Multi-module system for continuous tangential flow filtration

Abstract

A multi-stage multi-module tangential flow filtration (TFF) system for continuous concentration, filtration or liquid exchange suitable for use in biological separation, purification, concentration, or other industrial separation is disclosed. These systems include a plurality of modular TFF systems connected in series or in parallel, with each module at least one tangential flow filtration device, reflux tube section, feed tube, permeate tube, and circulation pump. In one aspect, raw material liquid can only be introduced into the first-stage module, and reflux liquid or permeate liquid of one module is connected to the next-stage module through a pipeline with a valve according to process requirements, so that fluid can sequentially flow through the modules. The water continuously flows out from the circulation of the last-stage module; in some aspects, the invention has the advantages of achieving continuous TFF operation, using optimized filters / conditions at each stage to improve efficiency, higher throughput than a single TFF system, and enabling continuous buffer exchange; configurations of the present invention include with / without process tanks and different combinations of operations.

Description

Technical Field

[0001] This invention involves advances in bioprocessing and filtration technologies. Specifically, it seeks to enhance large-scale biomanufacturing processes with an emphasis on producing cultured meat, alternative proteins, precision fermentation products, and other synthetic bio products. In some respects, innovations revolve around continuous processing systems and hybrid filtration approaches designed to optimize production efficiency while reducing environmental impact and costs. Beyond bioprocessing, these new approaches have broader applications in a variety of industries that require precise separation, concentration, and purification processes. Background Art

[0002] Large-scale biomanufacturing, such as cell-cultured meat, alternative proteins, precision fermentation, and synthetic biology-based mass production, is a promising solution for future sustainable development. Meat protein substitutes produced by large-scale biomanufacturing have attracted much attention due to their potential to provide an environmentally friendly source of food protein, which can alleviate greenhouse gas emissions, land use and water consumption, while improving animal welfare.

[0003] The production of cultured meat, alternative proteins, and synthetic bioproducts requires large bioproduction capacity. Continuous processing systems have become a viable and necessary approach to meet large-scale bioprocessing needs. Such systems offer increased production capacity, the ability to effectively manage peak production demands, and can eliminate the need for equipment required for batch production, such as intermediate storage tanks. These advantages can significantly reduce the capital expenditure required to expand production capacity, increase automation, and reduce production costs.

[0004] Tangential Flow Filtration (TFF), also known as Cross-Flow Filtration, is a separation and purification technology widely used in the biopharmaceutical field. Perfusion cell culture is a cell culture method that continuously supplies fresh culture medium in a bioreactor and removes metabolic waste and cell products. Combining TFF and perfusion cell culture can increase cell density and product yield. The following is an introduction to the technology: Tangential flow filtration reduces particle accumulation and blockage on the membrane surface by making the fluid flow parallel to the surface of the filter membrane, thereby extending the service life of the filter and improving filtration efficiency. Its main components include: • Filtration device / filter: selectively allows targeted substances to pass through and blocks other substances.

[0005] • Pump and piping system: Maintain fluid flow to provide a flushing effect on the membrane surface and provide transmembrane pressure as the driving force for filtration.

[0006] • Collection system: collects the permeate (filtrate) and retentate (concentrate).

[0007] Tangential flow filtration (TFF) is a key unit operation widely used in the manufacture of alternative proteins, large molecule drugs, vaccines, and various bioproduction processes, including separation, purification, concentration and liquid exchange. Its applications include cell separation in perfusion, removal of cell debris, product concentration and buffer exchange.

[0008] Current filtration processes, including TFF processes, are primarily batch-oriented. Conventional TFF systems require the use of a reflux tank, often referred to as a feed tank or feed vessel, to contain the incoming feed or feed. In the context of alternative protein and food and beverage production, the volumes involved can be large, reaching 100,000 liters or more. The construction of feed tanks, intermediate storage tanks, etc. alone can incur significant capital expenditures. Continuous production can avoid or reduce this expenditure.

[0009] In addition, large-scale TFF batch operations present various challenges, such as the need for powerful circulation pumps, process risks associated with large parallel filters, large-scale piping systems, limited operational flexibility of the system and equipment, and issues with equipment portability and interchangeability. These challenges also extend to the engineering complexity involved in designing and maintaining large tanks, pumps, valves, and related components in extensive TFF systems.

[0010] Perfusion cell culture technology Perfusion cell culture is a dynamic cell culture method that allows cells to continue to grow and produce target products under optimal conditions by continuously or intermittently adding fresh culture medium and removing waste fluid. Its advantages include: • High Cell Density: Maintain and increase cell density.

[0011] • Stable environment: Continuously provide fresh culture medium to stabilize the cell culture environment.

[0012] • High product yield: Improve the yield of target products through high cell density and stable culture conditions.

[0013] In perfusion cell culture, it is necessary to be able to effectively separate cells and culture medium in order to remove the nutrient-depleted culture medium from the reactor. Currently, the most common cell division technology is achieved through filtration technology. One advantage of filtration technology is that it can achieve 100% cell retention. In addition, cell circulation is driven through the filtration device by a cell-friendly pump, such as a magnetic levitation pump (Levitronix) for TFF or a diaphragm pump used in an Alternative flow Tangential flow (ATF) perfusion system.

[0014] Combining TFF or ATF with a bioreactor or fermenter to achieve perfusion cell culture or continuous fermentation has the following advantages: 1. Improve cell concentration and yield to achieve process intensification: TFF system can continuously remove waste liquid, maintain the nutrient content of culture medium, and support high-density cell culture. In this way, more cells and / or cell products can be produced per unit reactor density.

[0015] 2. Reduce contamination risk: The closed system reduces the risk of external contamination and ensures the sterility of the culture process.

[0016] 3. Stable production environment: Stable culture conditions and nutrient supply improve cell vitality, product uniformity, and yield.

[0017] 4. Economical and efficient: The production cost is reduced due to the reduced frequency of culture medium replacement and waste liquid treatment. The demand for bioreactors is reduced.

[0018] In order to reduce the overall biomanufacturing cost and improve production capacity, continuous manufacturing has become a promising and necessary solution. Continuous production has been successfully applied in industries such as petrochemicals. While replacing the cell culture medium, cell perfusion culture can continuously harvest cells and / or cell products, which can realize continuous cell culture or cell product production.

[0019] Continuous operation, especially in clarification of conditioned medium and volume reduction through membrane concentration, is of great value in large-scale therapeutic antibody production. For example, the titer of monoclonal antibody (Mab) production cell culture stage puts forward better capacity requirements for downstream separation and purification. Even in relatively small-scale biopharmaceutical production, continuous production has become a trend. Continuous production, especially continuous filtration including tangential flow filtration, is more critical in alternative food proteins, precision fermentation, and other large-scale bio-industries, because these industrial productions require at least hundreds of times more capacity.

