A polysulfone hollow filter membrane, a preparation method and application thereof

Abstract

The application provides a polysulfone hollow filter membrane and a preparation method and application thereof. The polysulfone hollow filter membrane comprises a main body, one side of the main body is an inner surface facing an inner cavity, the other side of the main body is an outer surface, the inner surface comprises a plurality of first large holes and a plurality of first small holes, and the inner surface comprises a plurality of cluster fibers for forming the first large holes and the first small holes. By controlling the SEM average diameter of the first large holes to be 5-8 mu m, the SEM average diameter of the first small holes to be 0.8-2 mu m, and the SEM average width of the cluster fibers to be 0.7-1.8 mu m, the polysulfone hollow filter membrane has good strength, can be used for perfusion culture, and when used for N-1 grade perfusion culture, a high cell density and a high cell survival rate are obtained.

Description

Technical Field

[0001] This invention relates to the field of membrane materials technology, and more specifically to a polysulfone-based hollow fiber membrane, its preparation method, and its application. Background Technology

[0002] In the biopharmaceutical field, alternating tangential flow (ATF) perfusion culture technology has become a core technology for high-density cell expansion and continuous production of biopharmaceuticals (recombinant proteins, monoclonal antibodies, viral vectors, etc.) due to its advantages such as high cell retention rate and long-term stable operation. The core working principle of ATF perfusion culture is to drive the liquid to reciprocate in an alternating flow within a hollow fiber membrane using a diaphragm pump. During forward flow (i.e., the process of liquid flowing from the bioreactor to the hollow fiber membrane), the hollow fiber membrane retains cells and removes metabolites. During reverse flow (i.e., the process of liquid returning from the hollow fiber membrane to the bioreactor), shear force dynamically washes the inner surface of the membrane, inhibiting the formation of a filter cake layer on the inner surface of the hollow fiber membrane, thus enabling continuous perfusion culture.

[0003] As can be seen from the core working principle of ATF perfusion culture, the structure of the hollow fiber membrane is one of the important factors determining whether perfusion culture can be sustained, cell density, and cell viability. Among many membrane substrates, polysulfone materials (including polysulfone, polyethersulfone, etc.) have become the preferred substrate for hollow fiber membranes used in ATF perfusion culture due to their advantages such as high mechanical strength, good chemical stability, high temperature resistance, and excellent biocompatibility. Currently, some polysulfone hollow fiber membranes are available on the market for ATF perfusion, but they still have some drawbacks in practical applications, such as low cell retention efficiency and easy formation of filter cake layers, making it impossible to maintain long-term continuous perfusion.

[0004] Therefore, there is an urgent need to develop more polysulfone hollow fiber membranes with different structures. When used for ATF perfusion culture, these membranes enable continuous perfusion culture, resulting in high cell density and high cell viability, thus meeting the needs of the biopharmaceutical industry for ATF perfusion culture technology. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a polysulfone-based hollow fiber membrane, its preparation method, and its application. By regulating the inner surface to include a first macropore, a first micropore, and clustered fibers for forming the first macropore and the first micropore, and further by controlling the diameter of the first macropore, the diameter of the first micropore, and the width of the clustered fibers within a reasonable range, the prepared hollow fiber membrane can be used for perfusion culture, and when used for N-1 stage perfusion culture, it achieves high cell density and high cell viability.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a polysulfone hollow fiber membrane, comprising a body, one side of which is an inner surface facing the inner cavity, and the other side of which is an outer surface. The inner surface includes a plurality of first macropores and a plurality of first micropores. The SEM average diameter of the first macropores is 5μm-8μm; the SEM average diameter of the first micropores is 0.8μm-2μm; the inner surface also includes a plurality of clustered fibers for forming the first macropores and first micropores; the SEM average width of the clustered fibers is 0.7μm-1.8μm.

[0007] Common perfusion cultures include N-1 stage perfusion and N-stage perfusion. N-1 stage perfusion involves only cell culture, with minimal protein expression during the culture process. N-stage perfusion includes both cell culture and protein expression (initially primarily cell culture with minimal protein expression, later involving both simultaneously). In N-1 stage perfusion cell culture, the feed solution mainly consists of cells, metabolites, and nutrient solution. As the feed solution passes through the inner cavity of the hollow fiber membrane, small molecules such as metabolites and nutrient solution permeate through the inner surface of the membrane, then through the membrane wall, and finally through the outer surface, while cells are retained on the inner surface of the membrane. If the inner surface structure of the hollow fiber membrane is not reasonable, cells are prone to deposit on the inner surface (achieving "contact" between the cells and the inner surface). The deposited cells cannot return to the feed solution in time under the action of shear force (achieving "anchoring" of the cells on the inner surface). The deposited cells will further adsorb other cells in the feed solution (achieving "growth" of the cells on the inner surface). Finally, under the action of transmembrane pressure difference, they will be continuously compacted to form an irreversible filter cake layer, causing the hollow fiber membrane to be unable to be used for perfusion culture continuously.

[0008] Through continuous research, we have discovered that when the inner surface is composed of a first macropore of a specific diameter, a first micropore of a specific diameter, and clusters of fibers of a specific width forming the macropore and micropore, it can effectively reduce the formation of the filter cake layer, allowing perfusion culture to continue, while obtaining high cell density and high cell viability. Specifically:

[0009] Firstly, by controlling the average SEM diameter of the first macropore to be 5μm-8μm, the mass transfer resistance of metabolites and other substances passing through the first macropore is small, which can increase the discharge rate of metabolites and other substances, and facilitate the timely discharge of metabolites. In addition, the diameter of cells is usually greater than 10μm. When cells pass through the first macropore, since the diameter of the cells is larger than the average SEM diameter of the first macropore, even if the cells enter the first macropore, only a small part of the cell volume enters the first macropore. The cells are in a loose state in the first macropore. Under the back flushing of a small shear force, the cells can return to the feed solution, avoiding the cells from clogging the first macropore, keeping the first macropore in a flowing state, and continuously maintaining the outflow of metabolites. It is also beneficial for cells to remain in the feed solution. If the average SEM diameter of the first macropore is too large, for example, greater than 8 μm, the volume of cells entering the macropore is large, requiring significant shear force to detach the cells. This excessive shear force can damage the membrane pores of the macropore, causing structural disruption on the inner surface and hindering the continuous perfusion culture. Furthermore, the excessive shear force can also damage the cells, generating more cell debris. This debris can enter the membrane through the inner surface pores, partially blocking them and reducing the membrane flux, thus slowing down the removal of metabolites and hindering the continuous cell culture. Conversely, if the average SEM diameter of the first macropore is too small, for example, less than 5 μm, it is essentially nonexistent. Metabolite removal is relatively slow, which is detrimental to effective cell culture and the acquisition of high-density cells. Therefore, the presence of a macropore with an average SEM diameter of 5 μm-8 μm is essential for improving cell density, cell viability, and ensuring continuous ATF perfusion.

[0010] Secondly, by controlling the inclusion of a first pore on the inner surface, with a SEM average diameter of 0.8μm-2μm (meaning the pore diameter is much smaller than the cell diameter), the volume of cells entering the first pore is smaller than that of the first macropore. This requires less shear force for backflushing to return the cells to the substrate. Simultaneously, the presence of the first pore increases the expulsion sites for metabolites, disperses the fluid, reduces concentration polarization, and helps reduce cell adsorption on the inner surface, thus reducing cell "contact" and "anchoring" at the source. It also serves as an entry point for removing the initial filter cake layer, requiring only a small shear force to detach it from the inner surface. This inhibits cell compaction on the inner surface, keeping the first pore in a flow-through state, facilitating timely expulsion of metabolites in conjunction with the first macropore. Without the first pore or with a too small SEM average diameter (e.g., less than 0.8μm), the expulsion rate of metabolites is relatively slow, which is detrimental to effective cell culture. Significant concentration polarization and shear force can lead to increased cell damage. The resulting cell debris can easily clog membrane pores, reducing membrane flux and the rate of metabolite removal, which is detrimental to cell culture. If the average SEM diameter of the first pore is greater than 2 μm, it is too large and cannot maintain the pressure resistance of the inner surface of the hollow fiber membrane, thus failing to ensure the continuous perfusion culture. The synergistic effect of the first pore and the first large pore allows metabolites to be removed in a timely manner, enabling continuous cell culture. In other words, the synergistic effect of the first large pore and the first small pore achieves a balance between cell retention and high-flux mass transfer of metabolites, providing core support for increasing cell density and maintaining cell viability.

[0011] Furthermore, while the average SEM width of the clustered fibers is relatively narrow at 0.7 μm-1.8 μm, the filter membrane of this invention is made of polysulfone, which possesses good strength. Therefore, even with a relatively narrow width, the clustered fibers can still support the stability of the first macropore and the first micropore during perfusion culture, ensuring continuous perfusion. Also, since the average width of the clustered fibers is much smaller than the cell diameter, the contact area between the cells and the clustered fibers is small when cells adsorb onto the surface. Similarly, the transmembrane pressure difference has not yet had time to compact the cells onto the clustered fibers, allowing the cells to detach from the clusters under relatively small shear forces. Fiber surface peeling allows cells to return to the feed solution and reduces the formation of filter cake. If the average SEM width of the clustered fibers is too small, for example, less than 0.7 μm, the compressive strength of the inner surface of the hollow fiber membrane is too low, which cannot guarantee the continuous perfusion culture. If the average SEM width of the clustered fibers is too large, for example, greater than 1.8 μm, the contact area between the cells and the clustered fibers increases, requiring a larger shear force to backwash the cells to detach from the clustered fibers. The larger shear force will cause damage to the inner surface structure and also damage the cells, producing more cell debris, causing blockage of the membrane pores, and failing to guarantee the continuous perfusion culture.

[0012] It is evident that when cells adsorb onto the first macropore, the first micropore, or the clustered fibers on the inner surface of the hollow fiber filter membrane of this invention, the contact area is relatively small. Under a small shear force, the cells can return to the feed solution. The transmembrane pressure difference is insufficient to compact the cells on the inner surface, preventing cell growth and thus reducing the formation of the filter cake layer at its source. The smaller shear force also helps protect the cells in the feed solution from damage. Furthermore, the synergistic effect of the first macropore, the first micropore, and the clustered fibers prevents cells from easily forming bridges and accumulating on the inner surface. In other words, this inner surface structure ensures the integrity of the hollow fiber filter membrane. The inner surface is less prone to forming a filter cake layer, providing a necessary foundation for continuous perfusion culture. The synergistic effect of the first macropore, the first micropore, the clustered fibers, and the polysulfone material results in good compressive strength of the inner surface, ensuring the continuity of perfusion culture. Furthermore, cells do not significantly clog the first macropore and the first micropore, resulting in less cell debris and minimal blockage of the membrane pores during perfusion culture. The synergistic effect of the first macropore and the first micropore allows for continuous removal of metabolites, minimizing residual metabolites in the culture system and ensuring continuous cell culture, thereby achieving high cell density and high cell viability.

[0013] In summary, the inner surface of the hollow fiber membrane includes a first macropore, a first micropore, and clustered fibers for forming the first macropore and the first micropore. With the synergistic effect of the first macropore having an average SEM diameter of 5μm-8μm, the first micropore having an average SEM diameter of 0.8μm-2μm, and the clustered fibers having an average SEM width of 0.7μm-1.8μm, it can be used for N-1 stage perfusion culture, while obtaining high cell density and high cell viability.

