Ultrafiltration membrane as well as preparation method and application thereof

Abstract

The invention discloses an ultrafiltration membrane as well as a preparation method and application thereof. Specifically, the ultrafiltration membrane provided by the invention comprises a cellulose ultrafiltration layer and a supporting layer, and the cellulose ultrafiltration layer is obtained by performing enzymatic degradation treatment on the surface of a non-porous cellulose membrane by adopting cellulase. Therefore, according to the ultrafiltration membrane provided by the invention, the surface of the cellulose ultrafiltration layer is provided with the uniformly distributed nanoscale pore channel structure, so that the separation precision of the ultrafiltration membrane can be improved. Various substances within a target molecular weight range can be accurately intercepted, and high-efficiency separation of micromolecular impurities and macromolecular bioactive substances can be realized, so that high purity of permeate liquid is ensured, and strict requirements on product purity in a biological manufacturing process are met.

Description

Technical Field

[0001] This application relates to a filtration membrane, and specifically to an ultrafiltration membrane and its preparation method and application. Background Art

[0002] In the synthetic biology industry, from the production of biopharmaceuticals to the synthesis of bio-based materials, the efficient purification of products is a key link to ensure product quality, safety, and performance. With the rapid development of biotechnology, the demand for high-purity biological products is increasing day by day. Whether it is pharmaceutical and health products such as monoclonal antibodies, vaccines, and recombinant proteins, or emerging bio-based materials such as biofuels and biodegradable polymers, complex impurity removal steps are inevitably involved in their manufacturing processes. These impurities include unreacted raw materials, by-products, cell debris, host cell proteins, and endotoxins, etc. Their presence not only affects the purity and activity of the final product but may also cause adverse consequences such as immune reactions.

[0003] As a pressure-driven membrane separation technology between microfiltration and reverse osmosis, ultrafiltration demonstrates unique advantages in the field of biomanufacturing purification by virtue of its ability to selectively retain substances according to molecular size, and has become a hot topic in recent research and applications. By setting a specific molecular weight cut-off, the ultrafiltration membrane allows solvents, small molecule solutes, and some bioactive molecules to pass through, while retaining macromolecular impurities, achieving preliminary or fine purification purposes, with simple operation and easy scale-up.

[0004] However, there are still many problems to be solved with the existing ultrafiltration membranes for biomanufacturing purification. Common ultrafiltration membranes on the market, such as those based on polyethersulfone (PES) and polyvinylidene fluoride (PVDF), are prepared from synthetic polymer materials. These synthetic materials have inherent defects in biocompatibility, are prone to adsorbing biomolecules, resulting in the loss of target products, reducing product yields, and may also cause biofouling during long-term use, shortening the service life of the membrane and increasing the cost of frequent cleaning or membrane module replacement.

[0005] In view of the urgent need for highly efficient, low-cost, and biocompatible ultrafiltration membranes in the field of biomanufacturing purification, it is urgent to develop a new cellulose ultrafiltration membrane to break through the bottleneck of the existing technology and promote the further development of the biomanufacturing industry. Summary of the Invention

[0006] This application aims to solve at least one of the technical problems in the related technologies to some extent. For this reason, one objective of this application is to propose an ultrafiltration membrane and its preparation method. The ultrafiltration membrane of this application has nano-scale pores with uniform pore size distribution, which can improve the separation accuracy of the ultrafiltration membrane, ensure efficient retention of substances with a molecular weight of 1 - 1000 kDa (kiloDalton), and has good flux, suitable for wide applications in the field of biomanufacturing purification.

[0007] Therefore, in the first aspect of the present application, the present application proposes an ultrafiltration membrane. According to an embodiment of the present application, the ultrafiltration membrane includes: a cellulose ultrafiltration layer and a support layer, and the cellulose ultrafiltration layer is obtained by subjecting the surface of a cellulose membrane without pores to enzymatic degradation treatment using cellulase. Thus, for the ultrafiltration membrane of the present application, the surface of its cellulose ultrafiltration layer has a uniformly distributed nano-scale pore structure, which can improve the separation accuracy of the ultrafiltration membrane. It can accurately intercept various substances within the target molecular weight range. Whether it is small molecule impurities or macromolecular bioactive substances, high-efficiency separation can be achieved, meeting the strict requirements for product purity in the biological manufacturing process. In addition, by optimizing the time of enzymatic degradation treatment and the concentration of cellulase, the size and density of the pores can be flexibly adjusted, thereby achieving high-throughput filtration. Cellulose itself has good biocompatibility, and after enzymatic hydrolysis treatment, its surface hydrophilicity is further enhanced, effectively reducing the adsorption of biomolecules, reducing the risk of membrane fouling, and extending the service life of the membrane.

[0008] According to an embodiment of the present application, the raw material of the cellulose membrane includes at least one of cellulose diacetate, cellulose triacetate, cellulose acetate butyrate, and cellulose acetate propionate.

[0009] According to an embodiment of the present application, the support layer is a non-woven fabric, and the material of the non-woven fabric includes at least one of polyethylene (PE), polypropylene (PP), polyacrylonitrile (PAN), polyethersulfone (PES), polysulfone (PSU), polyethylene terephthalate (PET), polytetrafluoroethylene (PTFE), and polyvinylidene fluoride (PVDF).

[0010] According to an embodiment of the present application, the thickness of the cellulose ultrafiltration layer is 10 μm - 1000 μm.

[0011] According to an embodiment of the present application, the thickness of the support layer is 10 μm - 500 μm.