[0020] At present, the role of the circulating pumps in the perfusion operation is crucial in the process of cell perfusion culture. They are responsible for transferring the live cell suspension from the bioreactor to the tangential flow filtration device, separating the cells and the culture medium by filtration, and then transferring the cell suspension from the filtration device to the bioreactor. The perfusion circulation pump must be able to ensure low shear, be cell-friendly, and avoid cell stress response or death caused by flow rate or pressure fluctuations. And it can ensure long-term sterile operation. This puts higher requirements on the design and manufacture of the pump, and there are currently large-scale perfusion pumps on the market that can meet this demand. So much so that at present, the largest perfusion reactor for animal cell culture is often limited to about 1,000 liters or less. If it exceeds 1,000 liters, multiple cell perfusion devices are often connected in parallel, or the perfusion cell filtration device is replaced midway. Multiple perfusion devices are connected in parallel, or the cell filtration device is replaced midway. While increasing costs, the complexity of the relevant pipelines and the complexity of operation are greatly increased, and the risk of contamination is increased. This has become a major bottleneck restricting cell perfusion technology in large-scale industrial production.

[0021] It is of great significance to achieve larger capacity through modular smaller filtration systems. In addition, contract development and manufacturing organizations (CDMOs) engaged in cultured meat projects often have difficulty in meeting fluctuating capacity requirements. In this case, it is of great significance to provide TFF systems that can flexibly adjust the processing volume in continuous operation.

[0022] While current single-pass tangential flow (SpTFF) technologies excel in continuous processing, they face limitations when high concentration factors or high concentration ratios are required. Achieving high concentration factors in single-pass tangential flow can be challenging, especially for large-scale manufacturing processes that require high filtration throughput and large sample flow rates. Importantly, SpTFF is not well suited to handling viscous samples often encountered in high-concentration applications. In addition, mitigating membrane fouling in high-throughput situations often requires increased recirculation rates, which necessitates an extended flow path to achieve high concentration ratios. This extended path introduces higher backpressure, increases robustness requirements for the pipeline, and creates challenges in system design, filter engineering, cost implications, and safety. Current single-pass tangential flow technologies are currently only suitable for single-use processes, as there is currently no simple and efficient solution for cleaning and regeneration of filter media for single-pass tangential flow filtration.

[0023] Although SpTFF facilitates continuous filtration operations, its application to buffer exchange (also known as buffer exchange or diafiltration) has proven inefficient.

[0024] The present invention innovatively introduces a modular TFF system that provides the combined advantages of traditional single-pass TFF and SpTFF. These TFF systems can also be called hybrid TFF systems, which can support cell separation operations in continuous cell perfusion culture and perform continuous bioprocessing for concentration and buffer exchange. The modular TFF system has the following characteristics: a. They support continuous tangential flow filtration process and realize tangential flow filtration operation with uninterrupted continuous flow.

[0025] b. They achieve higher throughput, which is difficult to achieve with single-pass TFF systems and current batch tangential flow operations.

[0026] c. They are able to perform all functions of a batch TFF process, including clarification, concentration, buffer exchange, and other operations.

[0027] d. They consist of relatively small-sized pumps, equipment and components that form system modules, which are easy to install, maintain and replace.

[0028] e. They adjust capacity by adding, replacing, or removing the number of system modules, thereby providing flexibility in processing volume and reliability of system operation.

[0029] f. In the field of cell perfusion, since multiple system modules are connected in series, it is not necessary for all modules to be connected to the bioreactor or fermenter, thereby reducing the structure of the reactor and the risk of contamination during the sterilization process.

[0030] The multi-module system shows great flexibility in workload distribution, allowing other modules to share the workload when one modular system needs maintenance, thus ensuring the continuity of the entire filtration process.

[0031] Notably, these modular filtration systems enable continuous tangential flow filtration and exchange, thus enabling end-to-end complete filtration processes. The disclosed filter designs are widely applicable to a variety of TFF filter formats, membrane pore sizes, and membrane materials, including ceramic, PVTF, PTFE, mPES, and nylon. This innovation has the potential to be used in a wider range of applications beyond continuous perfusion cell culture, including protein concentration, buffer exchange, and separation and purification operations in other industries.

[0032] In the context of the industrial development of synthetic biology, the prospects of alternative protein sources and precision fermentation, modular filtration systems have applications in both upstream cell culture and downstream purification, thus representing a transformative step towards process intensification, sustainable, and high-capacity production methods. Summary of the invention

[0033] The following summary summarizes the key innovations embodied in the systems, methods, devices and apparatus described herein. It is important to note that this summary provides a brief overview of the core features of the invention, and the detailed description of the invention disclosure is not intended to limit the scope of the claims.

[0034] In some of its embodiments, the present invention achieves the capacity of a large-scale tangential flow filtration device by configuring multiple relatively small modular TFF systems that are carefully connected in series to facilitate continuous processing. The core concept revolves around the connection and control of these modular TFF systems, where each system module contributes to the entire filtration process, thereby rationally distributing the functions of a large TFF system over multiple connected system modules.

[0035] In one aspect, the retentate loops of these smaller TFF systems are cleverly connected to maintain continuous flow of feed solution throughout the modules. The feed solution then flows in the retentate flow path. This continuous flow is achieved by directing a portion of the retentate from one module to the next module for further filtration, thereby achieving continuous, efficient and scalable processing capacity.

[0036] On the other hand, by using two multi-parallel modular systems at each stage, redundancy and maintenance issues can be seamlessly solved. This allows the workload to be redistributed to other connected modules, which will be operated by other parallel or serial systems when one modular system is performing membrane cleaning and regeneration, or replacing the membrane, thereby achieving uninterrupted filtration operation. This makes end-to-end continuous filtration operation possible.

[0037] In one non-limiting embodiment, continuous diafiltration can be achieved by introducing fresh buffer directly into the flow path of the retentate. The buffer is quickly mixed with the retentate before entering the subsequent module, ensuring accurate and continuous buffer exchange.

[0038] On the other hand, during the filtration process, as the filtration operation proceeds, the properties of the feed, such as concentration and viscosity, will also change, and the properties of the feed in different system modules may be different. The modular design allows different system modules to use different membranes for the properties of the material. The pore size, membrane material, and filtration area of ​​the filter membrane between different modules can be optimized according to the changes in the properties of the feed, thereby improving the filtration efficiency. The feed, reflux or permeate from one module can be used as the feed for the next one, providing adaptability to changes in the properties of the feed and to different process requirements.

[0039] In non-limiting examples, modules can be equipped with reflux tanks (or feed tanks) of different sizes, or without feed tanks. Feed tanks facilitate mixing but may increase hold-up volume, cleaning costs, and hardware costs.

[0040] Additionally, the system allows for the injection of buffers, or other liquids, such as those relevant to other formulations, into the feed for additional continuous operation.

[0041] Overall, the multi-stage modular system can perform a range of different filtration, mixing, formulation adjustment and other functions, including perfusion, cell collection, clarification, concentration, isotangential flow operation, and mixing functions. Its modular design not only ensures scalability by adjusting the number of modules, but also provides the convenience of pre-assembled closed disposable flow channels for rapid installation and implementation.