[0014] The "several first large holes" refers to two or more first large holes, and the "several first small holes" refers to two or more first small holes.

[0015] During the membrane fabrication process, in the direction perpendicular to the membrane thickness (the membrane of this invention is a hollow fiber membrane, and this direction is perpendicular to the radial direction), its various characteristics, such as pore distribution (including size, etc.), are roughly uniform and basically consistent. Therefore, a portion of the corresponding plane can be used as the measurement object. The method for measuring the SEM average diameter of the first macropore and the SEM average diameter of the first micropore is as follows: the inner surface of the filter membrane is characterized using a scanning electron microscope to obtain an ideal SEM image of the inner surface (preferably, the SEM image can clearly show the shape of the pores, etc.). A certain area of ​​the SEM image of the inner surface is selected (at a magnification that allows for the measurement of the diameter of the pores on the inner surface), for example, 40,000 μm. 2 (200μm*200μm), 10000μm 2 (100μm*100μm), the specific area size depends on the actual situation. First, use appropriate computer software (such as Matlab, NIS-Elements, etc.) or manually measure the longest line segment among the two line segments on the outer periphery of the connecting hole. Then measure the longest line segment among the two line segments on the outer periphery of the connecting hole that intersects the longest line segment perpendicularly as the diameter of the inner surface hole. If the diameter of the inner surface hole is greater than 3μm, it is the first large hole. If the diameter of the inner surface hole is not greater than 3μm, it is the first small hole. Preferably, the SEM diameters of more than 10 first large holes and more than 10 first small holes are measured respectively, and the average values ​​are calculated respectively, that is, the SEM average diameter of the first large hole and the SEM average diameter of the first small hole.

[0016] Clustered fibers refer to the clustered, interwoven, and overlapping fiber bundle structure formed on the inner surface (such as...). Figure 3 The SEM width (marked in Chinese) refers to the maximum distance of a single cluster of fibers along the length of the filter membrane. The specific measurement method is as follows: Select a certain area of ​​the inner surface electron micrograph (at a magnification sufficient to measure the width of the clustered fibers), for example, 400 μm. 2 (20μm*20μm), 100μm 2(10μm*10μm), the specific area size depends on the actual situation. Then, use appropriate computer software (such as Matlab, NIS-Elements, etc.) or manually measure the maximum distance of the clustered fibers in the length direction of the filter membrane. Preferably, measure the width of the clustered fibers at more than 10 different positions and calculate the average value, which is the SEM average width of the clustered fibers.

[0017] As a further improvement of the present invention, the initial water contact angle of the inner surface is 55°-75°; the initial water contact angle of the outer surface is 60°-85°; and the SEM average width of the clustered fibers is 0.8μm-1.5μm.

[0018] When the hollow fiber membrane of this invention is used in N-stage perfusion, because cells undergo simultaneous cell culture and protein expression in the later stages of culture (i.e., the feed solution includes cells, proteins, metabolites, nutrient solution, etc.), cells are trapped on the inner surface of the hollow fiber membrane, while proteins, metabolites, nutrient solution, etc., permeate through the inner surface of the hollow fiber membrane, then through the membrane wall, and finally through the outer surface of the hollow fiber membrane. Since proteins have high requirements for the hydrophilicity or hydrophobicity of the hollow fiber membrane when contacting it, if the protein comes into contact with a hollow fiber membrane with poor hydrophilicity, the protein is easily adsorbed onto the membrane, easily forming a filter cake layer, causing blockage of the membrane pores. The rate of metabolite excretion gradually slows down or even stops, making perfusion culture less efficient. Since continuous operation is not feasible and protein rejection is high, we adjust the initial water contact angle of the inner surface to 55°-75° and the initial water contact angle of the outer surface to 60°-85°. This means that both the inner and outer surfaces of the hollow fiber membrane are relatively hydrophilic. Because the hollow fiber membrane is integrally molded, the entire membrane is relatively hydrophilic, given that both the inner and outer surfaces are relatively hydrophilic. When protein in the feed solution passes through the hollow fiber membrane, the membrane adsorbs very little protein. Since the diameters of the first macropore and the first micropore are much larger than the size of the protein, the resistance encountered by the protein as it passes through the inner surface is extremely low, which helps to reduce the protein rejection rate.

[0019] Due to the good hydrophilicity of the inner surface, the clustered fibers adsorb very little protein. Even if a small amount is adsorbed, it can return to the feed solution and permeate through the first macropore or first micropore under relatively small shear force. This means that the opportunity for "contact" and "anchoring" between the protein and the clustered fibers is very limited, reducing the formation of a protein cake layer at the source. Furthermore, the good hydrophilicity of the inner surface results in weaker interaction forces between the cells and the inner surface. Combined with the synergistic effect of the SEM average diameter of the first macropore (5μm-8μm), the first micropore (0.8μm-2μm), and the clustered fiber width (0.8μm-1.5μm), the contact area between the cells and the inner surface is smaller, and the interaction forces are weaker. This allows the protein to permeate under even less shear force. When cells are returned to the feed solution, they are less likely to "grow" on the inner surface of the hollow fiber membrane. This significantly reduces the chance of filter cake formation for both proteins and cells. During perfusion culture, the smaller shear force has less impact on the inner surface of the membrane. Even though the average width of the clustered fibers is relatively small, the inner surface can still ensure the continuous perfusion culture. The smaller shear force causes less damage to proteins and cells, resulting in fewer cell debris in the feed solution and less protein adsorption by the membrane. This reduces the blockage of membrane pores by cell debris and proteins, meaning that the flux decreases less during perfusion culture, which is conducive to the continuous removal of metabolites and other substances. This makes the cell culture process smoother, forms a better virtuous cycle, and helps to improve cell density and cell viability.

[0020] When a hollow fiber membrane with a hydrophilic inner surface is used for N-1 stage perfusion, the interaction between cells and the first macropore, first micropore, and clustered fibers is weaker. This means that the contact area between cells and the inner surface is smaller and the interaction force is weaker. Therefore, only a smaller shear force is needed for the reverse flushing to return the cells to the feed solution. This prevents the formation of filter cake layer at the source. The smaller shear force also causes less damage to the cells, resulting in fewer cell fragments in the feed solution and less clogging of the membrane pores. This means that the flux of the filter membrane decreases less during perfusion culture, which is conducive to the continuous removal of metabolites and other substances. This also makes the culture process smoother and helps to improve cell density and cell viability.

[0021] The initial water contact angle of the inner surface and the initial water contact angle of the outer surface are tested using the same method. Water is used as the test liquid and a contact angle tester is used. When 10-100 μL of water is evenly dropped onto the test sample surface (the inner surface and the outer surface in this invention) for an instant (0.4s), a regular contact angle is formed. Multiple tests are conducted and the average value is taken. Of course, those skilled in the art can also obtain the above parameters through other testing methods. The above testing methods are for reference only.

[0022] As a further improvement of the present invention, the roughness of the inner surface is 3μm-15μm; the pore area ratio of the first macropore is 2%-15%.

[0023] The surface roughness of a filter membrane is an indicator of its smoothness. The smaller the surface roughness, the smoother the surface. In this invention, we control the roughness of the inner surface to 3μm-15μm, meaning the inner surface is relatively smooth. The smaller the inner surface roughness, the weaker the interaction forces between cells, proteins, and the inner surface. Combined with the optimal synergistic effect of the first macropore, first micropore, and clustered fibers on the inner surface, the interaction forces between cells and the inner surface are even smaller, and the contact area is smaller. Furthermore, the smaller contact angle of the inner surface further contributes to this effect. With this method, the interaction forces between cells and proteins and the inner surface are smaller, requiring only less shear force to flush them back into the feed solution. This further reduces the damage to cells and proteins caused by shear force. At the same time, the relatively smooth inner surface reduces frictional contact with cells and proteins, minimizing scratches and resulting in fewer cell fragments in the feed solution. The flux of the filter membrane decreases more slowly during perfusion culture, and metabolites can be continuously and rapidly discharged, which is beneficial for further improving cell density and cell viability.

[0024] The presence of the first macropore can increase the discharge rate of metabolites. The more macropores there are (i.e., the larger the pore area ratio), the faster the metabolites are discharged. However, as the number of macropores increases, the compressive strength of the inner surface will decrease accordingly. Therefore, by controlling the pore area ratio of the first macropore to 2%-15%, we can ensure that the compressive strength of the inner surface can guarantee the continuous use of the hollow fiber membrane for perfusion culture while effectively increasing the discharge rate of metabolites. This results in less metabolite residue in the feed solution, allowing cell culture to proceed more efficiently, achieving higher cell density and cell viability, while further reducing protein retention rate.

[0025] Roughness was measured using a Keyence VHX-7000 ultra-depth-of-field 3D microscope. Specifically, the filter membrane was placed on the sample stage under the microscope (with the inner surface facing the lens). The position and focus were adjusted until the texture on the filter membrane could be clearly observed. Then, "Depth Up", "Quick Synthesis 3D", "Execute Synthesis", "Display Synthesis", "3D Display", and "Surface Roughness" were selected in sequence. The test area was selected in the image, and the obtained Sa value is the roughness value. Multiple measurements were taken and the average value was taken.

[0026] The porosity of the largest hole refers to the ratio of the sum of the areas of all the largest holes to the area of ​​the inner surface, measured using ImageJ, as follows:

[0027] ① Open the image; Open the image (in this invention, it refers to the electron microscope image of the inner surface, preferably one that can clearly show the first large hole) in the image processing software ImageJ. File>Open, select the electron microscope image and click Open;

[0028] ② Set the scale; Use the line tool from the toolbar to draw a straight line within the scale area, making the drawn line the same length as the scale on the electron microscope image. Select Analyze > Set Scale, enter the length of the scale in "KnownDistance" (for example, enter 10), enter the unit in "Unitoflength" (for example, enter um), and select OK.

[0029] ③ Set measurement parameters; In Analyze>SetMeasurements, ensure that “Area” is checked, and then select OK;

[0030] ④ Select and measure the membrane pores; Use FreehandSelection from the toolbar to select the membrane pores. After selection, select Analyze > Tools > ROIManager. In the pop-up task box, click "Add". Repeat the above operation until all the first macropores in the electron microscope image are selected. Use the Shift key to select all the added data in the task box, click "Measure", and sum the results and divide by the area of ​​the electron microscope image to get the pore area ratio of the first macropore.

[0031] As a further improvement of the present invention, some adjacent first macropores are connected by fine fibers to form a long strip-shaped first macropore region, the SEM average length of the first macropore region is not greater than 30μm; the SEM average diameter of the fine fibers is 0.3μm-0.8μm.

[0032] The presence of the first macropore reduces mass transfer resistance to metabolites. We found that when some adjacent first macropores are connected by fine fibers to form a long strip-shaped first macropore region, meaning that some first macropores are perforated, the mass transfer resistance of metabolites, proteins, and other substances entering the first macropore region is even smaller. This allows the fluid to flow towards the outer surface more quickly, resulting in faster metabolite discharge and lower mass transfer resistance. The required transmembrane pressure difference is also smaller. Even if a small number of cells or proteins adsorbed on the inner surface do not return to the feed solution in time under shear force, the smaller transmembrane pressure difference will not compact the adsorbed cells or proteins on the inner surface in a short time. They can then return to the feed solution under shear force, thus limiting the growth of the filter cake layer. This allows the first macropores and first micropores to be in a better flow state. In other words, the metabolite discharge rate is faster throughout the perfusion culture process, the residual amount of metabolites in the culture system is less, cell culture proceeds more smoothly, and it is conducive to obtaining higher cell density and better viability, while further reducing protein retention rate.