[0012] According to an embodiment of the present application, the cut-off pore size of the ultrafiltration membrane is 0.5 nm - 50 nm.

[0013] According to an embodiment of the present application, the porosity of the ultrafiltration membrane is higher than 30%.

[0014] According to an embodiment of the present application, the ultrafiltration membrane has a cut-off standard molecular weight of 1 kDa - 1000 kDa.

[0015] According to an embodiment of the present application, the pure water flux of the ultrafiltration membrane is greater than 5 L·m -2 h -1 bar -1 。

[0016] In the second aspect of the present application, a method for preparing the ultrafiltration membrane described in the first aspect is proposed. According to an embodiment of the present application, the method includes: contacting a regenerated cellulose membrane with cellulase and performing enzymatic degradation treatment to obtain the ultrafiltration membrane. Thus, the preparation conditions of the method of the present application are mild, which will not cause damage to the cellulose material. At the same time, it avoids the energy consumption and equipment requirements brought by extreme conditions such as high temperature and high pressure, reduces the production cost, and is suitable for large-scale industrial production. At the same time, by simply adjusting the concentration of cellulase and the treatment time, the pore size, molecular weight cut-off, and flux of the membrane can be flexibly regulated, endowing the method with extremely high adjustability and wide applicability. In addition, the ultrafiltration membrane prepared by the method of the present application not only has high separation accuracy and can efficiently retain substances with a molecular weight in the range of 1-1000 kDa, but also has good flux, making it have broad application prospects in the field of biomanufacturing purification and being able to meet the stringent requirements in the purification process of different biological products.

[0017] According to an embodiment of the present application, the surface of the regenerated cellulose membrane is pore-free.

[0018] According to an embodiment of the present application, the regenerated cellulose membrane is prepared by the following method: mixing a cellulose ester and a solvent to obtain a casting solution; placing the casting solution on the surface of a support layer to form a liquid film on the surface of the support layer; allowing the liquid film to stand to form a dense layer on the surface of the support layer; subjecting the support layer with a dense layer formed on its surface to phase separation and coagulation treatment to obtain a film; and subjecting the film to hydrolysis treatment in an alkaline solution to obtain the regenerated cellulose membrane.

[0019] According to an embodiment of the present application, the contacting of the regenerated cellulose membrane with cellulase is carried out by immersing the regenerated cellulose membrane in a cellulase solution, and the concentration of the cellulase solution is 1 μg / g - 100 μg / g.

[0020] According to an embodiment of the present application, the pH value of the cellulase solution is 4 - 7.

[0021] According to an embodiment of the present application, the temperature of the enzymatic degradation treatment is 40°C - 55°C.

[0022] According to an embodiment of the present application, the time of the enzymatic degradation treatment is 5 min - 120 min.

[0023] According to an embodiment of the present application, the cellulose ester includes at least one of cellulose diacetate, cellulose triacetate, cellulose acetate butyrate, and cellulose acetate propionate.

[0024] According to an embodiment of the present application, the solvent includes at least one of acetone, N-methylpyrrolidone, N,N-dimethylacetamide, formamide, dioxane, acetic acid, propionic acid, and butyric acid.

[0025] According to an embodiment of the present application, the weight ratio of the cellulose ester to the solvent is (10 - 30):(70 - 90).

[0026] According to an embodiment of the present application, the support layer is a non-woven fabric, and the material of the non-woven fabric includes at least one of polyethylene (PE), polypropylene (PP), polyacrylonitrile (PAN), polyethersulfone (PES), polysulfone (PSU), polyethylene terephthalate (PET), polytetrafluoroethylene (PTFE), and polyvinylidene fluoride (PVDF).

[0027] According to an embodiment of the present application, the time of the static treatment is 30 s - 120 s.

[0028] According to an embodiment of the present application, the phase separation coagulation treatment is carried out by immersing the support layer with a dense layer formed on the surface into a coagulation bath, and the coagulation bath is water.

[0029] According to an embodiment of the present application, the time of the phase separation coagulation treatment is 10 s - 60 s.

[0030] According to an embodiment of the present application, the temperature of the phase separation coagulation treatment is 0°C - 40°C.

[0031] According to an embodiment of the present application, the alkaline solution is a 0.1 wt% - 10 wt% aqueous NaOH solution.

[0032] According to an embodiment of the present application, the temperature of the hydrolysis treatment is 30°C - 80°C.

[0033] According to an embodiment of the present application, the time of the hydrolysis treatment is 2 min - 60 min.

[0034] In the third aspect of the present application, the present application provides the use of the ultrafiltration membrane described in the first aspect or the ultrafiltration membrane prepared by the method described in the second aspect in the purification of biological products. As described above, the ultrafiltration membrane of the present application not only has high separation accuracy and can efficiently retain substances with a molecular weight in the range of 1 - 1000 kDa, but also has good flux, making it have broad application prospects in the field of biological manufacturing purification and can meet the stringent requirements in the purification process of different biological products.

