Membrane sealing layer and spacer ring for viral clearance chromatography devices

Abstract

The chromatography device has a housing with an inlet and an outlet. At least two media layers are disposed within the housing between the inlet and outlet, at least one of the layers having a functionalized layer. An optional spacer ring disposed between the two media layers forms an air gap therebetween. A non-functionalized sealing layer is disposed within the housing between the inlet and outlet as the last layer of media in the media stack within the housing as fluid passes from the inlet to the outlet through the media stack. A margin of the sealing layer contacting the housing is compressed by the housing to form a compression seal to prevent fluid from leaking past the compression seal to the outlet.

Description

[Background technology]

[0001] In the bioprocess industry, there are several examples of viral contamination of recombinant proteins, vaccines, and plasma products. The source of contamination can be either exogenous, i.e., raw materials, or environmental, or endogenous, i.e., expressed within cells or retrovirus-like particles (RVLPs). Cell lines such as Chinese hamster ovary fibroblast cell lines (CHO) contain retroviral sequences in their chromosomes, which can result in the loss of numerous RVLPs. To protect against viral contamination, biopharmaceutical manufacturers often employ multi-layered strategies that include testing the viral safety of raw materials, testing in-process and in-finish products, and implementing viral clearance technologies. [Overview of the project]

[0002] Biopharmaceutical manufacturers often rely on two or more virus clearance techniques, which may include virus inactivation by maintaining a low pH, using solvents or detergents, removing viruses by heat, radiation or ultraviolet light, precipitation, chromatography, and / or filtration. Chromatographic virus removal may involve the use of functionalized chemicals that adsorb viruses and virus-like particles (VLPs). Viruses and VLPs are typically 15–400 nm in size. Membrane chromatography devices intended for virus removal may utilize membranes coated, grafted, or otherwise functionalized with specific functionalized chemicals to adsorb viruses and VLPs. To ensure effective removal of viruses and VLP contaminants, membrane chromatography devices must be very well sealed at the interface between the membrane layer and the housing.

[0003] Experiments have shown that achieving a viral clearance of approximately 7 logarithmic reductions (LRVs) by sealing membranes containing functionalized chemicals is extremely difficult. 7 LRVs corresponds to 99.99999% viral removal. While regulatory bodies do not currently specify the amount of virus that needs to be removed during processing, the industry targets a cumulative removal of approximately 12–15 LRVs for endogenous viruses and approximately 6–8 LRVs for exogenous viruses, which can potentially be achieved with two or more viral clearance procedures. A single viral clearance procedure is generally considered effective if it achieves 4 LRVs or more.

[0004] In flat-sheet chromatography devices, the edges of the membrane or medium are often sealed by compression. This creates localized zones near the edges where the permeability of the membrane or medium can be reduced. In the case of membranes with functionalized chemicals, the membrane morphology changes when interacting with viral solutions. Therefore, devices with functionalized membranes may not be effectively sealed to the housing by compression as desired. This can result in a lower logarithmic reduction value when challenged with a viral solution.

[0005] The present invention relates to a sealing layer located as the last layer through which a fluid passes from inlet to outlet within a stack of at least two media layers inside a chromatography device. This sealing layer is in contact with the housing, and at least a portion of the periphery of the sealing layer forms a compression seal within the device. The sealing layer is a “non-functionalized” layer as defined herein. A “functionalized” layer as defined herein does not function well as a compression seal, and it has been found that when this is the last layer in contact with the housing for sealing, the LRV of the chromatography device is lower than that of a similar configuration chromatography device having the same functionalized layer structure, but differing in that a sealing layer is added as the last layer in contact with the housing.

[0006] Accordingly, in one embodiment, the present invention relates to a chromatography device having a housing having an inlet and an outlet; at least one functionalized media layer disposed between the inlet and the outlet inside the housing; a non-functionalized sealing layer disposed between the inlet and the outlet inside the housing as the final media layer in the media stack inside the housing as the fluid passes through the media stack from the inlet to the outlet; and a margin of the sealing layer in contact with the housing, which is compressed by the housing to form a compression seal, preventing the fluid from passing through the compression seal and leaking out.

[0007] The present invention also relates to spacer rings between medium layers within a chromatography device for increasing the dynamic binding capacity of the chromatography device. The function of the spacer rings is to provide a gap between the previous medium layer and the next medium layer in the direction of fluid flow within the chromatography device.

[0008] While we do not wish to be bound by theory, voids are thought to help prevent challenge fluids from tunneling prematurely through only the center of the medium by allowing the liquid to disperse more quickly to the edges of the medium. Voids can bring the fluid flow directly to the edges of the medium, rather than relying on capillary action to move the fluid to the edges of the medium. Furthermore, certain media, such as functionalized nonwovens, may swell upon contact with liquid, and this swelling can lead to undesirable tunneling that prevents the fluid from dispersing to the edges of the medium. By slightly separating the layers, voids can be provided that allow liquid leaving one medium layer to flow laterally before entering the next medium layer. Moreover, voids can provide space to accommodate the swelling of the functionalized medium, which, if not accommodated, can cause high compressive stress within the medium layers as adjacent medium layers swell relative to each other. Thus, the center of the medium may expand, and more fluid may flow through it.

[0009] Thus, in another embodiment, the present invention is directed to a chromatography device having a housing with an inlet and an outlet, and at least two media layers disposed between the inlet and the outlet inside the housing, wherein at least one of the media layers has a functionalized layer, and a spacer ring disposed between the two media layers to form a void therebetween.

[0010] When simultaneously presenting the problem of increasing both the dynamic binding capacity and the LRV of a chromatography device, it was particularly effective to use both at least one spacer ring and a sealing layer in contact with the housing as the last layer through which the fluid passes within the chromatography device.

[0011] Thus, in one embodiment, the present invention is directed to a chromatography device having a housing with an inlet and an outlet, and at least two media layers disposed between the inlet and the outlet inside the housing to form a media stack, wherein at least one of the media layers includes a functionalized layer, a spacer ring disposed between the two media layers to form a void therebetween, a non-functionalized sealing layer disposed between the inlet and the outlet inside the housing as the last media layer within the media stack in the housing when the fluid passes through the media stack from the inlet to the outlet, and a margin of the sealing layer in contact with the housing, which is compressed by the housing to form a compression seal to prevent the fluid from leaking through the compression seal to the outlet.

Brief Description of the Drawings

[0012] <000085> [Figure 1] A front view of one embodiment of a chromatography device. [Figure 2] A top view of the chromatography device of FIG. 1. [Figure 3] A bottom view of the chromatography device of FIG. 1. [Figure 4] A perspective view of the chromatography device of FIG. 1. [Figure 5] It is a cross-sectional view of a chromatography device along 5-5 of FIG. 2. [Figure 6] It is a cross-sectional view of the chromatography device shown in FIG. 5, showing another embodiment. [Figure 7] It is a cross-sectional view of the chromatography device shown in FIG. 6 before ultrasonic welding of the upper housing and the lower housing. [Figure 8] It is a view of a chromatography device having a media laminate including a layer of functionalized non-woven fabric, a layer of functionalized non-woven fabric, and a layer of functionalized membrane in the direction from the inlet to the outlet. [Figure 9] It is a view of a chromatography device having a media laminate including a layer of functionalized non-woven fabric, a layer of functionalized non-woven fabric, a spacer ring, and a layer of functionalized membrane from the inlet to the outlet. [[ID=​​​​​​​​​​​​​​​​This is a diagram of a chromatography device having a media laminate comprising layers of functionalized nonwoven fabric, spacer rings, layers of functionalized nonwoven fabric, spacer rings, layers of functionalized film, and a sealing layer from the inlet to the outlet. [Modes for carrying out the invention]

[0013] Throughout this document, values ​​expressed in range form should be interpreted flexibly to include not only the numerical value explicitly stated as the limit of the range, but also all individual numerical values ​​or subranges within that range, as if each numerical value and subrange were explicitly stated. For example, the range "approximately 0.1% to approximately 5%" or "approximately 0.1% to approximately 5%" should be interpreted to include not only approximately 0.1% to approximately 5%, but also the individual values ​​within the indicated range (e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%). The notation "approximately X to Y" is equivalent to "approximately X to approximately Y" unless otherwise indicated. Similarly, the notation "approximately X, Y, or approximately Z" is equivalent to "approximately X, approximately Y, or approximately Z" unless otherwise indicated.

[0014] In this document, the terms “a,” “an,” or “the” are used to include one or more unless otherwise indicated by the context. The term “or” is used to mean nonexclusive “or” unless otherwise indicated. The phrases “at least one of A and B” or “at least one of A or B” are synonymous with “A, B, or A and B.” In addition, any expressions or terms used herein that are not specifically defined should be understood to be for illustrative purposes only and not to be restrictive. Any use of section headings is intended to aid the reading of this document and should not be interpreted as restrictive, and the information related to a section heading may be found within or outside that particular section.

[0015] When used herein, the term “about” may allow for variability in a value or range. For example, within 10%, 5%, or 1% of the stated value or the limit of a stated range, and including the stated value or range itself.