[0042] The technical solution adopted by the present invention is as follows: A tangential flow filtration system, comprising: a. two or more modular tangential flow filtration systems connected in series, each modular system comprising at least one tangential flow filtration device, a reflux liquid pipe section, a feed pipe, a permeate pipe and a circulation pump; and b. Inter-system feed transfer pipeline, which is used to transport part or all of the feed from the reflux liquid flow path of the previous system module or from the permeate flow path to the next modular system for sequential processing.

[0043] Preferably, each modular system further comprises one or more of the following components: Liquid tank, feed pump, buffer pump, permeate pump, pressure sensor, flow meter, mixer, back pressure valve, analyte detector, exhaust valve and pipeline connector in the circulation flow path, spectrometer and conductivity meter for online monitoring.

[0044] Preferably, each system module includes redundant parallel modules, allowing the feed solution to bypass one of the modules during operation. The bypass can isolate one module to perform operations such as membrane replacement, cleaning, or regeneration.

[0045] Preferably, a method of tangential filtration operation comprises: a. Providing a tangential flow filtration system according to the above; b. continuously introducing feed into the first modular system; c. transferring the reflux from each modular system section to the next modular system for further processing; and d. Continuously collect the permeate and the final reflux liquid; Preferably, the method further comprises introducing a buffer into the reflux loop of each modular system for liquid replacement.

[0046] Preferably, the modular tangential flow filtration system sequentially performs two or more identical or different combinations of tangential flow filtration operations, wherein the tangential flow filtration operations preferably include cell separation filtration and / or clarification filtration and / or concentration and / or fluid exchange and other filtration or mixing operations.

[0047] Preferably, a perfusion bioreactor system comprises: a) bioreactor or fermenter systems; b) a tangential flow filtration system according to the above, comprising two or more modular tangential flow filtration systems connected in series, each modular system comprising at least one tangential flow filtration device, a retentate pipe section, a feed pipe, a permeate pipe and a circulation pump; the filtration device of the modular tangential flow filtration system is capable of retaining cells, such as cells or bacteria, and allowing culture medium components or smaller particle components to pass through; and c) a line for conveying the cell suspension from the bioreactor or fermenter to the modular tangential flow filtration (TFF) system, a line for recycling all, part or all of the cell suspension back to the bioreactor, and a line for collecting and directing the permeate.

[0048] Preferably, the tangential flow filtration device in the system uses a hollow fiber tube, a ceramic membrane tube, a stainless steel tube, or a polymer tubular membrane as a filtration medium, and has a lumen diameter of 0.5-12 mm, preferably 0.5-5 mm, and a molecular weight cutoff of 50 nanometers to 5 microns; Preferably, the tangential flow filtration device in the system uses a ceramic membrane tube or a polymer tubular membrane as a filtration medium, and has a tube lumen diameter of 2-12 mm and a molecular weight cutoff of 0.2-5 μm; Preferably, the tangential flow filtration device uses a ceramic membrane or a polymer material membrane tube with a flow channel size of 1.5-12 mm and a molecular weight cutoff of 0.5-2 microns.

[0049] Preferably, the insert is placed in the inner cavity of the membrane tube of the tangential flow filtration device to reduce the cross-sectional area, optimize the flow pattern of the feed liquid, and reduce the circulation flow rate of the feed liquid.

[0050] Preferably, the bioreactor produces cells, proteins produced by the cells or other bioproducts, and the cells are separated from the cell culture medium by tangential flow filtration.

[0051] Preferably, cell products, including but not limited to exosomes, proteins, viruses or other biological products, are extracted from the cell suspension by tangential flow filtration.

[0052] Preferably, the cells are separated from the cell culture medium by tangential flow filtration, wherein the cells are mammalian cells, such as CHO cells, HEK cells, VERO cells, or other animal cells, such as muscle cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The novel features believed characteristic of the illustrative embodiments are set forth in the appended claims. The illustrative embodiments and the preferred mode of use will be best understood by reference to the following detailed description when read in conjunction with the accompanying drawings, in which: Figure 1a A 4-stage modular TFF subsystem for continuous TFF operation is described, where a portion of the reflux from one modular system is used as feed to the next module or as final product, by-product or waste. Each TFF module is equipped with a TFF reflux tank, and the concentration or composition ratio of the feed varies between modules, while generally maintaining a constant feed rate during continuous operation.

[0054] Figure 1b A 4-stage modular TFF subsystem for continuous TFF operation is described, similar to Figure 1a , where a portion of the reflux from one modular system is fed to the next module or used as a final product or by-product. Each TFF module contains a TFF reflux tank, and the concentration or component ratio of the feed solution varies between modules. Here the module includes a permeate pump to regulate the permeate flow rate.

[0055] Figure 2a A 4-stage TFF system optimized for concentration operation is shown. Here, the reflux from the first module is used as feed to the second module. Not all TFF system modules have a reflux tank.

[0056] Figure 2b and Figure 2a Similarly, a 4-stage TFF system designed for concentration operation is illustrated. The reflux from the first module is used as the feed to the second module, and not all TFF system modules contain a reflux tank. This configuration includes an in-line mixer and permeate pump in the flow path, as well as a vent valve added to the reflux line. Figure 2b The system module in the system includes a permeate pump to regulate the permeate flow rate.

[0057] Figure 3a An overview of a 4-module TFF system designed for continuous concentration and buffer exchange in a mixed-mode TFF process. Each TFF module includes a reflux tank, and some modular systems have lines for direct introduction of fresh buffer into the feed tank. The first two modules can be used for concentration / filtration operations. The last two modules are equipped with buffer refill lines.

[0058] Figure 3b and Figure 3a Similarly, a 4-module TFF system is depicted that is designed for continuous concentration and buffer exchange in a mixed-mode TFF process. However, in this case, the TFF modules do not include a reflux tank, and fresh buffer is introduced directly into the reflux line. The absence of a reflux tank improves the efficiency of buffer exchange. An inline mixer is included in the recirculation flow path. The first two modules can be used for concentration / filtration operations. The second two modules are equipped with buffer refill lines.

[0059] Figure 4 A 4-module TFF system tailored for continuous concentration and intermittent liquid exchange processes was demonstrated. The first two systems are connected in series, and the second two are connected in parallel. In this configuration, the permeate from the first two stages is concentrated and then used as the feed for the next stage of batch liquid exchange. The latter liquid exchange operation includes two parallel system modules, which can perform liquid exchange or filtration operations simultaneously or in turn, thereby achieving continuous production.

[0060] Figure 5 The steps of a 4-module TFF system designed for two different continuous concentrations are outlined. The first two systems are connected in series, and the second two are also connected in series. The permeate from the first two modules is used as the feed for the next two modules for subsequent concentration or filtration process operations.

