A tangential flow ultrafiltration device and filtration method

Abstract

The present application relates to a kind of tangential flow ultrafiltration device and filtration method.The device includes wash bottle, feed liquid circulating bottle, three-way valve, peristaltic pump, pressure gauge, electronic scale, hollow fiber filter cartridge and waste liquid bottle and other components, these components are connected into a flow path system by silica gel pipe.The device uses innovative flow path design, allow sample to filter while washing, also can independently flush the system.The flow path system includes enrichment, wash filter, cleaning and backflush four flow paths, can be adjusted to adapt to different filtration requirements according to needs.The innovation of the present application is that it ingeniously combines a variety of flow path strategies, and the high flexibility of flow path design allows flexible adjustment and change of different flow path configurations according to needs.This provides an efficient and accurate solution for processing complex samples.At the same time, the present application also provides an effective filter cartridge regeneration method, further enhances the sustainability and service life of the device.Therefore, the present application has wide application prospect in biomedical and bioengineering fields.

Description

Technical Field

[0001] This invention relates to a batch separation and enrichment device, and more particularly to a tangential flow ultrafiltration device with an innovative flow path design, and its application in the batch preparation of extracellular vesicles from complex cell supernatants. Background Technology

[0002] Membrane separation technology utilizes a pressure difference as the driving force. By creating a pressure difference across a synthetic polymer membrane, components smaller than the membrane pore size pass through the pores, achieving separation. Simultaneously, components larger than the pore size are retained by the membrane, thus achieving separation. Based on the membrane's pore size or molecular weight cutoff, membrane separation technology can be categorized into microfiltration, ultrafiltration, nanofiltration, and reverse osmosis membranes. Ultrafiltration membranes typically have pore sizes between 2-50 nm and can be used to retain substances with molecular weight cutoffs ranging from 1-1000 kilodaltons (kDa), including but not limited to the separation of large molecules such as viruses, enzymes, proteins, and polysaccharides. Extracellular vesicles (EVs) are nanoscale lipid vesicles secreted by cells into the extracellular matrix, with particle sizes typically ranging from 30 nm to 1000 nm. Therefore, ultrafiltration technology has broad application potential in the separation of extracellular vesicles.

[0003] Currently, methods for isolating extracellular vesicles mainly include differential centrifugation, density gradient centrifugation, immunoprecipitation, polymer precipitation, size exclusion chromatography, and asymmetric flow field separation. While these methods have their own advantages and limitations in different aspects, they generally suffer from limited yield, long processing times, and low efficiency. Furthermore, these methods are typically only suitable for the isolation and purification of small samples, and cannot meet the needs of batch processing of large numbers of samples. In clinical treatment, the demand for EVs is enormous; the median dose for a 50 kg adult is approximately 1.5 × 10⁻⁶. 10 Therefore, there is an urgent need to develop a novel device or method for the batch preparation, separation, and enrichment of EVs.

[0004] Ultrafiltration membrane-based filtration methods are widely used for batch separation of target products. In traditional vertical filtration, the liquid flows from top to bottom through the membrane, easily forming a high-concentration gel and particle layer on the membrane surface, thus clogging the membrane pores. In contrast, tangential flow filtration generates transmembrane pressure by flowing the liquid tangentially across the membrane surface, pushing small particles and solution against the membrane while simultaneously circulating retained substances within the system, achieving sample concentration. Throughout the process, the liquid continuously flows across the membrane surface at a constant speed, simultaneously filtration and scouring the membrane surface, slowing down the rate of pore clogging and significantly improving filtration efficiency. Tangential flow filtration can not only process up to tens of liters of feed solution at a time using simple linear scale-up techniques, but its process flexibility also makes it the preferred solution for standardized batch production.

[0005] Currently, several companies produce tangential flow equipment, including international companies such as Pall, Repligen, Sartorius, and Cytiva, as well as domestic companies such as Huakan Bio and Huixin Bio. While commercial tangential flow equipment boasts high automation and versatility, its high price, relatively fixed design, and lack of flexibility make it less effective. Furthermore, current commercial tangential flow filtration equipment still faces the problem of membrane clogging, especially when processing complex samples such as cell supernatants containing serum, blood samples, and milk. These samples contain a large number of small protein impurities, causing a rapid decrease in membrane flux and a significant reduction in the removal rate of these impurities. Therefore, there is a need to develop a new, highly efficient separation device suitable for processing complex samples that can effectively avoid membrane flux decline and pore clogging. Summary of the Invention

