Membrane seals and spacer rings for virus removal chromatography devices

Abstract

The present invention provides a chromatographic separation device comprising: a housing having an inlet and an outlet; at least two dielectric layers disposed within the housing between the inlet and the outlet to form a dielectric stack, at least one of the dielectric layers comprising a functionalized layer; an optional spacer ring disposed between the two dielectric layers to form an air gap between the two dielectric layers; a non-functionalized sealing layer disposed within the housing between the inlet and the outlet, the non-functionalized sealing layer serving as the last dielectric layer in the dielectric stack when a fluid flows from the inlet to the outlet through the dielectric stack within the housing; an edge of the sealing layer in contact with the housing, the edge being compressed by the housing to form a compression seal portion to prevent the fluid from leaking through the compression seal portion to the outlet.

Description

Background Art

[0001] In the bioprocessing industry, there are several examples of viral contamination of recombinant proteins, vaccines, and plasma products. The source of contamination can be exogenous, i.e., raw materials, the environment; or endogenous, i.e., expressed in cells or retrovirus-like particles (RVLPs). Cell lines such as Chinese hamster ovary (CHO) contain retroviral sequences in their chromosomes, which can lead to the shedding of large amounts of RVLPs. To safely prevent viral contamination, biopharmaceutical manufacturers typically adopt a multi-layered strategy involving the viral safety of raw materials, in-process and final product testing, and the implementation of viral clearance technologies. Summary of the Invention

[0002] Biopharmaceutical manufacturers typically rely on one or more virus removal technologies, which can involve inactivating viruses by maintaining a low pH, using solvents or detergents, heating, irradiation or ultraviolet light, removing viruses by precipitation, chromatography and / or filtration. Removing viruses by chromatography can involve using functional chemicals to adsorb viruses and virus-like particles (VLPs). Viruses and VLPs are typically 15nm-400nm in size. Membrane chromatography separation devices designed to remove viruses can utilize membranes coated, grafted or otherwise functionalized with certain functional chemicals to adsorb viruses and VLPs. In order to ensure adequate removal of virus and VLP contaminants, the membrane chromatography separation device must be particularly well sealed at the interface between the membrane layer and the housing.

[0003] It has been found experimentally that sealing membranes with functional chemicals to achieve viral clearance of approximately 7 log reduction values (LRV) is extremely challenging. 7LRV corresponds to 99.99999% viral removal. While regulatory agencies currently do not specify the amount of virus that needs to be removed during processing, the industry aims for a cumulative removal of approximately 12LRV-15LRV of endogenous viruses and approximately 6LRV-8LRV of exogenous viruses, which may be achieved through more than one viral clearance step. A single viral clearance step is generally considered effective if greater than or equal to 4LRV is achieved.

[0004] In flat-plate chromatography separation devices, the edges of the membrane or medium are typically sealed by compression. This creates localized areas near the edges where the membrane or medium permeability can be reduced. In the case of membranes with functionalized chemicals, the morphology of the membrane changes as it interacts with the virus solution. Therefore, devices with functionalized membranes may not be able to effectively seal the housing by compression as desired. This can result in lower log reduction values when challenged with the virus solution.

[0005] The present invention resides in a sealing layer positioned as the last layer through which fluid flows from an inlet to an outlet in a stack of at least two layers within a chromatographic separation device. The sealing layer contacts the housing, and at least a portion of its periphery forms a compressive seal within the device. The sealing layer is a "non-functionalized" layer, as defined herein. It has been found that a "functionalized" layer, as defined herein, does not achieve a good compressive seal, and when the layer is the last layer in contact with the housing for sealing, the LRV of the chromatographic separation device is less than that of a similarly configured chromatographic separation device having the same functionalized layer configuration but with the addition of the sealing layer as the last layer in contact with the housing.