[0016] As used herein, the term “substantial” means a large or almost complete amount, such as at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or at least about 99.999%, or 100%. As used herein, the term “substantial” may mean that the amount of material present does not affect the material properties of the composition, such that the composition contains a small amount of material, such as about 0% to about 5% by weight, or about 0% to about 1% by weight, or about 5% by weight or less, or about 4.5% by weight, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001% by weight or less.

[0017] As used herein, “layer” means a material of a certain thickness through which a fluid being processed passes, and all material within the layer is formed from the same material. A layer may be a monolithic layer formed from the same material of a certain thickness. Alternatively, a layer may have one or more individual material plies that are stacked on top of each other within the layer to form the thickness of the layer. For example, one layer of a typical facial tissue is often a tissue paper material made from two individual tissue paper plies placed in face-to-face contact, and the two individual plies are generally held together by weak mechanical bonds in the form of crimp lines, so that they can be easily separated from each other.

[0018] As used herein, one or more “ply” refers to a single material of a certain thickness that can be processed into a layer by conventional deformation operations, such as, but not limited to, winding, folding, cutting, or stacking. Often, a ply is a material of a certain thickness after the forming process has been completed on a web manufacturing machine. One or more plies are then stacked to form a layer. For example, a nonwoven fabric can be produced as a single ply on a forming machine and wound onto a roll. The nonwoven roll can then be unwound and folded in half transversely by a folding plate as it passes through a deformation machine in the longitudinal direction, and then the two-ply layer can be cut by a cutting die into a disc to form a circular layer of nonwoven fabric material having two separate plies.

[0019] As used herein, “functionalized layer” is a layer that adsorbs target particles or molecules by attractive forces, such as electrostatic forces, resulting from the presence of one or more chemical moieties, ligands, or functional groups on the surface of the layer, distinct from the material forming the majority of the layer, primarily providing the structural shape and integrity of the layer. The chemical moieties, ligands, or functional groups are specifically intended to adsorb target particles or molecules onto the surface of the functionalized layer. The functionalized layer may be prepared by coating or grafting ligands, monomers, or polymers designed to molecularly adsorb target particles or molecules onto a porous layer. Alternatively, the functionalized layer may be prepared by providing a surface-modifying polymer or chemical moiety in the formulation used to prepare such a layer, which localizes on the surface of the layer during formation, so that chemical groups designed to adsorb target particles or molecules are present on the surface of the layer. In some embodiments, the attractive forces between functional groups on the surface of the functionalized layer are electrostatic forces, and the chemical moieties, ligands, or polymers present on the surface of the functionalized layer are electrostatically charged. The functionalized layer may have a positive charge and adsorb negatively charged particles, i.e., anion exchange chromatography, or the functionalized layer may have a negative charge and adsorb positively charged particles, i.e., cation exchange chromatography. In other embodiments, the attractive force may be van der Waals forces, and the target particles or molecules are adsorbed to the functional groups on the surface of the functionalized layer due to relative concentrations or deficiencies in polarizing or hydrogen bonding (i.e., hydrophobic interaction chromatography). Furthermore, the attractive force may include a combination of electrostatic and van der Waals forces (i.e., mixed-mode chromatography). Suitable functionalized materials for the functionalized layer of chromatography devices are manufactured by Pall, Millipore, and Sartorious and are sold under the brands Mustang® Q, NTARFLO® HD-Q, and Sartoband® Q. Suitable functionalized layers for use in chromatography devices may be nonwoven fabrics, films, or other suitable materials.Preferred functionalized nonwoven materials have been developed by 3M Company and are disclosed in U.S. Patent No. 9,821,276, entitled "Nonwoven Article Grafted with Copolymer." Preferred functionalized films have been developed by 3M Company and are disclosed in U.S. Patents No. 9,650,470 and 10,017,461, entitled "Method of Making Ligand Functionalized Substrates." All three of the patents mentioned are incorporated herein by reference in their entirety.

[0020] As used herein, “non-functionalized layer” is a layer that does not contain any coatings, grafts, or surface-localized adsorbent chemical moieties (e.g., electrostatically charged chemical moieties, ligands, or functional groups) that are different from the material that forms the majority of the layer.

[0021] As used herein, “media laminate” refers to all the material layers through which the fluid being processed passes as it moves from the inlet through the housing to the outlet.

[0022] As used herein, “membrane” refers to a synthetic liquid-permeable membrane comprising a material sheet having a plurality of pores or a network of interconnected pores that allow fluid to pass through the membrane. Such membranes generally include polymer membranes prepared by a phase inversion process in which a homogeneous solution of one or more polymers in a suitable solvent or combination of solvents undergoes phase separation to form a porous structure. Phase separation can be brought about by introducing a film of the homogeneous solution into a non-solvent liquid bath (known as diffusion-induced phase separation), introducing it into a non-solvent atmosphere (known as vapor-induced phase separation), or changing the temperature of the homogeneous solution (known as thermal-induced phase separation). Alternatively, pores can also be formed within the polymer sheet by a stretching process or an irradiation process (track-etched membrane). Membranes can have pore diameters of about 0.1 to about 20 micrometers (microporous membranes) or less than about 0.1 micrometers (ultramicroporous membranes). Suitable polymers for film formation include cellulose acetate, nitrocellulose, cellulose esters, polysulfones including bisphenol A polysulfone and polyethersulfone, polyacrylonitrile, polyamides (e.g., nylon-6 and nylon-6,6), polyimide, polyethylene, polypropylene, polytetrafluoroethylene, polyvinylidene fluoride, polyvinyl chloride, and ethylene-chlorotrifluoroethylene copolymers.

[0023] As used herein, “Dynamic Binding Capacity (DBC)” means the mass of target molecules captured by the medium layer from the challenge solution at a specified flow rate, depending on the layer projection area, with an endpoint defined as a specified concentration of target molecules detected in the device drain. Therefore, if the chromatography device has three medium layers between the inlet and outlet, the fluid contact surface area of ​​only one layer is used for the calculation.

[0024] As used herein, “challenge solution” means a solution of a precisely known concentration of the target molecule that can selectively bind to the membrane (medium) of the device.

[0025] In one embodiment, the target value of the challenge solution is 1 mg / mL of bovine serum albumin (BSA). A 25 mM Tris 50 mM NaCl aqueous solution is prepared by slowly dissolving 3.029 g of Tris base and 2.922 g of sodium chloride (NaCl) in 1 L of deionized (DI) water. The pH is adjusted to 8 by adding a small amount of concentrated hydrochloric acid (HCl) while measuring the pH. Approximately 300 mg of BSA is sprinkled onto the surface of the 25 mM Tris 50 mM NaCl buffer solution. The BSA can be dissolved in the buffer solution by slowly hydrating it for at least 1 hour. This solution is then placed in a sterile medium bottle through a 0.2 μm filter. The absolute concentration of BSA is determined by measuring the UV absorbance of the solution at 280 nm using the Lambert-Beer law and an extinction coefficient (ε)b of 0.667.

[0026] To determine the BSA DBC of a chromatography device, the solution is passed through the test device at a standard flow rate of 210 LMH (liters per square meter of membrane area per hour). The endpoint is determined by the breakthrough of the BSA challenge solution, indicated by the 10% absorbance of the effluent (based on the initial BSA solution defined as 100%) using UV detection at 280 nm. The dynamic binding capacity is then determined using the volume of the challenge solution that has passed through the test device before reaching the endpoint, and the mass of BSA in that volume is calculated. The dynamic binding capacity is obtained by dividing this mass by the effective membrane (medium) area, as shown in Equation 1.

number

[0027] In another embodiment, the challenge is 20 mM potassium chloride. Using the procedure described in U.S. Patent Application No. 67 / 783,319, filed December 21, 2018, entitled “Method For Testing A Chromatography Device Used For Ion Exchange,” which is incorporated entirely herein by reference, specifically the procedure starting on line 0096 of page 33 and ending on line 0019 of page 35, the challenge is tested on a chromatographic device using potassium chloride (Cl - Measure DBC.

[0028] In another embodiment, to determine LRV, the challenge solution has a target value of 1 × 10⁶ plaque-forming units (PFU) / mL of bacteriophage Phi-X 174. 8 It has at least 1 × 10 11 A stock of Phi-X 174 at PFU / mL is grown. A 50 mM Tris aqueous solution is prepared by slowly dissolving 6.057 g of Tris base in 1 L of deionized (DI) water. The pH is adjusted to 8 by adding a small amount of concentrated hydrochloric acid (HCl) while measuring the pH. The conductivity is confirmed to be 20 mS / cm and adjusted by adding a small amount of Tris or DI water. The Phi-X 174 stock is incubated in a 50 mM Tris-HCl buffer with pH 8 and conductivity of 20 mS / cm, at a rate of 1 × 10⁻¹⁶. 8 Dilute to a concentration of PFU / mL. Store a small aliquot of this virus challenge solution as the "input" sample for LRV calculation.

