during top washing and improving the top washing LRV of the virus removal membrane.

[0146] 5. When the virus removal membrane has a two-layer structure of a pre-filtration layer and a separation layer, based on the good self-supporting effect of the separation layer, the average pore size and porosity of the pre-filtration layer and the average diameter of the supporting fibers measured by SEM are adjusted to ensure that the pre-filtration layer can form a three-dimensional network structure with good support effect. This, together with the separation layer, enables the virus removal membrane to have good mechanical properties, thereby improving the flux and load of the virus removal membrane and reducing the amount of virus released during top washing, thus improving the top washing LRV.

[0147] 6. When the virus removal membrane has a three-layer structure of pre-filtration layer, separation layer and protective layer, based on the good self-supporting effect of the separation layer, by adjusting the pore structure and fiber structure of the pre-filtration layer and the protective layer, it is ensured that the pre-filtration layer and the protective layer can form a good structural reinforcement effect on the separation layer located therein. Thus, in synergy with the separation layer, the virus removal membrane has good mechanical properties, thereby improving the flux and load of the virus removal membrane, and reducing the amount of virus released during top washing, so as to improve the top washing LRV.

[0148] 7. When the virus removal membrane has a four-layer structure consisting of a pre-filtration layer, a separation layer, a porous support layer, and an anti-leakage layer, the introduction of a double retention layer structure allows the separation layer, porous support layer, and anti-leakage layer to work together to create discontinuous pore size variations in the membrane thickness. This results in discontinuous virus movement, thereby improving the virus retention effect of the virus removal membrane. Furthermore, by adjusting the pore structure and fiber structure of the pre-filtration layer and porous support layer, and working in conjunction with the separation layer which has good self-supporting properties, the possibility of deformation of the virus removal membrane during use can be greatly reduced. Attached Figure Description

[0149] Figure 1 This is a cross-sectional scanning electron microscope image of the virus-removing membrane prepared in Example 1 of this application, with a magnification of 10K×.

[0150] Figure 2 This is a scanning electron microscope image of the first outer surface of the virus-free membrane obtained in Example 1 of this application, with a magnification of 50K×.

[0151] Figure 3 This is a scanning electron microscope image of the second outer surface of the virus-free membrane obtained in Example 1 of this application, with a magnification of 50K×.

[0152] Figure 4 This is a cross-sectional scanning electron microscope image of the virus-removing membrane prepared in Example 7 of this application, with a magnification of 5K×.

[0153] Figure 5 This is a scanning electron microscope image of the first outer surface of the virus-free membrane obtained in Example 7 of this application, with a magnification of 10K×.

[0154] Figure 6This is a scanning electron microscope image of the second outer surface of the virus-free membrane obtained in Example 7 of this application, with a magnification of 50K×.

[0155] Figure 7 This is a cross-sectional scanning electron microscope image of the virus-free membrane near the first outer surface obtained in Example 9 of this application, with a magnification of 5K×.

[0156] Figure 8 This is a cross-sectional scanning electron microscope image of the virus-free membrane near the second outer surface obtained in Example 9 of this application, with a magnification of 5.4K×.

[0157] Figure 9 This is a scanning electron microscope image of the first outer surface of the virus-free membrane obtained in Example 9 of this application, with a magnification of 50K×.

[0158] Figure 10 This is a scanning electron microscope image of the second outer surface of the virus membrane obtained in Example 9 of this application, with a magnification of 20K×.

[0159] Figure 11 This is a cross-sectional scanning electron microscope image of the virus-free membrane prepared in Example 12 of this application, with a magnification of 4K×.

[0160] Figure 12 This is a cross-sectional scanning electron microscope image of the virus-removing membrane prepared in Example 16 of this application, with a magnification of 2K×.

[0161] Figure 13 This is a scanning electron microscope image of the first outer surface of the virus-free membrane obtained in Example 16 of this application, with a magnification of 5K×.

[0162] Figure 14 This is a scanning electron microscope image of the second outer surface of the virus membrane obtained in Example 16 of this application, with a magnification of 2K×. Detailed Implementation

[0163] The following is in conjunction with the appendix Figures 1-14 This application will be described in further detail.

[0164] To more clearly illustrate the overall concept of this application, detailed descriptions are provided below using examples. Unless otherwise specified, the raw materials and equipment used in the following examples and comparative examples are all available through conventional commercial channels. Specifically, a Hitachi S-5500 scanning electron microscope was used to characterize the structural morphology of the virus-removing membrane.