[0035] The additional aspects and advantages of the present application will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0037] Figure 1 Shows a schematic process diagram for preparing an ultrafiltration membrane according to an embodiment of the present application;

[0038] Figure 2 Shows a surface SEM image of the regenerated cellulose membrane 1 according to an embodiment of the present application, with a magnification of 55.7K;

[0039] Figure 3 Shows a surface SEM image of the cellulose ultrafiltration membrane 5 according to an embodiment of the present application, with a magnification of 20K;

[0040] Figure 4 Shows a surface SEM image of the cellulose ultrafiltration membrane 9 according to an embodiment of the present application, with a magnification of 20K. Detailed Description of the Invention

[0041] Reference to "embodiment" herein means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the present application. The phrase appears in various places in the specification and does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0042] For the sake of brevity, only some numerical ranges are specifically disclosed herein. However, any lower limit can be combined with any upper limit to form a range not explicitly recited; and any lower limit can be combined with other lower limits to form a range not explicitly recited, and similarly any upper limit can be combined with any other upper limit to form a range not explicitly recited. In addition, each individually disclosed point or single numerical value itself can be used as a lower limit or an upper limit and combined with any other point or single numerical value or combined with other lower limits or upper limits to form a range not explicitly recited.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "comprising" and "having" and any variations thereof in the specification and claims of this application and the above drawings are intended to cover non-exclusive inclusion.

[0044] As used herein, the term "comprising" or "including" is an open-ended expression, i.e., it includes the content specified in this application but does not exclude other aspects.

[0045] It should be noted that the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present application, unless otherwise specified, "a plurality of" means two or more than two.

[0046] Without special instructions, all embodiments and optional embodiments of the present application can be combined with each other to form new technical solutions.

[0047] As the most abundant natural polymer material on Earth, cellulose has many advantages such as wide sources, renewability, low cost, and excellent biocompatibility, making it a highly potential candidate material for preparing ultrafiltration membranes. Its unique chemical structure endows cellulose membranes with good hydrophilicity, which can effectively reduce the adsorption of biomolecules, lower the risk of biofouling, and extend the service life of the membrane. At the same time, the abundant hydroxyl groups on the cellulose molecular chain can introduce various functional groups through chemical modification to further optimize the performance of the membrane, such as adjusting the cut-off molecular weight and enhancing the anti-fouling ability.

[0048] In recent years, researchers have carried out extensive research on cellulose ultrafiltration membranes and tried various preparation methods to realize the application of cellulose ultrafiltration membranes in the field of biomanufacturing purification. However, some key technical problems still exist. The preparation method of cellulose membranes generally uses the solvent phase inversion method. The cellulose casting solution (usually cellulose or cellulose ether dissolved in an appropriate solvent system) undergoes phase separation through the exchange of solvent and non-solvent, thereby forming a solid membrane structure. By adjusting the parameters in the phase inversion process, the size and distribution of the membrane pore structure for separation are controlled, so as to adjust the cut-off molecular weight of the ultrafiltration membrane. This method is difficult to precisely control the microstructure of the membrane, resulting in uneven membrane pore size distribution and affecting the separation accuracy.

[0049] In view of the urgent need for high-efficiency, low-cost, and biocompatible ultrafiltration membranes in the field of biomanufacturing purification, as well as the problems existing in the current research and application of cellulose ultrafiltration membranes. The present application has developed a new type of ultrafiltration membrane, which uses cellulose as the ultrafiltration layer. The pore structure on the surface of the cellulose ultrafiltration layer is formed by enzymatic degradation treatment with cellulase, and the size and density of the pores can be flexibly adjusted by precisely controlling the time of enzymatic degradation treatment and the concentration of cellulase. Thus, the ultrafiltration membrane of the present application not only has high retention for substances with a molecular weight of 1 - 1000 Kd and good flux, but also has mild preparation conditions and is suitable for large-scale industrial production. The ultrafiltration membrane, its preparation method, and application will be introduced in detail below.

[0050] Ultrafiltration membrane

[0051] Therefore, in the first aspect of the present application, the present application proposes an ultrafiltration membrane. According to an embodiment of the present application, the ultrafiltration membrane includes: a cellulose ultrafiltration layer and a support layer, and the cellulose ultrafiltration layer is obtained by subjecting the surface of a cellulose membrane without pores to enzymatic degradation treatment using cellulase. Thus, for the ultrafiltration membrane of the present application, the surface of its cellulose ultrafiltration layer has a uniformly distributed nano-scale pore structure, which can improve the separation accuracy of the ultrafiltration membrane. It can accurately intercept various substances within the target molecular weight range. Whether it is small molecule impurities or macromolecular bioactive substances, efficient separation can be achieved, meeting the strict requirements for product purity in the biological manufacturing process. In addition, by optimizing the time of enzymatic degradation treatment and the concentration of cellulase, the size and density of the pores can be flexibly adjusted, thereby realizing high-throughput filtration. Cellulose itself has good biocompatibility, and after enzymatic hydrolysis treatment, its surface hydrophilicity is further enhanced, effectively reducing the adsorption of biomolecules, reducing the risk of membrane fouling, and extending the service life of the membrane.

[0052] In some embodiments of the present invention, the raw materials of the cellulose membrane include, but are not limited to, at least one of cellulose diacetate, cellulose triacetate, cellulose acetate butyrate, and cellulose acetate propionate. Thus, by using the above substances as the raw materials of the cellulose membrane, the cellulose membrane can have good solubility, good biocompatibility, and hydrophilicity.

[0053] In some embodiments of the present invention, the support layer is a non-woven fabric, and the material of the non-woven fabric includes at least one of polyethylene (PE), polypropylene (PP), polyacrylonitrile (PAN), polysulfone (PES), polysulfone (PSU), polyethylene terephthalate (PET), polytetrafluoroethylene (PTFE), and polyvinylidene fluoride (PVDF). Thus, the above materials not only have excellent mechanical properties and toughness, can provide good physical support for the ultrafiltration membrane, and prevent the membrane from deforming or rupturing due to pressure during the filtration process, but also have good chemical stability, can resist the erosion of common chemical reagents and biological liquids, and are suitable for a variety of different filtration application scenarios.

