Disposable TFF unit including a tangential flow filtration (TFF) system and an integrated pumping device

Abstract

To provide a tangential flow filtration system (TFF) and a disposable TFF system that includes an integrated pump apparatus.SOLUTION: A TFF system is provided that includes a clamp assembly into which a disposable TFF unit 100, two fluid reservoirs 200 and an air supply assembly 300 are integrated. Further, the disposable TFF unit includes a diaphragm pump assembly and a concentration membrane. Further, methods of using the TFF system that includes the disposable TFF are provided.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to and claims priority to U.S. Provisional Patent Application No. 62 / 616,486, filed January 12, 2018, the entire disclosure of which is incorporated herein by reference.

[0002] The presently disclosed subject matter relates generally to methods of protein production, and more specifically to a tangential flow filtration (TFF) system and a disposable TFF unit including an integrated pumping device. [Background technology]

[0003] Protein production is a complex process that involves some combination of the use of living cells, intracellular components, chemical reactions, separation methods and filtration. The end result of this process (and many intermittent steps) is usually a very dilute solution that contains the target protein and small molecules, such as salts, that cannot be removed in previous steps. To continue the purification process or to utilize the protein for scientific or industrial purposes, the solution usually requires concentration and buffer exchange. One of the most common methods for this is ultrafiltration.

[0004] Like standard filtration, ultrafiltration involves a medium (membrane) that is permeable only to some small components of the mixture. At the same time, the pore size of the membrane is selected so that it is impermeable to the protein of interest. By applying a force (usually pressure) to the starting mixture on the sample side of the membrane, the protein remains on the sample side of the membrane while the smaller components are forced to cross the membrane, thus concentrating it. The same principle is used for buffer exchange by resuspending the concentrated protein in the desired buffer and repeating the concentration process.

[0005] When the sample flow is unidirectional, across the membrane, such a filtration arrangement is called dead-end filtration. While simple to make and use, it has significant drawbacks. As the mixing sample relies exclusively on diffusion and convection, it creates a concentration gradient in the membrane. The concentration gradient creates conditions that reduce filtration flow and reduce protein stability. Most commonly, dead-end ultrafiltration devices use centrifugal force to create pressure in the membrane. For example, a solution is placed in a tube with a membrane that separates the solution from the lower empty part of the tube. The tube is spun in a centrifuge so that the solution and small molecules are pushed through the membrane, leaving less solution and larger protein molecules in the upper part. This method is lengthy for two main reasons: (1) it is not scalable due to the discrete size of the filtration device (i.e., the largest commonly used laboratory device has a capacity of 15 mL); and (2) it is difficult to monitor because checking the progress of the filtration requires stopping the centrifuge. Failure to check the solution to ensure that it is not overtaxed will result in proteins coming out of the solution and precipitating on the membrane. Furthermore, these centrifuge tubes and membranes cannot be effectively cleaned, and therefore are generally discarded after only one use.

[0006] In contrast to dead-end filtration, tangential flow filtration (TFF) devices have a constant recirculation flow parallel to the membrane on the sample side. TFF (also known as cross-flow filtration) is a type of filtration used in protein purification. In TFF, both pressure and flow are usually created using a combination of peristaltic pumps and flow splitters. The type of membrane used is the same as in dead-end filtration. However, the main advantage of TFF is that the surface of the membrane is always cleared of proteins and the solution is mixed so that there is a consistent concentration throughout. Since there is no concentration gradient, TFF is gentler on proteins and offers better flow characteristics. Other advantages of TFF include the ease of online monitoring and scalability. The membrane can be washed and reused or discarded, while the tubing and solution containers are washed after being used. The main disadvantage of a traditional TFF setup is the dead volume of tens of mL (caused by pumps and measurements), which is not suitable for laboratory-scale use.