[0033] However, if the average length of the SEM in the first macropore region is too long, the compressive strength of the inner surface will decrease, making it impossible to guarantee the continuous perfusion culture. Therefore, by controlling the average length of the SEM in the first macropore region to be no more than 30 μm, that is, the length of the first macropore region is slightly long, but not excessively long, and its compressive strength of the inner surface decreases slightly. Furthermore, by controlling the average diameter of the SEM in the fine fibers to be 0.3 μm-0.8 μm, the fine fibers act as "reinforcing ribs," stabilizing the first macropore region and dispersing the impact of shear force and transmembrane pressure difference on the first macropore region, so that the first macropore region has good compressive strength, and thus the hollow filter membrane has good strength, allowing the perfusion culture to be carried out for a long time, ensuring a higher cell density in the end.

[0034] The average SEM length of the first macropore region refers to the length of the first macropore region parallel to the circumferential direction of the membrane. It is measured by selecting a certain area of ​​the inner surface from the SEM image (enough to measure the length of the first macropore region), for example, 40000 μm. 2 (200μm*200μm), 10000μm 2 (100μm*100μm), the specific area size depends on the actual situation. Then, use appropriate computer software (such as Matlab, NIS-Elements, etc.) or manually measure the distance between the two vertices of the first macropore area in the circumferential direction and the tangents parallel to the length direction. Preferably, measure the length of more than 10 first macropore areas and calculate the average value, which is the SEM average length of the first macropore area.

[0035] The average diameter of the fine fibers was determined by selecting a specific area (e.g., 10000 μm) from the SEM image of the inner surface. 2 (100μm*100μm), 2500μm 2 (50μm*50μm), the specific area size depends on the actual situation. Then, use appropriate computer software (such as Matlab, NIS-Elements, etc.) or manually measure the diameter of the fine fiber. Preferably, measure the diameter of more than 10 fine fibers and calculate the average value, which is the SEM average diameter of the fine fiber.

[0036] As a further improvement of the present invention, the pore area ratio of the first small hole is 15%-35%; the porosity of the filter membrane is 60%-85%.

[0037] The first pore has a relatively small impact on the inner surface pressure resistance. Therefore, the pore area ratio of the first pore can be slightly larger. By controlling the pore area ratio of the first pore to 15%-35%, that is, the first pores are small and relatively numerous, with a relatively high distribution density. This allows for better fluid dispersion, resulting in less concentration polarization and less shear force generated by the feed solution itself. Simultaneously, the relatively large number of small first pores provides more entry points for cake removal on the inner surface. The initial cake layer requires less shear force to detach from the inner surface, reducing cell damage, resulting in less cell debris in the feed solution, less clogging of the filter membrane, and thus less flux drop during perfusion culture. Throughout the perfusion culture process, metabolites can be rapidly discharged, which is beneficial for obtaining cells with higher density and viability. The lower shear force also causes less damage to proteins, which helps to reduce protein retention. At the same time, the inner surface has good pressure resistance, and the distribution density of the first pore is relatively high, which can evenly distribute the transmembrane pressure and effectively delay the local thickening and compaction of the filter cake layer on the inner surface, so as to keep the transmembrane pressure difference stable and allow perfusion culture to be carried out for a longer period of time. The high distribution density of the first pore also facilitates the timely discharge of metabolites, resulting in less residual metabolites in the feed solution, allowing cell culture to proceed faster, and ultimately obtaining higher cell density and cell viability, and allowing perfusion culture to be carried out for a longer period of time.

[0038] Porosity is the volume ratio of the overall pores in a hollow fiber membrane. The higher the porosity, the greater the volume ratio of the overall pores in the membrane, meaning there are more flow paths for fluid. The porosity of this invention is 60%-85%, which is relatively high. This provides more flow paths for fluid, facilitating the passage of metabolites, proteins, and other substances that enter through the first large and small pores on the inner surface of the hollow fiber membrane. This means that metabolites are discharged more promptly, allowing for better cell culture. At the same time, the high porosity structure gives the hollow fiber membrane a large contaminant-holding capacity. Even if a small number of particles (such as cell debris) are trapped inside the hollow fiber membrane, there are still many flow paths, maintaining a high level of metabolite discharge and allowing cell culture to proceed at a relatively fast rate. Furthermore, at this porosity, the overall mechanical strength of the membrane is also good, allowing perfusion culture to continue for a longer period, increasing cell density and cell viability, and reducing protein retention.

[0039] The pore area ratio of the first small pore refers to the ratio of the sum of the areas of all the first small pores to the area of ​​the inner surface. The measurement method is the same as the measurement steps of the pore area ratio of the first large pore, except that the membrane pore selected by Freehand selection in step ④ is the first small pore.

[0040] Commonly used methods for testing porosity include mercury intrusion porosimetry, density method, and wet-dry film weighing method. Of course, those skilled in the art can also obtain the above parameters through other testing methods. The above testing methods are for reference only.

[0041] As a further improvement of the present invention, the inner surface further includes a plurality of fluid diversion holes, the fluid diversion holes being elongated, the length direction of the fluid diversion holes being perpendicular to the length direction of the filter membrane, the SEM average minor axis of the fluid diversion holes being 10μm-50μm; the ratio of the SEM average major axis of the fluid diversion holes to the SEM average minor axis of the fluid diversion holes being 1.5-4; and the pore area ratio of the fluid diversion holes being no greater than 10%.

[0042] Because the average minor axis of the fluid diversion orifice is relatively large, and the ratio of the average major axis to the average minor axis of the fluid diversion orifice is 1.5-4, the average major axis of the fluid diversion orifice is also relatively large. This means that the fluid diversion orifice itself is a large pore. When the fluid passes through the fluid diversion orifice, the flow resistance is smaller, which is conducive to the faster discharge of metabolites, proteins, etc., so that the perfusion culture can proceed more quickly. With the synergistic effect of the first large pore and the first small pore, the cell density and cell viability are higher, and the protein retention rate is reduced. At the same time, in order to ensure that the perfusion culture can continue, the pore area ratio of the fluid diversion orifice is controlled to be no more than 10%. The pore area ratio of the fluid diversion orifice is relatively small, and the distribution of the fluid diversion orifice is relatively discrete, which can still ensure the compressive strength of the inner surface. That is, under the synergistic effect of the above-mentioned fluid diversion orifice SEM average short diameter, the ratio of the fluid diversion orifice SEM average long diameter to the fluid diversion orifice SEM average short diameter, and the pore area ratio of the fluid diversion orifice within a reasonable range, cell density and cell viability are improved, protein retention rate is reduced, and the continuous perfusion culture is ensured.

[0043] The major axis of the fluid splitter orifice refers to the length of the orifice parallel to the circumferential direction of the membrane. It is measured by selecting a specific area (e.g., 1,000,000 μm) of the electron micrograph of the inner surface. 2 (1000μm*1000μm), 250000μm 2(500μm*500μm), the specific area size depends on the actual situation. Then, use appropriate computer software (such as Matlab, NIS-Elements, etc.) or manually to measure the distance between the two vertices of the fluid distribution hole in the circumferential direction and the tangents parallel to the length direction. Preferably, measure the major diameter of more than 10 fluid distribution holes and calculate the average value, which is the SEM average major diameter of the fluid distribution hole. The SEM average minor diameter of the fluid distribution hole refers to the maximum value of the length of the fluid distribution hole parallel to the length direction of the membrane. Use appropriate computer software (such as Matlab, NIS-Elements, etc.) or manually to measure the maximum value of the length of the fluid distribution hole in the length direction, which is the SEM minor diameter of the fluid distribution hole. Preferably, measure the minor diameter of more than 10 fluid distribution holes and calculate the average value, which is the SEM average minor diameter of the fluid distribution hole.

[0044] The length direction of the fluid diversion orifice is perpendicular to the length direction of the filter membrane, meaning that the angle between the line segment between the two vertices and the circumferential direction in the length measurement of the fluid diversion orifice is less than 20°.

[0045] The pore area ratio of the fluid diversion orifice is the ratio of the sum of the areas of all fluid diversion orifices to the area of ​​the inner surface. Its measurement method is the same as that of the pore area ratio of the first large orifice. The difference is that the membrane orifice selected by Freehand selection in step ④ is the fluid diversion orifice.

[0046] As a further improvement of the present invention, the complete bubble point of the IPA of the filter membrane is 8psi-20psi; the ratio of the complete bubble point of the IPA of the filter membrane to the initial bubble point of the IPA of the filter membrane is 1.05-1.30.

[0047] The complete bubble point of IPA reflects the overall pore size characteristics of the filter membrane. The smaller the complete bubble point, the larger the overall pore size of the filter membrane. The complete bubble point of IPA of the filter membrane of this invention is relatively small, which means that the overall pore size of the filter membrane is relatively large. It has a relatively wide flow path, and the flow resistance of metabolites and proteins is small when passing through the filter membrane. With the synergistic effect of the first large pore and the first small pore on the inner surface, metabolites and proteins can pass through quickly. At the same time, the overall pore size of the filter membrane is not very large, which makes the filter membrane have excellent strength. That is, when the complete bubble point of IPA of the filter membrane is 8psi-20psi, not only can cell culture be carried out more quickly, but the filter membrane also has excellent strength.

[0048] Furthermore, the initial bubble point of the IPA in the filter membrane reflects the maximum pore size of the membrane. The ratio of the complete bubble point of the IPA to the initial bubble point of the IPA in the filter membrane is close to 1, indicating that the overall pore size difference of the filter membrane is small and the pore size distribution is relatively uniform, that is, the pore channel uniformity is good. This is conducive to the relatively uniform flow rate of fluids flowing inside the membrane. The shear force experienced by proteins when passing through the membrane is small, resulting in less damage to the proteins and helping to reduce the protein rejection rate. At the same time, the relatively uniform flow rate also results in less shear force generated by the feed solution itself, reducing damage to cells and generating less cell debris. This helps the flow path of the filter membrane to remain relatively wide, allowing metabolites to be discharged at a relatively fast rate, which is conducive to faster cell culture and obtaining higher cell density and cell viability.

[0049] The methods for testing the complete bubble point and the initial bubble point are well known in the art, and the procedures for these tests are explained in detail, for example, in ASTM F316-03 (2019), which is incorporated herein by reference. The initial bubble point and the complete bubble point of the IPA for the filter membrane are both measured using a 60% (v / v) isopropanol aqueous solution as the test medium. The pressure at which the filter membrane begins to bubble is recorded as the initial bubble point, and the pressure at which the filter membrane begins to bubble continuously is recorded as the complete bubble point.

[0050] As a further improvement of the present invention, the pore size of the filter membrane first increases and then decreases in the thickness direction from the inner surface to the outer surface. The transition region where the pore size first increases and then decreases in the thickness direction from the inner surface to the outer surface is a fluid acceleration zone. The thickness of the fluid acceleration zone is 30μm-70μm. The closest distance between the fluid acceleration zone and the SEM of the inner surface is 10μm-30μm. The average pore size of the fluid acceleration zone in the SEM is 1.5μm-3.5μm.