[0061] Figure 6 A modular TFF system designed for continuous concentration operation was demonstrated, with two filters in series within each TFF system module to increase filtration efficiency and capacity. DETAILED DESCRIPTION

[0062] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The terms or words used in the specification and claims of the present invention should not be interpreted as being limited to their common meanings and dictionary meanings. Instead, they should be interpreted based on the meaning and concept of the present invention to comply with the scope of the invention based on the principle that the inventor can appropriately define terms to describe the present invention in the best manner.

[0063] It should be understood that the form of the present invention shown and described herein is as a preferred embodiment of the present invention, and therefore it does not express or limit the technical spirit and scope of the present invention. Therefore, it should be understood that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0064] Here, "filtration" and "concentration" are interchangeable, and what is concentrated here can be either the product or the impurities. Filtration "system module" and "module system" can be considered synonymous.

[0065] The terms "feed container" and "sample container" for filtration can be considered synonymous.

[0066] "Concentration factor" and "concentration fold" may be considered synonymous.

[0067] "Filtering device" and "filter" may be considered synonymous.

[0068] In the present disclosure, the term "exemplary" may be interpreted to mean an embodiment provided as an example.

[0069] According to one aspect, the present invention discloses a method of replacing a single larger TFF system and enabling continuous operation by combining smaller TFF systems in a modular manner to achieve greater processing capacity. Figure 1a and Figure 1b As shown, the incoming feed liquid is continuously added to the TFF system, and the permeate and final reflux liquid are continuously collected. In a preferred embodiment, a filtrate pump such as a filtrate pump for controlling the permeate or filtrate flow system may be included. Figure 1b shown.

[0070] Therefore, the modularly connected system may include two or more TFF modular systems, each of which may include at least one TFF filter (91, 92, 93 and 94 as shown in the figure) and a feed pipe for conveying the feed liquid to the inlet of the TFF filter, a reflux pipe for collecting the reflux liquid of the TFF filter and conveying the reflux liquid back to a feed liquid tank or also called a feed liquid container (110, 120, 130 or 140 as shown in the figure), a permeate pipe for collecting the permeate liquid from the permeate port of the TFF device, and a TFF pump (01, 02, 03, 04) for conveying the feed from the feed liquid tank to the inlet of the TFF filter through the feed pipe.

[0071] The feed, or sample 100, entering the system is introduced into the feed tank of the first modular system, such as Figure 1a and 1b As shown. Figure 1a and 1b It is shown that a portion of the reflux liquid is partially diverted to the next-stage TFF module after flowing through the TFF device as the feed for the next stage. In some embodiments, the feed for the next-stage TFF module can also be introduced from the feed liquid portion between the TFF pump and the TFF filter of the previous stage. In this way, the pressure generated by the pump can be used to transfer the feed liquid from the previous stage to the next stage, especially when the viscosity of the feed liquid is high. The disclosure of the present invention does not list all possible locations where fluid is extracted from one modular system to the next modular system as feed. For convenience and other considerations, other possibilities can be selected during implementation according to process requirements and sample parameters.

[0072] In operation, the sample or so-called feed 100 is loaded continuously or in batches into the feed container 81 of the first modular system. The sample then circulates in the first TFF system module flow path, while larger particles or molecules are retained and concentrated by the TFF filter, while small molecules and solutions can leave the retentate circulation flow path through the filter medium of the TFF filter 91. A portion of the retentate is directed to the next stage TFF module for further concentration, where the process is repeated for each TFF module in the next stage. The concentration factor at each stage will be the ratio of the feed flow rate to the feed flow rate flowing into the next stage TFF module, or the ratio of the feed flow rate to the flow rate of the final product or by-product.

[0073] In the illustrated diagram, there are four stages (also referred to as "stages"), each with a corresponding TFF filter (also referred to as a filtration device), 91, 92, 93 and 94, in order. Specifically, the incoming feed 100 is introduced into the feed container 81, and can reach the TFF filter 91 through the TFF pump 1 in the processing container, which is the first stage to produce the permeate 150. The first stage is also shown as having a pressure regulating valve 61, pressure sensors 21 and 29. The pressure sensors 21 and 29 are located before and after the TFF filter 91, respectively. The permeate 150 can be monitored by the permeate pressure sensor 25, and the flow rate can be monitored by the permeate flow meter 45. Each subsequent stage shown has a corresponding permeate, as shown in the figure at 160, 170 and 180, and the final reflux container 85 is shown. The permeate pressure can be measured in conjunction with the pressure sensors 25, 26, 27 and 28. Additional flow meters 46, 47 and 48 may be integrated to measure permeate flow rates corresponding to 160, 170 and 180 in the figure. Additional TFF pumps 2, 3 and 4 are shown.

[0074] Using a modular system, samples can be continuously loaded into the first-stage TFF system, and the final product can be collected from the last-stage TFF module and continuous operation can be achieved. The product can be further processed by the next step (chromatographic separation) without waiting for the processing of all samples, saving a total of operating time. The concentration multiple of the feed introduced into each TFF module can be calculated by the ratio of the flow rate of the introduced feed to the amount of reflux liquid diverted to the next-stage TFF system. The total concentration rate (concentration factor) is the product of the concentration multiples of each stage. In some other examples, after each filtration stage, a concentration monitoring device can be used to detect the concentration of the core feed component, as shown in Figures 11, 12, 13 and 14.

[0075] In some embodiments where the permeate flow rate is critical, permeate pumps 6, 7, 8 and 9 may be included to control the permeate flow rate, such as Figure 1bMultiple flow detectors can be integrated into the system shown in 49, 50, 51 and 52 to measure flow.

[0076] In some embodiments, additional equipment for pressure monitoring (e.g., 21, 22, 23, and 24 located before the TFF filter, and 29, 30, 31, and 32 located after the TFF filter) is used for pressure monitoring and subsequent control of feed, reflux pressure, permeate pressure, etc., flow monitoring and flow control. Additional feed liquid component monitoring devices can also be used for online concentration monitoring or monitoring of other process parameters. These devices may include ultraviolet detectors, conductivity detectors, pH meters, and thermometers, etc. Figure 1b 4 module TFF systems with additional sensors and filtrate pumps are shown. Additional sensors can be added to improve process monitoring and control. Adding additional components is within the expected scope of the present invention. For example, the volume or weight of the feed solution in each module is monitored by a weight measuring device (e.g., a scale or a load cell).

[0077] In some embodiments, the permeate flow rate is determined by flow meters as shown in the figure 49, 50, 51 and 52, and the total permeate volume is determined by integral calculation. As an alternative to using a flow meter, a permeate pump can be implemented to control the permeate flow rate.

[0078] In the present invention, the processing capacity of the system can be partially adjusted by increasing the number of system modules or increasing the membrane surface area and recirculation rate of each stage and other operating process parameters.