[0054] In some embodiments of the present invention, the thickness of the cellulose ultrafiltration layer is 10 μm - 1000 μm. For example, it can be 10 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, etc., or it can be a range composed of any of the above values. Thus, by making the thickness of the cellulose ultrafiltration layer within the above range, not only can the interception efficiency of the target molecular weight substances be improved, realizing efficient and accurate selective separation, but also the separation accuracy can be further optimized on the basis of ensuring good flux.

[0055] In some embodiments of the present invention, the thickness of the support layer is 10 μm - 500 μm. For example, it can be 10 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, etc., or it can be a range composed of any of the above values. Thus, by making the thickness of the support layer within the above range, it is possible to ensure that the support layer has sufficient mechanical strength, while not increasing unnecessary costs or resistance due to excessive thickness.

[0056] In some embodiments of the present invention, the cut-off pore size of the ultrafiltration membrane is 0.5 nm - 50 nm. For example, it can be 0.5 nm, 1.5 nm, 2 nm, 5 nm, 10 nm, 20 nm, 50 nm, etc., or it can be a range composed of any of the above values. Thus, it is possible to effectively retain substances within a specific molecular weight range (such as proteins, polysaccharides, virus particles, etc.), while allowing small molecule solutes and solvents to pass through, ensuring efficient separation and maintaining good filtration flux.

[0057] In some embodiments of the present invention, the porosity of the ultrafiltration membrane is higher than 30%. For example, it can be 30%, 40%, 50%, 60%, 70%, 80%, etc., or it can be a range composed of any of the above values. Thus, it is possible to make the ultrafiltration membrane have good filtration flux and separation efficiency.

[0058] In some embodiments of the present invention, the ultrafiltration membrane has a cut-off standard molecular weight of 1 kDa - 1000 kDa. For example, it can be 1 kDa, 5 kDa, 10 kDa, 50 kDa, 100 kDa, 200 kDa, 400 kDa, 600 kDa, 800 kDa, 1000 kDa, etc., or it can be a range composed of any of the above values. Thus, the ultrafiltration membrane of the present invention can be applied to the separation and purification of various bioactive substances.

[0059] In some embodiments of the present invention, the pure water flux of the ultrafiltration membrane is greater than 5 L·m -2 h -1 bar -1 . For example, it can be 6 L·m -2 h -1 bar -1 、10 L·m -2 h -1 bar -1 、50 L·m -2 h -1 bar -1 、100 L·m -2 h -1 bar -1 、500 L·m -2 h -1 bar -1 、1000 L·m-2 h -1 bar -1 、1500 L·m -2 h -1 bar -1 、2000 L·m -2 h -1 bar -1 、3000 L·m -2 h -1 bar -1 etc., or can be a range composed of any of the above values. In some embodiments of the present invention, the pure water flux of the ultrafiltration membrane is 10 - 2000 L·m -2 h -1 bar -1 。

[0060] Method for preparing the ultrafiltration membrane

[0061] In a second aspect of the present invention, the present invention provides a method for preparing the ultrafiltration membrane described in the first aspect. According to the embodiments of the present invention, referring to Figure 1 , the method includes:

[0062] S100: Mix cellulose ester and a solvent to obtain a casting solution;

[0063] In some embodiments of the present application, the cellulose ester includes at least one of cellulose diacetate, cellulose triacetate, cellulose acetate butyrate, and cellulose acetate propionate. Thus, by using the above cellulose ester, the prepared cellulose membrane can have good solubility, good biocompatibility and hydrophilicity, thereby reducing the adsorption of biomolecules, reducing the risk of membrane fouling, improving the anti-fouling ability and service life of the membrane, and meeting the requirements for high-precision separation in fields such as biomanufacturing.

[0064] In some embodiments of the present application, the solvent includes at least one of water, acetone, N-methylpyrrolidone, N,N-dimethylacetamide, formamide, dioxane, acetic acid, propionic acid, and butyric acid.

[0065] In some embodiments of the present application, the weight ratio of the cellulose ester to the solvent is (10 - 30):(70 - 90). For example, it can be 10:90, 15:85, 20:80, 25:75, 30:70, etc., or can be a range composed of any of the above values. Thus, by making the weight ratio of the cellulose ester to the solvent within the above range, it can be ensured that the cellulose ester is fully dissolved in the solvent to form a uniform casting solution, facilitating the subsequent membrane preparation process. If the content of cellulose ester is too high, it may lead to incomplete dissolution and form a non-uniform solution, affecting the quality of the membrane. While too low a content may lead to too fast evaporation of the solvent and it is difficult to form a stable membrane structure.

[0066] S200: Place the casting solution on the surface of the support layer to form a liquid film on the surface of the support layer;

[0067] In some embodiments of the present application, the support layer is a non-woven fabric, and the material of the non-woven fabric includes at least one of polyethylene (PE), polypropylene (PP), polyacrylonitrile (PAN), polyethersulfone (PES), polysulfone (PSU), polyethylene terephthalate (PET), polytetrafluoroethylene (PTFE), and polyvinylidene fluoride (PVDF). Thus, the above materials not only have excellent mechanical properties and toughness, can provide good physical support for the ultrafiltration membrane, and prevent the membrane from deforming or breaking due to pressure during the filtration process, but also have good chemical stability, can resist the erosion of common chemical reagents and biological liquids, and are suitable for a variety of different filtration application scenarios.

[0068] In some embodiments of the present application, the liquid film is formed by doctor-blading the casting solution on the surface of the support layer.