[0006] The present invention aims to solve the problem of easy clogging of filter membranes in existing tangential flow filtration equipment when processing complex samples, and provides a novel flow path ultrafiltration device that can effectively slow down the rate of membrane pore clogging, maintain the water flux of the filter membrane, and thus improve the efficiency of particle separation in the feed solution.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A tangential flow ultrafiltration device includes a feed circulation bottle, the outlet of which is connected to a first three-way valve. One branch of the first three-way valve is connected to a washing liquid bottle, and the other branch is connected to a first peristaltic pump. The first peristaltic pump is connected to a second three-way valve. Two branches of the second three-way valve are respectively connected to the inner and outer sides of the filter membrane of a hollow fiber filter element. A second pressure reducing valve is also provided on the outer side of the filter membrane of the hollow fiber filter element, and the second pressure reducing valve is connected to a second waste liquid bottle. The outlet of the inner side of the filter membrane of the hollow fiber filter element is connected to the first pressure reducing valve, which is then connected to a third three-way valve. One branch of the third three-way valve is connected to the first waste liquid bottle, and the other branch is connected to the feed circulation bottle.

[0009] Furthermore, a first pressure gauge is provided between the first peristaltic pump and the second three-way valve; a third pressure gauge is provided between the second pressure reducing valve and the second waste liquid bottle; and a second pressure gauge is provided between the first pressure reducing valve and the third three-way valve.

[0010] The present invention also provides the application of the aforementioned tangential flow ultrafiltration device in the enrichment and purification of biological samples.

[0011] Furthermore, an example of the biological sample is extracellular vesicles in complex cell supernatant.

[0012] This invention also provides a method for enriching and purifying biological samples, comprising the following steps:

[0013] Step 1, enrichment: Add the sample to be processed into the feed liquid circulation tank, turn on the peristaltic pump, and pump the sample from the feed liquid circulation tank into the hollow fiber filter for filtration. The feed liquid enters the feed liquid circulation bottle through the first pressure reducing valve, and the filtrate enters the second waste liquid bottle through the second pressure reducing valve. This cycle is repeated to separate and enrich the sample.

[0014] Step 2, washing and filtering simultaneously: When the mass change per unit time in the second waste liquid bottle gradually decreases, the washing liquid in the washing liquid bottle and the material liquid circulation bottle are simultaneously pumped into the hollow fiber filter element through the first three-way valve.

[0015] Step 3, Filter Cartridge Cleaning: In the later stage of filtration, one side of the feed liquid circulation tank is closed, and the other side of the washing liquid bottle is opened. First, the inner membrane surface of the filter cartridge is cleaned: the washing liquid in the washing liquid bottle enters from the inside of the hollow fiber filter membrane, and the washing liquid coming out from the inside of the hollow fiber filter membrane enters the first waste liquid bottle through the first pressure reducing valve. Second, the membrane pores of the filter cartridge are cleaned: the second pressure reducing valve is closed, and the washing liquid in the washing liquid bottle enters from the outside of the hollow fiber filter membrane, and the washing liquid flowing out from the inside of the hollow fiber filter membrane enters the first waste liquid bottle through the first pressure reducing valve.

[0016] Step 4: After restoring the filter cartridge flow rate, switch the flow path to the enrichment flow path to wash, filter, and enrich the solution in the feed circulation bottle;

[0017] Step 5: After separation and enrichment are completed, collect the solution in the feed circulation bottle.

[0018] Furthermore, after the filter element has been used, when the membrane flux decreases to less than 70% of the original flux, the filter membrane is regenerated.

[0019] Furthermore, the membrane regeneration step includes:

[0020] (1) Adjust the first three-way valve to open the liquid path on the side of the washing liquid bottle, close the second pressure reducing valve, pump in 2% Triton X-100, fill the entire system and the inner and outer spaces of the fiber with Triton X-100, and incubate at room temperature for 30 minutes;

[0021] (2) Adjust the second three-way valve to open the liquid path outside the filter membrane space, and open the second pressure reducing valve to allow 100 mL / 0.1 cm⁻¹ solution to flow. 2 The amount of cleaning water pumped into the membrane area is used to rinse and remove impurity particles from the space outside the filter membrane.

[0022] (3) Adjust the second three-way valve to open the liquid path within the filter membrane space, and close the second pressure reducing valve to allow 250 mL / 0.1 cm⁻¹ solution to be applied. 2 The amount of cleaning water pumped into the membrane area is used to rinse and remove impurity particles from the space inside the filter membrane.