[0165] Example 1

[0166] A process for preparing a cellulose virus-removing membrane with high virus rejection rate includes the following steps:

[0167] S1. Preparation of the casting solution: The casting solution comprises, by weight, 20 parts cellulose acetate, 35 parts good solvent, 4 parts non-solvent, and 0.5 parts inorganic salt. Specifically, the cellulose acetate is diacetate, the good solvent is acetone, the non-solvent is deionized water, and the inorganic salt is sodium sulfate. After the casting solution is prepared, it is cast onto the carrier to fully spread and form a liquid film.

[0168] S2. Pre-phase separation: The first outer surface of the liquid film away from the carrier is pre-phase separated with a first pre-phase separation liquid to obtain a nascent film. The first pre-phase separation liquid is an 80wt% aqueous solution of acetone, and the pre-phase separation time is 8s. After pre-phase separation, the first outer surface of the liquid film is initially separated and forms a porous structure.

[0169] S3. Solidification and Phase Separation: The nascent membrane obtained in step S2 is immersed in a coagulation bath to allow the casting solution in the nascent membrane to fully separate into two phases, forming a solvent-rich phase and a polymer-rich phase. The solvent-rich phase forms a porous structure, while the polymer-rich phase solidifies to form a porous fibrous structure. After the nascent membrane has fully separated and solidified, a film is obtained. The coagulation bath is an aqueous solution of a penetrant with a concentration of 1 wt%, and ethanol is selected as the penetrant.

[0170] S4. Post-treatment: The membrane is hydrolyzed in a 0.05 mol / L sodium hydroxide aqueous solution at 50°C until cellulose acetate is hydrolyzed into regenerated cellulose. The hydrolyzed membrane is then removed and washed with water until the pH is neutral, yielding a solid membrane. The solid membrane is then crosslinked in a crosslinking agent aqueous solution at pH 9.5. The crosslinking agent is epichlorohydrin, with a concentration of 10 wt%, a crosslinking time of 20 min, and a crosslinking temperature of 50°C.

[0171] Examples 2-4

[0172] The main difference between Examples 2-4 and Example 1 lies in the different process parameters in each step, as shown in the table below:

[0173]

[0174] Examples 5-13

[0175] The main difference between Examples 5-13 and Example 1 lies in the different process parameters in each step, as shown in the table below:

[0176]

[0177]

[0178] Example 14

[0179] A process for preparing a cellulose virus-removing membrane with high virus rejection rate includes the following steps:

[0180] S1. Preparation of the casting solution: The casting solution comprises, by weight, 20 parts cellulose acetate, 30 parts good solvent, 4 parts non-solvent, and 0.3 parts inorganic salt. Specifically, the cellulose acetate is diacetate, the good solvent is acetone, the non-solvent is deionized water, and the inorganic salt is sodium sulfate. After the casting solution is prepared, it is cast onto the carrier to fully spread and form a liquid film.

[0181] S2, Pre-phase separation, specifically includes pre-phase separation of the first outer surface of the liquid membrane away from the carrier with a first pre-phase separation liquid; and pre-phase separation of the second outer surface of the liquid membrane close to the carrier with a second pre-phase separation liquid to obtain a nascent membrane.

[0182] The first pre-phase separation liquid is an 80 wt% acetone aqueous solution, and the pre-phase separation time is 5 s; the second pre-phase separation liquid is a 50 wt% acetone aqueous solution, and the pre-phase separation time is 6 s; after pre-phase separation, the first and second outer surfaces of the liquid film both form a porous structure due to the initial phase separation.

[0183] S3. Solidification and Phase Separation: The nascent membrane obtained in step S2 is immersed in a coagulation bath to allow the casting solution in the nascent membrane to fully separate into two phases, forming a solvent-rich phase and a polymer-rich phase. The solvent-rich phase forms a porous structure, while the polymer-rich phase solidifies to form a porous fibrous structure. After the nascent membrane has fully separated and solidified, a film is obtained. The coagulation bath is an aqueous solution of a penetrant with a concentration of 1 wt%, and ethanol is selected as the penetrant.

[0184] S4. Post-treatment: The membrane is hydrolyzed in a 0.05 mol / L sodium hydroxide aqueous solution at 50°C until cellulose acetate is hydrolyzed into regenerated cellulose. The hydrolyzed membrane is then removed and washed with water until the pH is neutral, yielding a solid membrane. The hydrolyzed solid membrane is further crosslinked in a crosslinking agent aqueous solution at pH 9.5. The crosslinking agent is epichlorohydrin, with a concentration of 10 wt%, a crosslinking time of 20 min, and a crosslinking temperature of 50°C.