[0029] To determine the virus log reduction value of the chromatography device, this input and eluate are plate-cultured in the presence of the bacterium Escherichia coli. Add 50 μL of Escherichia coli 13076 host per 100 μL to each of the input dilutions or output dilutions in 5 mL test tubes. Add 2.5 mL of New Tryptone Broth Top Agar Medium (New Tryptone Broth supplemented with 0.6% agar) to each test tube, mix by rotation to ensure a well-mixed solution. Then pour the solution onto the surface of a New Tryptone agar plate, allow it to solidify, and incubate at 37 °C for 3 hours. After incubation, Phi-X 174 virus plaques are formed in the circular clearing areas within the colonies of the grown Escherichia coli. Then count the plaques on the resulting input plate and eluate plate, and use Equation 2 to determine the concentration (PFU / mL). C = average number of plaques × dilution factor ÷ volume plated (2)

[0030] The final virus clearance reduction value is determined using the concentration according to Equation 3. LRV = log 10 [(C Feed ×Vol Feed ) / (C Final ×Vol Final )] (3)

[0031] Membrane chromatography is a relatively new ion-exchange chromatography method that evolved from the needs of the bioprocess industry to overcome the limitations of conventional resin bead-based chromatography. Membrane chromatography devices contain a microporous medium with pores containing adsorbent sites capable of binding target proteins and / or viruses to VLPs depending on the functional chemicals and operating conditions. Because membrane chromatography devices rely on convective mass transfer, they can utilize higher flow rates without significant pressure drops, resulting in high throughput and reduced processing times. There are three main types of membrane-based chromatography devices: flat sheet, hollow fiber, and radial flow. Flat sheet chromatography devices are typically more common due to their larger adsorbent membrane volume.

[0032] Membrane chromatography devices often come in various sizes, depending on the stage of molecular development. Laboratory devices typically have a medium volume of approximately 0.08–3 mL. Scale-up or prototype devices typically have a medium volume of approximately 15–100 mL. Mass production devices typically have a medium volume of 200 mL or more. Note that other medium volumes can be provided according to customer needs.

[0033] One method for calculating the media volume is to multiply the effective filtration area (EFA) by the nominal media height or thickness. The nominal media height or thickness can be measured using calipers, and the EFA can be measured by filtering a dye solution through the device. The dye binds to the media, and after the dye breaks through to the outlet flow, the device is disassembled and the diameter of the dye-colored portion of the media layer(s) is measured to determine the average diameter. Alternatively, the colored area can be measured directly, for example, using an optical method. Using the average diameter of the dye-colored portion(s) (for two or more layers), the EFA is calculated using the formula for the area of ​​a circle. Alternatively, the EFA can be calculated by averaging the area of ​​each layer measured.

[0034] The present invention can be used with any desired volume of media and is particularly suitable for chromatography devices intended for viral clearance. Although the present invention is referred to as “chromatography devices” and “membrane chromatography devices,” all aspects of the present invention are equally applicable to composite media structures, including (but not limited to) functionalized membranes and other types of filtration and chromatography media such as hydrogel-functionalized nonwoven fabrics, cellulose and diatomaceous earth-based charged media, and activated carbon.

[0035] While the present invention can be used with any desired medium volume, the laboratory-scale chromatography device described in U.S. Patent Application No. 62 / 792,166, “Sample Size Chromatography Device,” filed on January 14, 2019, and incorporated herein by reference in its entirety, is particularly preferred. In one embodiment, a medium volume of 0.08 mL was used to minimize the amount of virus solution required for the example. The hold-up volume of this chromatography device was measured to be approximately 1.1 mL.

[0036] Laboratory-scale chromatography devices Referring here to Figures 1, 2, 3, 4, and 5, a preferred embodiment of the chromatography device 8 is shown. The device has a housing 10 formed by joining an upper housing 12 to a lower housing 14. The housing has an inlet 16, an outlet 18, and an optional vent 20. Between the inlet 16 and the outlet 18, a membrane or medium 22 is placed in a chamber 24, so that fluid from the inlet 16 enters the inner chamber 24, then passes through the medium 22 and exits from the outlet 18. The sealing layer, spacer ring, or combination thereof of the present invention can be used with any preferred housing having an inlet, an outlet, and a medium laminate placed between them for the passage of fluid.

[0037] Chamber 24 is in fluid communication with the inlet 16 and the vent 20 so that air inside the chamber 24 can be purged through the vent 20. A Luer lock connector (not shown) can be attached to the vent 20 and used as a valve to purge air from chamber 24 until the liquid from the inlet 16 begins to exit through the vent 20 and the valve is closed. In the illustrated embodiment, the membrane volume is 0.08 mL, but this can be easily changed by increasing or decreasing the diameter of the medium and adjusting the size of the housing to match that diameter.

[0038] As shown in Figure 1, at least one of the inlet 16 and the vent 20 can be positioned at a constant angle with respect to the longitudinal axis 26 of the housing 10, preferably both are positioned at a constant angle with respect to the longitudinal axis. As shown in the figure, the inlet 16 is positioned at angle α with respect to the longitudinal axis 26 of the housing, and the vent is positioned at angle β with respect to the lateral axis of the housing. This provides two advantages. Firstly, it provides sufficient clearance to use Luer lock connectors in both the inlet 16 and the vent 20 used, and provides a quick and convenient way to purge air from the chamber 24. In axially aligned inlets do not provide sufficient clearance to accommodate vents with positive lock seals, such as Luer lock connectors, in small-volume experimental devices.

[0039] Secondly, the angled inlet 16 directs the incoming fluid flow so that, as the fluid passes through the chamber 24, it strikes the upper surface of the medium at an angle other than 90 degrees, as indicated by the arrows in Figure 5. With an inlet aligned axially parallel to the longitudinal axis 26, the incoming fluid would directly impact the surface of the medium at approximately 90 degrees at the center of the medium. This design has a tunneling problem, where the supply solution "tunnels" through the center of the disk, leading to premature breakthrough. By angling the inlet 16 so that the incoming fluid flow has a tangential velocity component parallel to the upper surface of the medium, at least a portion of the incoming fluid will flow across at least a portion of the upper surface of the medium before flowing through it. This is very similar to pouring water from a bucket at an angle onto the floor to wash it, spreading the water along the floor away from the person emptying the bucket. Angling the inlet not only helps prevent tunneling but also helps to direct air from the chamber 24 towards the vent 20 and expel it from there. The angle of the inlet can be designed so that a certain volume of buffer or solute flowing into the device does not immediately penetrate the medium due to surface tension and edge effects, but instead flows over the medium along the chamber wall. This movement of the incoming fluid volume results in automatic redistribution and mixing within the chamber, thereby allowing for more uniform utilization of the medium's volume.

[0040] In various embodiments of this device, the angle α between the longitudinal axis 28 of the inlet and the longitudinal axis 26 of the housing may be about 10 to about 80 degrees, about 25 to about 65 degrees, or about 40 to about 50 degrees. In various embodiments of this device, the angle β between the longitudinal axis 30 of the vent and the longitudinal axis 26 of the housing may be about 10 to about 80 degrees, about 25 to about 65 degrees, or about 40 to about 50 degrees. Angle α may be the same as angle β, or smaller or larger than angle β. In addition, if only one of the inlet and vent is angled for Luer lock clearance, preferably the inlet is angled for the positive flow effect described above. In the illustrated embodiment, angle α was 45 degrees and angle β was 45 degrees so that the inlet and vent could be swapped and used for opposite functions as needed.

[0041] The upper housing 12 and the lower housing 14 are joined by ultrasonic welding to form a final liquid-tight housing while simultaneously providing edge sealing to the medium. Specifically, the force applied to the housing that acts to compress the upper and lower housing portions during assembly is controlled during ultrasonic welding, ensuring control of the compression of the medium regardless of changes in the medium's thickness. The unique welding process will be described in detail later. The housing 10 is generally circular, but any other suitable shape can be used.

[0042] As best seen in Figure 5, the upper housing 12 includes two cylindrical projections 32 extending from the upper surface 34 of the upper disc 36 on each side of the longitudinal axis 26 of the housing, positioned at an angle to the longitudinal axis of the housing, and forming a truncated V-shape between them. Each cylindrical projection has a tapered internal bore 38 that fits into a Luer lock taper and is in fluid communication with the chamber 24. A truncated hemispherical surface 40 is present inside the chamber 24 and is formed at the center of the upper disc 36 between the inlet and outlet, reducing the volume of the chamber. The tapered bores 38 of the inlet and vents are in fluid communication with a cylindrical passage 42 leading to the chamber 24, thus allowing fluid to pass through the tapered bores into the cylindrical passage and into the chamber of the assembled housing. The chamber, as shown, has a substantially cylindrical shape with a truncated hemispherical top surface. Other chamber shapes may be used, and generally, the overall size of the chamber is as small as possible to reduce the hold-up volume while still allowing fluid communication between the inlet, vents, chamber, and the surface of the medium.