[0023] (4) Adjust the flow path to the backwash flow path, at 1L / 0.1cm. 2 The membrane area is pumped into deionized water to backwash the system;

[0024] (5) Adjust the flow path to the cleaning flow path, using 500 mL / 0.1 cm 2 The membrane area is pumped into deionized water to perform a forward flush of the system;

[0025] (6) Repeat the above operation 2-3 times until the filter cartridge flow rate is restored to more than 90% of the original flow rate.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] To overcome the problem of tangential flow filtration devices easily clogging the filter membrane when processing complex samples, this invention uses multiple three-way valves to control the flow direction of the liquid path and sets up enrichment flow path, washing flow path, rinsing flow path and backflushing flow path according to different functions.

[0028] The function of the enrichment flow path is to separate and enrich EVs in the cell supernatant. Under the pressure of a peristaltic pump, the cell supernatant is filtered from bottom to top through a hollow fiber filter cartridge. During this process, particles smaller than the membrane pore size and the solution enter the second waste bottle, while particles larger than the membrane pore size flow back to the feed circulation bottle through the upper port of the filter cartridge under tangential force. This process continuously concentrates and enriches the cell supernatant, filtering out most of the glucose, amino acids, peptides, nucleic acids, and some small proteins.

[0029] The function of the washing and filtration path is to process proteins in complex cell supernatants. Even with tangential flow filtration, the enrichment factor of the solution increases over time, leading to an increase in the concentration of circulating proteins and the formation of a gel layer on the inner surface of the filter membrane, which can clog the membrane pores. Therefore, this invention adds a washing bottle before the feed circulation bottle to hold PBS filtered through a 0.22μm membrane. By adjusting the first three-way valve, the sample solution and PBS are simultaneously pumped into the hollow fiber filter cartridge, allowing for simultaneous washing and filtration, which improves filtration efficiency while slowing down the rate of membrane pore clogging. This flow path design is inspired by washing clothes; running water is significantly more efficient than static water.

[0030] The purpose of the cleaning flow path is to rinse the gel layer that has already formed inside the membrane. When the gel layer has formed, even a wash-and-filter approach cannot effectively remove impurities adhering to the inner surface of the membrane. In this case, by adjusting the first three-way valve, only clean PBS is pumped in to rinse the impurities on the inner surface of the membrane, and by adjusting the third three-way valve, the waste liquid from rinsing the filter cartridge is collected in the first waste liquid bottle. This effectively removes impurities from the inner surface of the membrane and avoids ineffective dilution of the sample.

[0031] The backflushing flow path addresses the issue of impurity particles embedded within the membrane pores. Building upon the cleaning flow path, fresh PBS is pumped into the space outside the filter membrane by adjusting the second three-way valve and closing the second pressure-reducing valve. Under pressure, the solution is forced from the external space into the internal space of the filter membrane, simultaneously carrying away impurity particles from the pores. This design effectively clears membrane clogging, thereby increasing the filter membrane flux to some extent.

[0032] To overcome the problem of membrane clogging, this invention designs the aforementioned flow path, providing a targeted solution. From avoiding membrane clogging to mitigating it, and then to restoring membrane pore flux, this invention offers multiple strategies, providing a more powerful solution for the batch preparation of EVs in complex samples.

[0033] Furthermore, because the pores of the membrane are not straight-through and contain many irregular microstructures, some small proteins become trapped within these microstructures, making them difficult to remove effectively by both forward and reverse flushing. Therefore, chemical treatment to restore membrane flux is a better option. This invention explores the components in the cell supernatant and designs an effective filter cartridge regeneration method. By pumping 2% Triton X-100 into the inner and outer spaces of the fiber and incubating at room temperature for 30 minutes, repeating this process three times, followed by rinsing with sufficient deionized water, the water flux of the filter cartridge can be effectively restored. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0035] Figure 1 Schematic diagram of existing commercial tangential flow filtration equipment

[0036] Figure 2 Schematic diagram of the pressure exerted on the solute during tangential flow filtration.

[0037] Figure 3 Schematic diagram of the tangential flow ultrafiltration device of the present invention

[0038] Figure 4 Schematic diagram of the enrichment flow path of the tangential flow ultrafiltration device of the present invention

[0039] Figure 5 Schematic diagram of the washing flow path of the tangential flow ultrafiltration device of the present invention

[0040] Figure 6 Schematic diagram of the cleaning flow path of the tangential flow ultrafiltration device of the present invention

[0041] Figure 7 Schematic diagram of the backwash flow path of the tangential flow ultrafiltration device of the present invention

[0042] In the schematic diagram of the enrichment, filtration, cleaning and backflushing flow paths, the arrows represent the direction of liquid flow.