[0185] Examples 15-17

[0186] The main difference between Examples 15-17 and Example 14 lies in the different process parameters in each step, as shown in the table below:

[0187]

[0188] Example 18

[0189] A process for preparing a cellulose virus-removing membrane with high virus rejection rate includes the following steps:

[0190] S1. Preparation of the casting solution: The casting solution comprises, by weight, 25 parts cellulose acetate, 35 parts good solvent, 4 parts non-solvent, and 0.5 parts inorganic salt. Specifically, the cellulose acetate is diacetate, the good solvent is acetone, the non-solvent is deionized water, and the inorganic salt is sodium sulfate. After the casting solution is prepared, it is cast onto the carrier to fully spread and form a liquid film.

[0191] S2, Pre-phase separation, specifically includes pre-phase separation of the first outer surface of the liquid membrane away from the carrier with a first pre-phase separation liquid; and pre-phase separation of the second outer surface of the liquid membrane close to the carrier with a third pre-phase separation liquid to obtain a nascent membrane.

[0192] The first pre-phase separation liquid is an 80 wt% acetone aqueous solution, and the pre-phase separation time is 6 s; the third pre-phase separation liquid is a 30 wt% acetone aqueous solution, and the pre-phase separation time is 6 s, and the temperature of the third pre-phase separation liquid is 7 ℃ lower than the temperature of the liquid film; after pre-phase separation, the first and second outer surfaces of the liquid film both form a porous structure due to the initial phase separation.

[0193] S3. Solidification and Phase Separation: The nascent membrane obtained in step S2 is immersed in a coagulation bath to allow the casting solution in the nascent membrane to fully separate into two phases, forming a solvent-rich phase and a polymer-rich phase. The solvent-rich phase forms a porous structure, while the polymer-rich phase solidifies to form a porous fibrous structure. After the nascent membrane has fully separated and solidified, a film is obtained. The coagulation bath is an aqueous solution of a penetrant with a concentration of 1 wt%, and ethanol is selected as the penetrant.

[0194] S4. Post-treatment: The membrane is hydrolyzed in a 0.05 mol / L sodium hydroxide aqueous solution at 50°C until cellulose acetate is hydrolyzed into regenerated cellulose. The hydrolyzed membrane is then removed and washed with water until the pH is neutral to obtain a solid membrane. The hydrolyzed solid membrane is further crosslinked in a crosslinking agent aqueous solution at pH 9.5. The crosslinking agent is epichlorohydrin, with a concentration of 10 wt%, a crosslinking time of 20 min, and a crosslinking temperature of 50°C.

[0195] Examples 19-21

[0196] The main difference between Examples 19-21 and Example 18 lies in the different process parameters in each step, as shown in the table below:

[0197]

[0198] Comparative Example

[0199] Comparative Example 1

[0200] A process for preparing a cellulose virus-removing membrane with high virus rejection rate includes the following steps:

[0201] S1. Preparation of the casting solution: The casting solution comprises, by weight, 20 parts cellulose acetate, 35 parts good solvent, 4 parts non-solvent, and 0.5 parts inorganic salt. Specifically, the cellulose acetate is diacetate, the good solvent is acetone, the non-solvent is deionized water, and the inorganic salt is sodium sulfate. After the casting solution is prepared, it is cast onto the carrier to fully spread and form a liquid film.

[0202] S2. Pre-phase separation: The first outer surface of the liquid film away from the carrier is pre-phase separated using a first pre-phase separation liquid to obtain a nascent film. The first pre-phase separation liquid is a 60wt% acetone aqueous solution, and the pre-phase separation time is 1.5s. After pre-phase separation, the first outer surface of the liquid film undergoes initial phase separation and forms a porous structure.

[0203] S3. Solidification and Phase Separation: The nascent membrane obtained in step S2 is immersed in a coagulation bath to allow the casting solution in the nascent membrane to fully separate into two phases, forming a solvent-rich phase and a polymer-rich phase. The solvent-rich phase forms a porous structure, while the polymer-rich phase solidifies to form a porous fibrous structure. After the nascent membrane has fully separated and solidified, a film is obtained. The coagulation bath is an aqueous solution of a penetrant with a concentration of 6.0 wt%, and ethanol is selected as the penetrant.