[0043] As used herein, the upper housing 12 is a relative term for convenience and, in one embodiment, is the housing portion having both an inlet 16 and a vent 20 into the chamber 24. Similarly, the lower housing is a relative term for convenience. A first housing portion may be used instead of the upper housing, and a second housing portion may be used instead of the lower housing. Throughout the specification, any element described by the term “upper” is replaceable with “first,” and any element described by the term “lower” is replaceable with “second.”

[0044] As seen in Figures 5, 6, and 7, a compression extension 46 extends from the lower surface 44 of the upper disk 36 of the upper housing 12, which in some embodiments is a protruding ring structure. If the periphery of the medium is a geometric shape other than circular, such as a square or hexagon, the compression extension will assume the same corresponding shape as the periphery of the medium. The compression extension 46, working in cooperation with the boss 60 supporting the medium 22, compresses the margin or periphery of the medium to a distance X, as shown. This seals the margin or periphery of the medium, preventing fluid leakage from the chamber 24 and bypassing it to the outlet 18 around the margin or periphery of the medium. Sufficient compression is necessary to prevent leakage, but if the medium is compressed excessively, too much medium area will be lost due to compression, and the performance of the experimental device may differ significantly from that of a scale-up or mass-production device using the same medium. Therefore, the height of the compression extension protruding from the lower surface 44, along with how firmly the upper and lower housings are pressed together during ultrasonic welding, controls the distance X and the resulting edge compression of the media disk 22. During the actual ultrasonic welding, the welding energy is set to control the relative edge compression of the media. The housing is depicted with the compression extension 46 extending from the lower surface of the upper housing and combined with the boss 60 of the lower housing, but these two components are interchangeable, and the compression extension 46 may extend from the lower housing and the boss 60 may be located in the upper housing. In an alternative embodiment, the boss 60 may protrude from the inner peripheral surface of the housing. For example, the two extensions can be used to press the periphery of the media together and seal the periphery.

[0045] An interlock weld extension 48 protrudes from the lower surface 44 of the upper housing 12, to be welded to the lower housing 14. In some embodiments, the interlock weld extension is also a protruding ring having a chamfered tip 49 for use during the ultrasonic welding process. The interlock weld extension is located outside the compression extension, further away from the longitudinal axis of the housing. The interlock weld extension 48 initially abuts a step 51 in an optional recess 53 within the lower housing, as seen in Figure 7. The central part of the housing has a height Y' before welding, and the compression extension has a height X'. Due to the chamfer and step, the final height Y of the housing can be changed by increasing the amount of ultrasonic energy applied to the housing during welding. This affects the final dimension X of the compressed edge. The more energy applied during the ultrasonic welding process, the lower the height 51 of the step becomes, and the deeper the chamfered tip 49 slides into the recess 53. Compare Figure 7 with Figure 6. Thus, even when the parts are completely welded to each other, the final height of the capsule Y can be changed, thereby changing the compression distance X at the edge. Increasing the applied energy allows the chamfered tip 53 and the interlock weld extension 49 to slide deeper into the recess 53, resulting in greater compression at the edge of the medium and a decrease in height X, thus reducing the final height Y of the assembly. The opposite is true when less welding energy is applied, resulting in a larger final height Y of the assembly, less compression at the edge, and a larger dimension X. The housing is illustrated with an interlock weld extension 48 extending from the lower surface 44 of the upper housing 12 and a step 51 of the lower housing 14, but the interlock weld extension 48 may extend from the lower housing 14, and the step 51 may be on the upper housing. The cross-sectional profile of the interlock weld extension and the shape of its periphery can be adjusted for various housing shapes. As shown, a ring shape is preferred for the circular medium 22.

[0046] In one embodiment, a circular media laminate is used, and the compression extension and interlock weld extension are protruding rings as described below. Extending from the lower surface 44 of the upper disk 36 are a first protruding ring 46 of the compression extension for compressing the margin or periphery of the media, and a second protruding ring 48 of the interlock weld extension positioned inside the outer diameter 50 of the upper disk 36. The longitudinal length of the first protruding ring is selected to clamp and seal the margin or periphery of the media. The longitudinal length of the second protruding ring is selected to allow dimension X to vary within a height range while maintaining media sealing along the margin or periphery, and to ultrasonically weld and fit with the feature portion of the lower housing. In media with significant thickness variations, the protruding rings may need to have varying longitudinal lengths to avoid over-compressing the media and degrading performance, or to avoid failure of sealing along the edges to prevent bypass. As shown in the figure, the first and second protruding rings have tapered sidewalls with a thicker base and a narrower tip. Other cross-sectional shapes can also be used. The first side wall 52 of the first protruding ring 46 forms part of the side wall of the chamber 24 below the inlet and vent.

[0047] As best seen in Figure 5, the lower housing 14 has a third protruding ring 52 and a fourth protruding ring 54 extending from the upper surface of the lower disc 56. These protruding rings are optional but preferred. A valley or recess 53 is formed between the two rings, and an interlocking weld extension for ultrasonic welding attachment to the lower housing is located inside it. The outer sidewall of the fourth protruding ring 52 forms the majority of the outer sidewall of the assembled housing and may optionally be knurled or have spaced longitudinal ribs 58 along the periphery to enhance grip when handling the housing 10. The inner sidewalls of the third and fourth protruding rings are inclined to match the taper of the second protruding ring of the upper housing in order to nest the two housing portions. By nesting the interlock welded extension 48 between the third protruding ring (52) and the fourth protruding ring (54), greater structural integrity is achieved for the welded housing, and the assembled housing can better resist lateral forces on the housing without damaging the ultrasonic welding. In addition, the inner surface of the third protruding ring 52 functions as a guide and centering device for positioning the circular medium 22 on the boss 60 when assembling the components, as is best seen in Figure 7.

[0048] The inside of the fourth protruding ring, located at the base of its innermost side wall, is a circular boss 60 that supports the periphery of a circular medium disk 22 positioned at the center of the lower housing. The fourth protruding ring generally guides the medium into place, centering it on the support boss 60. The distance X from the top surface 62 of the boss to the tip 64 of the first protruding ring 46 (compression extension) is selected to influence the required compression of the medium, providing a fluid seal along the margin or periphery of the medium disk, and to control this distance during the ultrasonic welding process to best match the performance of the experimental device to that of a scale-up or mass-production device. Below the circular boss is an optional circular dish-shaped recess 66 that acts as a funnel for directing the filtered fluid toward the outlet. A cylindrical projection 32, parallel to and concentric with the longitudinal axis 26 of the housing, extends from the bottom surface 68 of the lower disk 56. This cylindrical projection has a tapered internal bore 38 that fits the Luer lock taper and is in fluid communication with a dish-shaped recess 66. The tapered internal bore 38 at the outlet is in fluid communication with a cylindrical passage 42 that leads to the dish-shaped recess, and thus allows fluid to pass from the dish-shaped recess through the cylindrical passage and through the tapered bore to the outside of the housing.

[0049] One unique feature of this housing design is the combination of media clamping and sealing of the margin or periphery of the media within the device, as described above. Typically, chromatography devices have O-rings or washers to seal and compress the media. One feature of this design is the incorporation of media clamping between the upper and lower housings via the tip of the first protruding ring and a circular boss, as shown in Figure 5. This provides a simple and straightforward method for controlling media compression and can adapt to variations in the thickness of the chromatography media, as the housing portions are ultrasonically welded together under a constant load, and the final welded height may vary with variations in the thickness of the media stack. This design of the device assembly, combined with ultrasonic welding of the upper and lower housing portions, ensures that the edge effect remains constant despite variations in the thickness of the media stack, providing a method for predictable device capacity and pressure drop.

[0050] Typical laboratory-scale chromatography devices commercially available today are manufactured by assembling a two-piece housing (inlet and outlet housing sections) with an internal medium placed between the housing sections, compressing the assembled housing to a certain height, and then using an overmolding process. The overmolding process involves compressing the two housing sections to a final specified height, and then injecting molten plastic onto the outside of the housing assembly to form a fluid-tight housing that maintains a predetermined height before the molten plastic is applied. During this process, several tons of force can be applied to the chromatography medium, resulting in a significant and large compressed margin or peripheral zone. This large compressed margin or peripheral zone degrades the performance of the chromatography device, as described above. During the overmolding process, significant compressive force is required on the assembled housing sections to contain the molten plastic and prevent burrs. A predetermined mold height is selected to prevent burrs. Therefore, thicker medium laminates experience greater compression than thinner medium laminates, leading to significant performance variations in small-volume chromatography devices. This approach to fabricating chromatography devices is not very versatile because it requires multiple molds in the overmolding process to ensure consistent compression of the chromatography medium layer at the margin or periphery across mediums of varying thicknesses.