[0006] Therefore, in one embodiment, the present invention is a chromatographic separation device comprising: a shell having an inlet and an outlet; at least one functionalized media layer, the at least one functionalized media layer being disposed between the inlet and the outlet inside the shell; a non-functionalized sealing layer, the non-functionalized sealing layer being disposed between the inlet and the outlet inside the shell, the non-functionalized sealing layer serving as the last media layer in the media stack as the fluid flows from the inlet to the outlet through the media stack within the shell; and an edge of the sealing layer in contact with the shell, the edge being compressed by the shell to form a compression seal portion to prevent the fluid from leaking through the compression seal portion to the outlet.

[0007] The present invention also provides a spacer ring between dielectric layers in a chromatographic separation device, which is used to increase the dynamic binding capacity of the chromatographic separation device. The function of the spacer ring is to provide an air gap between the front dielectric layer and the rear dielectric layer in the direction of fluid flow in the chromatographic separation device.

[0008] Without wishing to be bound by theory, it is believed that the air gaps allow liquid to disperse more quickly to the edges of the media, thereby helping to prevent challenging fluids from prematurely tunneling through only the center of the media. Air gaps allow fluids to flow directly to the edges of the media, rather than relying on capillary action to move the fluid to the edges. Additionally, certain media, such as functionalized nonwovens, can swell upon contact with liquids, and this swelling can lead to undesirable tunneling, thereby preventing the fluid from dispersing to the edges of the media. By spacing the layers only slightly apart, air gaps can be provided that allow liquid exiting one media layer to flow laterally before entering the next. Furthermore, air gaps can provide space to accommodate the swelling of the functionalized media, which otherwise could cause high compressive stresses in adjacent media layers as the media layers swell relative to each other. Consequently, the center of the media can expand and cause more liquid to flow through it.

[0009] Therefore, in another embodiment, the present invention is a chromatographic separation device comprising: a housing having an inlet and an outlet; at least two dielectric layers disposed between the inlet and the outlet inside the housing, at least one of the dielectric layers comprising a functionalized layer; and a spacer ring disposed between the two dielectric layers to form an air gap between the two dielectric layers.

[0010] When faced with the problem of increasing both the dynamic binding capacity and the LRV of a chromatographic separation device, it is 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 fluid flows in the chromatographic separation device.

[0011] Therefore, in one embodiment, the present invention is a chromatographic separation device comprising: a shell having an inlet and an outlet; at least two media layers, the at least two media layers being arranged between the inlet and the outlet inside the shell to form a media stack, at least one of the media layers comprising a functionalized layer; a spacer ring, the spacer ring being arranged between the two media layers to form an air gap between the two media layers; a non-functionalized sealing layer, the non-functionalized sealing layer being arranged between the inlet and the outlet inside the shell, and when the fluid flows from the inlet to the outlet through the media stack in the shell, the non-functionalized sealing layer serves as the last media layer in the media stack; and an edge of the sealing layer in contact with the shell, the edge being compressed by the shell to form a compression seal portion to prevent the fluid from leaking to the outlet through the compression seal portion. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is a front view of one embodiment of a chromatographic separation device.

[0013] Figure 2 for Figure 1 Top view of the chromatographic separation device.

[0014] Figure 3 for Figure 1 Bottom view of the chromatographic separation device.

[0015] Figure 4 yes Figure 1 Perspective view of a chromatographic separation apparatus.

[0016] Figure 5 for Figure 2 A cross-sectional view of the chromatographic separation device taken at 5-5 in FIG.

[0017] Figure 6 Another embodiment is shown Figure 5 Cross-sectional view of the chromatographic separation device depicted in .

[0018] Figure 7 Yes Figure 6A cross-sectional view of the chromatography separation device is shown before ultrasonic welding of the upper and lower shells.

[0019] Figure 8 The invention relates to a chromatographic separation device having a media stack in a direction from an inlet to an outlet, the media stack comprising a functionalized nonwoven layer, a functionalized nonwoven layer and a functionalized membrane layer.

[0020] Figure 9 The invention relates to a chromatographic separation device having a media stack from an inlet to an outlet. The media stack comprises a functionalized nonwoven layer, a functionalized nonwoven layer, a spacer ring and a functionalized membrane layer.