[0051] Because the pore size of the filter membrane increases and then decreases along its thickness from the inner to the outer surface, the first passage is through the expanding channels. This results in less mass transfer resistance for fluids such as metabolites, requiring a smaller transmembrane pressure difference to allow fluids to pass through the first large and small pores on the inner surface and enter the membrane. This smaller transmembrane pressure difference also hinders the formation of a robust filter cake layer on the inner surface. Particles (such as cells) adsorbed on the inner surface can return to the feed solution under less shear force, allowing for longer perfusion cultures. Furthermore, the lower shear force causes less cell damage, which is beneficial for improving cell viability. The increased shear force results in less cell debris, which facilitates more efficient cell culture. Furthermore, as metabolites and other fluids pass through the region with the increased pore size, the mass transfer resistance is lower, allowing for faster flow. When passing through the region with the decreased pore size, the resulting micro-turbulence promotes fluid flow towards the outer surface. In other words, the overall fluid velocity across the membrane cross-section is relatively fast. This is especially true for N-1 stage perfusion, where metabolites can be discharged more quickly, resulting in less residual metabolites in the feed solution. This is more conducive to cell culture and improves cell density and viability.

[0052] Because the average pore size of the SEM in the fluid acceleration zone is relatively large (1.5μm-3.5μm), the flow path of the fluid through the acceleration zone is wide, resulting in low resistance and rapid removal of metabolites and proteins, which is beneficial for rapid cell culture and achieving higher cell densities. Furthermore, the thickness of the fluid acceleration zone (30μm-70μm) is moderate, providing a reasonably wide flow path, especially in the later stages of cell culture when cell growth and metabolite production are rapid. This wider flow path facilitates timely removal of metabolites, ensuring that even in the later stages of cell culture, the residual amount of metabolites in the solution remains low, maintaining a relatively fast culture rate and contributing to higher cell densities. Simultaneously, the thickness of the fluid acceleration zone is not excessive, and the closest distance to the SEM on the inner surface of the fluid acceleration zone is 10μm-30μm, resulting in excellent strength of the hollow fiber membrane itself. Therefore, under the synergistic effect of these characteristics, higher cell density and cell viability are achieved through perfusion culture.

[0053] The fluid acceleration zone is located in the transition region where the pore size first increases and then decreases in the thickness direction from the inner surface to the outer surface. The location of the fluid acceleration zone is determined by using a scanning electron microscope (SEM) to characterize the cross-section of the entire membrane, for example, obtaining SEM images of the membrane cross-section at magnifications of 300, 400, or 500. The transition region Z1 where the pore size first increases and then decreases is identified. Then, region Z1 is continuously photographed, for example, obtaining multiple SEM images of the membrane cross-section at magnifications of 2000, 3000, 5000, 6000, or 8000. The pore size of the multiple SEM images of the membrane cross-section obtained in region Z1 is measured. The method is to select multiple pores on the same plane, measure their SEM pore size, and calculate the average value to obtain the average SEM pore size of different planes. The plane with the largest average SEM pore size value is plane P1, and plane P1 corresponds to S... The average SEM aperture is D1. Then, the membrane cross-section between plane P1 and the inner surface is continuously photographed to obtain multiple SEM images of the membrane cross-section with magnifications of 2000, 3000, 5000, 6000, or 8000. Next, the average SEM aperture of the corresponding plane is measured step by step from plane P1 towards the inner surface using the same method until the average SEM aperture value is 0.5D1. The plane with this average SEM aperture of 0.5D1 is plane P2, which is the fluid acceleration zone near the inner surface. The other plane P3 of the fluid acceleration zone (located between plane P1 and the outer surface) is determined using the same method. The average SEM aperture of plane P3 is also 0.5D1. The distance between plane P2 and plane P3 is the thickness of the fluid acceleration zone. The distance between plane P2 and the inner surface is the closest SEM distance between the fluid acceleration zone and the inner surface. The thickness of the fluid acceleration zone and the closest SEM distance between the fluid acceleration zone and the inner surface are measured by measuring the distance between plane P2 and plane P3, and the distance between plane P2 and the inner surface, on a cross-sectional SEM image of the membrane using computer software (such as Matlab, NIS-Elements, etc.) or manually. The average pore size of the fluid acceleration zone is determined by characterizing the area between planes P2 and P3 using a scanning electron microscope at a certain magnification, such as 3000x, 5000x, or 8000x. Points (preferably more than 10 points) at different planar positions are uniformly selected as measurement objects, and the corresponding SEM pore sizes are measured using appropriate computer software (such as Matlab, NIS-Elements, etc.) or manually. The average value is then calculated to obtain the average SEM pore size of the fluid acceleration zone.

[0054] As a further improvement of the present invention, the outer surface includes a plurality of second holes, the SEM average diameter of the second holes being 0.3μm-0.8μm; and the pore area ratio of the second holes being 30%-50%.

[0055] The second pore on the outer surface is the final area for the discharge of metabolites and proteins. The relatively small but numerous second pores on the outer surface reduce the mass transfer resistance during the discharge of metabolites and proteins, ensuring their rapid discharge and allowing cell culture to proceed more quickly, achieving higher cell density. This also improves the pressure resistance of the outer surface, resulting in better overall strength of the hollow fiber membrane, which is essential for continuous perfusion culture. In synergy with the first large and small pores, the mass transfer resistance of metabolites and proteins through the membrane is further reduced, requiring a smaller transmembrane pressure difference and causing less damage to cells and proteins. This contributes to improved cell viability and reduced protein retention.

[0056] The SEM average diameter of the second hole refers to the equivalent diameter of the second hole (i.e., the diameter of a circle with the same SEM average area as the second hole). The specific measurement method is as follows:

[0057] ① Open the image; Open the image (in this invention, it refers to the electron microscope image of the outer surface, such as an electron microscope image with a magnification of 5000) in the image processing software ImageJ. File>Open, select the electron microscope image and click Open;

[0058] ② Set the scale; Use the line tool from the toolbar to draw a straight line within the scale area, making the drawn line the same length as the scale on the electron microscope image. Select Analyze > Set Scale, enter the length of the scale in "KnownDistance" (for example, enter 10), enter the unit in "Unitoflength" (for example, enter um), and select OK.

[0059] ③ Set measurement parameters; In Analyze>SetMeasurements, ensure that “Area” is checked, and then select OK;

[0060] ④ Select and measure the membrane pores; Use FreehandSelection from the toolbar to select the membrane pores. After selecting, select Analyze > Tools > ROIManager. In the pop-up task box, click "Add". Repeat the above operation. It is preferable to select more than 10 membrane pores. Use the Shift key to select all the added data in the task box, click "Measure", calculate the average area of ​​the selected second pore, and then calculate the radius length r using the formula S (average area of ​​membrane pores) = πr². 2r is the equivalent diameter of the second pore.

[0061] The porosity of the second hole refers to the ratio of the sum of the areas of all second holes on the outer surface to the area of ​​the outer surface. It can be measured using ImageJ. Specifically,

[0062] ① Open the image and adjust the pixels: Open the electron microscope image of the outer surface in the image processing software ImageJ. File>Open, select the electron microscope image and click Open, then select Image>Type>8-bit to convert the image to an 8-bit grayscale image;

[0063] ② Measure the pore area ratio of the outer surface: In Image>Adjust>Threshold, adjust the scale in the dialog box that appears so that all the membrane pores are filled. The percentage obtained is the pore area ratio of the outer surface. Select at least 5 electron micrographs of different outer surfaces as the measurement objects and take the average value.

[0064] As a further improvement of the present invention, the cell density obtained when the filter membrane is used for cell perfusion culture is not less than 3*10⁻⁶. 7 The filter membrane has a cell density of at least 90% and a cell viability of at least 90%; the tensile strength of the filter membrane is at least 3.5 N / mm². 2 The water flux of the filter membrane is greater than 600 LMH / psi; the thickness of the filter membrane is 120 μm-180 μm.

[0065] Cell density refers to the number of cells per unit volume in the feed solution. It is an important parameter in perfusion culture. Higher cell density indicates better cell growth, which is beneficial for improving protein synthesis capacity per unit volume and increasing protein titer. When the hollow fiber filter membrane of this invention is used for perfusion culture, its cell density can reach no less than 3*102. 7 It has cells / mL and a good cell density.

[0066] Cell viability refers to the proportion of live cells. The higher the cell viability, the fewer apoptotic cells occur during perfusion culture. When the hollow fiber filter membrane of this invention is used for perfusion culture, its cell viability is not less than 90%, which is a good cell viability, and relatively few cells undergo apoptosis during the culture process.

[0067] Mechanical strength tests showed that the tensile strength of the hollow fiber filter membrane prepared by this invention is not less than 3.5 N / mm². 2 It has excellent mechanical properties, which is beneficial for the continuous perfusion culture.

[0068] The sample was stretched at a constant speed (50 mm / min, 30 mm between the upper and lower clamps) using a tensile testing machine at room temperature until it broke, and the tensile strength was measured. This process was repeated 3 times and the average value was taken as the final tensile strength.

[0069] Permeation flux, also known as permeation rate or simply flux, refers to the amount of material that passes through a unit area of ​​a hollow fiber membrane per unit time under a certain operating pressure. The magnitude of the flux reflects the rate at which metabolites are expelled; a higher flux indicates a faster rate of metabolite expulsion. The polysulfone-based hollow fiber membrane of this invention has a water flux greater than 600 LMH / psi, exhibiting a high water flux. This indicates that when used for perfusion culture, the hollow fiber membrane facilitates rapid metabolite expulsion, allowing metabolites to pass through the membrane quickly. This enables continuous perfusion culture, resulting in high-density cell production and significant economic benefits.

[0070] For the test method of water flux of hollow fiber membrane, please refer to paragraphs 0150-0153 of the specification of patent CN119607899A.

[0071] The filter membrane of the present invention has a thickness of 120μm-180μm, which is relatively thick and conducive to the continuous perfusion culture. The measurement method is to measure the distance between the inner and outer surfaces on the cross-sectional electron microscope image of the membrane using computer software (such as Matlab, NIS-Elements, etc.) or manually.

[0072] A second aspect of the present invention provides a method for preparing a polysulfone-based hollow fiber membrane as described above, comprising the following steps:

[0073] Step 1: Preparation of casting solution and core solution:

[0074] The casting solution comprises the following components by weight: 12-18 parts of polysulfone, 8-20 parts of pore-forming agent, and 55-70 parts of first organic solvent;

[0075] The core fluid is composed of a second organic solvent, water, and glycerol, wherein the water content in the core fluid is 5%-25% by mass, and the glycerol content is 5%-10% by mass.

[0076] The polysulfone-based material includes polysulfone A with a weight-average molecular weight of 50,000-80,000 and polysulfone B with a weight-average molecular weight of 120,000-150,000, wherein the mass ratio of polysulfone B to polysulfone A is 0.5-0.8.

[0077] The porogen is one or more of polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol and polyethyleneimine, the molecular weight distribution of the porogen is 3-8, and the weight average molecular weight of the porogen is 10,000-50,000.

[0078] The temperature of the core liquid is 5°C-15°C higher than the temperature of the casting liquid;

[0079] Step 2: Spinning:

[0080] The casting solution and core solution prepared in step one are extruded together from the spinneret to form a molded product with an inner surface and an outer surface.

[0081] Step 3: Phase Separation:

[0082] The molded product obtained in step two is immersed in a coagulation bath to perform complete phase separation and form a biofilm I.