[0007] Therefore, there is a need for a small-scale TFF device that is efficient, expendable, has low dead volume, and can be used and monitored in an automated manner to reduce the effort associated with the ultrafiltration step of protein production. Summary of the Invention

[0008] Disclosed herein are devices, systems and methods for tangential flow filtration (TFF) applications. In one embodiment, a TFF system for concentrating a protein solution is described, comprising a TFF unit having a diaphragm pump assembly and a concentration membrane, the TFF unit being configured to concentrate protein from a protein-containing solution, an air supply assembly operably connected to the TFF unit, the TFF unit being adapted to control air flow, and a fluid reservoir assembly operably connected to the TFF unit, the TFF unit being adapted to control liquid flow. The air supply assembly is connected to the TFF unit at one end, and the fluid reservoir assembly is connected to the TFF unit at the opposite end.

[0009] The TFF unit is disposable and can be adapted for limited use and small volume concentration. The TFF unit has multiple layers. In one embodiment, the multiple layers of the TFF unit have air channels, fluid channels and flow ports for liquid flow control and air flow control, whereby the diaphragm pump assembly and the concentration membrane are between the outer layers of the multiple layers, and a chamber is formed between the multiple layers adjacent to the diaphragm pump assembly.

[0010] In one embodiment, the plurality of layers includes four acrylic layers that are laser bonded together, with the diaphragm pump assembly and the concentration membrane being between two middle layers of the plurality of layers.

[0011] The diaphragm pump assembly includes a valve component and a diaphragm component configured to aspirate or dispense a protein-containing solution through a flow port. The valve component has groups of air-actuated valves including a group of three air-actuated valves at one end of the diaphragm pump assembly and a group of two air-actuated valves at an opposite end of the diaphragm pump.

[0012] The diaphragm component includes a plurality of diaphragms. Further, the diaphragm component is a dome-shaped portion that is larger than the valve component.

[0013] The air supply assembly of the TFF system includes an air supply unit and a mass of solenoids configured to apply positive or negative pressure through the air supply unit to the diaphragm pump assembly.

[0014] The fluid reservoir assembly includes two fluid reservoirs, one for containing a protein-containing solution and the other for containing a buffer solution, each of the two fluid reservoirs includes a cap with a liquid flow connector and an air flow connector attached.

[0015] The liquid flow connector includes two tubes extending down to the contained liquid, one of the two tubes is used to aspirate liquid from the fluid reservoir and the other of the two tubes is used to return liquid to the fluid reservoir, and the air flow connector is common between the two fluid reservoirs.

[0016] The TFF system further includes a clamp assembly for holding the TFF unit, an air supply assembly, and a fluid reservoir assembly. The TFF system further includes a volume sensor for measuring a liquid level in the fluid reservoir assembly, a waste slide configured to direct the filtration product, and a waste container connected to the waste slide and configured to collect the directed filtration product.

[0017] The TFF system also includes a unit holder and a clamp handle, the unit holder having a slot for aligning the TFF unit, and the clamp handle configured to urge the unit holder and the TFF unit into the clamp assembly so that the TFF unit is connected to and sealed with the fluid reservoir assembly and the air supply assembly.

[0018] A method for making a TFF system for concentrating a protein solution includes the steps of providing components for making a TFF system, the components including a disposable TFF unit, a fluid reservoir assembly, an air supply assembly, and a clamp assembly; assembling the TFF system with the components; providing a user interface connected to the TFF system; setting parameters for a filtration process using the user interface; and initiating the filtration process.

[0019] The assembly steps include inserting the fluid reservoir assembly into the clamp assembly, securing the disposable TFF unit to the clamp assembly, and connecting the TFF disposable unit to the air supply assembly and the fluid reservoir assembly.

[0020] The method of the present invention also includes introducing a volume of a buffer solution and a volume of a protein-containing solution into the fluid reservoir assembly, whereby the filtration process includes priming the TFF system with the buffer solution and concentrating the protein from the protein-containing solution.

[0021] A method for concentrating a protein solution by TFF includes providing a TFF system including a TFF unit having a diaphragm pump assembly and a concentration membrane, the TFF unit configured to concentrate a protein from a protein-containing solution, an air supply assembly operably connected to the TFF unit, and a fluid reservoir operably connected to the TFF unit, the fluid reservoir containing a buffer and a protein-containing solution; setting pump sequence parameters for processing the protein-containing solution; beginning processing of the protein-containing solution by initiating a preparatory pump sequence; continuing to process the protein-containing solution by initiating a concentration pump sequence; continuing to process the protein-containing solution by initiating a diafiltration pump sequence; and collecting protein residue from the protein solution.