[0204] S4. Post-treatment: The membrane is hydrolyzed in a 0.05 mol / L sodium hydroxide aqueous solution at 50°C until cellulose acetate is hydrolyzed into regenerated cellulose. The hydrolyzed membrane is then removed and washed with water until the pH is neutral to obtain a solid membrane. The hydrolyzed solid membrane is further crosslinked in a crosslinking agent aqueous solution at pH 9.5. The crosslinking agent is epichlorohydrin, with a concentration of 10 wt%, a crosslinking time of 20 min, and a crosslinking temperature of 50°C.

[0205] Comparative Example 2

[0206] A process for preparing a cellulose virus-removing membrane with high virus rejection rate includes the following steps:

[0207] S1. Preparation of the casting solution: The casting solution comprises, by weight, 10 parts cellulose acetate, 25 parts good solvent, 3 parts non-solvent, and 0.3 parts inorganic salt. Specifically, the cellulose acetate is diacetate, the good solvent is acetone, the non-solvent is deionized water, and the inorganic salt is sodium sulfate. After the casting solution is prepared, it is cast onto the carrier to fully spread and form a liquid film.

[0208] S2. Pre-phase separation: The first outer surface of the liquid film away from the carrier is pre-phase separated with a first pre-phase separation liquid to obtain a nascent film. The first pre-phase separation liquid is an 80wt% aqueous solution of acetone, and the pre-phase separation time is 6s. After pre-phase separation, the first outer surface of the liquid film is initially separated and forms a porous structure.

[0209] S3. Solidification and Phase Separation: The nascent membrane obtained in step S2 is immersed in a coagulation bath to allow the casting solution in the nascent membrane to fully separate into two phases, forming a solvent-rich phase and a polymer-rich phase. The solvent-rich phase forms a porous structure, while the polymer-rich phase solidifies to form a porous fibrous structure. After the nascent membrane has fully separated and solidified, the resulting membrane is obtained. The coagulation bath is an aqueous solution of a penetrant with a concentration of 0.05 wt%, and ethanol is selected as the penetrant.

[0210] S4. Post-treatment: The membrane is hydrolyzed in a 0.05 mol / L sodium hydroxide aqueous solution at 50°C until cellulose acetate is hydrolyzed into regenerated cellulose. The hydrolyzed membrane is then removed and washed with water until the pH is neutral to obtain a solid membrane. The hydrolyzed solid membrane is further crosslinked in a crosslinking agent aqueous solution at pH 9.5. The crosslinking agent is epichlorohydrin, with a concentration of 10 wt%, a crosslinking time of 20 min, and a crosslinking temperature of 50°C.

[0211] Performance testing and data logging

[0212] 1. Structural Characterization

[0213] The required data can be obtained by characterizing the morphology of the virus-removing membranes obtained in each embodiment using scanning electron microscopy. Examples 1-13 and Comparative Examples 1-2 all have a two-layer membrane structure (a macroporous pre-filtration layer and a microporous separation layer). The morphological parameters of each membrane layer in Examples 1-13 and Comparative Examples 1-2 are recorded in the table below:

[0214]

[0215]

[0216] In the table above, the unit for each layer thickness is μm, the unit for each layer average pore diameter (i.e., the average pore diameter measured by SEM) is nm, the unit for each layer porosity is %, the unit for the average pore diameter variation gradient (i.e., the average pore diameter variation gradient measured by SEM) is nm / μm, the unit for the thickness variation rate is %, and the unit for the fiber diameter (i.e., the average diameter measured by SEM) is nm.

[0217] By observing the cross-sectional electron microscope images of Examples 1 to 13, it was found that the average pore size of the pre-filter layer of the virus removal membrane changes at a relatively high rate along the direction of liquid flow, and then at a relatively low rate. That is, there is a sudden region where the average pore size of the pre-filter layer decreases rapidly on the side near the first outer surface.

[0218] The morphological parameters of the overall membrane and both side surfaces of Examples 1-13 and Comparative Examples 1-2 are recorded in the following table:

[0219]

[0220]

[0221] In the table above, the unit for thickness is μm, the units for porosity and pore area ratio are %, the unit for first fiber diameter (i.e., average diameter measured by SEM) is nm, and the unit for average pore diameter (i.e., average pore diameter measured by SEM) is nm.

[0222] Examples 14-17 are three-layer membrane structures (macropore pre-filtration layer, micropore separation layer, and mesopore protective layer). The morphological parameters of each membrane layer in Examples 14-17 are recorded in the following table:

[0223]

[0224] In the table above, the unit for each layer thickness is μm, the unit for each layer average pore diameter (i.e., the average pore diameter measured by SEM) is nm, the unit for each layer porosity is %, the unit for the average pore diameter variation gradient (i.e., the average pore diameter variation gradient measured by SEM) is nm / μm, the unit for the thickness variation rate is %, and the unit for the fiber diameter (i.e., the average diameter measured by SEM) is nm.