[0079] The feed tank for each TFF module can provide a mixing device for the feed flow. These tanks are shown as 81, 82, 83, 84 and 85. The adaptability of each modular system provides flexibility. The present invention discloses a system configuration that does not require a TFF process tank for each TFT module, which has the advantages of reducing the equipment cost associated with the storage tank and the connecting pipe; reducing the hold-up volume, reducing the footprint of the entire equipment, etc.

[0080] In order to eliminate air bubbles in the flow path, the flow path can be equipped with an exhaust valve, such as Figure 1b 71, 72, 73 and 74 shown. If necessary, an air sterilizing filter can also be installed on the exhaust pipe as a bacterial isolation barrier to meet the requirements of cell perfusion and other aseptic operations.

[0081] In some embodiments, the process tanks used in each modular system can be eliminated in situations where a small containment volume is required. These process tanks include Figure 1a and 1b Part or all of the process tanks shown at 81, 82, 83 and 84. Figure 2a and2b Shown is a TFF modular system configuration that does not use a feed tank, as opposed to traditional batch TFF operations that require a feed tank.

[0082] In this embodiment, the incoming feed liquid 100 is combined with the reflux liquid of the first stage, such as Figure 2a and 2b. Part of the reflux liquid is then introduced into the reflux liquid pipeline of the next stage, mixed with the reflux liquid, and then enters the inlet of the next stage TFF device through TFF circulation pump 1. The reflux liquid is mixed with the feed liquid introduced from the previous stage and transported to the inlet of the TFF device through circulation pumps 2, 3 or 4, respectively, and this is repeated, as shown in FIG. Figure 2a and 2b shown.

[0083] The configuration disclosed in the present invention not only has the potential to reduce the cost of system hardware, but also provides great advantages for high-value product processing because the hold-up volume of the flow path can be minimized and the final product yield can be increased while reducing the volume of cleaning fluid or buffer required to manage cleaning and filter regeneration.

[0084] In the recirculation flow path, Figure 2b Static mixers at various stages such as 34, 35, 36 and 37 can be used to enhance the mixing of the feed liquid and the reflux liquid to ensure that the feed stream is properly mixed. In addition, pressure and flow monitoring devices and concentration monitoring devices can be implemented in the flow path as needed. The incoming feed liquid is processed in the TFF modular system and the final reflux liquid is connected to the final collection container. The permeate stream from each TFF modular system can be collected and pooled for further processing or disposal.

[0085] For microfiltration applications, a filtrate pump can be added to each modular system for permeate control. Figure 2a and 2b 6, 7, 8, and 9 show gas outlets integrated into the various stages in order to eliminate possible air bubbles.

[0086] Conventional tangential flow filtration (TFF) liquid exchange operation is an intermittent process. The present disclosure provides a method for continuous liquid exchange using multiple TFF modular systems. The feed liquid continuously enters some modular systems connected, and the product, by-product, or waste liquid is continuously discharged. In addition, the present disclosure provides a multi-stage TFF system configuration, which can perform different types of TFF operations in a continuous mode, including concentration and buffer exchange.

[0087] refer to Figure 3a and 3b, illustrating a system configuration for continuous concentration and liquid exchange. In both figures, the first two modules perform feed concentration, and the subsequent two modules perform buffer exchange in a continuous operation. FIG. 3 shows two stages of concentration and liquid exchange, and each of the two concentration and liquid exchange operations shows a two-stage operation. The incoming feed / sample 100 is loaded into the first stage and concentrated, and a portion of the retentate is introduced into the second stage, where it can be further concentrated.

[0088] After the desired volume reduction / concentration ratio is achieved, a portion of the retentate from the second stage is passed to the third stage for buffer exchange. Buffer 230 is added and mixed with the retentate from the third TFF module and the incoming feed stream from the previous TFF module system into the process tank 83. The mixed feed is then delivered to the inlet of the TFF filter 93 of the third modular system. A portion of the retentate from the third modular system is delivered to the feed container to be mixed with fresh buffer 240 and recycled back to the filter 94. Another portion of the retentate is delivered to the next stage for buffer exchange in the next stage. This illustration is non-limiting and more stages can be integrated for concentration or buffer exchange as needed.

[0089] According to one aspect, the weight or volume of the feed in the process vessel can be determined by a balance or other level or weight monitoring device. The recirculation flow rate can be controlled by the speed of the pump. The permeate flow rate of each stage can be monitored by a flow meter or permeate weight monitoring device, or controlled by a permeate pump. The feed flow rate from the previous stage can be determined by a flow meter or controlled by a pump.

[0090] Although Figure 3a and 3b Two different TFF operations are listed, concentration and buffer exchange, but the present invention allows for additional concentration / filtration and buffer exchange operations before or after the above stages shown. Examples include a buffer exchange operation with the addition of a second buffer, and a second concentration step to further reduce the volume. All of these can be performed continuously.

[0091] In some embodiments, a recirculation tank as shown in Figure 3 may not be used. Fresh buffer may be introduced and combined with the feed and reflux from the previous stage, e.g. Figure 3b 300 and 310 in FIG. If additional mixing is required, a static mixer can be implemented in the feed line after the circulation pump, as shown in FIG. 35, 36 and 37.

[0092] In some embodiments, the buffer flow rate can be controlled to be the same as the permeate rate of each stage. It can be achieved by buffer injection pump or valve control. In one aspect, the flow rate can be controlled and synchronized by using a multi-channel pump with two synchronized channels, and the different channels of the pump can deliver the same flow rate.

[0093] In continuous operation, the feed flow rate from the previous stage is set as the flow rate of the reflux liquid introduced into the feed operation of the next stage to achieve steady-state operation. In some embodiments, if the flow rate of the incoming buffer is equal to the flow rate of the permeate, the addition flow rate of the buffer can be passively controlled to match the permeate flow rate to achieve constant volume buffer replacement. There are many other ways to achieve constant volume liquid replacement, and the present invention only discloses several possible reasons.

[0094] The retentate from the buffer exchange can be collected after the last buffer exchange operation.

[0095] Figure 3 shows only one combination of concentration and one buffer exchange. Other combinations of TFF operations are possible, and it is possible to use a greater number of modular systems to achieve higher concentration factors and more thorough buffer exchanges.

[0096] The module system of the present invention can be flexibly configured to meet different process and capacity requirements and can be connected in series or in parallel. Figure 4 A 4-module TFF system for continuous concentration and batch exchange is shown. In this configuration, the permeate from the first two stages of series concentration operation is collected and then used as feed for the next stage of concentration or exchange operation. The next stage of concentration or exchange operation is operated here by parallel modular systems. The approach of combining multiple modular TFF system methods to achieve modular operation can be extended to any other scenario that can benefit from continuous TFF operation. In some embodiments, batch exchange is still required to save buffer consumption, because continuous multi-stage exchange may require relatively more buffer.