[0022] Having described the presently disclosed subject matter in general terms, reference is now made to the accompanying drawings, which are not necessarily drawn to scale. [Brief description of the drawings]

[0023] [Figure 1] 1 depicts a schematic perspective view of an example of a presently disclosed disposable TFF unit according to one embodiment of the present subject matter. [Diagram 2] 1 depicts an exploded view of an example of a presently disclosed disposable TFF unit in accordance with one embodiment of the present subject matter. [Diagram 3]1 depicts an exploded view of a disposable TFF unit combining a pair of fluid reservoirs and an air supply assembly according to one embodiment of the present subject matter. [Figure 4A-4B] 1A-1D depict plan and cross-sectional views, respectively, of a presently disclosed disposable TFF unit according to one embodiment of the present subject matter. [Diagram 5] 1 depicts various views of an example of a diaphragm pump assembly of the presently disclosed disposable TFF unit in accordance with one embodiment of the present subject matter. [Figure 6] 1 shows a schematic diagram of a disposable TFF unit and an example of a basic filtration operation of a TFF system according to one embodiment of the present subject matter. [Figure 7] 1 depicts a block diagram of the presently disclosed TFF system including three modes of operation (or pumping sequences) for performing a filtration cycle according to one embodiment of the present subject matter. [Figure 8A] 8 illustrates a block diagram showing the preparatory pump sequence of the TFF system shown in FIG. [Figure 8B] FIG. 8 illustrates a block diagram showing the concentrate pump sequence of the TFF system shown in FIG. [Figure 8C] 8 illustrates a block diagram showing the diafiltration pump sequence of the TFF system shown in FIG. [Figure 9] 1 depicts a schematic perspective view of a portion of the presently disclosed TFF system, including an air supply assembly and a clamping assembly, according to one embodiment of the present subject matter. [Figure 10] 1 depicts a schematic perspective view of a portion of the presently disclosed TFF system, including an air supply assembly, a clamping assembly, a volume sensor, a waste slide, and a waste container, according to one embodiment of the present subject matter. [Figure 11] 1 depicts an exploded view of all the components of the presently disclosed TFF system, including the presently disclosed disposable TFF unit according to one embodiment of the present subject matter. [Figure 12] 1 depicts a side view of the presently disclosed TFF system when fully assembled, including the presently disclosed TFF system according to one embodiment of the present subject matter. [Figure 13] 1 depicts a flow chart of an embodiment of a method for using a TFF system including a disposable TFF unit. [Figure 14] 1 depicts a flow chart of an embodiment of a method for using a TFF system including a disposable TFF unit. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The presently disclosed subject matter will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all, embodiments of the presently disclosed subject matter are shown. Like numerals refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter described herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Consequently, it should be understood that the presently disclosed subject matter is not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0025] In some embodiments, the presently disclosed subject matter provides a disposable tangential flow filtration (TFF) unit including a TFF system and an integrated pump device. That is, a TFF system is provided including a clamp assembly, two fluid reservoirs, and an air supply assembly that integrates the disposable TFF unit. Further, the disposable TFF unit includes a diaphragm pump assembly and a concentration membrane.

[0026] Embodiments of the presently disclosed TFF units are disposable and use small volumes to utilize the TFF method without the need for extensive cleaning after each use. Thus, the presently disclosed disposable TFF units offer advantages over conventional TFF systems, which imply relatively large volumes (>1 L), are bulky, and must be cleaned after each use.

[0027] 1 and 2, which are schematic perspective and exploded views, respectively, of an embodiment of a disposable TFF unit 100 of the presently disclosed TFF system. As depicted, the disposable TFF unit 100 includes an integrated diaphragm pump assembly 110 for concentrating a protein solution with an integrated concentration membrane 120 by a process known as tangential flow filtration (TFF). TFF is a process in which a protein solution flows past a membrane under pressure such that small, non-protein molecules are filtered as the filtration product, and proteins and less solution are retained in the system as a retentate.