[0051] On the other hand, ultrasonic bonding can involve compressing the assembly of the inlet housing, outlet housing, and internal chromatography medium laminate to a specified force rather than a fixed height. Once this predetermined force is reached, the ultrasonic welding process begins, and the vibrational energy applied to the energy director causes localized melting and bonding. In this process, the force experienced by the edges of the chromatography medium is only a few pounds (orders of magnitude smaller than the forces observed during the overmolding process). The margin or peripheral zone compressed by ultrasonic bonding is small, and the actual amount of compression can be controlled by the energy applied during the ultrasonic welding process, thereby resulting in variations in the final height of the assembled housing. Furthermore, since the initiation of welding is triggered by a set force, normal variations in medium thickness do not significantly affect the size of the margin or peripheral compression zone. Thicker medium results in a higher housing height, and thinner medium results in a lower housing height. The welding process self-compensates for variations in medium thickness. Thus, this design provides a simple method for ensuring consistent device performance.

[0052] This approach to fabricating chromatography devices is highly versatile and can accommodate chromatography media of various thicknesses. While significant variations in the thickness of the media stacks of different products may necessitate varying longitudinal lengths for the compression extensions and interlock weld extensions, nominal variations due to manufacturing tolerances are easily handled, resulting in chromatography devices with consistently high performance.

[0053] Referring here to Figure 6, another embodiment of the chromatography device is shown. As indicated by the arrow, a baffle 70 extends from the ceiling of the chamber, which redirects the inflow flow in a direction more parallel to the longitudinal axis of the housing. In one embodiment, the baffle is a fifth protruding ring having a side wall 72, which is roughly parallel to the longitudinal axis 26 and extends longitudinally long enough to redirect the inflow flow as described, but short enough to prevent interference between the swollen medium and the baffle feature. The outer diameter of the fifth protruding ring is small enough to fit between the inlet cylindrical passage 42 and the vent, and the fifth protruding ring nominally starts just inside where these passages intersect the chamber 24, as shown. In some embodiments, the longitudinal height of the fifth protruding ring is about the same size as the diameter of the Luer connector, for example, about 3 mm to about 6 mm.

[0054] Depending on the surface tension and wettability of the chromatography medium, baffles 70 may be required. The baffles help direct the inflow of buffer or solute along the chamber sidewalls towards the compressed margins or periphery of the medium. Because the permeability of the medium may be reduced in this region, the buffer or solute tends to redistribute towards the center of the medium disk and pass through the medium. This phenomenon is another way to eliminate the tunneling described above.

[0055] The cylindrical projections forming the inlet, outlet, and vent can be sized to mate with a tapered Luer lock connector. To facilitate the use of the Luer lock connector, the outer surface of these cylindrical projections 32 may have two opposing lateral tabs 80, which extend from the outer diameter of the cylindrical projection and are located near the distal end of the cylindrical projection. The tabs engage with the threads of the male Luer lock connector. Optionally, other fluid connectors, such as hose barbs, can also be used to introduce and remove fluid from the chromatography device.

[0056] Chromatography devices are preferably injection-molded from a suitable material. Preferably, this material is readily ultrasonically welded so that the upper and lower housings can be bonded together in a fluid-tight manner. Suitable materials for the housings include thermoplastic resins such as acetal (POM), acrylic (PMMA), acrylonitrile butadiene styrene (ABS), polycarbonate (PC), polyethylene (LD / HDPE), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), and polypropylene (PP).

[0057] Alternatively, other means can be used to secure the upper housing to the lower housing, such as a liquid-tight threaded connection, like those used in typical water pipes. The upper and lower housings can be made of materials suitable for threaded connections, such as plastic or metal.

[0058] Alternatively, the housings may be 3D printed using a 3D printer. In this case, the housings can be joined together using an adhesive such as epoxy or acrylic to form a liquid-tight seal.

[0059] Spacer ring As seen in Figures 9, 10, 11, 13, 14, and 15, in some embodiments, one or more spacer rings 86 may be used in the chromatography device 8 as an optional choice. The function of the spacer ring is to provide a gap between the previous medium layer and the next medium layer in the direction of fluid flow within the chromatography device. While we do not wish to be constrained by theory, it is thought that the gap helps to prevent tunneling through the center of the medium by allowing the liquid to disperse more quickly to the edges of the medium. The gap can bring the fluid flow directly to the edges of the medium rather than relying on capillary action to move the fluid to the edges of the medium.

[0060] Certain media, such as functionalized nonwoven fabrics, may swell upon contact with liquids. This swelling, combined with edge compression sealing, can lead to undesirable tunneling. By slightly separating the layers, voids can be provided that allow liquid leaving one media layer to flow laterally before entering the next.

[0061] Therefore, the optimal spacer height depends on the expected and possible media swelling. In non-swelling media layers, void heights and spacer heights as low as 0.001 inches work well, but generally, larger void and spacer heights are used in functionalized nonwovens, which tend to swell more.

[0062] In some embodiments, the longitudinal height of the spacer ring between media layers can be 0.001, 0.005, 0.010, 0.020, or 0.030 inches or more. The maximum height of the spacer ring is often limited by the overall length of the housing and the height of the internal chamber in which the media layers forming the media stack can be arranged. If the spacer ring is too tall, the thickness of the media layers that can be arranged in the chromatography device may be reduced. Typically, the spacer ring has a longitudinal height of 1.0, 0.90, 0.80, 0.60, or 0.50 inches or less between media layers. The range between this range of heights is within the scope of the present invention. Particularly preferred spacer rings have a longitudinal height of 0.030 to 0.050 inches between media layers.

[0063] The spacer ring may have an outer diameter, an inner diameter, a central aperture or opening, and a height, and may be similar in structure to a washer, bushing, or short tube. The outer diameter is often the same size as the outer diameter of the compression seal of the medium. This often corresponds to the outer diameter of the compression extension 46, as seen in Figures 5 and 9.

[0064] The inner diameter can be smaller than the compression sealing inner diameter of the medium, but in that case, more of the medium surface is covered, reducing the volume and increasing the compression edge effect in the medium margin. Therefore, as seen in Figures 5 and 9, the inner diameter is approximately the same size as, or slightly smaller than, the inner diameter of the medium compression extension 46.

[0065] In many cases, the width of the spacer ring matches the width of the compression extension 46, as shown in Figure 9, for example. This allows for sufficient sealing area without negating edge effects or loss of device capacitance.

[0066] The optimal outer diameter, inner diameter, and width of the spacer ring can be determined based on the specific housing design. Since the housing diameter increases or decreases for various sizes of chromatography devices, the spacer ring diameter is adjusted accordingly. In one embodiment, the outer diameter of the spacer ring was approximately 1.1 inches, the inner diameter approximately 0.9 inches, and the width 0.1 inches.

[0067] The number of spacer rings can vary, and is often one less than the number of media layers in the chromatography device. Thus, a two-layer device may have one spacer ring between the two media layers in the media stack, and a three-layer device may have two spacer rings in the media stack: one between the first and second media layers and one between the second and third media layers. Typically, it is important to space functionalized nonwoven layers from subsequent layers because they swell, but it is less important to space functionalized film layers from subsequent layers (as they typically do not swell) if the overall height of the media stack is a concern to fit it within a chosen housing.

[0068] Spacer rings can be constructed from many different materials. The choice of material often depends on the reactivity of the fluid being processed to the material. The same material suitable for molding the housing is also suitable for the spacer ring. Generally, the material selected for the housing is also used for the spacer ring. Suitable materials for molding spacer rings include thermoplastic resins such as acetal (POM), acrylic (PMMA), acrylonitrile butadiene styrene (ABS), polycarbonate (PC), polyethylene (LD / HDPE), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), and polypropylene (PP). These thermoplastic materials are easily injection moldable and can be used to manufacture spacer rings of suitable dimensions.

[0069] In many embodiments, the housing has a compression extension 46 and a boss 60, between which the media laminate is compressed to create an edge seal. In some embodiments, the distal portions of these surfaces in contact with the media are relatively flat or planar, as shown in Figure 5. In other embodiments, the distal portions of these surfaces in contact with the media are provided with clamping projections. As shown in Figure 8, the distal end of the compression extension 46 of the upper housing is longer on the side adjacent to the internal chamber 24 and shorter on the side facing outward from the housing. Similarly, the circular boss 60 in the lower housing also has clamping projections extending from its surface. Although not shown, the spacer ring 86 may have clamping projections located on either or both of these surfaces instead of having a smooth upper and a smooth lower surface as shown.

[0070] The function of the clamping projections is to bite into the medium and prevent it from slipping out of the edge compression zone. Preferably, the clamping projections are positioned to compress the margin or periphery of the medium more strongly in areas closer to the internal chamber 24 and less strongly in areas further away from the internal chamber. This creates a weakly compressed zone of the medium facing outward from the housing, which needs to be further compressed and pulled under the clamping projections as the medium swells, but swelling is unlikely to occur.

[0071] The clamping protrusions may be continuous rings, ridges, steps, or other features projecting from the distal surface of a compression extension, boss, or spacer ring. Alternatively, the clamping protrusions may be discontinuous surfaces composed of short segments or projections that clamp or bite into the surface of the medium. Clamping protrusions have been found effective in enhancing the sealing of the medium, particularly functionalized nonwoven layers that tend to swell during use in chromatography devices and detach from the compression zone at the edge of the housing.