[0225] The morphological parameters of the membrane as a whole and on both sides in Examples 14-17 are recorded in the following table:

[0226]

[0227] In the table above, the unit for thickness is μm, the units for porosity and pore area ratio are %, the unit for first fiber diameter (i.e., average diameter measured by SEM) is nm, and the unit for average pore diameter (i.e., average pore diameter measured by SEM) is nm.

[0228] Examples 18-21 are four-layer membrane structures (macropore pre-filtration layer, micropore separation layer, mesopore porous support layer, and micropore anti-leakage layer). The morphological parameters of each membrane structure in Examples 18-21 are recorded in the following table:

[0229]

[0230] In the table above, the unit for each layer thickness is μm, the unit for each layer average pore diameter (i.e., the average pore diameter measured by SEM) is nm, the unit for each layer porosity is %, the unit for the average pore diameter variation gradient (i.e., the average pore diameter variation gradient measured by SEM) is nm / μm, the unit for the thickness variation rate is %, and the unit for the fiber diameter (i.e., the average diameter measured by SEM) is nm.

[0231] The morphological parameters of the membrane as a whole and on both sides in Examples 18-21 are recorded in the following table:

[0232]

[0233] In the table above, the unit for thickness is μm, the units for porosity and pore area ratio are %, the unit for first fiber diameter (i.e., average diameter measured by SEM) is nm, and the unit for average pore diameter (i.e., average pore diameter measured by SEM) is nm.

[0234] II. Virus filtration efficiency

[0235] 2.1 Initial LRV

[0236] The virus-removing membranes prepared in each example or comparative example were used as samples for virus challenge testing. The initial LRV detection method for the virus-removing membranes followed the guidance document TR41 issued by the PDA. During the test, PP7 bacteriophage was used as the intercepted virus, the feed stream was immunoglobulin IVIG, and the buffer system was PBS. The feed liquid was pressurized to 30 psi during the virus challenge test. The initial LRV was calculated by detecting the titer of PP7 bacteriophage in the challenge solution and filtrate; the protein yield was calculated by detecting the protein concentration in the challenge solution and filtrate; and the flux was calculated by recording the flow rate and time.

[0237] 2.2 Top Wash LRV

[0238] When initially detecting the LRV of the virus-free membrane, filtration was stopped when the flow rate of the feed solution decreased by 75% or when only a small amount of feed solution remained. The pressure was then slowly released to completely depressurize the container holding the feed solution, and the remaining feed solution was poured out. Approximately 30 mL of buffer solution was added, and the container was allowed to stand. After a 5-minute pressure interruption, the container was pressurized to 30 psi and filtered. The collected filtrate was the wash buffer. The PP7 phage titer in the wash buffer was measured, and the wash LRV was calculated.

[0239] III. Colloidal Gold Capture Test

[0240] The virus-removing membranes prepared in each embodiment or comparative example were used as the test objects. Two samples were cut from each sample, with the distance between the two samples not exceeding 1 cm, and were named Sample 1 and Sample 2.

[0241] 3.1 Peak capture location of colloidal gold at 20nm before top washing

[0242] Sample 1 was subjected to a 20nm colloidal gold capture test, and the brightness of the cross-section of the virus-removing membrane containing the 20nm colloidal gold was measured. The maximum peak of the spectrum is the peak capture location of the 20nm colloidal gold. The side of the separation layer away from the pre-filter layer is the bottom surface of the separation layer, and the distance between the maximum peak of the spectrum on the cross-section of the virus-removing membrane and the bottom surface of the separation layer is D0.

[0243] 3.2 Peak capture location of colloidal gold at 20nm after top washing

[0244] Using sample 1, a 20 nm colloidal gold capture test was performed. Filtration was stopped when the flow rate of colloidal gold decreased by 75% or when only a small amount of colloidal gold remained. The pressure was then slowly released to completely relieve the pressure inside the container holding the colloidal gold, and the remaining colloidal gold was poured out. Approximately 30 mL of buffer solution was added, and the container was allowed to stand. After a 30-minute pressure interruption, pressure was applied for top-wash filtration. After top-washing, the brightness of the cross-section of the virus-removing membrane containing the captured 20 nm colloidal gold was measured. The maximum peak of the spectrum is the peak capture location of the 20 nm colloidal gold. The side of the separation layer furthest from the pre-filter layer is the bottom surface of the separation layer. The distance between the maximum peak of the spectrum on the cross-section of the virus-removing membrane and the bottom surface of the separation layer is D1.