[0028] In one embodiment, the disposable TFF unit 100 is made of multiple layers. For example, the disposable TFF unit 100 includes four layers 106 (i.e., layers 106a, 106b, 106c, and 106d). In one example, the four layers 106 are acrylic layers. However, in other embodiments, the four layers 106 can be any material not inconsistent with the objectives of the present disclosure. Furthermore, the disposable TFF unit 100 can be formed by laser bonding the layers 106 together. For example, the laser bonding process can use a dye that absorbs a specific wavelength of light output by a laser to heat the bonding portions of the two layers and melt them together.

[0029] 2, the diaphragm pump assembly 110 and the concentrator membrane 120 are provided between the outer layers 106a and 106d, and more specifically between layers 106b and 106c, which are the middle two layers of the disposable TFF unit between the outer layers 106a and 106d. The layers 106 include interference features that create a seal that allows air to actuate the valves and diaphragms to pump liquid around the disposable TFF unit 100 and over the concentrator membrane 120. In particular, the disposable TFF unit 100 is formed with multiple layers 106 having various air channels 101, various fluid channels 102, the concentrator membrane 120, and the diaphragm pump assembly 110. The diaphragm pump assembly 110 (see FIG. 5) includes an actuated valve component including a group of air actuated valves 111, and a diaphragm component including a diaphragm 112 for pumping liquid. Input / output flow ports (through holes) 103 are included in the connections for air flow control. Additionally, layer 106 includes input / output flow ports (through holes) 104 that can be used in fluid ports for liquid flow control.

[0030] 3, an exploded view of one embodiment of a disposable TFF unit 100 combining a fluid reservoir assembly including a pair of fluid reservoirs 200 and an air supply assembly 300. In this example, the disposable TFF unit 100 is connected to the air supply assembly 300 at one end and two fluid reservoirs 200 at the opposite end. The fluid reservoirs 200 are, for example, two 50 mL Falcon tubes. In one example, one fluid reservoir 200 contains a protein solution while the other fluid reservoir 200 contains a buffer solution. Each of the fluid reservoirs 200 includes a custom cap 210, a standard Falcon tube 220, and a small liquid tube (not shown) that is attached to the cap 210 and extends down to the liquid. Two types of gaskets (e.g., tube gasket 230 and air supply gasket 320) are used to seal the connections of the disposable TFF unit 100, the fluid reservoirs 200, and the air supply assembly 300. The air supply assembly 300 includes an air supply unit 310 and a number of solenoids 330 that are used to actuate various portions of the disposable TFF unit 100 .

[0031] 4A and 4B, which are plan and cross-sectional views, respectively, of the disposable TFF unit 100. That is, FIG. 4B is a cross-sectional view of the disposable TFF unit 100 taken along line AA in FIG. 4A. Now referring again to FIG. 5, various views of an embodiment of a diaphragm pump assembly 110 are shown. The diaphragm pump assembly 110 includes a group of actuated valves 111 and a diaphragm 112, as shown in FIG. 5. For example, the diaphragm pump assembly 110 includes a group of air-actuated valves 111 (e.g., five air-actuated valves 111) and one diaphragm 112. However, various combinations of air-actuated valves 111 and diaphragms 112 can be used to provide more or less control over how liquid is circulated within the disposable TFF unit 100. For example, the presence of multiple diaphragms 112 to generate pumping action can provide a more consistent flow compared to using only a single diaphragm 112.