[0072] sealing layer As shown in Figure 12, the sealing layer 88 is located as the last layer through which the liquid passes from inlet to outlet within the stack of at least two layers inside the chromatography device. This sealing layer is in contact with the housing, and at least a portion of the margin or periphery of this layer forms a compression seal within the chromatography device. This is the most important layer for sealing the housing within the chromatography device because it is the last compression seal through which the liquid can or has passed (the bottom layer within the medium stack) as the fluid exits the housing.

[0073] This sealing layer is a non-functionalized layer as defined herein. The functionalized layer as defined herein does not function well as a compression seal with the housing, and when this is the last layer of the media laminate for sealing, the LRV of the chromatography device has been found to be lower than that of a similar configuration chromatography device having the same media laminate structure but differing in that a non-functionalized medium sealing layer is added as the last layer of the media laminate that contacts the housing.

[0074] The sealing layer is a non-functionalized porous medium and can have a relatively smooth surface and sufficient rigidity due to its thickness to support the functionalized layer above it, preventing it from sagging and slipping out of the edge seal due to the pressure generated by the differential pressure created by the flow across the medium layer during use. The sealing layer may be a film or a nonwoven fabric layer. Since films often have a smooth surface roughness, in preferred embodiments the sealing layer is a film. The sealing layer may comprise a multi-component material having one ply film and another medium such as a scrim layer. Alternatively, the sealing layer may comprise two or more plies of the film or other medium.

[0075] If the sealing layer is a membrane, it can also be formed from any suitable membrane-forming material, such as polyamide (including nylon-6 or nylon-6,6), polysulfone (including bisphenol A polysulfone and polyethersulfone), polypropylene, polyethylene, and fluorinated polymers including polyvinylidene fluoride and polytetrafluoroethylene. The membrane may be a supported membrane, meaning that the membrane is cast on a porous support layer such as a nonwoven layer, spunbond layer, fabric layer, or scrim. Alternatively, the membrane may be an unsupported membrane, meaning that the membrane is formed from the membrane-forming material without the help of a support layer. The membrane may be a symmetrical membrane, meaning that the average pore diameter between the two outer principal surfaces of the membrane is substantially the same at any given location. Alternatively, the membrane may be an asymmetrical or gradient membrane, meaning that the average pore diameter in the region near one principal surface of the membrane is substantially larger than the average pore diameter in the region near the opposite principal surface of the membrane. Furthermore, the membrane may be a multizone membrane, meaning that the membrane includes a thickness-penetrating zone between the main outer surfaces of the membrane, and this zone has a different average pore diameter from the average pore diameter of another thickness-penetrating zone located between the main outer surfaces of the membrane. In some embodiments, the sealing layer includes a supported nylon-6,6 membrane as described in U.S. Patent No. 6,264,044, issued on July 24, 2001, which is incorporated herein by reference in its entirety.

[0076] In one embodiment, the sealing layer was a nylon-6,6 film having an average pore diameter of 0.8 micrometers. The thickness of the sealing layer was in the range of 17 to 20 mil. Other suitable materials for the sealing layer include nylon-6,6 films having an average pore diameter of 0.8 micrometers and a thickness of 8.5 to 10.0 mil, nylon-6,6 films having an average pore diameter of 0.65 micrometers and a thickness of 6.0 to 7.0 mil, nylon-6,6 films having an average pore diameter of 0.2 micrometers and a thickness of 6.0 to 7.0 mil, and nylon-6,6 films having a pore diameter in the range of 0.2 to 1.2 micrometers and a thickness of 13.0 to 15.4 mil.

[0077] In various embodiments of the present invention, the film for the sealing layer may have a minimum pore diameter within the film of 0.1 to 5.0, 0.1 to 3.0, or 0.2 to 1.2 micrometers and a thickness of 6.0 to 20.0 mil.

[0078] As seen in the examples, the following materials were found not to increase the LRV of the chromatography device and did not function as suitable sealing layers: spunbond nonwoven fabric with an average thickness of 9 mil; polyethersulfone film with an average pore size of 0.2 micrometers and a thickness of 100-120 micrometers; and the above spunbond nonwoven fabric, both of which were ultrasonically bonded to the exit shell of the experimental device to provide a rigid support structure.

[0079] While we do not wish to be constrained by theory, the sealing layer is considered to provide consistent sealing because it does not undergo morphological changes like the functionalized layer when in contact with the challenge solution. Furthermore, when a film is used as the sealing layer, the medium often does not allow any significant tangential flow, improving sealing performance. In many cases, the sealing layer can be formed from the same medium used to create the functionalized layer, but without the coating or grafting treatment for functionalization. Therefore, it can have a relatively similar thickness and pore distribution to one of the functionalized layers in the chromatography device. In a preferred embodiment, the functionalized film is used as one of the chromatography medium layers, and the same precursor film before functionalization is used as the sealing layer.

[0080] Chromatographic devices with a medium or membrane volume of approximately 0.08 mL were evaluated using a spacer ring, a sealing layer, or both. The upper and lower housings and spacer ring (height 0.050 inches) were injection molded using polypropylene random copolymer at a mass melt flow rate (MFR) of 9.0 g / 10 min. The selected chromatographic media had three main components: an anion exchange nonwoven fabric, an anion exchange membrane, and a sealing layer. The anion exchange nonwoven fabric layer consisted of a 4-ply polypropylene nonwoven fabric with a covalently bonded quaternary ammonium functionalized polymer. The anion exchange membrane layer consisted of a 3-ply highly porous polyamide membrane with a covalently bonded guanidinium functionalized polymer. In some embodiments, this was followed by a polyamide unfunctionalized membrane used as a sealing layer within the assembled housing capsule.

[0081] Examples shown in Figures 8 to 15 To assemble the chromatography device, the medium components are punched out to obtain a disk with a diameter of 1.0625 inches. The disk is placed inside the protruding ring 54 within the lower housing 14. A spacer ring 86 is added between the functionalized film and the nonwoven fabric layer. The upper housing is placed on top of the medium so that the protruding ring 48 of the upper housing slides between the protruding rings 52 and 54 of the lower housing. This assembly is inverted and placed in a nest or fixture so that the outer surface 68 of the lower housing 14 can contact the ultrasonic horn. To weld these parts, a Branson 20kHz ultrasonic welding machine (model 2000xdt), a Black Booster, and a horn with a gain of 2.5x are used. Fixed parameters were set to an air pressure of 80 psi, a descent rate of 10%, an amplitude of 80%, a welding time of 2 seconds, and a trigger force of 200 lbf to initiate welding. The welding energy was varied in the range of 200 to 600 joules to obtain samples with consistent compression levels depending on the number of spacer rings and sealing layers. The housing assembly with the chromatography medium is placed in a nest directly below the horn so that the longitudinal axis 26 of the housing is aligned with the axis of the ultrasonic horn. When the welding process is initiated, the horn descends over the lower housing, compressing the housing and medium assembly until a force of 200 lbf is reached. At this point, the shear energy director is under compression. The horn begins to vibrate, delivering a set amount of energy to the plastic energy director, causing localized melting and joining. After the welding duration, the horn retracts, leaving the welded chromatography device in the nest.

[0082] In the case of the polyethersulfone (PES) sample, the PES film and a thin spunbond nonwoven fabric layer are placed inside the lower housing 14 within the protruding ring 54, as shown below, so that the spunbond nonwoven fabric layer is in contact with the lower housing 14. The lower housing is positioned in the lower nest so that the PES film can come into contact with the ultrasonic horn. A Branson 20kHz ultrasonic welding machine (model 2000xdt), a gold booster, and a horn with a gain of 1.5x are used to weld the film and the supporting nonwoven fabric layer. Fixed parameters were set to an air pressure of 10 psi, a descent rate of 10%, an amplitude of 80%, a welding time of 0.05 seconds, and a trigger force of 10 lbf to initiate welding. It was found that a welding energy of 100 J was sufficient to obtain good, uniform welding without damaging the film.

[0083] All chromatography device samples were autoclaved at 121°C for 30 minutes using a PreVac cycle. After autoclaving, the samples were left at room temperature and fully cooled before testing.

[0084] To evaluate the viral clearance performance of the chromatography device, a Cole Parmer MasterFlex Peristaltic pump was set up with Master Flex tubing and a base disinfected with 0.5M sodium hydroxide (NaOH) for at least 30 minutes. After disinfection, the sterile chromatography device was connected to the peristaltic pump. Sterile sample chloride (Cl) - The DBC was measured as described above in accordance with U.S. Patent Application No. 67 / 783,319. Subsequently, the chromatography device was flushed with 15 mL of 50 mM Tris-HCl buffer having a pH of 8 and a conductivity of 20 mS / cm. Next, 15 mL of the viral challenge solution was perfused through the chromatography device and the eluate was collected. The input and eluate were plate-cultured and the LRV was calculated using Equations 2 and 3.