[0245] The performance parameters of the virus-removing membranes prepared in each embodiment and comparative example are recorded in the following table:

[0246]

[0247]

[0248] It should be noted that since the virus-removing membranes in the various embodiments and comparative examples of this application are all regenerated cellulose membranes, the protein adsorption rate is low. The virus-removing membranes prepared in each embodiment can achieve a protein yield of not less than 98%.

[0249] Furthermore, for filter membranes with relatively low initial LRV but higher than 5, such as those in Examples 2 and 4, membrane filters with LRV > 8 or even LRV > 10 can be obtained by stacking and connecting two virus-removing membranes in series, making them suitable for materials with higher filtration requirements. That is, the virus-removing membrane in this application is not limited to single-layer use. In actual use, a single-layer virus-removing membrane can be selected for use, or two or more virus-removing membranes can be used in series to obtain the desired virus removal effect, depending on actual needs.

[0250] in conclusion:

[0251] Examples 1-13, which feature a two-layer structure for the virus membrane, all achieve high initial LRV and top-wash LRV. Furthermore, due to their high throughput, they can improve filtration efficiency while ensuring good virus removal.

[0252] By comparing the technical solutions of Example 5 and Example 13, it can be seen that, with similar film thickness, separation layer thickness, porosity, etc., the initial LRV of the two is not significantly different, indicating that the change rate of separation layer thickness has a relatively small impact on the initial LRV; while the top wash LRV of Example 13, which has a higher change rate of separation layer thickness, is obviously lower.

[0253] Examples 14-17, which use a three-layer deviring membrane, also achieved high initial LRV and top-wash LRV, and the flux of the three-layer deviring membrane did not show a significant decrease compared to the two-layer deviring membrane. This may be because the introduction of the protective layer acts as a reinforcing rib, reducing the compressive deformation of the pore structure of the deviring membrane under filtrate pressure.

[0254] Examples 18-21, with a four-layer devirulence membrane structure, exhibited significantly higher initial LRV and top-wash LRV. This is likely due to their unique dual-retention layer structure disrupting the continuous movement of the virus, thereby improving the virus filtration efficiency of the devirulence membrane. Furthermore, although a dual-retention layer structure was introduced, the porous support layer and the impermeable layer structure with specific pore structures worked synergistically to reduce the impact on flux; therefore, the flux decrease in Examples 18-21 was less pronounced.

[0255] Comparative Example 1, which also uses a two-layer virus removal membrane, has a larger membrane thickness, a higher proportion of the separation layer, and a smaller average pore size of the pre-filtration layer, resulting in lower throughput and making it unsuitable for practical application.

[0256] Comparative Example 2, which also features a two-sided viral removal membrane, has a smaller membrane thickness, a lower proportion of the separation layer, and a larger average pore size in the pre-filtration layer. As a result, although it has a larger flux, its virus filtration effect is poor, and the safety of the biological agent cannot be guaranteed.

[0257] Furthermore, pressure resistance tests were conducted on all samples from Examples 1-21, and their pressure resistance was greater than 30 psi. Under 30 psi pressure, they were able to stably and rapidly filter the phase solution. Additionally, during integrity testing, they withstood 50 psi pressure without affecting the membrane pores. This demonstrates that the virus-removing membranes of the embodiments in this application can operate normally for extended periods under a relatively high pressure of 30 psi, significantly improving upon the current problem of cellulose-based virus-removing membranes being unable to withstand high pressure.

[0258] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A cellulose virus-removing membrane with high virus rejection rate, comprising a porous body having non-directional tortuous pathways within the porous body, one side surface of the porous body being a first outer surface, and the other side surface of the porous body being a second outer surface, characterized in that: The porous body includes a pre-filter layer and a separation layer for trapping viruses. One side of the pre-filter layer is a first outer surface, and the separation layer is located on the side of the pre-filter layer near the second outer surface. The pre-filter layer and the separation layer are connected by continuous fibers. The average pore size measured by SEM of the pre-filter layer is greater than the average pore size measured by SEM of the separation layer. The thickness of the separation layer is 5–60 μm, and the average pore size of the separation layer measured by SEM is 25–85 nm. The thickness ratio of the separation layer to the porous body is 40-95%, and the porosity of the virus removal membrane is 15-50%. For PP7 bacteriophage, the top wash LRV of the devirulent membrane is not less than 4, and the ratio of the top wash LRV of the devirulent membrane to the initial LRV is not less than 0.