[0021] Figure 10 The invention relates to a chromatographic separation device having a media stack from an inlet to an outlet. The media stack comprises a functionalized nonwoven layer, a spacer ring, a functionalized nonwoven layer and a functionalized membrane layer.

[0022] Figure 11 The invention relates to a chromatographic separation device having a media stack from an inlet to an outlet. The media stack comprises a functionalized nonwoven layer, a spacer ring, a functionalized nonwoven layer, a spacer ring and a functionalized membrane layer.

[0023] Figure 12 The invention relates to a chromatographic separation device having a media stack from an inlet to an outlet. The media stack comprises a functionalized nonwoven layer, a functionalized nonwoven layer, a functionalized membrane layer and a sealing layer.

[0024] Figure 13 The invention relates to a chromatographic separation device having a media stack from an inlet to an outlet. The media stack comprises a functionalized nonwoven layer, a functionalized nonwoven layer, a spacer ring, a functionalized membrane layer and a sealing layer.

[0025] Figure 14 The invention relates to a chromatographic separation device having a media stack from an inlet to an outlet. The media stack comprises a functionalized nonwoven layer, a spacer ring, a functionalized nonwoven layer, a functionalized membrane layer and a sealing layer.

[0026] Figure 15 The invention relates to a chromatographic separation device having a media stack from an inlet to an outlet. The media stack comprises a functionalized nonwoven layer, a spacer ring, a functionalized nonwoven layer, a spacer ring, a functionalized membrane layer and a sealing layer. DETAILED DESCRIPTION

[0027] Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the values explicitly listed as the limits of the range but also all individual values or subranges encompassed within the range, as if each value and subrange were explicitly listed. For example, the range "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also individual values (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%) within the indicated range. Unless otherwise indicated, the expression "about X to Y" has the same meaning as "about X to about Y." Similarly, unless otherwise indicated, the expression "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z."

[0028] In this document, unless the context clearly indicates otherwise, the terms "a", "an", or "the" are used to include one or more than one. Unless otherwise indicated, the term "or" is used to refer to a non-exclusive "or". The expression "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B". In addition, it should be understood that the words or terms used herein and not otherwise defined are for illustrative purposes only and are not limiting. Any use of section headings is intended to facilitate understanding of the document and should not be construed as limiting; information related to a section heading may appear within or outside that particular section.

[0029] As used herein, the term "about" can allow for a certain degree of variability in values or ranges, for example, within 10%, within 5%, or within 1% of the stated value or limit of the stated range, and includes the specifically stated value or range.

[0030] As used herein, the term "substantially" refers to a majority or majority, such as at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. As used herein, the term "substantially free" can mean free of or having an insignificant amount such that the amount of material present does not affect the material properties of the composition containing the material, such that the composition contains from about 0% to about 5% by weight of the material, or from about 0% to about 1% by weight, or about 5% by weight or less, or less than or equal to about 4.5%, 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.

[0031] As used herein, a "layer" refers to a thickness of material through which a fluid to be treated flows, wherein the material in the layer is entirely formed of the same material. A layer can be a monolithic layer formed from a thickness of the same material. Alternatively, a layer can have one or more discrete plies of material stacked one on top of the other within the layer to form its thickness. For example, a conventional facial tissue layer is typically a tissue material made from two separate tissue layers placed in face-to-face contact, and the two separate plies can be easily separated from each other because the two separate plies are typically held together by a weak mechanical bond in the form of a crimped wire.

[0032] As used herein, " ply or plies " are the materials of the single thickness that can be processed by conventional conversion processing operation (such as but not limited to winding, folding, cutting or stacking in layers).Usually, ply is the material thickness after completing the forming process on the web manufacturing machine.Thereafter, one or more plies can be stacked to form a layer.For example, nonwoven can be made into a single ply and wound into a roll on a former.Thereafter, when the nonwoven roll is longitudinally passed through the conversion processing machine, the nonwoven roll can be unfolded and folded in half horizontally by folding plate, and then by cutting die, double-layer lamella is cut into disk, to form the circular layer of the nonwoven material with two discontinuous plies.