[0083] Step 4: Cleaning and drying:

[0084] The biofilm I obtained in step three is washed with pure water and then dried to obtain a polysulfone-based hollow fiber membrane.

[0085] As a further improvement of the present invention, the casting solution further comprises 1-3 parts by weight of sulfonated polyether sulfone; the weight-average molecular weight of the sulfonated polyether sulfone is 20,000-50,000; the polysulfone is at least one of bisphenol A polysulfone, polyether sulfone and polyphenylene sulfone.

[0086] As a further improvement of the present invention, the coagulation bath in step three includes a first coagulation bath and a second coagulation bath. During the phase separation process in the first coagulation bath, the filter membrane is simultaneously stretched, and the stretching rate is 2%-5%. The temperature of the casting solution is 20℃-40℃.

[0087] As a further improvement of the present invention, the first coagulation bath is composed of a third organic solvent and water, wherein the mass content of the third organic solvent is 75%-95%; the second coagulation bath is composed of isopropanol and water, wherein the mass content of isopropanol is 0%-30%; the time of the molded article in the first coagulation bath is 10s-25s, and the time in the second coagulation bath is 100s-250s; the first organic solvent, the second organic solvent, and the third organic solvent are all at least one of dimethylformamide, dimethylacetamide, N-methylpyrrolidone, and dimethyl sulfoxide.

[0088] In preparing the polysulfone-based hollow fiber membrane of the present invention, a casting solution is first prepared. The casting solution includes a film-forming polysulfone substance, a pore-forming agent, and a first organic solvent for dissolving the polysulfone substance. The polysulfone substance includes polysulfone A with a weight-average molecular weight of 50,000-80,000 and polysulfone B with a weight-average molecular weight of 120,000-150,000, and the mass ratio of polysulfone B to polysulfone A is 0.5-0.8. Because polysulfone A has a relatively low weight-average molecular weight, relatively short molecular chains, and relatively low chain segment entanglement, it is beneficial for a relatively fast phase separation rate during subsequent phase separation, providing a basis for forming the first small pores on the inner surface. Polysulfone B, on the other hand, has a relatively high weight-average molecular weight, which facilitates faster phase separation during subsequent phase separation processes, thus providing a basis for forming the first small pores on the inner surface. The relatively longer sub-chains and higher degree of chain segment entanglement facilitate a slower phase separation rate during subsequent phase separation processes, providing a foundation for the formation of the first macropores and fluid diversion pores on the inner surface. Furthermore, the addition of polysulfone B enhances the overall strength of the filter membrane. Simultaneously, the molecular chains of polysulfone B tend to stretch and extend along the phase interface, intertwining and adhering with the chains of polysulfone A. Under a controlled mass ratio of 0.5-0.8 for polysulfone B and polysulfone A, this provides skeletal support for the formation of clustered fibers. The polysulfone material is at least one of bisphenol A polysulfone, polyethersulfone, and polyphenylene sulfone. Polysulfone A and polysulfone B can be the same polysulfone material or different polysulfone materials. Polysulfone-based substances; furthermore, the porogen is one or more of polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, and polyethyleneimine. The porogen has a wide molecular weight distribution (3-8) and a weight-average molecular weight of 10,000-50,000. That is, the porogen contains both substances with relatively low and relatively high molecular weights. In the casting solution near the core liquid, the porogen with relatively low molecular weight has better compatibility with the core liquid and a faster exchange rate, i.e., a faster phase separation rate, which is beneficial for forming the first micropores on the inner surface. On the other hand, the substances with relatively high molecular weight have relatively poor compatibility with the substances in the core liquid. A slower exchange rate is beneficial for forming the first macropores and fluid distribution pores on the inner surface. By adjusting the mass composition of polysulfones, pore-forming agents, and the first organic solvent (selected from at least one of dimethylformamide, dimethylacetamide, N-methylpyrrolidone, and dimethyl sulfoxide), the casting solution can be made to have a suitable viscosity. The viscosity of the casting solution also has a certain influence on the structure and performance of the final hollow fiber membrane, such as the pore composition of the hollow fiber membrane. This provides a basis for the final hollow fiber membrane to have a first macropore of suitable diameter, a first micropore of suitable diameter, and clustered fibers of suitable width on its inner surface, which can be effectively used in perfusion culture.

[0089] The composition of the core fluid primarily affects the structure of the inner surface and surrounding area of ​​the hollow fiber membrane. The core fluid used in the extrusion of the hollow fiber membrane of this invention consists of a second organic solvent (selected from at least one of dimethylformamide, dimethylacetamide, N-methylpyrrolidone, and dimethyl sulfoxide), water, and glycerol. The low water content in the core fluid helps to slow down the phase separation rate on the inner surface. Simultaneously, the presence of glycerol in the core fluid further slows down the phase separation rate on the inner surface. Combined with the composition of the casting solution and the fact that the core fluid temperature is 5°C-15°C higher than the casting solution temperature, different parts of the inner surface of the casting solution exhibit different phase separation rates. The parts with relatively fast phase separation rates form the first small pores on the inner surface, while the parts with relatively slow phase separation rates... The formation of a first macropore, or a first macropore and a fluid diversion pore, occurs when the glycerol content is higher, such as when the glycerol mass content is 8%-10%, the mass ratio of polysulfone B to polysulfone A is 0.5-0.6, and the molecular weight distribution of the porogen is 7-8. This results in a very slow phase separation rate in some areas of the inner surface, forming a fluid diversion pore. In other words, the inner surface consists of a first macropore, a fluid diversion pore, and a first micropore. However, when the glycerol mass content is less than 8% but greater than or equal to 5%, the mass ratio of polysulfone B to polysulfone A is greater than 0.6 but less than or equal to 0.8, and the molecular weight distribution of the porogen is greater than or equal to 3 but less than 7, no fluid diversion pores are formed on the inner surface. In other words, the inner surface consists of a first macropore and a first micropore. Preferably, the temperature of the casting solution is 20℃-40℃, which is a moderate temperature. Since the temperature of the core solution is 5℃-15℃ higher than that of the casting solution, the temperature of the core solution is also moderate. Under the synergistic effect of the composition of the casting solution and the composition of the core solution, the pore area ratio of the first large pore and the pore area ratio of the first small pore are within the preferred range.

[0090] The second step is to extrude the prepared casting solution and core solution together from the spinneret. The casting solution forms a molded product with an inner surface and an outer surface in the nozzle, namely a hollow filter membrane. The extruded hollow filter membrane has an inner surface facing the inner cavity and an outer surface opposite to the cavity.

[0091] Step 3, phase separation, involves directly immersing the molded product obtained in step 2 into a coagulation bath to achieve complete phase separation.

[0092] The resulting membrane I after phase separation was washed with pure water and then dried to obtain a polysulfone-based hollow fiber membrane.

[0093] Preferably, the casting solution further comprises 1-3 parts by mass of sulfonated polyether sulfone. By controlling the weight-average molecular weight of the sulfonated polyether sulfone to be 20,000-50,000, the sulfonated polyether sulfone has a moderate molecular chain length and contains highly polar sulfonic acid groups. The sulfonated polyether sulfone has good compatibility with polysulfone substances, can be dispersed in polysulfone substances and fixed on the skeleton. During the phase separation process of the casting solution, the sulfonated polyether sulfone will not dissolve, which is beneficial to improving the stability of the hydrophilicity of the obtained hollow fiber membrane and reducing the initial water contact angle of the hollow fiber membrane.

[0094] Preferably, the coagulation bath in step three includes a first coagulation bath and a second coagulation bath. During the phase separation process in the first coagulation bath, the filter membrane is simultaneously stretched. During stretching, the molecular chains of polysulfones in the casting solution align along the stretching direction (i.e., the length direction of the membrane), and the membrane pores preferentially expand perpendicular to the stretching direction. Therefore, the formed macropores can be connected by fine fibers into a long, strip-shaped macropore region. When the inner surface contains fluid diversion pores, these pores are long and strip-shaped with their length direction perpendicular to the length direction of the filter membrane. By controlling the stretching rate, the SEM of the first macropore region is optimized. The average length is within a reasonable range, and the ratio of the average major diameter to the average minor diameter of the fluid distribution orifice via SEM is controlled within a suitable range. The first coagulation bath consists of a third organic solvent (selected from at least one of dimethylformamide, dimethylacetamide, N-methylpyrrolidone, and dimethyl sulfoxide) and water. By controlling the composition of the first coagulation bath, the phase separation rate on the outer surface is faster than that on the inner surface. Before polysulfone A and polysulfone B can diffuse, i.e., while still in a relatively uniform state, phase separation begins on the outer surface. The SEM average length of the second pores formed on the outer surface is... The average diameter is relatively small, and the pore area ratio is moderate. The first coagulation bath gradually enters the interior of the membrane through the pores formed on the outer surface. The farther away from the outer surface, the less the first coagulation bath enters, the slower the phase separation rate, and the larger the formed pores. This creates a portion where the pore diameter decreases from the inner surface to the outer surface in the thickness direction. Because the phase separation rate of the core liquid on the inner surface and the nearby casting liquid is slower, its rate of entry into the membrane interior through the inner surface is also slower. The farther away from the inner surface, the less core liquid enters, and the slower the phase separation rate is relative to the inner surface. The resulting membrane pores are also larger, forming a portion where the pore size initially increases from the inner surface to the outer surface in the thickness direction. Phase separation is achieved within the first coagulation bath time of 10-25 seconds. This means that, under the combined influence of the composition of the casting solution, the temperature difference between the casting solution and the core solution, the composition of the core solution, and the composition of the first coagulation bath, the filter membrane of this invention exhibits a structure where the pore size initially increases and then decreases from the inner surface to the outer surface in the thickness direction. This forms the fluid acceleration zone of this invention, and the closest distance between the fluid acceleration zone and the inner surface (SEM image) and the average pore size of the fluid acceleration zone (SEM image) are within the preferred range. The second coagulation bath consists of isopropanol and water. By controlling the isopropanol content to be relatively low, it serves to solidify the phase-separated filter membrane. By controlling the time in the second coagulation bath to be 100-250 seconds, a longer time, sufficient solidification is achieved, resulting in an improvement in the overall strength of the filter membrane.

[0095] A third aspect of the invention provides an application of a polysulfone hollow fiber membrane for: (a) cell amplification; (b) harvesting of monoclonal antibodies; (c) harvesting of recombinant proteins; (d) harvesting of vaccines; and (e) harvesting of viral vectors.

[0096] Compared with the prior art, the present invention has the following beneficial technical effects:

[0097] 1. This invention scientifically regulates the inner surface of a polysulfone-based hollow fiber membrane to include a first macropore, a first micropore, and clustered fibers for forming the first macropore and the first micropore. By controlling the SEM average diameter of the first macropore, the SEM average diameter of the first micropore, and the SEM average width of the clustered fibers within a reasonable range, the resulting polysulfone-based hollow fiber membrane can be used for perfusion culture. When used for N-1 stage perfusion culture, it yields high cell density and high cell viability.

[0098] 2. The present invention further improves upon the invention by controlling the initial water contact angle of the inner surface and the initial water contact angle of the outer surface of the polysulfone hollow filter membrane to be relatively small, i.e. relatively hydrophilic. When the polysulfone hollow filter membrane is used for N-1 stage perfusion culture and N stage perfusion culture, the cell density is higher and the cell viability is higher. When used for N stage perfusion culture, the protein retention rate is lower.