[0032] In operation, one embodiment of the disposable TFF unit 100 pumps liquid using air actuated positive displacement. A mass of solenoids 330 (see FIG. 3) is used through the air supply unit 310 to apply positive or negative pressure to one side of the air actuated valves 111 and the diaphragm 112 to open or close them. The air actuated valves 111 are a group of small areas that allow or prohibit flow between two flow ports (through holes). In one embodiment, three actuated valves 111 are grouped at one end of the diaphragm pump assembly 110 and two actuated valves 111 are grouped at the opposite end. The diaphragm 112 is a dome-shaped portion of the diaphragm pump assembly 110 that is larger than the actuated valves 111 and that moves in and out of the chamber 107 adjacent to the diaphragm 112 (see FIG. 4) and draws or dispenses liquid through the flow ports (through holes). The driving air pressure applied to the air-actuated valves 111 and diaphragms 112 through the air channels 101 is higher than the internal pressure to create an equal pressure difference between the vacuum, the internal pressure and the driving pressure. Different sequences of opening and closing the air-actuated valves 111 and diaphragms 112 are used to pump liquid from either liquid reservoir 200 through the fluid channels 102 and in various ways throughout the disposable TFF unit 100.

[0033] Additionally, Figure 6 shows a schematic diagram of an embodiment of a basic filtration operation supported by the disposable TFF unit 100 and / or TFF system 105 described hereinafter with reference to Figures 7-12. In Figure 6, a combination of circulation and internal pressure above atmospheric pressure forces a volume of solution through the membrane, leaving proteins and remaining solution behind. The solution that passes through the membrane is referred to as the "filtration product." The remaining solution that does not pass through the membrane is referred to as the "retentate."

[0034] FIG. 7 shows a block diagram of one embodiment of a TFF system 105, including the layout of all of the diaphragm pump assembly 110, the concentrate membrane 120, the protein fluid reservoir 200, and the buffer fluid reservoir 200. Additionally, the TFF system 105 provides three modes of operation (or pump sequences) for performing a filtration cycle. For example, FIG. 8A, FIG. 8B, and FIG. 8C show block diagrams illustrating the preparatory pump sequence, the concentrate pump sequence, and the diafiltration pump sequence, respectively, of the TFF system 105 shown in FIG. 7. In FIG. 8A, FIG. 8B, and FIG. 8C, the arrows indicate the direction of flow for each of the pump sequences.

[0035] The solutions in the two fluid reservoirs 200 are pumped in a number of ways. Air channel 101 from air supply unit 310 pressurizes the entire liquid system (i.e., Falcon tube 220 and disposable TFF unit 100) to the desired pressure. A combination of circulation and internal pressure above atmospheric pressure forces the solution through the membrane (i.e., filtration product) while the proteins and remaining solution (i.e., retentate) remain behind, as previously shown and described in FIG. 6.

[0036] Now, referring back to FIG. 3, there are two liquid connections and one air connection on each of the caps 210. The caps 210 of the fluid reservoirs 200 each typically have two small diameter tubes (not shown) attached to them that extend down to the liquid. Generally, one connection / tube is used to aspirate liquid from the fluid reservoir 200 and the other is used to return liquid to the fluid reservoir 200. Depending on the particular pump sequence, these steps will vary. The air connection to each tube is common between them and pressurizes the air and liquid in both tubes, pressurizing the channels of the disposable TFF unit 100.

[0037] Typically, one fluid reservoir 200 contains a protein solution and the other fluid reservoir 200 contains a buffer solution, but both fluid reservoirs 200 can contain a protein solution. The buffer solution is used to first prime the TFF system 105 (see FIG. 8A) to remove air bubbles from the fluid channels 102. The protein solution is circulated under pressure until it is concentrated to a desired amount (see FIG. 8B). The buffer solution is used to diafilter or dilute the protein (see FIG. 8C) if desired, before another concentration cycle. If not, the TFF system 105 pumps the remaining protein solution back to the protein fluid reservoir 200 and notifies the user that the cycle is over. In some embodiments, the disposable TFF unit 100 is intended to be used for up to three 50 mL concentration cycles of the same type of protein solution, while in other embodiments, the disposable TFF unit 100 can be used for up to four, five, or six 50 mL concentration cycles of the same type of protein solution. After such use, the disposable TFF unit 100 is discarded and the cap 210 is either discarded or cleaned.