[0033] As used herein, a "functionalized layer" is a layer that will attract target particles or molecules by an attractive force (such as electrostatic force) due to the presence of one or more chemical moieties, ligands or functional groups at the surface of the layer, which are different from the material of the body forming the layer, which primarily provides its structural shape and integrity. The chemical moieties, ligands or functional groups are specifically designed to attract target particles or molecules to the surface of the functionalized layer. The functionalized layer can be formed by coating or grafting a porous layer with ligands, monomers or polymers designed to molecularly attract target particles or molecules. Alternatively, the functionalized layer can be formed by providing a surface-modified polymer or chemical moiety in a formulation for preparing such a layer, which is located at the surface of the layer during its formation, resulting in the presence of chemical groups designed to attract target particles or molecules on the surface of the layer. In some embodiments, the attractive force between the functional groups on the surface of the functionalized layer is an electrostatic force, and the chemical moieties, ligands or polymers present on the surface of the functionalized layer are electrostatically charged. The functionalized layer can have a positive charge and attract negatively charged particles, i.e., anion exchange chromatography, or the functionalized layer can have a negative charge and attract positively charged particles, i.e., cation exchange chromatography. In other embodiments, the attraction can be a van der Waals force, and the target particles or molecules are attracted to the functional groups on the functionalized layer surface by relatively concentrated or scarce polarizable or hydrogen bonding parts (i.e., hydrophobic interaction chromatography). In addition, the attraction can include a combination of electrostatic force and van der Waals force (i.e., mixed mode chromatography). Functionalized materials suitable for the functionalized layer in chromatographic separation devices are manufactured by Pall, Millipore, and Sartorious and sold under the following trademarks: Q. HD-Q and Q. The functionalized layer suitable for use in a chromatographic separation device can be a nonwoven, a membrane, or other suitable material. Preferred functionalized nonwoven materials are manufactured by 3M Company and disclosed in U.S. Patent No. 9,821,276, entitled "Nonwoven Article Grafted with Copolymer." Preferred functionalized membranes are manufactured by 3M Company and disclosed in U.S. Patent Nos. 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.

[0034] As used herein, a "non-functionalized layer" is a layer that has no coated, grafted, or surface-located attractive chemical moieties (eg, electrostatically charged chemical moieties, ligands, or functional groups) that are different from the material forming the bulk of the layer.

[0035] As used herein, a "media stack" is all of the layers of material through which a fluid to be treated flows within a housing as the fluid moves through the housing from an inlet to an outlet.

[0036] As used herein, " film " refers to a synthetic liquid permeable membrane, which includes a material sheet, in which a plurality of holes or interconnected pore networks are provided so that fluid can pass through the membrane. Such membranes include polymer membranes typically prepared by a phase inversion process, wherein a uniform solution of one or more polymers in a suitable solvent or solvent combination is subjected to phase separation to form a porous structure. Phase separation can be achieved by introducing a membrane of a uniform solution into a non-solvent bath (called diffusion-induced phase separation) or a non-solvent atmosphere (called vapor-induced phase separation) or by changing the temperature of the uniform solution (called heat-induced phase separation). Alternatively, holes can be formed in a polymer sheet by a stretching process or by an irradiation process (track etching membrane). The membrane can have a pore size (microporous membrane) with a diameter of about 0.1 micron to about 20 microns or a pore size (superporous membrane) less than about 0.1 micron. Suitable polymers for forming membranes include cellulose acetate, nitrocellulose, cellulose esters, polysulfones (including bisphenol A polysulfone and polyethersulfone), polyacrylonitrile, polyamides (e.g., nylon-6 and nylon-6,6), polyimides, polyethylene, polypropylene, polytetrafluoroethylene, polyvinylidene fluoride, polyvinyl chloride, and ethylene-chlorotrifluoroethylene copolymers.