[0085] The BSA DBC chromatography device was tested using the Akta Pure System (GE Healthcare Life Sciences). Protein breakthrough was detected using inline UV monitoring at 280 nm. First, the chloride (Cl) of the sterile sample was tested. - The DBC was measured as described above in accordance with U.S. Patent Application No. 67 / 783,319. Subsequently, the chromatography device was subjected to 1.94 mL / min / cm³ of 25 mM Tris 50 mM NaCl buffer medium having pH 8. 2 The device was then flushed. Next, the chromatography device was challenged by adding a BSA solution up to approximately 1 mg / mL to the aforementioned buffer until 10% breakthrough occurred. To establish the endpoint of the test, the absorbance of the BSA challenge solution was measured at 280 nm, and the 10% breakthrough value was calculated. The dynamic binding capacity (DBC) of the chromatography device at 10% breakthrough was calculated using Equation 1.

[0086] Referring to Figures 8 to 15, various combinations of the aforementioned medium layer, spacer ring, and encapsulation film are shown in various chromatography devices.

[0087] contrast Figure 8 shows a chromatography device having a medium laminate containing layers 82 of functionalized nonwoven fabric (FNW), 82 of functionalized nonwoven fabric, and 84 of functionalized film (FM) in the inlet-outlet direction. No spacer rings or sealing films are used, and this represents a control device for the following configuration. This device uses 4.07Cl - / cm 2 Cl - DBC, 14.73 mg / cm³ 2 It was found to have a BSA DBC and an LRV of 3.83.

[0088] Spacer ring Figure 9 shows a chromatography device having a medium laminate containing a functionalized nonwoven fabric layer 82, a spacer ring 86, and a functionalized film layer 84 from the inlet to the outlet. This device uses 5.74Cl - / cm 2 Cl - DBC, 19.42 mg / cm³ 2 It was found to have a BSA DBC and a 4.28 LRV.

[0089] Figure 10 is a diagram of a chromatography device having a medium laminate including a layer of functionalized nonwoven fabric 82, a spacer ring 86, another layer of functionalized nonwoven fabric 82, and a layer of functionalized film 84 from the inlet to the outlet. This device uses 5.28Cl - / cm 2 Cl - DBC, 18.04 mg / cm³ 2 It was found to have a BSA DBC and a 4.22 LRV.

[0090] Figure 11 shows a chromatography device having a medium laminate comprising a functionalized nonwoven fabric layer 82, a first spacer ring 86, another functionalized nonwoven fabric layer 82, a second spacer ring 86, and a functionalized film layer 84 from the inlet to the outlet. This device uses 6.21Cl - / cm 2 Cl - DBC, 20.23 mg / cm³ 2 It was found to have a BSA DBC and an LRV of 4.47.

[0091] sealing layer Figure 12 shows a chromatography device having a medium laminate containing a functionalized nonwoven fabric layer 82, a functionalized nonwoven fabric layer 82, a functionalized film layer 84, and a sealing layer 88 from the inlet to the outlet. This device uses 3.27Cl - / cm 2 Cl - DBC, 13.34 mg / cm³ 2 It was found to have a BSA DBC and an LRV of 6.74.

[0092] Encapsulation layer and spacer Figure 13 shows a chromatography device having a medium laminate comprising a functionalized nonwoven fabric layer 82, a spacer ring 86, a functionalized film layer 84, and a sealing layer 88 from the inlet to the outlet. This device uses 5.42Cl - / cm 2 Cl - DBC, 19.79 mg / cm³ 2 It was found to have a BSA DBC and an LRV of 7.62.

[0093] Figure 14 shows a chromatography device having a medium laminate comprising a functionalized nonwoven fabric layer 82, a spacer ring 86, another functionalized nonwoven fabric layer 82, a functionalized film layer 84, and a sealing layer 88 from the inlet to the outlet. This device uses 4.48Cl - / cm 2 Cl - DBC, 17.22 mg / cm³ 2 It was found to have a BSA DBC and a 7.00 LRV.

[0094] Figure 15 shows a chromatography device having a medium laminate comprising a functionalized nonwoven fabric layer 82, a first spacer ring 86, another functionalized nonwoven fabric layer 82, a second spacer ring 86, a functionalized film layer 84, and a sealing layer 88 from the inlet to the outlet. This device uses 5.82Cl - / cm 2 Cl - DBC, 19.93 mg / cm³ 2 It was found to have a BSA DBC and an LRV of 7.74. [Table 1]

[0095] Comparing the control in Figure 8 with Figures 9-11, it can be seen that adding one or more spacer rings without a sealing layer improves the dynamic binding capacity by 22.5% to 52.6%. See Table 3. Here, the performance is significantly improved without adding any further functionalized media! Comparing the control in Figure 8 with Figure 12, the addition of a sealing layer improves LRV by 75.8%. This is a significant improvement in the viral clearance of the chromatography device. The chromatography device in Figure 15, when using two spacer rings in combination with a sealing layer, showed a 43.0% increase in CL-DBC and a 102% increase in LRV compared to the control device in Figure 8.

[0096] Examples of additional chromatograph layers To demonstrate the effect of the sealing layer on the viral clearance performance of chromatography devices, samples with different sealing layers were prepared. The LRV values ​​for the various embodiments discussed herein are summarized in Table 2. [Table 2]

[0097] Figure 15 illustrates a structure in which a sealing layer 88 is used as the final layer of a chromatography device adjacent to the outlet, and is in compression sealing contact with the housing to prevent bypass around the sealing layer. This chromatography device has a 2-ply functionalized nonwoven fabric layer 82, a 50 mil thick spacer ring 86, a 2-ply second functionalized nonwoven fabric layer 82, another 50 mil thick spacer ring 86, a 3-ply functionalized film layer 84, and a sealing layer 88 as the fluid moves from the inlet to the outlet.

[0098] Figure 11 illustrates a structure in which a functionalized film is used as the final layer of a chromatography device adjacent to the outlet, and is in compression sealing contact with the housing to prevent bypass. This chromatography device has a 2-ply functionalized nonwoven fabric layer 82, a 50 mil thick spacer ring 86, a 2-ply second functionalized nonwoven fabric layer 82, another 50 mil thick spacer ring 86, and a 3-ply functionalized film layer 84 as the fluid moves from the inlet to the outlet. The same materials and weights of materials used in the embodiment of Figure 15 are used.

[0099] Referring to Table 2, the logarithmic reduction value (LRV) of the structure in Figure 15 is at least 6.49, while the LRV of Figure 11 is only 4.47. When a functionalized film is used as the last layer of a chromatography device and is in sealing contact with the housing, viruses are likely to bypass the functionalized layer, easily leak through the underside of the medium in compression contact with the housing, and pass through to the exit, reducing the device's LRV. On the other hand, when a sealing layer is used at this position immediately below the functionalized layer, an improvement in compression sealing occurs, resulting in a remarkable increase of 45.2% in LRV. This is a remarkable performance improvement achieved without adding any functionalized material to the device. In various embodiments, the LRV of a medium stack in a chromatography device when a sealing layer is used can increase by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% compared to the same medium stack in a control chromatography device without a sealing layer.

[0100] Considerations regarding additional spacers Figures 9, 10, 11, 13, 14, and 15 illustrate various structures in which at least one spacer ring is positioned between two layers within a chromatography device. In some embodiments, only a single spacer ring is used, while in other embodiments, two spacer rings are used. Each chromatography device has a 2-ply functionalized nonwoven fabric layer, a 2-ply second functionalized nonwoven fabric layer, and a 3-ply functionalized film layer as the fluid moves from the inlet to the outlet. As shown, one or more spacer rings are positioned between the various layers.

[0101] Figure 8 shows a control chromatography device having a 2-ply functionalized nonwoven fabric layer, a second 2-ply functionalized nonwoven fabric layer, and a 3-ply functionalized film layer as the fluid moves from the inlet to the outlet. The same materials and weights of materials used for the layers are those used in the examples in Figures 9, 10, and 11. Figure 12 shows a chromatography device having a nylon 6,6 film with an average pore size of 0.8 micrometers as a sealing layer. The thickness of the sealing layer ranged from 17 to 20 mil. Figures 13, 14, and 15 illustrate various structures in which, in addition to incorporating a sealing layer, at least one spacer ring is placed between the two layers in the chromatography device. [Table 3]