[0043] The labels in the diagram represent:

[0044] 1-Washing solution bottle; 2-Supply circulation bottle; 3-First three-way valve; 4-First peristaltic pump; 5-First pressure gauge; 6-Second three-way valve; 7-Hollow fiber filter element; 8-First pressure reducing valve; 9-Second pressure gauge; 10-Third three-way valve; 11-First waste liquid bottle; 12-Second pressure reducing valve; 13-Third pressure gauge; 14-Second waste liquid bottle; 15-Second peristaltic pump;

[0045] These accompanying drawings are used to illustrate the different parts and flow paths of the invention in more detail and to help the reader better understand the embodiments of the invention. The reference numerals in the drawings denote different components and elements for reference in the description. These illustrations will help those skilled in the art understand the structure and function of the invention. Detailed Implementation

[0046] To more clearly describe the objectives, technical solutions, and advantages of the present invention, a detailed and complete description will be provided below with reference to the accompanying drawings of the embodiments of the present invention. It should be understood that the described embodiments are only a part of the examples of the present invention, and not all of them. Based on these examples, those skilled in the art can obtain other embodiments without creative effort, and these embodiments are also included within the protection scope of the present invention.

[0047] Comparative Example 1

[0048] like Figure 1 As shown, existing commercial tangential flow filtration equipment typically consists of the following components: a hollow fiber filter element 7, whose inlet is connected to a feed circulation bottle 2 via a first peristaltic pump 4, and whose outlet is connected back to the feed circulation bottle 2 via a first pressure reducing valve 8; waste liquid enters a second waste liquid bottle 14; and a washing liquid bottle 1 is connected to the feed circulation bottle 2 via a second peristaltic pump 15. A first pressure gauge 5 is provided between the first peristaltic pump 4 and the hollow fiber filter element 7; a second pressure gauge 9 is provided between the first pressure reducing valve 8 and the feed circulation bottle 2; and a third pressure gauge 13 is provided between the filtrate outlet of the hollow fiber filter element 7 and the second waste liquid bottle 14.

[0049] The cell supernatant in the feed circulation bottle 2, under the action of the first peristaltic pump 4, passes through the first pressure gauge 5 and enters the hollow fiber filter element 7 from bottom to top for separation and enrichment. For example... Figure 2 As shown, particles within the filter element are simultaneously subjected to tangential forces parallel to the filter membrane and normal forces perpendicular to it. The tangential force helps reduce particle accumulation on the membrane's inner surface, while the normal force promotes the permeation of solvent and particles smaller than the membrane pore size. Tangential flow filtration significantly slows down the accumulation rate of impurity particles on the membrane's inner surface. To better remove impurities from samples, most commercial tangential flow devices inject fresh washing solution into the feed circulation bottle 2 at a flow rate matching the rate at which solvent is removed from the sample. This flow path structure is currently the main design scheme for commercial tangential flow devices and has a certain degree of practicality. However, when processing more complex samples, even with the tangential force, a large number of particles will still accumulate on the membrane's inner surface over time, forming a gel layer. The concentration difference between the gel layer and the central flow causes back diffusion of particles, thus drastically reducing the filter element's filtration efficiency.

[0050] Example 1

[0051] The structure of the tangential flow ultrafiltration device in this embodiment is as follows: Figure 3 As shown.

[0052] The outlet of the feed circulation bottle 2 is connected to a first three-way valve 3. One branch of the first three-way valve 3 is connected to the washing liquid bottle 1, and the other branch is connected to a first peristaltic pump 4. The first peristaltic pump 4 is connected to a second three-way valve 6. A first pressure gauge 5 is installed between the first peristaltic pump 4 and the second three-way valve 6. The two branches of the second three-way valve 6 are respectively connected to the inner and outer sides of the filter membrane of the hollow fiber filter element 7. A second pressure reducing valve 12 is also installed on the outer side of the filter membrane of the hollow fiber filter element 7. The second pressure reducing valve 12 is connected to a second waste liquid bottle 14. A third pressure gauge 13 is installed between the second pressure reducing valve 12 and the second waste liquid bottle 14.

[0053] The liquid circulation outlet of the hollow fiber filter element 7 is connected to a first pressure reducing valve 8, which is connected to a third three-way valve 10. A second pressure gauge 9 is provided between the first pressure reducing valve 8 and the third three-way valve 10. One branch of the third three-way valve 10 is connected to a first waste liquid bottle 11, and the other branch is connected to a liquid circulation bottle 2.