[0037] As used herein, "dynamic binding capacity (DBC)" refers to the mass of target molecules captured by a media layer from a challenge solution as a function of the layer's projected area at a specified flow rate, where the endpoint is defined as a specified concentration of target molecules detected in the device effluent. Thus, if a chromatographic separation device has three media layers between the inlet and outlet, only the pre-fluid contact surface area of one layer is used in the calculation.

[0038] As used herein, "challenge solution" refers to a solution with a precisely known concentration of target molecules that can selectively bind to the membrane (media) of the device.

[0039] In one embodiment, the challenge solution has a target value of 1mg / mL bovine serum albumin (BSA). A 25mM Tris 50mM sodium chloride (NaCl) aqueous solution is prepared by slowly dissolving 3.029g Tris base and 2.922g NaCl in 1L deionized (DI) water. When measuring pH, a small amount of concentrated hydrochloric acid (HCl) is added to adjust the pH to 8. Approximately 300mg of BSA is sprayed on the surface of the 25mM Tris 50mM NaCl buffer solution. BSA is dissolved in the buffer solution by slow hydration for at least 1 hour. The solution is then passed through a 0.2 μm filter into a sterile medium bottle. The absolute concentration of BSA is determined using Beers' law by measuring the UV absorbance of the solution at 280nm, with 0.667 as the extinction coefficient (ε) b.

[0040] To determine the BSA DBC of the chromatographic separation device, the solution was heated to 210 LMH (L / m 2 The test device was flowed under standard flow conditions (100% surface area before the medium / hour). The endpoint was determined by the penetration of the BSA challenge solution, as indicated by the 10% absorbance value of the effluent (based on the initial BSA solution, defined as 100%) using UV detection at 280nm. The dynamic binding capacity was then determined by using the volume of the challenge solution that passed through the test device before reaching the endpoint condition and calculating the mass of BSA in that volume. The dynamic binding capacity is this mass divided by the effective membrane (medium) area, as shown in Formula 1.

[0041] (1

[0042] In another embodiment, the challenge substance is 20 mM potassium chloride. - )DBC is measured using the procedure described in U.S. patent application serial number 67 / 783,319, entitled “Method For Testing A Chromatography Device Used For Ion Exchange,” filed on December 21, 2018, which is incorporated herein by reference in its entirety, but particularly as described on page 33, line 0096, through page 35, line 00109.

[0043] In another embodiment, the challenge solution has 1×10 8 The target value of 1×10 plaque forming units (PFU) / mL of phage Phi-X 174 was used to determine the LRV. Initially, at least 1×10 11PFU / mL of Phi-X 174 stock solution. Prepare a 50mM Tris aqueous solution by slowly dissolving 6.057g of Tris base in 1L of deionized (DI) water. When measuring the pH, add a small amount of concentrated hydrochloric acid (HCl) to adjust the pH to 8. Check the conductivity to ensure it is 20mS / cm and adjust it by adding a small amount of Tris or DI water. Dilute the Phi-X 174 stock solution to 1×10 in 50mM Tris-HCl buffer solution with a pH of 8 and a conductivity of 20mS / cm. 8 A small aliquot of this viral challenge solution was saved as the "input" sample for LRV calculation.

[0044] In order to measure the viral logarithm reduction value of the chromatographic separation device, the input solution and the elution solution are plated in the presence of various dilutions of bacterial intestinal bacteria (Escherichia coli, E.coli). In a 5mL test tube, 50 μL of the E. coli 13076 host are added to each input or output dilution of every 100 μL. 2.5mL of nutrient broth top agar (supplemented with 0.6% agar nutrient broth) is added to each test tube, and then mixed via a rotary motion to ensure that the solution is fully mixed. The solution is then poured onto the surface of the nutrient agar plate and hardened, then incubated at 37°C for three hours. After incubation, Phi-X 174 virus plaques form circular transparent areas in the bacterial lawn of E. coli growth. The plaques of the gained input plate and elution plate are then counted to determine concentration (PFU / mL) using Formula 2.