7.

2. The cellulose virus-removing membrane with high virus rejection rate according to claim 1, characterized in that: The side of the separation layer away from the pre-filter layer is the bottom surface of the separation layer. The virus-removing membrane containing 20nm colloidal gold is top-washed. After top-washing, the distance between the peak part of the virus-removing membrane containing 20nm colloidal gold and the bottom surface of the separation layer is D1, where D1 is 0.2 to 5μm.

3. The cellulose virus-removing membrane with high virus rejection rate according to claim 2, characterized in that: In the cross-section of the virus-removing membrane, the distance between the peak region of the 20nm colloidal gold capture and the bottom surface of the separation layer is D0, where D0 is 0.5–5μm.

4. The cellulose virus-removing membrane with high virus rejection rate according to claim 3, characterized in that: D1 is less than D0, and the difference between D0 and D1 is 0.1 to 3 μm.

5. The cellulose virus-removing membrane with high virus rejection rate according to claim 1, characterized in that: The average pore size variation gradient of the separation layer measured by SEM is 0–3 nm / μm; for PP7 bacteriophage, the ratio of the top wash LRV to the initial LRV of the virus-removing membrane is 0.85–0.

95.

6. The cellulose virus-removing membrane with high virus rejection rate according to claim 1, characterized in that: The porosity of the separation layer is 6-30%, and the thickness variation rate of the separation layer is no higher than 10%.

7. The cellulose virus-removing membrane with high virus rejection rate according to claim 1, characterized in that: The flux of the virus removal membrane is greater than 35 L·h -1 ·m -2 @30psi; the protein yield of the viral membrane is not less than 98%.

8. The cellulose virus-removing membrane with high virus rejection rate according to claim 1, characterized in that: The first outer surface includes a plurality of long strip-shaped and interconnected first fibers. The average diameter of the first fibers measured by SEM is 70-650 nm. Adjacent and interconnected first fibers surround each other to form a first hole.

9. A cellulose virus-removing membrane with high virus rejection rate according to any one of claims 1 to 8, characterized in that: The separation layer includes a second outer surface with second pores. The average pore diameter of the second pores measured by SEM is 15–35 nm, and the pore area ratio of the second outer surface is 2–15%. The fibers of the separation layer are long strips, and the average fiber diameter of the separation layer measured by SEM is 20–50 nm.

10. The cellulose virus-removing membrane with high virus rejection rate according to claim 9, characterized in that: The thickness ratio of the separation layer to the porous body is 8-25%, and the porosity of the virus removal membrane is 20%-60%.

11. The cellulose virus-removing membrane with high virus rejection rate according to claim 10, characterized in that: The average aperture of the first hole on the first outer surface measured by SEM is 400-5000 nm, the ratio of the average aperture of the first hole and the second hole measured by SEM is 15-200, and the area ratio of the holes on the first outer surface is 8-45%.

12. The cellulose virus-removing membrane with high virus rejection rate according to claim 10, characterized in that: The SEM measurement average pore size of the pre-filter layer is 160-600 nm, the porosity of the pre-filter layer is 35%-75%, and the SEM measurement average pore size of the pre-filter layer gradually decreases from the side near the first outer surface to the side near the second outer surface. The pore size of the pre-filter layer near the first outer surface decreases at a greater rate than the pore size of the pre-filter layer near the second outer surface.

13. The cellulose virus-removing membrane with high virus rejection rate according to claim 12, characterized in that: The average aperture of the first hole measured by SEM is 300–4500 nm, the ratio of the average aperture of the first hole to the second hole measured by SEM is 10–150, and the area ratio of the holes on the first outer surface is 5–40%.

14. The cellulose virus-removing membrane with high virus rejection rate according to claim 12, characterized in that: The SEM measurement average pore size of the pre-filter layer is 150-500 nm, the porosity of the pre-filter layer is 30-70%, and the SEM measurement average pore size of the pre-filter layer gradually decreases from the side near the first outer surface to the side near the second outer surface. The pore size of the pre-filter layer near the first outer surface decreases at a greater rate than the pore size of the pre-filter layer near the second outer surface.

15. A cellulose virus-removing membrane with high virus rejection rate according to any one of claims 1 to 8, characterized in that: A protective layer is also provided on the side of the separation layer away from the pre-filter layer, and the protective layer and the separation layer are transitioned by continuous fibers. The protective layer includes a second outer surface. The average pore size measured by SEM of the protective layer is greater than that of the separation layer but smaller than that of the pre-filter layer. The thickness of the porous body is 30–80 μm, and the ratio of the thickness of the protective layer to the thickness of the porous body is 5–20%.