[0099] 3. The polysulfone hollow fiber membrane of the present invention is integrally molded using a casting liquid, which has the advantages of simple preparation method, better manufacturing cost, environmental friendliness, and suitability for industrial production. Attached Figure Description

[0100] Figure 1 This is a scanning electron microscope (SEM) schematic diagram of the inner surface of the polysulfone hollow filter membrane prepared in Example 2, with a magnification of 100×.

[0101] Figure 2 This is a further magnified scanning electron microscope (SEM) schematic diagram of the inner surface of the polysulfone hollow filter membrane prepared in Example 2, with a magnification of 500×.

[0102] Figure 3 This is a further magnified scanning electron microscope (SEM) schematic diagram of the inner surface of the polysulfone hollow filter membrane prepared in Example 2, with a magnification of 5000×.

[0103] Figure 4 This is a scanning electron microscope (SEM) schematic diagram of the cross-section of the polysulfone hollow filter membrane prepared in Example 2, with a magnification of 500×. Detailed Implementation

[0104] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0105] Example 1

[0106] Step 1: Preparation of casting solution and core solution

[0107] The casting solution comprises the following components by weight: 12 parts of bisphenol A polysulfone with a weight average molecular weight of 60,000, 6 parts of bisphenol A polysulfone with a weight average molecular weight of 140,000, 1 part of sulfonated polyether sulfone with a weight average molecular weight of 30,000, 8 parts of polyethylene glycol with a weight average molecular weight of 30,000 and a molecular weight distribution of 7, and 70 parts of dimethylformamide. The temperature of the casting solution is 40°C.

[0108] The core fluid is composed of dimethylformamide, water and glycerol, wherein the mass content of water in the core fluid is 25%, the mass content of glycerol is 8%, and the temperature of the core fluid is 55℃;

[0109] Step 2: Spinning

[0110] The casting liquid and core liquid prepared in step one are extruded together from a two-hole spinneret to form a molded product with an inner surface and an outer surface.

[0111] Step 3: Phase Separation

[0112] The molded product obtained in step two is immersed in a first coagulation bath at 25°C for 10 seconds, and then immersed in a second coagulation bath at 25°C for 100 seconds to obtain membrane I. During the phase separation process in the first coagulation bath, the filter membrane is simultaneously stretched with a stretching rate of 2%. The first coagulation bath consists of 75% dimethylformamide and 25% water by mass, and the second coagulation bath consists of 20% isopropanol and 80% water by mass.

[0113] Step 4: Washing and Drying

[0114] The biofilm I obtained in step three is washed with pure water and then dried at room temperature to obtain a polysulfone hollow filter membrane.

[0115] Example 2

[0116] Step 1: Preparation of casting solution and core solution

[0117] The casting solution comprises the following components by weight: 10.1 parts polyethersulfone with a weight average molecular weight of 50,000, 5.9 parts polyethersulfone with a weight average molecular weight of 130,000, 2 parts sulfonated polyethersulfone with a weight average molecular weight of 40,000, 13 parts polyvinylpyrrolidone with a weight average molecular weight of 10,000 and a molecular weight distribution of 8, and 65 parts dimethylacetamide. The temperature of the casting solution is 35°C.

[0118] The core fluid is composed of dimethylacetamide, water and glycerol, wherein the mass content of water in the core fluid is 20%, the mass content of glycerol is 9%, and the temperature of the core fluid is 45℃;

[0119] Step 2: Spinning

[0120] The casting liquid and core liquid prepared in step one are extruded together from a two-hole spinneret to form a molded product with an inner surface and an outer surface.

[0121] Step 3: Phase Separation

[0122] The molded product obtained in step two is immersed in a first coagulation bath at 25°C for 15 seconds, and then immersed in a second coagulation bath at 25°C for 150 seconds to obtain a raw film I. During the phase separation process in the first coagulation bath, the filter membrane is stretched with a stretching rate of 3%. The first coagulation bath consists of 85% dimethylacetamide and 15% water by mass, and the second coagulation bath is water.

[0123] Step 4: Washing and Drying

[0124] The biofilm I obtained in step three is washed with pure water and then dried at room temperature to obtain a polysulfone hollow filter membrane.

[0125] Example 3

[0126] Step 1: Preparation of casting solution and core solution

[0127] The casting solution comprises the following components by weight: 9 parts polyphenylsulfone with a weight average molecular weight of 80,000, 5 parts polyphenylsulfone with a weight average molecular weight of 150,000, 3 parts sulfonated polyethersulfone with a weight average molecular weight of 20,000, 16 parts polyvinyl alcohol with a weight average molecular weight of 50,000 and a molecular weight distribution of 8, and 60 parts N-methylpyrrolidone. The temperature of the casting solution is 25°C.

[0128] The core fluid is composed of N-methylpyrrolidone, water and glycerol, wherein the mass content of water in the core fluid is 10%, the mass content of glycerol is 9%, and the temperature of the core fluid is 33℃;

[0129] Step 2: Spinning

[0130] The casting liquid and core liquid prepared in step one are extruded together from a two-hole spinneret to form a molded product with an inner surface and an outer surface.

[0131] Step 3: Phase Separation

[0132] The molded product obtained in step two is immersed in a first coagulation bath at 25°C for 20 seconds, and then immersed in a second coagulation bath at 25°C for 200 seconds to obtain membrane I. During the phase separation process in the first coagulation bath, the filter membrane is stretched with a stretching rate of 4%. The first coagulation bath consists of 90% N-methylpyrrolidone and 10% water by mass, and the second coagulation bath consists of 30% isopropanol and 70% water by mass.

[0133] Step 4: Washing and Drying

[0134] The biofilm I obtained in step three is washed with pure water and then dried at room temperature to obtain a polysulfone hollow filter membrane.

[0135] Example 4

[0136] Step 1: Preparation of casting solution and core solution

[0137] The casting solution comprises the following components by weight: 8 parts polyethersulfone with a weight average molecular weight of 70,000, 4 parts polyphenylsulfone with a weight average molecular weight of 120,000, 2.5 parts sulfonated polyethersulfone with a weight average molecular weight of 50,000, 20 parts polyethyleneimine with a weight average molecular weight of 20,000 and a molecular weight distribution of 7, and 55 parts dimethyl sulfoxide. The temperature of the casting solution is 20°C.

[0138] The core fluid is composed of dimethyl sulfoxide, water and glycerol, wherein the mass content of water in the core fluid is 5%, the mass content of glycerol is 10%, and the temperature of the core fluid is 25℃;

[0139] Step 2: Spinning

[0140] The casting liquid and core liquid prepared in step one are extruded together from a two-hole spinneret to form a molded product with an inner surface and an outer surface.

[0141] Step 3: Phase Separation

[0142] The molded product obtained in step two is immersed in a first coagulation bath at 25°C for 25 seconds, and then immersed in a second coagulation bath at 25°C for 250 seconds to obtain membrane I. During the phase separation process in the first coagulation bath, the filter membrane is simultaneously stretched with a stretching rate of 5%. The first coagulation bath consists of 95% dimethyl sulfoxide and 5% water by mass, and the second coagulation bath consists of 10% isopropanol and 90% water by mass.

[0143] Step 4: Washing and Drying

[0144] The biofilm I obtained in step three is washed with pure water and then dried at room temperature to obtain a polysulfone hollow filter membrane.

[0145] The structure, overall porosity, initial bubble point of IPA, ratio of the complete bubble point of IPA to the initial bubble point of IPA of the filter membrane, tensile strength, water flux, thickness, initial water contact angle of the inner and outer surfaces, roughness of the inner surface, cell density, cell viability, and protein retention rate of the polysulfone hollow fiber membranes prepared in Examples 1-4 were characterized. The specific results are shown below:

[0146] Table 1 - Overall characteristics of the air filter membrane in Examples 1-4

[0147] sample Example 1 Example 2 Example 3 Example 4 Porosity / % 63.4 77.2 79.5 84.2 Thickness / μm 120 145 165 180 IPA full foaming point / psi 20 16 11 8 The ratio of the complete bubble point of IPA to the initial bubble point of IPA in the filter membrane 1.18 1.07 1.28 1.14 Water flux / LMH / psi 807 912 1074 1398 Tensile strength / N / mm2 5.8 5.6 5.0 4.7 Cell density / ×10⁷ cells / mL 12.4 13.1 13.7 14.2 Cell viability / % 96.9 97.2 97.6 98.2 Protein retention rate / % 18.7 17.6 19.7 16.4

[0148] Table 2 - Characteristics of the inner surfaces in Examples 1-4

[0149] sample Example 1 Example 2 Example 3 Example 4 SEM average diameter of the first macropore / μm 5.1 5.7 6.5 7.8 The pore area ratio of the largest hole / % 2.4 7.7 11.8 14.3 SEM average length of the first macropore region (μm) 20.7 25.4 26.8 28.9 SEM average diameter of fine fibers / μm 0.34 0.44 0.67 0.78 SEM average diameter of the first aperture (μm) 0.9 1.1 1.5 1.9 The porosity of the first small hole / % 15.7 28.7 30.3 34.6 SEM average width of clustered fibers / μm 0.8 1.0 1.3 1.5 SEM average short diameter of fluid split orifice / μm 10.7 13.9 25.4 46.2 The ratio of the SEM mean major diameter to the SEM mean minor diameter of the fluid distribution orifice. 1.8 2.4 3.1 3.9 Pore ​​area ratio of fluid diversion orifice / % 3.4 7.1 8.2 8.8 Initial water contact angle / ° 74 68 56 61 Roughness / μm 14.7 9.4 3.2 5.3

[0150] Table 3 - Other features of Examples 1-4

[0151] sample Example 1 Example 2 Example 3 Example 4 Initial water contact angle of outer surface / ° 84 81 62 74 SEM average diameter of the second pore / μm 0.31 0.49 0.61 0.77 The porosity of the second hole is % 31.7 44.8 46.7 48.9 Thickness of the fluid acceleration zone / μm 31.2 57.7 62.1 68.7 SEM closest distance (μm) from the fluid acceleration zone to the inner surface 11.7 22.8 25.4 29.1 SEM average pore size in the fluid acceleration zone (μm) 1.6 2.6 3.1 3.5

[0152] The results above show that the polysulfone-based hollow fiber membranes prepared in Examples 1-4 have excellent tensile strength, not less than 4.5 N / mm². 2 It can be used for long-term perfusion culture; it also has excellent water flux, exceeding 800 LMH / psi, and its cell density can reach 10 × 10⁻⁶ when used for N-stage or N-1 stage perfusion culture. 7 The cell count is above 95% and the cell viability is above 95%. When used for N-stage perfusion culture, the protein retention rate is below 20%.