[0038] 9, 10, 11, and 12, an example of a TFF system 105 including the presently disclosed disposable TFF unit 100 is shown. FIG. 9 is a schematic perspective view of a portion of the TFF system 105 including the air supply assembly 300 and the clamp assembly 400. FIG. 10 is a schematic perspective view of a portion of the TFF system 105 including the air supply assembly 300 and the clamp assembly 400, and further including one or more volume sensors 410, a waste slide 420, and a waste container 430. FIG. 11 is an exploded view of all the components of one embodiment of the TFF system 105. FIG. 12 is a side view of one embodiment of the TFF system 105 when fully assembled.

[0039] The TFF system 105 includes a clamp assembly 400, pressure and vacuum pumps, electronics, a touch screen for setting parameters for operation, and other components not shown. In one embodiment, the clamp assembly 400 is a consumable clamp assembly. The clamp assembly 400 is an aluminum structure used to hold two fluid reservoirs 200, a disposable TFF unit 100, an air supply assembly 300, and a volume sensor 410 that measures the liquid level in the fluid reservoirs 200, and a waste slide 420 that collects the filtration product and directs it to a waste container 430.

[0040] 9, 10, 11, and 12 show the progression of parts of the clamp assembly 400 to clarify the number of parts of one embodiment of the overall TFF system 105. FIG. 9 shows the clamp assembly 400 and the air supply assembly 300. FIG. 10 shows the volume sensor 410, unit holder 440, clamp handle 450, waste slide 420, and waste container 430 added to the assembly. FIG. 11 shows the fluid reservoir 200 and disposable TFF unit 100, which are preferably installed in that order in the clamp assembly 400. As shown in FIG. 12, the disposable TFF unit 100 is held in the unit holder 440 with matching slots that keep the disposable TFF unit 100 aligned. The clamp handle 450 is then rotated 90° toward the rear of the clamp assembly 400. Rotating the clamp handle 450 uses a cam-type actuation to urge the unit holder 440, and therefore the disposable TFF unit 100, downward so that the disposable TFF unit 100 contacts and seals against the air delivery assembly 300 and fluid reservoir 200. The unit holder 440 is spring loaded so that when the clamp handle 450 is pulled up, the unit holder 440 springs back up.

[0041] 13, there is shown a flow chart of an embodiment of a method for using the TFF system 105, namely, method 500. Method 500 may include, but is not limited to, one or more of the following steps:

[0042] In step 510, the components of the TFF system 105 are provided. For example, a disposable TFF unit 100, a fluid reservoir 200, an air supply assembly 300, and a clamp assembly 400 including a volume sensor 410, a waste slide 420, and a waste container 430 are provided. In one embodiment, one fluid reservoir 200 contains a protein solution while the other fluid reservoir 200 contains a buffer solution. In another embodiment, both fluid reservoirs 200 contain a protein solution.

[0043] In step 515, the components of the TFF system 105 are assembled. For example, a user inserts two fluid reservoirs 200 into the clamp assembly 400. The user then inserts a disposable TFF unit 100 into the clamp assembly 400. The user then engages the clamp handle 450 to secure the disposable TFF unit 100 to the clamp assembly 400. The user then operably connects the TFF disposable unit 100 to the air delivery assembly 300 and the fluid reservoirs 200.

[0044] In step 520, the user interacts with a user interface, such as a touch screen, of the TFF system 105 to set parameters for the filtration.

[0045] In step 525, a user interacts with the user interface of the TFF system 105 to begin the filtration process.

[0046] Upon completion of method 500, the user reverses the order of operations in step 515 to remove the components of TFF system 105 and discards disposable TFF unit 100 or saves it for later use.

[0047] 14, there is shown a flow chart of another embodiment of a method for using the TFF system 105, namely method 600. Method 600 can include, but is not limited to, one or more of the following steps.

[0048] In step 610, the components of the TFF system 105 are provided. For example, a disposable TFF unit 100, a fluid reservoir 200, an air supply assembly 300, and a clamp assembly 400, further including a volume sensor 410, a waste slide 420, and a waste container 430, are provided. In one embodiment, one fluid reservoir 200 contains a protein solution while the other fluid reservoir 200 contains a buffer solution. In another embodiment, both fluid reservoirs 200 contain a protein solution.