[0054] This invention connects the washing solution bottle 1 and the feed solution circulation bottle 2 directly to the first peristaltic pump 4 via a first three-way valve 3. By adjusting the first three-way valve 3, the washing solution and sample can be pumped into the hollow fiber filter element simultaneously or separately. This flow path design not only enables simultaneous sample washing and filtration but also allows for independent rinsing of the system.

[0055] Both the wash bottle 1 and the feed circulation bottle 2 are custom-designed, with a dead volume of 3 mL and a 3.2 mm inner diameter threaded interface at the bottom, connecting to a 3.2 mm inner diameter silicone tube via a Luer adapter. The wash bottle primarily holds PBS filtered through a 0.22 μm membrane. Its main functions are threefold: first, to dilute the concentrated feed solution in the feed circulation bottle, slowing the accumulation of impurities on the membrane surface; second, to flush away impurities accumulated on the inner surface of the hollow fiber membrane, preventing pore blockage; and third, to provide fluid impact force for the backflushing flow path, clearing blocked membrane pores. The feed circulation bottle holds the feed solution to be filtered and collects the final product.

[0056] The three-way valve is a disposable medical sterile valve with Luer male connectors at both ends and a Luer female connector at one end. Its main function is to control the flow direction of fluid. There are three three-way valves in the system, which are used to regulate the flow direction of the fluid at the sample inlet, hollow fiber filter inlet, and reflux port, respectively.

[0057] The peristaltic pump applies pressure to the fluid in a non-contact manner, propelling it into the hollow fiber filter element for filtration. Combined with the first three-way valve, the peristaltic pump has three main functions: first, it pumps the sample solution from the feed circulation bottle into the hollow fiber filter element for tangential flow filtration; second, it simultaneously pumps in the sample solution and washing solution to wash the EVs and filter out impurities in the sample solution; and third, it pumps in only the washing solution to rinse the inner surface of the hollow fiber filter membrane and impurities within the membrane pores.

[0058] The pressure gauges have a range of 0 to 87 psi and an accuracy of 0.5% of the maximum range. Specifically, the first pressure gauge (5) measures the pressure in the flow path before the hollow fiber filter element enters, the second pressure gauge (9) measures the pressure in the return line, and the third pressure gauge (13) measures the pressure in the filtrate line. By monitoring changes in the line pressure, it is ensured that the hollow fiber filter membrane is within a safe pressure range (<30 psi), thus preventing damage to the hollow fiber filter membrane due to excessive pressure.

[0059] The electronic scales have an accuracy of ±0.1g, and there are two scales in the system. Scale 1 is used to monitor the liquid weight in the circulating storage tank in real time, while scale 2 is used to monitor the liquid weight of the filtrate in the second waste bottle in real time. By monitoring these weight changes, it can be determined whether the flux of the hollow fiber filter element has changed and whether the membrane pores have become clogged.

[0060] The hollow fiber filter element used in this device is Repligen's D02-E500-05-N. The filter element consists of 36 hollow fiber membranes with a pore size of 500 kDa, made of modified polyethersulfone, encased in a plastic shell. The membrane area is 115 cm². 2 The maximum pressure it can withstand is 30 psi, and the dead volume of the filter cartridge is 2 mL. Liquid flows upwards along the inner surface of the membrane. Particles larger than the membrane pore size cannot pass through the pores and flow back to the reservoir through the outlet at the top of the filter cartridge; particles smaller than the pore size pass through the pores and flow out through the outlet on the side of the filter cartridge. Through circulating filtration, EVs in the cell supernatant can be rapidly enriched, while glucose, amino acids, peptides, nucleic acids, and some small proteins are removed from the supernatant, reducing sample complexity.

[0061] The waste liquid bottle is a 500mL blue-mouth glass bottle. The second waste liquid bottle 14 is used to collect the filtrate filtered by the filter element, while the first waste liquid bottle 11 is used to collect impurities from rinsing the inner surface of the hollow fiber filter membrane and the waste liquid discharged from the backwash flow path. The main function of the added first waste liquid bottle is to prevent the solution in the feed liquid circulation tank from being ineffectively diluted.

[0062] During assembly, according to Figure 3 The schematic diagram shows the setup of a tangential flow ultrafiltration device. The various components are connected using silicone tubing with Luer connectors, while the tank and pressure gauge are connected to the silicone tubing via external threaded adapters. After the device is assembled, the pressure holding method is used to test the overall system's airtightness and the integrity of the hollow fiber filter element.