[0102] Referring to Table 3, the control BSA dynamic binding capacity in Figure 8 was 14.73 mg / cm², and the chloride capacity was 4.07 mL. The various spacer ring structures in Figures 9, 10, and 11 increased the capacity by 22.5% to 52.6% under two tests. The various spacer ring structures with a sealing layer in Figures 13, 14, and 15 increased the capacity by 10.1% to 43.2% under two tests. This is a significant increase in capacity compared to the control chromatography device in Figure 8, considering that no additional material was added to the layer. As shown above, when using one or more spacers, the DBC of the medium laminate of the chromatography device can increase by at least 10%, 20%, 30%, 40%, or 50% compared to the same medium laminate of a control chromatography device without spacers, when testing either the Cl-DBC or BSA DBC of the chromatography device. Examples of embodiments of the present invention are listed in the following sections [Aspect 1] to [Aspect 26]. [Aspect 1] A housing having an entrance and an exit, At least one functionalization medium layer disposed between the inlet and outlet inside the housing, As the fluid passes through the medium laminate from the inlet to the outlet, a non-functionalized sealing layer is disposed between the inlet and the outlet inside the housing as the final medium layer within the medium laminate within the housing, A margin of the sealing layer in contact with the housing, which is compressed by the housing to form a compression seal, preventing the fluid from passing through the compression seal and leaking out to the outlet; A chromatography device equipped with the following features. [Aspect 2] The chromatography device according to embodiment 1, wherein the sealing layer includes a film. [Aspect 3] The chromatography device according to embodiment 2, wherein the sealing layer film contains a precursor having the same material structure as the functionalized medium layer before functionalization. [Aspect 4] The chromatography device according to embodiment 2 or 3, wherein the sealing layer film includes a multizone film having various pore sizes in different zones. [Aspect 5] The chromatography device according to embodiment 2 or 3, wherein the sealing layer film compromises a symmetrical film having substantially constant pore size. [Aspect 6] The chromatography device according to embodiment 2, 3, 4, or 5, wherein the sealing layer film comprises a polyamide. [Aspect 7] The chromatography device according to embodiment 6, wherein the sealing layer film contains nylon 6,6. [Aspect 8] The chromatography device according to embodiment 2, 3, 4, 5, 6, or 7, wherein the sealing layer film includes a supported film cast on a porous support layer. [Aspect 9] The chromatography device according to embodiment 2, 3, 4, 5, 6, 7, or 8, wherein the sealing layer film has an average pore diameter of 0.1 to 5.0 micrometers. [Aspect 10] The chromatography device according to embodiment 2, 3, 4, 5, 6, 7, 8, or 9, wherein the sealing layer film has a thickness of 6 to 20 mil. [Aspect 11] A chromatography device according to embodiment 2, 3, 4, 5, 6, 7, 8, 9, or 10, wherein the LRV of the medium stack in the chromatography device when using the sealing layer is increased by at least 20 percent compared to the same medium stack in a control chromatography device without the sealing layer. [Aspect 12] The chromatography device according to embodiment 11, wherein the LRV increases by at least 70 percent. [Aspect 13] A housing having an entrance and an exit, Displaced between the inlet and outlet inside the housing, at least two media layers forming a media laminate, wherein at least one of the media layers includes a functionalization layer, A spacer ring is placed between the two media layers to form a gap between them, As the fluid passes through the medium laminate from the inlet to the outlet, a non-functionalized sealing layer is disposed between the inlet and the outlet inside the housing as the final medium layer within the medium laminate in the housing, A margin of the sealing layer in contact with the housing, which is compressed by the housing to form a compression seal, preventing the fluid from passing through the compression seal and leaking out to the outlet; A chromatography device equipped with the following features. [Aspect 14] The chromatography device according to embodiment 13, wherein the media laminate comprises a functionalized nonwoven fabric layer, a functionalized film layer, and the spacer ring disposed between the functionalized nonwoven fabric layer and the functionalized film layer. [Aspect 15] The chromatography device according to embodiment 13, wherein the media laminate comprises a functionalized nonwoven fabric layer, another functionalized nonwoven fabric layer, and the spacer ring disposed between the functionalized nonwoven fabric layer and the other functionalized nonwoven fabric layer. [Aspect 16] The chromatography device according to embodiment 13, wherein the media laminate comprises a functionalized nonwoven fabric layer, a first spacer ring, another functionalized nonwoven fabric layer, a second spacer ring, and a functionalized film layer. [Aspect 17] The chromatography device according to embodiment 13, 14, 15, or 16, wherein the functionalized layer is a functionalized nonwoven fabric. [Aspect 18] The chromatography device according to embodiment 13, 14, 15, 16, or 17, wherein the spacer ring has a height, and the height is 0.030 inches to 0.050 inches. [Aspect 19] A chromatography device according to embodiment 13, 14, 15, 16, 17, or 18, wherein the spacer ring comprises a rigid material selected from the group consisting of acetal (POM), acrylic (PMMA), acrylonitrile butadiene styrene (ABS), polycarbonate (PC), polyethylene (LD / HDPE), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), and polypropylene (PP). [Aspect 20] A chromatography device according to embodiment 13, 14, 15, 16, 17, 18, or 19, wherein when the spacer ring is used, the DBC of the medium stack in the chromatography device is increased by at least 10% compared to the same medium stack in a control chromatography device without the spacer ring, when either the Cl-DBC or BSA DBC of the chromatography device is tested. [Aspect 21] The chromatography device according to embodiment 13, 14, 15, 16, 17, 18, 19, or 20, wherein the sealing layer includes a film. [Aspect 22] The chromatography device according to embodiment 13, 14, 15, 16, 17, 18, 19, 20, or 21, wherein the sealing layer film comprises a polyamide. [Aspect 23] A chromatography device according to embodiment 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, wherein the sealing layer film includes a supported film cast on a porous support layer. [Aspect 24] The chromatography device according to embodiment 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, wherein the sealing layer film has an average pore diameter of 0.1 to 5.0 micrometers. [Aspect 25] The chromatography device according to embodiment 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24, wherein the sealing layer film has a thickness of 6 to 20 mil. [Aspect 26] A chromatography device according to embodiment 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, wherein the LRV of the medium stack in the chromatography device when using the sealing layer is increased by at least 20 percent compared to the same medium stack in a control chromatography device without the sealing layer.

Claims

1. A housing having an entrance and an exit, At least two media layers forming a media stack, At least one media layer of the media laminate includes at least one functionalized media layer disposed between the inlet and outlet inside the housing, The media laminate comprises at least two media layers, where at least one media layer includes a non-functionalized sealing layer positioned between the inlet and the outlet inside the housing, as the final media layer within the media laminate as the fluid passes through the media laminate from the inlet to the outlet. An edge of the sealing layer that contacts the housing, configured to be compressed by the housing to form a compression seal between the sealing layer and the housing, and preventing the fluid from passing through the compression seal and leaking out to the outlet, A chromatography device equipped with the following features.

2. The chromatography device according to claim 1, wherein the sealing layer includes a film.

3. The chromatography device according to claim 2, wherein the sealing layer film comprises a precursor having the same material structure as the functionalized medium layer before functionalization.

4. The chromatography device according to claim 2 or 3, wherein the sealing layer film includes a multizone film having various pore sizes in different zones.

5. The chromatography device according to claim 2 or 3, wherein the sealing layer film comprises a symmetrical film having substantially constant pore sizes.

6. The chromatography device according to claim 2, 3, 4, or 5, wherein the sealing layer film comprises a polyamide.

7. The chromatography device according to claim 2, 3, 4, 5, or 6, wherein the sealing layer film includes a supported film cast on a porous support layer.

8. The chromatography device according to claim 2, 3, 4, 5, 6, or 7, wherein the LRV of the medium stack in the chromatography device when the sealing layer is used is increased by at least 20 percent compared to the same medium stack in a control chromatography device without the sealing layer.

9. The chromatography device according to claim 8, wherein the LRV is increased by at least 70 percent.

10. A housing having an entrance and an exit, Displaced between the inlet and outlet inside the housing, at least two media layers forming a media laminate, wherein at least one of the media layers includes a functionalization layer, A spacer ring is placed between the two media layers, forming a gap between them, As the fluid passes through the medium laminate from the inlet to the outlet, a non-functionalized sealing layer is disposed between the inlet and the outlet inside the housing as the final medium layer within the medium laminate in the housing, An edge of the sealing layer that contacts the housing, configured to be compressed by the housing to form a compression seal between the sealing layer and the housing, and preventing the fluid from passing through the compression seal and leaking out to the outlet, A chromatography device equipped with the following features.

11. The chromatography device according to claim 10, wherein the media laminate comprises a functionalized nonwoven fabric layer, a functionalized film layer, and the spacer ring disposed between the functionalized nonwoven fabric layer and the functionalized film layer.

12. The chromatography device according to claim 10, wherein the media laminate comprises a functionalized nonwoven fabric layer, another functionalized nonwoven fabric layer, and the spacer ring disposed between the functionalized nonwoven fabric layer and the other functionalized nonwoven fabric layer.

13. The chromatography device according to claim 10, wherein the media laminate comprises a functionalized nonwoven fabric layer, a first spacer ring, another functionalized nonwoven fabric layer, a second spacer ring, and a functionalized film layer.

14. The chromatography device according to claim 10, 11, 12, or 13, wherein the functionalized layer is a functionalized nonwoven fabric.

15. The chromatography device according to claim 10, 11, 12, 13, or 14, wherein when the spacer ring is used, the DBC of the medium stack in the chromatography device is increased by at least 10% compared to the same medium stack in a control chromatography device without the spacer ring, when either the Cl-DBC or BSA DBC of the chromatography device is tested.

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