[0097] The modular system of the present invention can provide the possibility of flexible configuration to meet different process and capacity requirements. Figure 5 The figure shows a combination of four TFF module systems to complete two different filtration operations. The filtrate produced by the first two system modules is collected and introduced into the third system, and then the third and fourth systems are used for series concentration and filtration operations. Figure 5As shown, the output of the stage including TFF filters 91 and 92 passes through 500 as shown in the figure to the next stage. Here, the two modules at the back generally adopt the filter device with a molecular weight cut-off different from the previous two modules. For example, the molecular weight cut-off of the two stages at the back is one-tenth or less of the previous two stages. This configuration is illustrative, and therefore, this embodiment is to illustrate the application of a wide range of continuous TFF systems, which can be applied to any other TFF applications, making continuous filtration processes possible.

[0098] In some other embodiments, the multi-module TFF system can be used for two different filtrations. One such case is a process involving two-step TFF filtration, such as cell separation to collect conditioned medium containing cell products, and a subsequent filtration operation to concentrate, enrich, and purify the product produced by the cells, such as a protein product expressed by yeast. In such an embodiment, cells or cell debris are removed by a first TFF unit operation, and then the permeate 500 containing the product is concentrated in a second TFF unit operation. Each TFF unit operation can employ the multi-module TFF system, wherein feed is continuously loaded into the system and retentate is continuously collected after the TFF operation, such as Figure 5 In this example, the permeate from the first modular system (cells and some cell debris have been removed) is collected as feed to the second modular system (for protein concentration). Figure 5 Configurations using process vessels are shown, but process and system configurations are possible that do not employ process vessels in each modular system.

[0099] The filtration devices of the multiple modules included in the modular system shown do not have to be the same, and can be optimized according to the changes in the composition, volume or viscosity of the feed liquid. The TFF device used in each module is not limited to a specific type, it can be a ceramic, metal, polymer-based, disposable device or a reusable device. The pumps used in each TFF modular system do not have to be exactly the same. For example, in concentration or other filtration applications, the processing capacity of the configuration of the first-stage module can be higher, or suitable for processing low-concentration feed liquids, and the processing capacity of the subsequent modules can be smaller, but suitable for processing higher concentrations, smaller volumes, or higher viscosity feed liquids. Such a filter and flow path configuration helps to reduce the retention volume of the flow path to obtain a higher yield. The membrane type, molecular weight cutoff, flow channel design of the filter, and surface area of ​​each step here can be the same or different.

[0100] In some embodiments, the pore size or molecular weight cutoff of the filter media used in the two TFF steps is different. Taking the harvest application as an example, antibody production, in which, in the first concentration unit operation to remove cells or cell fragments, a hollow fiber membrane filter with a total surface area of ​​about 100 to 120 square meters, a molecular weight cutoff of 0.2 to 0.45 microns, and a membrane inner diameter of 1 mm can be used to separate cells from a 20,000 L bioreactor and concentrate cells from a concentration of 4% to more than 30%. In the second step, a flat sheet membrane package with a total surface area of ​​75 to 100 square meters and a molecular weight cutoff of 30kd can be used to concentrate the filtrate from 20,000L to 2000L in a continuous operation mode to enrich the secreted proteins of the cells. The separation of cells or cell fragments in the first step can use a 3-stage serial modular system, and the membrane area of ​​each stage can be 3-4 polyethersulfone hollow fiber membrane filters in parallel, and the area of ​​a single filter can be from 7-11 square meters. Here, each stage can use Levitronix 2000 magnetic suspension pump as circulation pump. And the second step concentration can also use 3 modular systems in series. Here, each stage can also use flat membrane packages with a total area of ​​25 square meters. The material of the filter membrane in the membrane package can be modified cellulose membrane or modified polyethersulfone membrane. Here, Quattroflow 20kSU can be used as circulation pump. Here, the choice of pump and filter is a solution. There is also the possibility of using other pumps, membranes of other materials, or filters with different filtration areas.

[0101] Another application of the invention is the concentration and washing of E. coli cells in the context of cell and gene therapy. In this case, the E. coli suspension can be added to the multi-modular concentration system. The suspension of bacteria becomes increasingly concentrated in the latter stage compared to the previous stage.

[0102] In one embodiment, hollow fiber membrane filtration with a 0.2 micron pore size cutoff and 1 mm inner diameter can be used for concentration in the initial stage with a larger surface area, and ceramic membranes with a flow channel diameter of 2 mm or more can be used for further concentration and cell washing in subsequent stages.

[0103] In another embodiment, the size of the TFF filters in each module may be the same or different.

[0104] Another application related to virus concentration is to facilitate the separation and purification of viruses in cell and gene therapy, such as the concentration of lentivirus or adenovirus and the concentration and exchange of culture medium after clarification. The first stage of concentration is to use a hollow fiber membrane filter with a surface area of ​​about 800 square centimeters, a molecular weight cutoff of 100 kd, and an inner diameter of 0.5 mm as the first stage to reduce the volume from 150 liters to 10 liters; the second stage is to use a hollow fiber membrane filter with a surface area of ​​about 800 square centimeters to reduce the volume from 10 liters to 500 ml; if necessary, a 115 The square centimeter membrane is used for the third concentration volume reduction and buffer exchange. Through these operation steps, the final volume can be continuously reduced to 15-100 ml. Continuous operation by three system modules in series can reduce the operation time by 50% or more compared to two or three step batch operation, as well as pre-washing the membrane buffer.

[0105] Some embodiments may illustrate sequential filter designs.In each TFF modular system, one or more TFF devices / filters may be implemented to achieve a desired concentration ratio. Figure 6 Two TFF devices are shown connected in series, with the outlet of one filter connected to the inlet of the next filter (e.g., 98 and 91, 97 and 92, 96 and 93, and 94 and 95). In some other applications, these filters can be connected in parallel.

[0106] The module design disclosed in the present invention only includes some embodiments. In other embodiments, each TFF system module can use one or more filtration devices. Figure 6 It is shown that each module system uses two filter devices connected in series. In some other embodiments, multiple filter devices can be connected in parallel, or a hybrid structure including parallel and series connection can be adopted.

[0107] Figure 1 to Figure 6 Only the important piping / connecting lines between these modular systems are shown. Others may have additional valves and piping not included for cleaning, collection, flushing and other operations, such as drain valves and piping, sampling ports, or even additional piping to connect the feed lines between each modular system for flexible operation.

[0108] In the disclosed multi-module TFF system, in addition to increasing the membrane surface area and TFF feed circulation flow rate at each stage, the processing volume can also be adjusted by increasing the number of filtration system modules in series or parallel. This approach has great flexibility in process development. For example, several smaller TFF modular systems can be used in series to process samples that usually require larger systems. In particular, this is particularly useful if the smaller system is easy to operate and install. Due to the limitations of clean plant facilities, such as door size, tool limitations, ceiling size, etc., it may not be easy or even impossible to install and transfer larger filtration systems. This advantage is of great significance in using existing pumps, pipelines and other equipment to achieve large-scale bioproduction capacity.

[0109] The steps in this implementation may also include many system optimizations and adjustments, as well as additional online or offline process analytical techniques that may be applied to process monitoring and control.