[0063] The specific steps for the device airtightness test are as follows:

[0064] (1) Adjust the first three-way valve 3 to open the liquid path on one side of the liquid circulation bottle 2, wet the components, and ensure that the entire system is filled with liquid solution, especially the space inside and outside the fiber. Then, drain the liquid in the circulation path and close the second pressure reducing valve 12.

[0065] (2) Close the first pressure reducing valve 8, start the pump, and continuously inject air into the inlet to raise the first pressure gauge 5 to 10 psi, then stop pumping. Next, close the first three-way valve 3 to keep the hollow fiber membrane cavity under positive pressure.

[0066] (3) Monitor pressure changes over time. If the pressure decreases at a rate exceeding 0.5 psi / min / (m²), 2 (Membrane area) There may be a leak in the system piping. Please check and tighten the piping, then repressurize to 10 psi and check for pressure drop again until it meets requirements.

[0067] The specific steps for testing the airtightness of hollow fiber filter elements are as follows:

[0068] (1) Adjust the first three-way valve 3 to open the liquid path on one side of the liquid circulation bottle 2, wet the components, and drain the liquid in the circulation path. Place the pipe at the permeate end below the liquid surface to ensure that the space outside the fiber membrane is completely submerged in water.

[0069] (2) Close the first pressure reducing valve 8, start the pump, increase the system inlet pressure to 10 psi, then stop pumping, close the second three-way valve 6, and keep the hollow fiber membrane cavity under positive pressure.

[0070] (3) Observe the frequency of bubbles appearing at the permeate end. If the membrane is damaged, bubbles will appear quickly; otherwise, it indicates that the membrane is intact.

[0071] After confirming the device is airtight and the filter membrane of the filter element is intact, adjust the first three-way valve 3 to open the liquid path of the washing solution bottle 1, and adjust the pump speed to 50% of the process flow rate, using approximately 1L / 0.1cm. 2 The entire system was flushed with Yibao water from the membrane area, and relevant data were recorded.

[0072] The specific steps for standard water flux testing are as follows:

[0073] (1) Adjust the first three-way valve 3 to open the liquid path on one side of the liquid circulation bottle 2, set the membrane injection flow rate, and discharge the reflux into the container until no air bubbles are discharged from the reflux end.

[0074] (2) Fully open the second pressure reducing valve 12 and increase the TMP (membrane pressure) to 10 psi by adjusting the first pressure reducing valve 8 to ensure that no air bubbles are discharged from the permeation end, and measure the permeation rate R of the permeation end in kg / min.

[0075] (3) Calculate the water flux WP, in units of LMH / psi, using the formula WP=600R / (TMP*A), where TMP is the transmembrane pressure in psi and A is the membrane area in cm². 2 .

[0076] (4) Depending on the actual situation, different water temperatures can be used to measure the water flux, or the water temperature can be fixed as needed and the water flux WP can be measured.

[0077] After recording the water flux through the membrane, to ensure that the membrane's pH and salt ion state are as close as possible to the initial state of the treated sample, approximately 500 mL / 0.1 cm⁻¹ was used. 2 A buffer solution of membrane area is used to rinse the hollow fiber membrane module to reduce membrane fouling and product loss.

[0078] The specific steps for enriching and purifying EVs from complex cell supernatants containing serum are as follows:

[0079] (1) According to the following Figure 4 The flow path scheme shown is used to construct a complete enrichment flow path by adjusting the various three-way valves and pressure reducing valves. For example... Figure 4 As indicated by the arrows, the fluid flow direction is as follows: Feed circulation bottle 2 --- First three-way valve 3 --- First peristaltic pump 4 --- Second three-way valve 6 --- Hollow fiber filter element 7 --- First pressure reducing valve 8 --- Third three-way valve 10 --- Feed circulation bottle 2. Another branch of the hollow fiber filter element 7 (located outside the filter membrane) --- Second pressure reducing valve 12 --- Second waste liquid bottle 14.

[0080] (2) The cell supernatant to be filtered was added to the feed circulation tank 2 for separation and enrichment. To maintain the integrity of EVs, the transmembrane pressure was limited to no more than 3 psi. To improve filtration efficiency and slow down the rate of membrane pore blockage, the maximum injection flow rate was set to 100 mL / min while limiting the transmembrane pressure. During circulation filtration, the mass change per unit time in the second waste bottle 14 was closely monitored.