[0069] S300: Let the liquid film stand to form a dense layer on the surface of the support layer;

[0070] In some embodiments of the present application, the standing time is 30s - 120s. For example, it can be 30s, 50s, 70s, 90s, 100s, 110s, 120s, etc., or it can be a range composed of any of the above values. Thus, by making the standing time within the above range, the solvent on the surface of the liquid film in contact with the air can volatilize sufficiently, so that the cellulose ester concentration on the surface of the liquid film increases, and a dense layer is formed.

[0071] In some embodiments of the present application, the prepared casting solution is evenly doctor-bladed on the non-woven fabric support layer to form a liquid film. When the liquid film is exposed to air, the solvent on its surface in contact with the air (i.e., the upper surface) will gradually volatilize. This process causes the cellulose ester concentration on the upper surface of the liquid film to gradually increase, thereby promoting the formation of a dense structure on this surface.

[0072] S400: Perform a phase separation and solidification treatment on the support layer with a dense layer formed on its surface to obtain a film;

[0073] In some embodiments of the present application, the fractional phase solidification treatment is carried out by immersing the support layer with a dense layer formed on its surface into a coagulation bath, and the coagulation bath is water. During the process of immersing the support layer into the coagulation bath, the cellulose ester solution in the liquid film contacts the water, promoting the concentration gradient-driven diffusion of water and solvent molecules. This diffusion process causes the cellulose ester molecules to gradually solidify and precipitate. Since the cellulose ester concentration on the upper surface of the liquid film is relatively high, during the phase separation process, the cellulose ester molecular chains are closely packed, forming a dense pore-free surface structure. This structure results in an extremely low pure water flux of the membrane, usually lower than 0.5 LMH (liters per square meter per hour) under a pressure of 1 bar.

[0074] In some embodiments of the present application, the time of the fractional phase solidification treatment is 10 s - 60 s. For example, it can be 10 s, 20 s, 30 s, 40 s, 50 s, 60 s, etc., or it can be a range composed of any of the above values. Thus, by making the time of the fractional phase solidification treatment within the above range, the cellulose ester molecules can be fully precipitated and solidified to form a uniform membrane structure.

[0075] In some embodiments of the present application, the temperature of the fractional phase solidification treatment is 0 °C - 40 °C. For example, it can be 0 °C, 10 °C, 20 °C, 30 °C, 40 °C, etc., or it can be a range composed of any of the above values. Thus, by making the temperature of the fractional phase solidification treatment within the above range, the cellulose ester molecular chains can be closely packed during the phase separation process to form a membrane with a dense pore-free structure.

[0076] S500: Place the formed film in an alkaline solution for hydrolysis treatment to obtain the regenerated cellulose membrane;

[0077] In some embodiments of the present application, the alkaline solution is an aqueous solution of NaOH with a concentration of 0.1 wt% - 10 wt%. For example, it can be an aqueous solution of NaOH with a concentration of 0.1 wt%, 1 wt%, 2 wt%, 5 wt%, 8 wt%, 10 wt%, etc., or it can be a range composed of any of the above values. Thus, during the hydrolysis treatment of the formed film in the alkaline solution, the dense cellulose ester on the film surface can undergo hydrolysis and be converted from cellulose ester to cellulose, that is, the regenerated cellulose membrane is obtained. During this process, the surface still maintains a dense pore-free structure, specifically manifested as an almost unchanged low pure water flux, usually lower than 0.5 LMH (liters per square meter per hour).

[0078] In some embodiments of the present application, the temperature of the hydrolysis treatment is 30°C - 80°C. For example, it can be 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, etc., or it can be a range composed of any of the above values. Thus, by making the temperature of the hydrolysis treatment within the above range, not only can it ensure that the cellulose ester is fully hydrolyzed within a reasonable time to be converted into regenerated cellulose, but also the cellulose molecular chains formed after the hydrolysis of the cellulose ester can be closely packed to maintain the dense and pore-free structure on the membrane surface.

[0079] In some embodiments of the present application, the time of the hydrolysis treatment is 2 min - 60 min. For example, it can be 2 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, etc., or it can be a range composed of any of the above values. Thus, by making the time of the hydrolysis treatment within the above range, it can ensure that the cellulose ester reacts fully with the alkaline solution and is thus completely converted into regenerated cellulose.

[0080] S600: Contact the regenerated cellulose membrane with cellulase for enzymatic degradation treatment to obtain the ultrafiltration membrane;

[0081] In some embodiments of the present application, the contact of the regenerated cellulose membrane with cellulase is carried out by immersing the regenerated cellulose membrane in a cellulase solution. Immerse the prepared regenerated cellulose membrane with a pore-free surface in the cellulase solution. The cellulase will specifically adsorb onto the cellulose surface and then decompose the cellulose into water-soluble glucose, thereby achieving the degradation of cellulose. During this process, the cellulase in the solution adsorbs onto the surface of the dense regenerated cellulose membrane and degrades the cellulose molecules on the membrane surface, and then forms a nano-scale pore structure on the membrane surface. By increasing the concentration of cellulase and prolonging the immersion time of the cellulose membrane in the solution, the size and density of the pore structure on the surface of the regenerated cellulose membrane can be increased, thereby improving the flux of the membrane and increasing its molecular weight cut-off.