[0045] C = average number of plaques × dilution ÷ plating volume (2)

[0046] This concentration was used to determine the final reduction in viral clearance according to Equation 3.

[0047] LRV=log 10 [(C 供给 ×Vol 供给 ) / (C 最终 ×Vol 最终 )] (3)

[0048] Membrane chromatography is a relatively new method of ion exchange chromatography that evolved from the need of the bioprocessing industry to overcome the limitations of conventional resin-bead based chromatography. Membrane chromatography devices comprise a microporous medium with pores that contain adsorption sites that can bind target proteins and / or viruses and VLPs depending on the functional chemistry and operating conditions. Because membrane chromatography devices rely on convective mass transfer, higher flow rates can be used without significant pressure drops, resulting in higher throughput and reduced processing time. There are three main types of membrane-based chromatography devices: flat plate, hollow fiber, and flowthrough. Flat plate chromatography devices are generally more popular because they have a larger adsorption membrane volume.

[0049] Membrane chromatography separation devices come in a variety of sizes, typically associated with the development stage of a molecule. Laboratory devices typically have media volumes ranging from approximately 0.08 mL to 3 mL. Large-scale or prototype devices typically have media volumes ranging from approximately 15 mL to 100 mL. Commercial production devices typically have media volumes greater than or equal to 200 mL. It should be noted that other media volumes are available depending on customer needs.

[0050] One way to calculate the volume of the media is to multiply the effective filter area (EFA) by the nominal media height or thickness. The nominal media height or thickness can be measured using a caliper, and the EFA can be measured by filtering a dye solution through the device. The dye is bound to the media, and after the dye penetrates into the outlet stream, the device is cut open and the diameter of the dye stain in the (one or more) layers of the media is measured to determine the average diameter, or the dye area can be measured directly using, for example, optical methods. The average diameter of the dye stain (if more than one layer) is used to calculate the EFA using the formula for the area of a circle, or the measured area of each layer can be averaged to derive the EFA.

[0051] The present invention can be used with any desired media volume and is particularly suitable for use in chromatographic separation devices for viral removal. Although the present invention refers to a "chromatographic separation device" and a "membrane chromatography separation device," all aspects of the invention are equally applicable to mixed media configurations including functionalized membranes and other types of filtration and chromatographic separation media, such as, but not limited to, hydrogel functionalized nonwovens, cellulose and diatomaceous earth based dielectrics, and activated carbon.

[0052] While the present invention can be used with any desired media volume, a laboratory-scale chromatographic separation device as described in U.S. Patent Application Serial No. 62 / 792,166, filed January 14, 2019, entitled "Sample Size Chromatography Device," and incorporated herein by reference in its entirety, is particularly suitable. In one embodiment, a media volume of 0.08 mL is used to minimize the amount of virus solution required for the sample. The holdup volume of the chromatographic separation device was measured to be approximately 1.1 mL.

[0053] Laboratory-grade chromatography separation device

[0054] Now see Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 and Figure 5 , shows a preferred example of a chromatographic separation device 8. 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. A membrane or media 22 in a chamber 24 is disposed between the inlet 16 and the outlet 18 such that fluid from the inlet 16 enters the interior chamber 24, then passes through the media 22 and exits the outlet 18. The seals, spacer rings, or combinations thereof of the present invention may be used with any suitable housing having an inlet, an outlet, and a media stack disposed between the inlet and the outlet for fluid flow therethrough.

[0055] The chamber 24 is in fluid communication with the inlet 16 and the vent 20 so that any air in the chamber 24 can be purged out of the vent 20. A Luer lock connector (not shown) can be attached to the vent 20 and acts as a valve to purge air from the chamber 24 until liquid from the inlet 16 begins to leave the vent 20 and the valve closes. 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 media and adjusting the size of the housing to that diameter.