[0081] (3) When the mass change per unit time in the second waste liquid bottle 14 gradually decreases, it indicates that the degree of particle accumulation on the inner surface of the membrane begins to affect the flow rate of the filter element. At this time, adjust the first three-way valve 3 to the following position: Figure 5 The washing and filtration flow path is shown. The filtration process involves washing and filtering simultaneously while circulating the filtration solution to maintain a constant filtration flux. Figure 5 and Figure 4 In contrast, the difference is that the washing solution bottle 1 and the material circulation bottle 2 simultaneously pump the washing solution and the sample into the hollow fiber filter element through the first three-way valve 3.

[0082] (4) In the later stages of filtration, the enrichment factor of the solution is relatively large, which easily leads to the formation of a gel layer on the inner surface of the membrane, resulting in a sharp decrease in the flux of the filter element. At this time, for impurity particles on the inner membrane surface, the flow path can be adjusted to... Figure 6 The cleaning flow path shown uses clean PBS to rinse the inner surface of the membrane to remove impurity particles and the gel layer. Figure 6 As shown, the liquid flow direction at this time is as follows: washing liquid bottle 1---first three-way valve 3---first peristaltic pump 4---second three-way valve 6---bottom inlet of hollow fiber filter element 7---first pressure reducing valve 8---third three-way valve 10---first waste liquid bottle 11.

[0083] In addition, for impurity particles within the membrane pores, the flow path can be adjusted to such a degree. Figure 7 The structure shown is used for backwashing to remove impurity particles from the membrane pores. For example... Figure 7 As shown, the liquid flow direction at this time is as follows: washing liquid bottle 1---first three-way valve 3---first peristaltic pump 4---second three-way valve 6---side inlet of hollow fiber filter element 7---first pressure reducing valve 8---third three-way valve 10---first waste liquid bottle 11.

[0084] (5) After restoring the filter cartridge flow rate, switch the flow path to the enrichment flow path to wash and enrich the solution in the feed circulation bottle 2. Depending on the purity requirements of the sample, perform different levels of washing and filtration. The higher the purity requirement, the larger the volume of washing solution used and the longer the time. Typically, using five times the original sample volume of washing solution can remove approximately 95% of the ions.

[0085] (6) After separation and enrichment, collect approximately 2-3 mL of the solution from circulation bottle 2. Then, pump air into the tubing to purge the solution and collect approximately 4-5 mL of the discharged solution. Generally, highly concentrated samples adhere throughout the system, especially to the inner surface of the membrane. Rinse 2-3 times with clean PBS and collect the washing solution. Finally, collect approximately 10-30 mL of sample, which should be milky white or transparent.

[0086] For biological samples with complex compositions, the membrane flux may decrease to less than 70% of the original flux after use. If continued use of the filter cartridge is desired, the membrane can be regenerated using 2% Triton X-100, with the specific steps as follows:

[0087] (1) Adjust the first three-way valve 3 to open the liquid path on the side of the washing liquid bottle 1, close the second pressure reducing valve 12, pump in 2% Triton X-100, so that the entire system and the inner and outer space of the filter membrane of the filter element are filled with liquid, and incubate at room temperature for 30 minutes.

[0088] (2) Adjust the second three-way valve 6 to open the liquid path outside the filter membrane space, and open the second pressure reducing valve 12 to allow 100 mL / 0.1 cm⁻¹ pressure to be released. 2 A certain amount of water is pumped into the membrane to rinse and remove impurities and particles from the space outside the filter membrane.

[0089] (3) Adjust the second three-way valve 6 to open the liquid path in the filter membrane space, and close the second pressure reducing valve 12, allowing 250 mL / 0.1 cm⁻¹ water to be dispensed. 2 A certain amount of water is pumped into the membrane to rinse and remove impurities and particles from the space inside the filter membrane.

[0090] (4) Adjust the flow path to Figure 7 The backwash flow path in the middle is 1L / 0.1cm 2 Deionized water is pumped into the membrane area to backwash the system.

[0091] (5) Adjust the flow path to Figure 6 The cleaning flow path in the middle is 500mL / 0.1cm. 2 Deionized water is pumped in at a rate equal to the membrane area to perform a forward flush of the system.

[0092] (6) Repeat the above operation 2 to 3 times, and the flow rate of the filter element can usually be restored to more than 90% of the original flow rate.

[0093] (7) Finally, fill the filter cartridge with 0.1M NaOH solution and store it in a refrigerator at 4°C.

[0094] In addition to the methods mentioned in the above embodiments, the structure of the device can also be changed in the following ways:

[0095] In another embodiment, the molecular weight cutoff can be changed: the molecular weight cutoff of the hollow fiber filter element can be changed from 500 kDa to 300 kDa or 100 kDa for experimentation to accommodate different molecular size requirements.