[0049] In step 615, the components of the TFF system 105 are assembled. For example, a user inserts two fluid reservoirs 200 into the clamp assembly 400. The user then inserts a disposable TFF unit 100 into the clamp assembly 400. The user then engages the clamp handle 450 to secure the disposable TFF unit 100 to the clamp assembly 400. The user then operatively connects the disposable TFF unit 100 to the air supply assembly 300 and the fluid reservoirs 200.

[0050] In step 620, processing of the protein solution begins by initiating a preparatory pump sequence, for example as shown in FIG. 8A.

[0051] In step 625, processing of the protein solution continues by initiating a concentrate pump sequence, for example as shown in FIG. 8B.

[0052] In step 630, processing of the protein solution continues by initiating a diafiltration pump sequence, for example as shown in FIG. 8C.

[0053] In step 635, the protein residues are collected from the protein solution.

[0054] Upon completion of method 600, the user reverses the order of operations in step 615, removes the components of TFF system 105, and discards disposable TFF unit 100 or saves it for later use.

[0055] Following long-standing patent law convention, the terms "a," "an," and "the" refer to "one or more" when used in this application, including the claims. Thus, for example, a reference to "an object" includes a plurality of objects unless the context clearly indicates the contrary (e.g., a plurality of objects).

[0056] Throughout this application and the claims, the terms "comprise" and "comprising" are used in a non-exclusive sense, except where the context requires otherwise. Similarly, the term "include" and its grammatical variations are intended to be non-limiting, such that the recitation of items in a list does not exclude other similar items that may be substituted for or added to the listed items.

[0057] For purposes of this specification and the appended claims, unless otherwise noted, all numbers expressing amounts, sizes, dimensions, proportions, shapes, forms, parameters, percentages, quantities, properties, and other numerical values ​​used in the specification and claims are to be understood as being modified in all instances by the term "about," even though the term "about" may not be expressly expressed in a value, amount, or range. Thus, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are not and need not be precise, but can be approximated and / or larger or smaller as desired, reflecting tolerances, conversion factors, rounding, errors, and the like, as well as other factors known to those of ordinary skill in the art depending upon the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term "about," when referring to a value, is meant to include variations from the specified amount of ±100%, in some embodiments ±50%, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1%, as such variations are appropriate for practicing the disclosed methods or using the disclosed compositions.

[0058] Additionally, the term "about," when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including every number in the range, and modifying that range by extending to boundaries above and below the numerical values ​​set forth. The recitation of numerical ranges by endpoints includes, for example, integers, including fractions thereof, contained within that range (e.g., recitation of 1 to 5 includes 1, 2, 3, 4, 5 and fractions such as 1.5, 2.25, 3.75, 4.1, etc.) and any range within that range.

[0059] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it should be understood that certain changes and modifications may be practiced by those skilled in the art within the scope of the appended claims.

Claims

1. 1. A tangential flow filtration (TFF) system for concentrating a protein solution, comprising: a TFF unit having multiple layers, the TFF unit having a diaphragm pump assembly and a concentration membrane configured to concentrate proteins from a protein-containing solution; an air supply assembly operably connected to one end of the TFF unit, the TFF unit adapted to control air flow; and A tangential flow filtration system comprising: a fluid reservoir assembly operably connected to another end of the TFF unit at an end opposite the air supply assembly, the TFF unit adapted to control liquid flow, the fluid reservoir assembly including two fluid reservoirs: a first fluid reservoir for containing a protein-containing solution and a second fluid reservoir for containing a buffer solution.

2. 10. The TFF system of claim 1, wherein the TFF unit is disposable and adapted for use for up to three 50 mL concentration cycles of the same type of protein-containing solution.

3. the layers of the TFF unit have air channels, fluid channels, and flow ports for liquid flow control and air flow control; the diaphragm pump assembly and the concentration membrane are between an outer layer of the plurality of layers; a chamber is formed between the diaphragm pump assembly and the adjacent layers; 10. The TFF system of claim 1, wherein the diaphragm pump assembly draws fluid into or dispenses fluid from the chamber.