[0153] The culture method for N-1 level perfusion in this invention is as follows (taking CHO cell culture and γ-ray irradiation sterilization as an example):

[0154] The prepared polysulfone-based hollow fiber membranes were assembled into hollow fiber membrane modules using standard processes and sterilized by gamma irradiation. After sterilization, the membrane modules were thoroughly rinsed, and integrity and pressure holding tests were performed sequentially. Only after passing the tests could the modules be connected to the aseptic culture system. Basic culture medium was added to the sterilized bioreactor and preheated to the target culture temperature. The qualified hollow fiber membrane modules were connected to the bioreactor piping, and the ATF (Alternating Tangential Flow) system and peristaltic pump were started to ensure thorough wetting of the membrane modules. Before formal culture, the operating parameters of the bioreactor were calibrated, and then CHO cell suspension was inoculated at the set initial inoculation density. The feeding program and ATF system were started simultaneously and adjusted to the target perfusion flow rate to formally commence CHO cell perfusion culture. During the culture process, the cell density and cell viability in the reactor were monitored in real time. When the cell density decreased, the culture was stopped, and the maximum detected cell density was taken as the cell density of the prepared polysulfone-based hollow fiber membrane. The cell viability corresponding to this cell density was taken as the cell viability of the prepared polysulfone-based hollow fiber membrane.

[0155] The N-stage perfusion culture method in this invention is as follows (taking CHO cells expressing IgG protein and γ-ray irradiation sterilization process as an example):

[0156] The prepared polysulfone hollow fiber membranes were assembled into hollow fiber membrane modules using standard processes and sterilized by gamma irradiation. After sterilization, the membrane modules were thoroughly rinsed, and integrity and pressure holding tests were performed sequentially. Only after passing the tests could the modules be connected to the aseptic culture system. Basic culture medium was added to the sterilized bioreactor and preheated to the target culture temperature. The qualified hollow fiber membrane modules were connected to the bioreactor piping, and the ATF (Alternating Tangential Flow) system and peristaltic pump were started to ensure thorough wetting of the membrane modules. Before formal culture, the bioreactor's operating parameters were calibrated, and then CHO cell suspension was inoculated at the set initial inoculation density. The feeding program and ATF system were simultaneously started and adjusted to the target perfusion flow rate to formally commence CHO cell perfusion culture. When the cell density and cell viability reached the cooling trigger threshold, the cooling program was executed to allow the CHO cells to enter the stage of efficient expression of the target protein. The timing of harvesting the feed solution was determined according to the established process specifications and real-time cell status. The cell density and cell viability at which the cell density and cell viability reach the cooling trigger threshold are the cell density and cell viability of the prepared polysulfone hollow filter membrane. The protein retention rate at the time of liquid harvest is the protein retention rate of the prepared polysulfone hollow filter membrane.

[0157] The cell density and cell viability of the sample can be obtained by placing the obtained cell solution sample into an automated cell counter.

[0158] The absorbance of the sample from the reactor after the N-stage perfusion was measured on a biochemical analyzer and was A. 原液 The absorbance of the harvested single-protein solution sample was measured on a biochemical analyzer and found to be A. 透过 The protein rejection rate is 1-A 透过 / A 原液 .

[0159] Example 5

[0160] Polysulfone-based hollow fiber membranes were prepared according to the preparation method of Example 2, except that dimethylacetamide in the casting solution in step one was replaced with N-methylpyrrolidone at a mass ratio of 70 parts, the casting solution temperature was 15°C, the core solution contained 10% glycerol and 16% water at a mass ratio, and the core solution temperature was 20°C. The resulting polysulfone-based hollow fiber membrane had a first macropore area ratio of 16.7%, a first micropore area ratio of 40.3%, and a porosity of 86.4%. Compared to Example 2, the tensile strength of the hollow fiber membrane obtained in Example 5 decreased to 3.7 N / mm. 2 The perfusion culture time is slightly shorter (and the corresponding cell density and cell viability are also slightly lower).

[0161] Example 6

[0162] Polysulfone-based hollow fiber membranes were prepared according to the preparation method of Example 2, except that the mass fraction of dimethylacetamide in the casting solution in step one was 63 parts, the temperature of the casting solution was 45°C, the mass content of glycerol in the core solution was 8%, the mass content of water was 25%, and the temperature of the core solution was 55°C. The resulting polysulfone-based hollow fiber membrane had a first macropore pore area fraction of 1.6%, a first micropore pore area fraction of 13.2%, and a porosity of 53.1%. Compared to Example 2, the water flux of the hollow fiber membrane obtained in Example 6 was reduced to 634 LMH / psi, and there were fewer metabolic waste removal channels. When used for N-stage perfusion, the cell density decreased to 8.4 × 10⁻⁶. 7 The cell count / mL decreased to 92.7%, while the protein retention rate increased to 25.4%.

[0163] Example 7

[0164] Polysulfone-based hollow fiber membranes were prepared according to the preparation method of Example 2, except that the stretching in the first coagulation bath in step three was omitted, and the dimethylacetamide in the first coagulation bath in step three was replaced with N-methylpyrrolidone, with an N-methylpyrrolidone mass content of 80% and a water mass content of 20%. The resulting polysulfone-based hollow fiber membrane did not have its first macropores connected into a single macropore region. Compared to Example 2, the water flux of the hollow fiber membrane obtained in Example 7 was reduced to 712 LMH / psi, and the corresponding number of metabolite discharge channels was reduced. When used for N-stage perfusion, the cell density decreased to 9.5 × 10⁻⁶. 7 The cell count / mL decreased, cell viability decreased to 94.3%, and protein retention increased to 31.1%.

[0165] Example 8

[0166] Polysulfone-based hollow fiber membranes were prepared according to the preparation method of Example 2. The difference was that, during the phase separation process in the first coagulation bath of step three, the membrane was simultaneously stretched by a stretching rate of 6%, and the dimethylacetamide in step one was replaced with dimethylformamide, with 66 parts by mass of dimethylformamide. The final polysulfone-based hollow fiber membrane had a SEM average length of 33.2 μm for the first macropore region. Compared to Example 2, the tensile strength of the hollow fiber membrane obtained in Example 8 decreased to 4.2 N / mm. 2 The perfusion culture time is slightly shorter (and the corresponding cell density and cell viability are also slightly lower).

[0167] Example 9

[0168] Polysulfone-based hollow fiber membranes were prepared according to the preparation method of Example 2, except that the casting solution in step one included the following components by weight: 9 parts polyethersulfone with a weight average molecular weight of 50,000, 7 parts polyethersulfone with a weight average molecular weight of 130,000, 2 parts sulfonated polyethersulfone with a weight average molecular weight of 40,000, 13 parts polyethylene glycol with a weight average molecular weight of 10,000 and a molecular weight distribution of 6, and 65 parts dimethylacetamide. The glycerol content in the core solution was 7%. The resulting polysulfone-based hollow fiber membrane did not contain fluid diversion pores. Compared to Example 2, the water flux of the hollow fiber membrane obtained in Example 9 was reduced to 687 LMH / psi, and the corresponding number of metabolite discharge channels was reduced. When used for N-stage perfusion, the cell density was reduced to 8.8 × 10⁻⁶. 7 The cell count / mL decreased to 93.5%, while the protein retention rate increased to 36.4%.

[0169] Example 10

[0170] Polysulfone-based hollow fiber membranes were prepared according to the preparation method of Example 2, except that the casting solution in step one included the following components by weight: 9.8 parts polyethersulfone with a weight average molecular weight of 50,000, 6.2 parts polyethersulfone with a weight average molecular weight of 130,000, 2 parts sulfonated polyethersulfone with a weight average molecular weight of 40,000, 13 parts polyethylene glycol with a weight average molecular weight of 10,000 and a molecular weight distribution of 3, and 65 parts dimethylacetamide. The glycerol content in the core solution was 5%. The resulting polysulfone-based hollow fiber membrane did not contain fluid diversion pores. Compared to Example 2, the water flux of the hollow fiber membrane obtained in Example 10 was reduced to 671 LMH / psi, and the corresponding number of metabolite discharge channels was reduced. When used for N-stage perfusion, the cell density was reduced to 8.5 × 10⁻⁶. 7 The cell count / mL decreased to 93.1%, while the protein retention rate increased to 41.5%.

[0171] Example 11

[0172] Polysulfone-based hollow fiber membranes were prepared according to the preparation method of Example 2. The difference was that the casting solution in step one included the following components by weight: 10.1 parts polyethersulfone with a weight-average molecular weight of 70,000, 5.9 parts polyethersulfone with a weight-average molecular weight of 150,000, 2 parts sulfonated polyethersulfone with a weight-average molecular weight of 40,000, 13 parts polyvinylpyrrolidone with a weight-average molecular weight of 10,000 and a molecular weight distribution of 8, and 65 parts dimethylacetamide. The first coagulation bath in step three consisted of 50% dimethylacetamide and 50% water by weight. The resulting polysulfone-based hollow fiber membrane had a SEM average diameter of 0.26 μm for the second pores, a pore area ratio of 26.3%, and an SEM average pore size of 1.2 μm in the fluid acceleration zone. Compared to Example 2, the water flux of the hollow fiber membrane obtained in Example 11 was reduced to 703 LMH / psi, with fewer metabolic pathways, resulting in a lower cell density of 9.2 × 10⁻⁶ cells when used for N-stage perfusion. 7 The cell count / mL decreased to 94.5%, while the protein retention rate increased to 34.8%.

[0173] Example 12

[0174] Polysulfone-based hollow fiber membranes were prepared according to the preparation method of Example 2, except that the casting solution in step one included the following components by weight: 10.1 parts polyethersulfone with a weight average molecular weight of 60,000, 5.9 parts polyethersulfone with a weight average molecular weight of 120,000, 2 parts sulfonated polyethersulfone with a weight average molecular weight of 40,000, 13 parts polyvinylpyrrolidone with a weight average molecular weight of 30,000 and a molecular weight distribution of 8, and 65 parts dimethylacetamide. The first coagulation bath in step three was dimethylacetamide. The SEM average diameter of the second pore of the resulting polysulfone-based hollow fiber membrane was 0.92 μm, the pore area ratio of the second pore was 57.4%, and the SEM average pore size in the fluid acceleration zone was 4.1 μm. Compared to Example 2, the tensile strength of the hollow fiber membrane obtained in Example 12 decreased to 4.0 N / mm. 2 The perfusion culture time is slightly shorter (and the corresponding cell density and cell viability are also slightly lower).

[0175] Example 13

[0176] Polysulfone-based hollow fiber membranes were prepared according to the preparation method of Example 2. The difference was that the casting solution in step one included the following components by weight: 11.4 parts polyethersulfone with a weight-average molecular weight of 50,000, 6.6 parts polyethersulfone with a weight-average molecular weight of 130,000, 13 parts polyvinylpyrrolidone with a weight-average molecular weight of 10,000 and a molecular weight distribution of 8, and 65 parts dimethylacetamide. The resulting polysulfone-based hollow fiber membrane had an initial water contact angle of 89°C on its inner surface and 97°C on its outer surface. The average SEM width of the clustered fibers was 0.7 μm. Compared to Example 2, the hollow fiber membrane obtained in Example 13 had slightly lower hydrophilicity, and its cell density decreased to 5.6 × 10⁻¹⁰ when used for N-1 stage perfusion. 7 The cell count / mL decreased, and the cell viability dropped to 91.3%.

[0177] Example 14

[0178] Polysulfone-based hollow fiber membranes were prepared according to the preparation method of Example 2. The difference was that the casting solution in step one included the following components by weight: 12 parts polyethersulfone with a weight-average molecular weight of 50,000, 6 parts polyethersulfone with a weight-average molecular weight of 130,000, 13 parts polyvinylpyrrolidone with a weight-average molecular weight of 10,000 and a molecular weight distribution of 8, and 65 parts dimethylacetamide. The final polysulfone-based hollow fiber membrane had an average SEM width of 1.8 μm. Compared to Example 2, the hollow fiber membrane obtained in Example 14 showed a cell density reduction to 5.4 × 10⁻¹⁰ when used for N-1 stage perfusion. 7 The cell count / mL decreased, and the cell viability dropped to 90.8%.