[0096] In another embodiment, valve replacement: the three-way valve in the device can be replaced with a valve or part of other materials or with similar function to meet specific application requirements.

[0097] In another embodiment, the bottle positions can be adjusted by moving or changing the relative positions of the 1-wash solution bottle and the 2-feed solution circulation bottle, as long as the function of the two bottles is not changed, so as to better adapt to the experimental environment.

[0098] In another embodiment, the number of washing bottles can be increased by adding x (x≥1) washing bottles in series or parallel with the 2-feed circulation bottles, for changes to the feed buffer system or further cleaning functions, to meet different experimental needs.

[0099] In another embodiment, the device can be linearly expanded: to handle larger sample volumes, the device can be linearly expanded proportionally, including increasing parameters such as the volume of the feed circulation bottle, the diameter of the silicone tube, the specifications of the Luer connector, and the membrane area of ​​the hollow fiber filter element, in order to increase processing capacity.

[0100] In addition, in another embodiment, the four flow path schemes can be freely set as needed, without being restricted by the order of use, so as to achieve more flexible operation and adapt to different experimental procedures.

[0101] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention should be considered equivalent alternatives and are included within the protection scope of the present invention.

Claims

1. A tangential flow ultrafiltration device, characterized in that: The system includes a liquid circulation bottle, the outlet of which is connected to a first three-way valve. One branch of the first three-way valve is connected to a washing liquid bottle, and the other branch is connected to a first peristaltic pump. The first peristaltic pump is connected to a second three-way valve. The two branches of the second three-way valve are respectively connected to the inner and outer sides of the filter membrane of a hollow fiber filter element. A second pressure reducing valve is provided at the outer outlet of the filter membrane of the hollow fiber filter element, and the second pressure reducing valve is connected to a second waste liquid bottle. The inner outlet of the filter membrane of the hollow fiber filter element is connected to a first pressure reducing valve, which is then connected to a third three-way valve. One branch of the third three-way valve is connected to the first waste liquid bottle, and the other branch is connected to the liquid circulation bottle.

2. The tangential flow ultrafiltration device according to claim 1, characterized in that: A first pressure gauge is installed between the first peristaltic pump and the second three-way valve; a second pressure gauge is installed between the first pressure reducing valve and the third three-way valve; and a third pressure gauge is installed between the second pressure reducing valve and the second waste liquid bottle.

3. The application of a tangential flow ultrafiltration device according to claim 1 or 2 in the enrichment and purification of biological samples.

4. The application according to claim 3, characterized in that, The biological sample includes at least one of extracellular vesicles, viruses, and proteins.

5. A method for enriching and purifying biological samples, using the tangential flow ultrafiltration device as described in claim 1, comprising the following steps: Step 1, enrichment: Add the sample to be processed into the feed circulation bottle, turn on the peristaltic pump, and pump the sample from the feed circulation bottle into the hollow fiber filter for filtration. The feed liquid enters the feed circulation bottle through the first pressure reducing valve, and the filtrate enters the second waste bottle through another branch of the hollow fiber filter through the second pressure reducing valve. This cycle is repeated to separate and enrich the sample. Step 2: Wash and filter simultaneously. When the mass change per unit time in the second waste liquid bottle gradually decreases, the washing liquid in the washing liquid bottle and the material liquid circulation bottle are simultaneously pumped into the hollow fiber filter element through the first three-way valve. Step 3, Cleaning: In the later stage of filtration, the feed circulation bottle is closed; first, the inner membrane surface of the filter element is cleaned: the washing liquid in the washing bottle enters from the inside of the hollow fiber filter membrane, and the washing liquid coming out from the inside of the hollow fiber filter membrane enters the first waste liquid bottle through the first pressure reducing valve, and the filtrate enters the second waste liquid bottle through the second pressure reducing valve; second, the membrane pores of the filter element are cleaned: the second pressure reducing valve is closed, the washing liquid in the washing bottle enters from the outside of the hollow fiber filter membrane, and the washing liquid flowing out from the inside of the hollow fiber filter membrane enters the first waste liquid bottle through the first pressure reducing valve; Step 4: After the filter cartridge flow rate is restored, switch the flow path to the enrichment flow path to enrich the solution in the feed circulation bottle; Step 5: After separation and enrichment are completed, collect the solution in the feed circulation bottle.

6. The method as described in claim 5, characterized in that: When the membrane flux drops to less than 70% of the original flux after the filter cartridge has been used, the filter membrane should be regenerated.

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