[0082] In some embodiments of the present application, the concentration of the cellulase solution is 1 μg / g - 100 μg / g. For example, it can be 1 μg / g, 5 μg / g, 10 μg / g, 20 μg / g, 50 μg / g, 80 μg / g, 100 μg / g, etc., or it can be a range composed of any of the above values. Among them, the concentration of the cellulase solution directly affects the contact frequency and reaction rate between the enzyme and cellulose. A higher enzyme concentration can provide more active sites, making it easier for the cellulose molecular chains to be degraded, thereby forming larger-sized pores. On the contrary, a lower enzyme concentration will form smaller pores. In some embodiments of the present application, the concentration of the cellulase solution is 40 μg / g - 50 μg / g. For example, it can be 40 μg / g, 42 μg / g, 45 μg / g, 48 μg / g, 50 μg / g, etc., or it can be a range composed of any of the above values.

[0083] In some embodiments of the present application, the pH value of the cellulase solution is 4 - 7. For example, it can be 4, 4.5, 5, 5.5, 6, 6.5, 7, etc., or it can be a range composed of any of the above values. Thus, within this pH value range, the cellulase solution can make the cellulase have a relatively high activity, so as to efficiently bind to cellulose molecules and catalyze the degradation reaction. In some embodiments of the present application, the pH value of the cellulase solution is 4.5 - 5.5. For example, it can be 4.5, 4.7, 5, 5.2, 5.5, etc., or it can be a range composed of any of the above values.

[0084] In some embodiments of the present application, the temperature of the enzymatic degradation treatment is 40°C - 55°C. For example, it can be 40°C, 42°C, 45°C, 47°C, 50°C, 52°C, 55°C, etc., or it can be a range composed of any of the above values. Thus, within the above temperature range, the activity of the cellulase is relatively high, and it can rapidly degrade cellulose molecules to form nano-scale pores. This not only improves the rate of the enzymatic hydrolysis reaction but also can achieve the required pore structure in a relatively short time.

[0085] In some embodiments of the present application, the time of the enzymatic degradation treatment is 5 min - 120 min. For example, it can be 5 min, 10 min, 15 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, 120 min, etc., or it can be a range composed of any of the above values. Thus, by regulating the time of the enzymatic degradation treatment, the degradation degree of cellulose can be effectively controlled. This process enables the adjustment of the size and density of the pores on the membrane surface, and thus has a direct impact on the flux and retention performance of the membrane. In some embodiments of the present application, the time of the enzymatic degradation treatment is 5 min - 50 min, 5 min - 30 min.

[0086] Use

[0087] In the third aspect of the present application, the present application proposes the use of the ultrafiltration membrane described in the first aspect or the ultrafiltration membrane prepared by the method described in the second aspect in the purification of biological products. As described above, the ultrafiltration membrane of the present application not only has a high separation accuracy and can efficiently retain substances with a molecular weight in the range of 1 - 1000 kDa, but also has a good flux, making it have a broad application prospect in the field of biological manufacturing purification and can meet the stringent requirements in the purification process of different biological products.

[0088] The solution of the present application will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. For those without specific technical or conditions noted in the embodiments, the techniques or conditions described in the literature in the field or according to the product specifications are followed. For reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchase.

[0089] Example 1: Preparation of regenerated cellulose membrane 1

[0090] A solution containing 15% cellulose diacetate and 85% acetone was blade-coated onto the surface of PET non-woven fabric to form a liquid film. After staying in the air for 30 s, it was immersed in pure water at 20 °C and stayed for 10 s to solidify into a film. Then it was hydrolyzed in a 0.5% NaOH solution at a hydrolysis temperature of 60 °C for 10 min. After washing with pure water, regenerated cellulose membrane 1 with a dense and pore-free surface was obtained.

[0091] Example 2: Preparation of cellulose ultrafiltration membrane 1

[0092] The regenerated cellulose membrane prepared in Example 1 was immersed in a 2 μ / g cellulase solution with a solution pH of 5.5, a temperature of 45 °C, and a time of 10 min. After washing with pure water, cellulose ultrafiltration membrane 1 was obtained.

[0093] Example 3: Preparation of cellulose ultrafiltration membrane 2

[0094] The regenerated cellulose membrane prepared in Example 1 was immersed in a 2 μ / g cellulase solution with a solution pH of 5.5, a temperature of 45 °C, and a time of 25 min. After washing with pure water, cellulose ultrafiltration membrane 2 was obtained.

[0095] Example 4: Preparation of cellulose ultrafiltration membrane 3

[0096] The regenerated cellulose membrane prepared in Example 1 was immersed in a 2 μ / g cellulase solution with a solution pH of 5.5, a temperature of 45 °C, and a time of 50 min. After washing with pure water, cellulose ultrafiltration membrane 3 was obtained.

[0097] Example 5: Preparation of regenerated cellulose membrane 2

[0098] A solution containing 20% cellulose diacetate, 60% acetone, and 20% DMAC (N,N-dimethylacetamide) was blade-coated onto the surface of PP / PE non-woven fabric to form a liquid film. After staying in the air for 60 s, it was immersed in pure water at 20 °C and stayed for 30 s to solidify into a film. Then it was hydrolyzed in a 1% NaOH solution at a hydrolysis temperature of 50 °C for 5 min. After washing with pure water, regenerated cellulose membrane 2 with a dense and pore-free surface was obtained.

[0099] Example 6: Preparation of cellulose ultrafiltration membrane 4

[0100] The regenerated cellulose membrane prepared in Example 5 was immersed in a 10 μ / g cellulase solution with a solution pH of 5.0, a temperature of 45 °C, and a time of 10 min. After washing with pure water, the cellulose ultrafiltration membrane 4 was obtained.