4. the plurality of layers includes four laser bonded layers; 4. The TFF system of claim 3, wherein the diaphragm pump assembly and the concentration membrane are between two middle layers of the plurality of layers.

5. 4. The TFF system of claim 3, wherein the diaphragm pump assembly includes a valve component and a diaphragm component configured to aspirate or dispense a protein-containing solution through the flow port.

6. 6. The TFF system of claim 5, wherein the valve components have groups of pneumatically actuated valves including a group of three pneumatically actuated valves at one end of the diaphragm pump assembly and a group of two pneumatically actuated valves at an opposite end of the diaphragm pump.

7. The TFF system of claim 5 , wherein the diaphragm component includes a plurality of diaphragms.

8. 6. The TFF system of claim 5, wherein the diaphragm component is a dome-shaped portion that is larger than the valve component.

9. 6. The TFF system of claim 5, wherein the air supply assembly includes an air supply unit and a mass of solenoids configured to apply positive or negative pressure through the air supply unit to the diaphragm pump assembly.

10. 10. The TFF system of claim 1, wherein each of the two fluid reservoirs includes a cap having a liquid flow connector and an air flow connector attached thereto.

11. the liquid flow connector includes two tubes extending downwardly into the protein-containing solution and the buffer solution; one of the two tubes is used to aspirate the protein-containing solution or the buffer solution from the fluid reservoir; the other of the two tubes is used to return the protein-containing solution or the buffer to the fluid reservoir; The TFF system of claim 10 , wherein the air flow connector is common between the two fluid reservoirs.

12. 10. The TFF system of claim 1, further comprising a clamp assembly for holding the TFF unit, the air supply assembly, and the fluid reservoir assembly.

13. 13. The TFF system of claim 12, further comprising a volume sensor for measuring a liquid level in the fluid reservoir assembly, a waste slide configured to direct a filtration product, and a waste container connected to the waste slide and configured to collect the directed filtration product.

14. In addition, it is equipped with a unit holder and a clamp handle. the unit holder has a slot for aligning the TFF unit; 14. The TFF system of claim 13, wherein the clamp handle is configured to urge the unit holder and the TFF unit into the clamp assembly such that the TFF unit is connected to and sealed with the fluid reservoir assembly and the air supply assembly.

15. (a) providing components for making a TFF system, the components including a disposable TFF unit, a fluid reservoir assembly, an air supply assembly, and a clamp assembly; the TFF unit having a diaphragm pump assembly and a concentration membrane and configured to concentrate proteins from a protein-containing solution; the air supply assembly is operably connected to one end of the TFF unit; providing a component, the fluid reservoir assembly being operably connected to the other end of the TFF unit; (b) assembling the TFF system with the components; (c) providing a user interface connected to the TFF system; (d) setting parameters of a filtration process using the user interface; (e) initiating said filtration process.

16. 16. The method of claim 15, wherein the assembling step comprises the steps of: inserting the fluid reservoir assembly into the clamp assembly; securing the disposable TFF unit to the clamp assembly; and connecting the TFF disposable unit to the air supply assembly and the fluid reservoir assembly.

17. further comprising introducing into said fluid reservoir assembly a volume of a buffer solution and a volume of a protein-containing solution; 16. The method of claim 15, wherein the filtration process comprises priming the TFF system with the buffer and concentrating the protein from the protein-containing solution.

18. (a) a TFF unit having a diaphragm pump assembly and a concentration membrane, the TFF unit configured to concentrate a protein from a protein-containing solution; an air supply assembly operably connected to one end of the TFF unit; providing a TFF system comprising: a fluid reservoir operably connected to another end of the TFF unit at an end opposite the air supply assembly, the fluid reservoir containing a buffer and a protein-containing solution; (b) setting pump sequence parameters for processing the protein-containing solution; (c) beginning processing of the protein-containing solution by initiating a warm-up pump sequence; (d) continuing to process the protein-containing solution by initiating a concentrate pump sequence; (e) continuing to process the protein solution by initiating a diafiltration pump sequence; (f) collecting protein residues from said protein solution.

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