[0110] In one variation, the feed tank can be aseptically adapted, including a vent valve for evacuation and an attached sterile air filter. At the top of the retentate line, an exhaust valve can be implemented to evacuate the TFF flow path at the start of the process. An air filter with a 0.2 micron pore size cutoff can be used to establish a bacterial isolation barrier and maintain aseptic operation of the tangential flow filtration loop.

[0111] In a cell perfusion application, the modular system shown in Figure 2 can be optimized and adjusted for perfusion applications in larger-scale reactors. The pump here can be a Levitronix magnetic levitation pump (such as Puralev 600 or PuraLev 2000 SU), and the tangential flow filtration device here can be a hollow fiber membrane filter with a molecular weight cutoff of 0.2–0.5 microns. It can also be a macroporous membrane tube filtration device with a molecular weight cutoff greater than 1.0 microns, such as Repligen's TFDF filter. The feed liquid can be a cell suspension from a bioreactor, such as CHO cells. It should be emphasized that the concentrated cells, in perfusion applications, are returned to the bioreactor rather than a separate container. The permeate here is a nutrient-depleted culture medium, which can contain cell-produced products, such as antibodies or viruses.

[0112] During cell perfusion operation, the reflux rate of tangential flow filtration can be 10% or less, and most of the feed liquid enters the next system or returns to the bioreactor. After passing through multiple module systems, the cell concentration of the cell suspension can be increased by 6% or more.

[0113] In applications that require aseptic operation or high cleanliness requirements, such as cell perfusion, multiple modular flow path systems connected in sequence can include multiple system components, such as valves, pump heads, and pressure sensors. The flow paths of these TFF modular systems can be prefabricated or sterilized in advance to form a closed system for aseptic processing. The flow paths of the feed liquid across different modular systems can be composed of smaller modules, which can be assembled and installed at the application site. The connection between these smaller modules can be achieved through mechanisms such as quick connectors, ferrules, and pipe welding, and aseptic operation techniques can be used if necessary.

[0114] The operation of a tangential flow filtration system, which consists of multiple modular systems, including starting and stopping the system, can be relatively complex.

[0115] As an example, the following is an illustration of a method for performing a filtration or concentration step using a plurality of modular systems. The operation of opening the filtration system modules in a sequential order is adopted here.

[0116] a) In this exemplary initial step, the feed to be treated is first introduced into the feed container of the first system module, and then the feed begins to circulate without sending the reflux to the next module system. Initially, the feed can fill the tangential flow filtration flow path with the permeate split closed. Then the permeate valve of the first system is opened, and the concentration (or filtration) operation begins, while the incoming feed is continuously supplied to the first module, and when the permeate leaves the flow loop of the first system, the incoming feed is continuously concentrated as the filtration proceeds.

[0117] b) When the filtration operation of the first module system reaches the required concentration multiple, the reflux diverter valve can be opened to allow a portion of the reflux liquid to enter the second stage system from the first stage filtration system. The flow rate target of the reflux liquid can be controlled as the feed flow rate divided by the target concentration multiple.

[0118] c) The next step is similar to the previous two steps. First, the valve that diverts the reflux liquid to the next module system is closed, and the feed liquid from the reflux liquid flow path of the previous stage is received. When the received volume is sufficient to fill the flow path, the circulation and concentration operation of the second system begins. When the second-stage system reaches the required concentration multiple, it may be necessary to open the reflux liquid diversion channel to allow a portion of the reflux liquid to enter the next-stage filtration module.

[0119] d) The previous process can be repeated until the last stage system is also started. When the last stage reaches the target concentration multiple, part of the concentrated reflux liquid is collected or discharged as the final product, by-product, or waste. At this time, the module exchange system can enter the continuous filtration production, the feed liquid continuously enters the first stage system, and finally the concentrated product and permeate are continuously exported from the system.

[0120] e) When the incoming feed is exhausted, additional flushing fluid, or buffer, can be added to flush the system to increase yield if necessary. After flushing, the channel that transfers the reflux to the next stage can be closed and the circulation pump of the first system can be turned off. The remaining feed from the first stage system can also be transferred to the next stage system through a separate pipeline (not shown in the figure for simplicity). The flushing buffer and remaining feed can be transported to the next stage system to displace the product out of the system.

[0121] f) The previous process can be repeated until the last stage of the system, the final system can be closed by installing a conventional tangential flow filtration system.

[0122] The operations disclosed here are presented only for the filtration or concentration step. When a process step involves a buffer exchange, similar procedures may be required to start and shut down the system. Typically, the first system is started up first, and then the buffer exchange is started until the desired number of buffer exchanges is reached, and then the feed solution is transferred to the next stage.

[0123] Here, only one procedure for starting the system is listed to demonstrate the operation of the system. There are many other ways to start the system. For example, one of the alternative methods of starting the process is to introduce the feed solution sequentially at the beginning of the process until the feed circulation flow channels of all module systems are completely filled. In this way, all TFF modular systems can be filled with lower concentration feed. Then, starting from the first-stage system, the circulation pump of each system and the corresponding permeate valve can be started sequentially to start the filtration operation and collect the filtrate.

[0124] During startup, the feed liquid is allowed to be introduced into the system from the feed pipe of the previous stage, and each stage begins the concentration operation. However, the final reflux liquid is not collected as a product, by-product, or waste until the feed liquid of the last module reaches the final concentration requirement. When the last stage reaches the final concentration requirement, the reflux liquid diversion valve of the last system module is opened to allow the collection of the final concentrated feed liquid and start continuous operation. At the beginning of the process, the flow rate of the final product collection may be higher and can be reduced as the membrane is fouled.

[0125] The method described above is universally applicable to modular system configurations, whether or not a modular filtration system is equipped with a feed container.

[0126] This is just an example. Different applications may have different operating steps. It is entirely possible to make adjustments and changes to the operating steps on this basis. These steps should fall within the scope of the present invention. Here, only a simple filtration operation process is listed, focusing on the concentration ratio of each stage. In specific implementations, more parameter monitoring can be designed to monitor the permeation flow, changes in the components in the feed liquid, changes in the properties of the feed liquid, etc. The present invention cannot list them one by one, but they should be considered within the scope of the present invention.

[0127] Like other process operations, modular multi-stage tangential flow filtration systems can be part of an end-to-end continuous production process. For end-to-end filtration processes, all modular systems can be pre-designed and configured and assembled at the production site. When using a one-time flow path, the complete flow path can be broken down into different flow path components, which can be assembled on site to match the modular system and shell to achieve continuous concentration and / or liquid exchange.

[0128] In some embodiments, one or more modules can be incorporated into the current batch process, such as for post-culture cell clarification, virus or protein concentration, etc., for continuous or semi-continuous operation. In addition, the number of system modules can be customized and optimized based on specific processing volume, filtration flux, feed and liquid conditions, filtration and liquid exchange, etc.