[0101] Example 7: Preparation of cellulose ultrafiltration membrane 5

[0102] The regenerated cellulose membrane prepared in Example 5 was immersed in a 10 μ / g cellulase solution with a solution pH of 5.0, a temperature of 45 °C, and a time of 15 min. After washing with pure water, the cellulose ultrafiltration membrane 5 was obtained.

[0103] Example 8: Preparation of cellulose ultrafiltration membrane 6

[0104] The regenerated cellulose membrane prepared in Example 5 was immersed in a 10 μ / g cellulase solution with a solution pH of 5.0, a temperature of 45 °C, and a time of 20 min. After washing with pure water, the cellulose ultrafiltration membrane 6 was obtained.

[0105] Example 9: Preparation of regenerated cellulose membrane 3

[0106] A solution containing 20% cellulose diacetate, 48% acetone, and 32% formamide was scrape-coated onto the surface of a PP / PE non-woven fabric to form a liquid film. After staying in the air for 60 s, it was immersed in pure water at 40 °C and stayed for 30 s to solidify into a film. Then it was hydrolyzed in a 1% NaOH solution at a hydrolysis temperature of 50 °C and a hydrolysis time of 15 min. After washing with pure water, the regenerated cellulose membrane 3 with a dense and pore-free surface was obtained.

[0107] Example 10: Preparation of cellulose ultrafiltration membrane 7

[0108] The regenerated cellulose membrane prepared in Example 9 was immersed in a 50 μ / g cellulase solution with a solution pH of 5.0, a temperature of 45 °C, and a time of 6 min. After washing with pure water, the cellulose ultrafiltration membrane 7 was obtained.

[0109] Example 11: Preparation of cellulose ultrafiltration membrane 8

[0110] The regenerated cellulose membrane prepared in Example 9 was immersed in a 50 μ / g cellulase solution with a solution pH of 5.0, a temperature of 45 °C, and a time of 15 min. After washing with pure water, the cellulose ultrafiltration membrane 8 was obtained.

[0111] Example 12: Preparation of cellulose ultrafiltration membrane 9

[0112] The regenerated cellulose membrane prepared in Example 9 was immersed in a 60 μ / g cellulase solution with a solution pH of 5.0, a temperature of 45 °C, and a time of 22 min. After washing with pure water, the cellulose ultrafiltration membrane 9 was obtained.

[0113] Comparative Example 1

[0114] A solution containing 20% cellulose diacetate and 80% acetone was scrape-coated onto the surface of PET non-woven fabric to form a liquid film. After staying in the air for 5 s, it was immersed in pure water at 5 °C and stayed for 30 s to solidify into a film. Then it was hydrolyzed in a 0.5% NaOH solution at a hydrolysis temperature of 60 °C for 10 min. After washing with pure water, a cellulose ultrafiltration membrane was obtained.

[0115] Comparative Example 2

[0116] A solution containing 15% cellulose diacetate and 85% DMCA was scrape-coated onto the surface of PP / PE non-woven fabric to form a liquid film. After staying in the air for 30 s, it was immersed in pure water at 50 °C and stayed for 30 s to solidify into a film. Then it was hydrolyzed in a 0.5% NaOH solution at a hydrolysis temperature of 60 °C for 10 min. After washing with pure water, a cellulose ultrafiltration membrane was obtained.

[0117] Membrane performance parameter testing method:

[0118] 1. Method for measuring the pure water flux of the membrane: The prepared membrane was cut into membrane pieces with a certain diameter (according to the requirements corresponding to the ultrafiltration cup), loaded into the ultrafiltration cup, and the weight of pure water flowing out of the ultrafiltration cup through the membrane piece was measured under a certain pressure. The pure water flux (J) was calculated according to the formula: J = V / (T*A*P), where V is the volume of pure water permeated, T is the measurement time, A is the membrane area, and P is the pressure adopted;

[0119] 2. Method for measuring the surface pore size distribution of the membrane: The ultrafiltration cup was used to measure the separation effect of the ultrafiltration membrane on dextran or PEG solutions with different molecular weights. The rejection coefficient for solutes with different molecular weights was calculated according to the formula: RE = (1 - Cp / Cf)×100%, where Cp is the solute concentration in the permeate and Cf is the original solute concentration in the ultrafiltration solution; The relationship between the molecular weight (MW) of the solute and the Stokes radius was used:

[0120] Logrs = -1.3363 + 0.395logM W

[0121] Through this formula, the Stokes radius corresponding to each solute can be obtained. Plotting the solute rejection coefficient (RE) against the corresponding Stokes radius (rs) will result in an approximately linear relationship. By performing linear regression on these data points, a straight line can be obtained, and thus the standard deviation (σ) of the membrane surface pore size distribution can be calculated. The smaller σ is, the more uniform the pore size distribution.

[0122] The pure water flux and surface pore size distribution of the cellulose ultrafiltration membranes prepared in Examples 1-12 and Comparative Examples 1 and 2 were tested according to the above method, and the experimental results are shown in Table 1. It can be seen from the data in the table that compared with Comparative Examples 1 and 2, the σ value of the cellulose ultrafiltration membrane prepared in the present invention is smaller, and the rejection rate is higher than 95%. This indicates that the pore size distribution of the cellulose ultrafiltration membrane prepared in the present invention is relatively uniform, and the rejection efficiency is relatively high. Further analysis of the data in Examples 2-4, 6-8, and 10-12 found that as the enzymatic hydrolysis treatment time was prolonged and the cellulase concentration was increased, the pure water flux showed a gradually increasing trend. This shows that the pore size of the holes on the surface of the cellulose ultrafiltration membrane increases accordingly. Thus, it can be seen that by regulating the enzymatic hydrolysis treatment time and the cellulase concentration, the pore size of the holes on the surface of the cellulose ultrafiltration membrane can be effectively regulated.