16. The cellulose virus-removing membrane with high virus rejection rate according to claim 15, characterized in that: The average aperture of the first hole measured by SEM is 300–4500 nm, and the average aperture of the second hole measured by SEM is 100–500 nm. The ratio of the average aperture of the first hole to the average aperture of the second hole measured by SEM is 2–25.

17. The cellulose virus-removing membrane with high virus rejection rate according to claim 15, characterized in that: The average pore size of the protective layer, as measured by SEM, is 50–450 nm, and the porosity of the protective layer is 20–55%.

18. A cellulose virus-removing membrane with high virus rejection rate according to any one of claims 1 to 8, characterized in that: The separation layer is further provided with a porous support layer on the side away from the pre-filtration layer, and the porous support layer is further provided with a leak-proof layer on the side away from the separation layer. The separation layer, the porous support layer and the leak-proof layer are connected by continuous fibers. The average pore size measured by SEM of the porous support layer is greater than the average pore size measured by SEM of the separation layer and the leak-proof layer, but smaller than the average pore size measured by SEM of the pre-filter layer. The average pore size of the porous support layer measured by SEM is 50–400 nm, and the average pore size of the anti-seepage layer measured by SEM is 25–35 nm.

19. The cellulose virus-removing membrane with high virus rejection rate according to claim 18, characterized in that: The average pore size measured by SEM of the pre-filter layer is 120–500 nm, the average pore size measured by SEM of the separation layer is 25–35 nm, and the ratio of the average pore size measured by SEM of the separation layer to the average pore size measured by SEM of the leak-proof layer is 0.75–1.

3.

20. A cellulose virus-removing membrane with high virus rejection rate according to claim 18, characterized in that: The thickness of the porous body is 25-100 μm, the ratio of the thickness of the separation layer to the thickness of the porous body is 5-25%, the ratio of the thickness of the porous support layer to the thickness of the porous body is 5-25%, and the ratio of the thickness of the leak-proof layer to the thickness of the porous body is 2-5%.

21. The preparation process of the cellulose virus-removing membrane with high virus rejection rate according to any one of claims 1 to 20, characterized in that: The process includes the following steps: S1. The casting solution is prepared and cast onto a carrier to form a liquid film; the casting solution comprises the following components by weight: 10-30 parts cellulose acetate; 20-50 parts good solvent; 3-6 parts non-solvent; 0.1-1 parts inorganic salt; The good solvent is at least one selected from acetone, dioxane, dimethylacetamide, N-methylpyrrolidone, acetic acid, propionic acid, butyric acid, and valeric acid; The non-solvent is water; The cation of the inorganic salt is one or more of sodium, potassium, calcium, and magnesium, and the anion of the inorganic salt is one or more of sulfate, sulfite, or carbonate. S2. Pre-phase separation: The first outer surface of the liquid film is pre-separated with a first pre-phase separation liquid to obtain a primary film; the first pre-phase separation liquid is a 60-90 wt% acetone aqueous solution, and the pre-phase separation time is 2-15 s. S3. Solidification and phase separation: The nascent membrane is immersed in a coagulation bath for phase separation and solidification to obtain a film. The coagulation bath includes water and a penetrant. The concentration of the penetrant is 0.1-5 wt%. The penetrant is at least one of ethanol, n-propanol, isopropanol, n-butanol, n-pentanol, and sec-pentanol. S4. Post-treatment: The film is placed in a sodium hydroxide solution for hydrolysis, and after hydrolysis, it is washed to form a solid film; the solid film is further placed in a crosslinking agent for crosslinking treatment, and after the crosslinking treatment is completed and the film is washed, the finished film is obtained. The crosslinking agent is at least one of haloepoxide, dihaloalkanes and dihaloalcohols.

22. The preparation process of the cellulose virus-removing membrane with high virus rejection rate according to claim 21, characterized in that: In step S2, the second outer surface is further pre-separated with a second pre-separation liquid, which is a 40-60 wt% acetone aqueous solution, and the pre-separation time is 2-10 s.

23. The preparation process of the cellulose virus-removing membrane with high virus rejection rate according to claim 21, characterized in that: In step S2, the second outer surface is further pre-separated with a third pre-separating liquid, which is a 20-40 wt% acetone aqueous solution, and the temperature of the third pre-separating liquid is 5-10°C lower than that of the liquid film. The pre-separation time is 2-10 seconds.

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