[0179] The results from Examples 5-14 show that when the pore area ratio of the first macropore, or the pore area ratio of the first micropore, or the porosity of the filter membrane, or the SEM average length of the first macropore region, or the SEM average diameter of the second pore, or the pore area ratio of the second pore, or the SEM average pore diameter of the fluid acceleration zone, or the SEM average width of the clustered fibers, or the initial water contact angle of the inner surface, or the initial water contact angle of the outer surface of the polysulfone hollow filter membrane are not within the preferred range, or the first macropores on the inner surface are not connected to form the first macropore region, or the inner surface does not contain fluid diversion pores, the resulting hollow filter membrane has acceptable tensile strength, can be used in perfusion culture, and has acceptable water flux, with a cell density of 5×10⁻¹⁰ when used for N-1 stage perfusion or for N-stage perfusion. 7 The cell count is above 90% and the cell viability is above 90%; when used for N-level perfusion, the protein rejection rate is below 50%.

[0180] Comparative Example 1

[0181] Polysulfone-based hollow fiber membranes were prepared according to the preparation method of Example 2. The difference was that the casting solution in step one included the following components by weight: 18 parts polyethersulfone with a weight average molecular weight of 50,000, 13 parts polyvinylpyrrolidone with a weight average molecular weight of 8,000 and a molecular weight distribution of 2, and 65 parts dimethylacetamide. The core solution consisted of water and dimethylacetamide, with water comprising 35% by weight. The resulting polysulfone-based hollow fiber membrane did not contain a first macropore or clustered fibers. Compared to Example 2, the hollow fiber membrane obtained in Comparative Example 1 showed a cell density reduction to 1.2 × 10⁻¹⁰ when used for N-1 stage perfusion culture. 7 The cell count / mL decreased, and the cell viability dropped to 82.1%.

[0182] Comparative Example 2

[0183] Polysulfone-based hollow fiber membranes were prepared according to the preparation method of Example 2, except that the casting solution in step one included the following components by weight: 18 parts polyethersulfone with a weight average molecular weight of 130,000, 13 parts polyvinylpyrrolidone with a weight average molecular weight of 55,000 and a molecular weight distribution of 2, and 65 parts dimethylacetamide. The core solution consisted of glycerol and water, with glycerol accounting for 8% of the total mass. The resulting polysulfone-based hollow fiber membrane did not contain the first pore or clustered fibers. Compared to Example 2, the tensile strength of the hollow fiber membrane obtained in Comparative Example 2 decreased to 2.1 N / mm. 2 It cannot be used for perfusion culture.

[0184] Comparative Example 3

[0185] Polysulfone-based hollow fiber membranes were prepared according to the preparation method of Example 2, except that the casting solution in step one included the following components by weight: 12.8 parts polyethersulfone with a weight average molecular weight of 50,000, 5.2 parts polyethersulfone with a weight average molecular weight of 130,000, 13 parts polyvinylpyrrolidone with a weight average molecular weight of 10,000 and a molecular weight distribution of 8, and 65 parts dimethylacetamide. The core solution temperature was 60°C. The SEM average width of the clustered fibers in the resulting polysulfone-based hollow fiber membrane was 0.5 μm. Compared to Example 2, the tensile strength of the hollow fiber membrane obtained in Comparative Example 3 decreased to 2.6 N / mm. 2 It cannot be used for perfusion culture.

[0186] Comparative Example 4

[0187] Polysulfone-based hollow fiber membranes were prepared according to the preparation method of Example 2. The difference was that the casting solution in step one included the following components by weight: 9.8 parts polyethersulfone with a weight-average molecular weight of 50,000, 8.2 parts polyethersulfone with a weight-average molecular weight of 130,000, 13 parts polyvinylpyrrolidone with a weight-average molecular weight of 10,000 and a molecular weight distribution of 8, and 65 parts dimethylacetamide. The core solution temperature was 15°C. The final polysulfone-based hollow fiber membrane had an average SEM width of 2.3 μm. Compared to Example 2, the hollow fiber membrane obtained in Comparative Example 4 showed a cell density reduction to 1.6 × 10⁻⁶ when used for N-1 stage perfusion culture. 7 The cell count / mL decreased, and the cell viability dropped to 83.3%.

[0188] The results of Comparative Examples 1-4 show that when the prepared polysulfone hollow fiber membranes do not contain a first macropore, or do not contain a first micropore, or the average SEM width of the clustered fibers is too small, or the average SEM width of the clustered fibers is too large, the prepared hollow fiber membranes can either not be used for perfusion culture, or when used for N-1 stage perfusion culture, the cell density is less than 3 × 10⁻⁶. 7 When the number of cells / mL is below 90%, the cell viability also decreases to below 90%, resulting in low industrial practical value.

[0189] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A polysulfone-based hollow fiber membrane, comprising a body, one side of which is an inner surface facing the inner cavity, and the other side of which is an outer surface, characterized in that: The inner surface includes a plurality of first large holes and a plurality of first small holes, wherein the SEM average diameter of the first large holes is 5μm-8μm; The average diameter of the first aperture on SEM is 0.8 μm-2 μm; The inner surface also includes a plurality of clustered fibers for forming the first large pore and the first small pore; The average width of the clustered fibers, as measured by SEM, is 0.7 μm to 1.8 μm.

2. The polysulfone-based hollow fiber membrane according to claim 1, characterized in that: The initial water contact angle of the inner surface is 55°-75°; The initial water contact angle of the outer surface is 60°-85°; The average width of the clustered fibers, as measured by SEM, is 0.8 μm to 1.5 μm.

3. The polysulfone-based hollow fiber membrane according to claim 1 or 2, characterized in that: The roughness of the inner surface is 3μm-15μm; The pore area ratio of the first large hole is 2%-15%.

4. The polysulfone-based hollow fiber membrane according to claim 1 or 2, characterized in that: Some adjacent first macropores are connected by fine fibers to form a long strip-shaped first macropore region, and the SEM average length of the first macropore region is no greater than 30 μm. The average diameter of the fine fibers, as measured by SEM, is 0.3 μm to 0.8 μm.

5. The polysulfone-based hollow fiber membrane according to claim 1 or 2, characterized in that: The pore area ratio of the first small hole is 15%-35%; The porosity of the filter membrane is 60%-85%.

6. The polysulfone-based hollow fiber membrane according to claim 1 or 2, characterized in that: The inner surface also includes a plurality of fluid diversion holes, which are elongated and perpendicular to the length direction of the filter membrane. The average minor axis of the fluid diversion holes, as measured by SEM, is 10 μm-50 μm. The ratio of the SEM average major diameter to the SEM average minor diameter of the fluid diversion orifice is 1.5-4. The pore area ratio of the fluid diversion orifice is no greater than 10%.

7. The polysulfone-based hollow fiber membrane according to claim 1 or 2, characterized in that: The complete bubble point of the IPA of the filter membrane is 8 psi-20 psi; The ratio of the complete bubble point of the IPA of the filter membrane to the initial bubble point of the IPA of the filter membrane is 1.05-1.

30.

8. The polysulfone-based hollow fiber membrane according to claim 1 or 2, characterized in that: The pore size of the filter membrane increases and then decreases along the thickness direction from the inner surface to the outer surface. The transition region where the pore size increases and then decreases along the thickness direction from the inner surface to the outer surface is the fluid acceleration region, and the thickness of the fluid acceleration region is 30μm-70μm. The closest distance between the fluid acceleration zone and the SEM image of the inner surface is 10μm-30μm; The average SEM pore size of the fluid acceleration zone is 1.5 μm-3.5 μm.

9. The polysulfone-based hollow fiber membrane according to claim 1 or 2, characterized in that: The outer surface includes a plurality of second holes, the SEM average diameter of which is 0.3μm-0.8μm; The area ratio of the second hole is 30%-50%.

10. The polysulfone-based hollow fiber membrane according to claim 1, characterized in that: The filter membrane, when used for cell perfusion culture, achieves a cell density of no less than 3*102. 7 cells / mL, cell viability not less than 90%; The tensile strength of the filter membrane is not less than 3.5 N / mm. 2 ; The water flux of the filter membrane is greater than 600 LMH / psi; The thickness of the filter membrane is 120μm-180μm.

11. A method for preparing a polysulfone-based hollow fiber membrane as described in any one of claims 1-10, characterized in that: Includes the following steps: Step 1: Preparation of casting solution and core solution: The casting solution comprises the following components by weight: 12-18 parts of polysulfone, 8-20 parts of pore-forming agent, and 55-70 parts of first organic solvent; The core fluid is composed of a second organic solvent, water, and glycerol, wherein the water content in the core fluid is 5%-25% by mass, and the glycerol content is 5%-10% by mass. The polysulfone-based material includes polysulfone A with a weight-average molecular weight of 50,000-80,000 and polysulfone B with a weight-average molecular weight of 120,000-150,000, wherein the mass ratio of polysulfone B to polysulfone A is 0.5-0.

8. The porogen is one or more of polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol and polyethyleneimine, the molecular weight distribution of the porogen is 3-8, and the weight average molecular weight of the porogen is 10,000-50,000. The temperature of the core liquid is 5°C-15°C higher than the temperature of the casting liquid; Step 2: Spinning: The casting solution and core solution prepared in step one are extruded together from the spinneret to form a molded product with an inner surface and an outer surface. Step 3: Phase Separation: The molded product obtained in step two is immersed in a coagulation bath to perform complete phase separation and form a biofilm I. Step 4: Cleaning and drying: The biofilm I obtained in step three is washed with pure water and then dried to obtain a polysulfone-based hollow fiber membrane.

12. The method for preparing a polysulfone-based hollow fiber membrane according to claim 11, characterized in that: The casting solution further comprises 1-3 parts by weight of sulfonated polyether sulfone; The weight-average molecular weight of the sulfonated polyether sulfone is 20,000 to 50,000. The polysulfone is at least one of bisphenol A polysulfone, polyethersulfone, and polyphenylsulfone.

13. The method for preparing a polysulfone-based hollow fiber membrane according to claim 11, characterized in that: The coagulation bath in step three includes a first coagulation bath and a second coagulation bath. During the phase separation process in the first coagulation bath, the filter membrane is simultaneously stretched, and the stretching rate is 2%-5%. The temperature of the casting solution is 20℃-40℃.

14. The method for preparing a polysulfone-based hollow fiber membrane according to claim 13, characterized in that: The first coagulation bath consists of a third organic solvent and water, wherein the mass content of the third organic solvent is 75%-95%; The second coagulation bath consists of isopropanol and water, wherein the mass content of isopropanol is 0%-30%; The time for the molded article in the first coagulation bath is 10s-25s, and the time in the second coagulation bath is 100s-250s; The first organic solvent, the second organic solvent, and the third organic solvent are all at least one of dimethylformamide, dimethylacetamide, N-methylpyrrolidone, and dimethyl sulfoxide.

15. An application of a polysulfone-based hollow fiber membrane as described in any one of claims 1-10, characterized in that: The filter membrane is used for: (a) Cell expansion; (b) Harvest of monoclonal antibodies; (c) Harvesting of recombinant proteins; (d) The harvest of vaccines; (e) Harvesting of viral vectors.

Images