[0123] Scanning electron microscopy (SEM) analysis was performed on the regenerated cellulose membrane 1 prepared in Example 1, the cellulose ultrafiltration membrane 5 prepared in Example 7, and the cellulose ultrafiltration membrane 9 prepared in Example 12, and the results are respectively as Figure 2 , Figure 3 and Figure 4 shown. From Figure 2 , it can be observed that the surface of the regenerated cellulose membrane prepared in Example 1 presents a dense structure, and no obvious holes are seen. However, when the regenerated cellulose membrane was subjected to enzymatic hydrolysis treatment with the cellulase solution, as Figure 3 and Figure 4 shown, a rich hole structure was formed on the surface of the cellulose ultrafiltration membrane. Further analysis found that as the cellulase concentration increased and the enzymatic hydrolysis time prolonged, the pore size of the holes showed a gradually increasing trend. This indicates that the enzymatic hydrolysis treatment not only successfully induced a hole structure on the surface of the cellulose membrane, but also the size of the holes can be effectively controlled by regulating the enzymatic hydrolysis conditions, thereby realizing the optimization of the performance of the cellulose ultrafiltration membrane.

[0124] Table 1

[0125]

[0126]

[0127] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present application. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present application.

Claims

1. An ultrafiltration membrane, characterized in that: include: The invention relates to a cellulose ultrafiltration layer and a supporting layer. The cellulose ultrafiltration layer is obtained by enzymatically degrading the surface of a non-porous cellulose membrane with cellulase.

2. The ultrafiltration membrane according to claim 1, characterized in that The raw material of the cellulose film includes at least one of cellulose diacetate, cellulose triacetate, cellulose acetate butyrate, and cellulose acetate propionate; And / or, the support layer is a non-woven fabric, and the material of the non-woven fabric includes at least one of polyethylene, polypropylene, polyacrylonitrile, polyether sulfone, polysulfone, polyethylene terephthalate, polytetrafluoroethylene, and polyvinylidene fluoride.

3. The ultrafiltration membrane according to claim 2, characterized in that The thickness of the cellulose ultrafiltration layer is 10 μm-1000 μm; And / or, the support layer has a thickness of 10 μm-500 μm.

4. The ultrafiltration membrane according to claim 3, characterized in that The ultrafiltration membrane has a pore size cutoff of 0.5 nm to 50 nm; And / or, the porosity of the ultrafiltration membrane is higher than 30%; And / or, the ultrafiltration membrane has a cut-off standard molecular weight of 1 kDa-1000 kDa; And / or, the pure water flux of the ultrafiltration membrane is greater than 5 L·m -2 h -1 bar -1 .

5. A method for preparing the ultrafiltration membrane according to any one of claims 1 to 4, characterized in that: include: The regenerated cellulose membrane is contacted with cellulase and subjected to enzymatic degradation treatment to obtain the ultrafiltration membrane.

6. The method according to claim 5, characterized in that The surface of the regenerated cellulose membrane is non-porous; And / or, the regenerated cellulose film is prepared by the following method: The cellulose ester and the solvent are mixed to obtain a casting solution; Placing the casting liquid on the surface of the support layer to form a liquid film on the surface of the support layer; Allowing the liquid film to stand for a period of time to form a dense layer on the surface of the support layer; The support layer with a dense layer on the surface is subjected to phase separation solidification treatment to obtain a film; The film is placed in an alkaline solution for hydrolysis treatment to obtain the regenerated cellulose film.

7. The method according to claim 5, characterized in that The contacting of the regenerated cellulose membrane with the cellulase is carried out by immersing the regenerated cellulose membrane in a cellulase solution, wherein the concentration of the cellulase solution is 1 μ / g-100 μ / g, and the pH value of the cellulase solution is 4-7; and / or, the temperature of the enzymatic degradation treatment is 40°C-55°C; And / or, the time of the enzymatic degradation treatment is 5 min-120 min.

8. The method according to claim 6, characterized in that The cellulose ester includes at least one of cellulose diacetate, cellulose triacetate, cellulose acetate butyrate, and cellulose acetate propionate; and / or, the solvent comprises at least one of water, acetone, N-methylpyrrolidone, N,N-dimethylacetamide, formamide, dioxane, acetic acid, propionic acid, and butyric acid; and / or, the weight ratio of the cellulose ester to the solvent is (10-30):(70-90); And / or, the support layer is a non-woven fabric, and the material of the non-woven fabric includes at least one of polyethylene, polypropylene, polyacrylonitrile, polyether sulfone, polysulfone, polyethylene terephthalate, polytetrafluoroethylene, and polyvinylidene fluoride.

9. The method according to claim 6, characterized in that The static treatment time is 30s-120s; And / or, the phase separation solidification treatment is performed by immersing the support layer with the dense layer formed on the surface into a solidification bath, and the solidification bath is water; And / or, the phase separation solidification treatment time is 10s-60s; And / or, the temperature of the phase separation solidification treatment is 0°C-40°C; And / or, the alkaline solution is a 0.1 wt%-10 wt% NaOH aqueous solution; And / or, the temperature of the hydrolysis treatment is 30°C-80°C; And / or, the hydrolysis treatment time is 2 min-60 min.

10. Use of the ultrafiltration membrane according to any one of claims 1 to 4 or the ultrafiltration membrane prepared by the method according to any one of claims 5 to 9 in the purification of biological products.

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