[0129] For biopharmaceutical products, the modular system included in the present invention can be used in conjunction with pre-assembled and pre-sterilized disposable flow including disposable pumps, filters, sensors and flow paths. The use of disposable flow paths can simplify installation and avoid steps such as cleaning validation. For different requirements, reusable pumps and sensors can be integrated with disposable flow paths and filters. The modular design can flexibly choose between reusable and disposable to balance cost and reusability.

[0130] It can be envisioned that aspects of the present invention represent a new filtration system design and configuration that improves upon conventional tangential flow filtration systems. Although it appears as an assembly consisting of a pump, filter, piping, and sensor, its essence is to achieve continuous filtration operation and increase throughput through a combination of multiple modular systems. This system design can help address the issues of throughput, efficiency, and flexibility that are not achievable in current batch and single-pass tangential flow filtration systems.

[0131] The present invention uses "concentration" to describe the filtration separation operation. The components concentrated here can be products, by-products, or process wastes. In many places, the concentration operation and the filtration operation are the same.

[0132] Furthermore, although preferred embodiments have been described for purposes of illustration, those skilled in the art will recognize that there are numerous possibilities for changes, additions, and adaptations to the design of the modular system, the manner in which the modular system is combined, and the convenient application of the system without departing from the spirit and scope of the invention as defined by the appended claims.

[0133] Therefore, the applicant considers that the relevant modifications, combinations and integrations included in the scope and intention of the disclosed invention are all within the scope of the disclosure of the present invention. The use of singular terms should be understood to include the plural, unless otherwise specified or the context is clear. Conjunctions connect any combination of disjunctive clauses and conjunction clauses. Therefore, terms such as "and / or" should be broadly understood as "and", "or", "and / or", except where limited by the context.

[0134] Industrial Applications

[0135] The present invention is applied in the field of continuous bioprocessing, which is critical for the production of biological products such as cultured meat, alternative proteins, and large-scale biopharmaceutical production. These industries require extremely large-scale production capacity, which is costly using traditional batch processes. The present invention introduces modular, multi-stage tangential flow filtration, which can exponentially increase production capacity while utilizing existing scale pumps and pipes, valves and other fittings to achieve continuous concentration, filtration and buffer exchange and other intensive process operations. This breakthrough system combines the advantages of single-pass and batch tangential flow filtration to achieve flexible, efficient and scalable continuous processing. Key applications that benefit include perfusion bioreactors, cell harvesting and washing, protein purification, and large-scale manufacturing of biopharmaceutical products. Implementation of these processes can greatly reduce capital and operating costs compared to large-scale batch processing systems. In addition, the modular design allows for tailored optimization and precise scaling for each application. The present invention is expected to become a widely supported continuous manufacturing platform technology for a variety of bioproduction industries with unprecedented throughput requirements.

Claims

1. A tangential flow filtration system, characterized in that: include: a. Two or more modular tangential flow filtration systems connected in series, each modular system comprising at least one tangential flow filtration device, a reflux liquid pipe section, a feed pipe, a permeate pipe and a circulation pump; and b. Inter-system feed transfer pipeline, which is used to transport part or all of the feed from the reflux liquid flow path of the previous system module or from the permeate flow path to the next modular system for sequential processing.

2. The tangential flow filtration system according to claim 1, characterized in that: Each modular system also includes one or more of the following components: Liquid tank, feed pump, buffer pump, permeate pump, pressure sensor, flow meter, mixer, back pressure valve, analyte detector, exhaust valve and pipeline connector in the circulation flow path, spectrometer and conductivity meter for online monitoring.

3. The tangential flow filtration system according to claim 1, characterized in that: Each system module includes redundant parallel modules, which allow the feed liquid to bypass one of the modules during operation. The bypass can isolate a module for operations such as membrane replacement, cleaning, or regeneration.

4. A method of tangential filtration operation, characterized in that: include: a. Providing a tangential flow filtration system according to claim 1; b. continuously introducing feed into the first modular system; c. transferring the reflux from each modular system section to the next modular system for further processing; and d. Continuously collect the permeate and the final reflux liquid; Preferably, the method further comprises introducing a buffer into the reflux loop of each modular system for liquid replacement.

5. The method of tangential filtration operation according to claim 4, characterized in that The modular tangential flow filtration system sequentially performs two or more identical or different tangential flow filtration operations in combination, wherein the tangential flow filtration operations preferably include cell separation filtration and / or clarification filtration and / or concentration and / or fluid exchange and other filtration or mixing operations.

6. A perfusion bioreactor system, characterized in that: include: a) bioreactor or fermenter systems; b) the tangential flow filtration system according to claim 1, comprising two or more modular tangential flow filtration systems connected in series, each modular system comprising at least one tangential flow filtration device, a retentate pipe section, a feed pipe, a permeate pipe and a circulation pump; the filtration device of the modular tangential flow filtration system is capable of retaining cells, such as cells or bacteria, and allowing culture medium components or smaller particle components to pass through; and c) a pipeline for transferring the cell suspension from the bioreactor or fermenter to the modular tangential flow filtration (TFF) system, a pipeline for recycling all or part of the cell suspension back to the bioreactor, and a pipeline for collecting and directing the permeate; Preferably, the cell suspension is transferred from the bioreactor or fermenter to the first stage modular tangential flow filtration system, and the last stage system recycles all or part of the cell suspension back to the bioreactor circuit.

7. The perfusion bioreactor system according to claim 6, characterized in that: The tangential flow filtration device in the system uses hollow fiber tubes, ceramic membrane tubes, stainless steel tubes, and polymer liquid tubular membranes as filtration media, and has a tube lumen diameter of 0.5-12 mm, preferably 0.5-5 mm, and a molecular weight cutoff of 50 nanometers to 5 microns; Preferably, the tangential flow filtration device in the system uses a ceramic membrane tube or a polymer tubular membrane as a filtration medium, and has a tube lumen diameter of 2-12 mm and a molecular weight cutoff of 0.2-5 μm; Preferably, the tangential flow filtration device uses a ceramic membrane or a polymer material membrane tube with a flow channel size of 1.5-12 mm and a molecular weight cutoff of 0.5-2 microns.

8. The perfusion bioreactor system according to claim 7, characterized in that: The insert is placed in the inner cavity of the membrane tube of the tangential flow filtration device to reduce the cross-sectional area, optimize the flow pattern of the feed liquid, and reduce the circulation flow rate of the feed liquid.

9. The perfusion bioreactor system according to claim 6, characterized in that: The bioreactor produces cells, proteins produced by the cells, or other biological products, and separates the cells from the cell culture medium by tangential flow filtration; Preferably, cell products, including but not limited to exosomes, proteins, viruses or other biological products, are extracted from the cell suspension by tangential flow filtration.

10. The perfusion bioreactor system according to claim 6, characterized in that: The cells are separated from the cell culture medium by tangential flow filtration, wherein the cells are mammalian cells, such as CHO cells, HEK cells, VERO cells, or other animal cells, such as muscle cells.

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