[0056] like Figure 1As shown, at least one of the inlet 16 and the vent 20 can be arranged at an angle to the longitudinal axis 26 of the housing 10, and preferably both are arranged at an angle to the longitudinal axis. As shown, the inlet 16 is arranged at an angle α to the housing longitudinal axis 26, and the vent is arranged at an angle β to the housing longitudinal axis. This produces two beneficial effects. First, it provides enough clearance for the use of the Luer lock connector on both the inlet 16 and the vent 20 to be used, thereby providing a quick and convenient method for purging air from the chamber 24. Axially aligned inlets do not provide enough clearance to include a vent with a reliable locking (positive lock) seal (such as a Luer lock connector) in small volume laboratory devices.

[0057] Second, the angled inlet 16 directs the incoming fluid flow as it passes through the chamber 24 to Figure 5 The arrow in the figure indicates that the fluid impinges on the upper surface of the medium at an angle other than 90 degrees. When the inlet is axially aligned parallel to the longitudinal axis 26, the incoming fluid impinges on the surface of the medium directly at the center of the medium at approximately 90 degrees. This design presents a tunneling problem, causing the feed solution to "tunnel" through the center of the disk, resulting in premature penetration. Angling the inlet 16 so that the incoming fluid stream has a tangential velocity component parallel to the upper surface of the medium will cause at least some of the incoming fluid to flow over at least a portion of the upper surface of the medium before flowing through the medium. This is unlike the action of throwing a bucket of water onto the floor at an angle to rinse the floor, causing the water to spread along the floor away from the person emptying the bucket. Angling the inlet not only helps prevent tunneling but also helps drive air from chamber 24 toward and away from vent 20. The angular position of the inlet can be designed so that a 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 top of the medium and into the chamber walls. This movement of the incoming fluid volume results in automatic redistribution and mixing within the chamber, providing a more even utilization of the media capacity.

[0058] In various embodiments of the device, the angle α between the longitudinal axis 28 of the inlet and the longitudinal axis 26 of the housing can be about 10 degrees to about 80 degrees, about 25 degrees to about 65 degrees, or about 40 degrees to about 50 degrees. In various embodiments of the device, the angle β between the longitudinal axis 30 of the vent and the longitudinal axis 26 of the housing can be about 10 degrees to about 80 degrees, about 25 degrees to about 65 degrees, or about 40 degrees to about 50 degrees. Angle α can be the same as, less than, or greater than angle β. Additionally, if only one of the inlet and the vent is angled for the Luer lock gap, it is preferred that the inlet be angled for the positive flow effect described above. In the illustrated embodiment, angle α is 45 degrees and angle β is 45 degrees, so that the inlet and vent can be interchanged as needed and used for opposite functions.

[0059] Upper shell 12 and lower shell 14 are designed to be ultrasonically welded together to form a final, liquid-tight shell while also providing an edge seal for the media. Specifically, the forces applied to the shell during assembly to compress the upper and lower shell sections are controlled, while ultrasonic welding reliably controls media compression regardless of variations in media thickness. This unique welding process is discussed in greater detail below. Shell 10 is generally circular, but any other suitable shape may be employed.

[0060] like Figure 5 As best shown, the upper housing 12 includes two cylindrical protrusions 32 extending from an upper surface 34 of an upper disc 36 on either side of the housing's longitudinal axis 26 and arranged at an angle to the housing's longitudinal axis, forming a truncated V-shape between the upper surface and the housing's longitudinal axis. Each cylindrical protrusion has a tapered internal bore 38 to accommodate a Luer lock taper and provide fluid communication with the chamber 24. A truncated hemispherical surface 40 is present within the interior of the chamber 24 and is molded into the center of the upper disc 36 between the inlet and outlet to reduce the volume of the chamber. The tapered bores 38 of the inlet and vent are in fluid communication with a cylindrical passage 42 leading to the chamber 24, allowing fluid to pass through the tapered bores into the cylindrical passage and into the chamber of the assembled housing. The chamber is generally cylindrical in shape, with a truncated hemispherical upper surface as shown. Other chamber shapes may be used, and generally, the overall size of the chamber is kept as small as possible to reduce hold-up volume while still allowing fluid communication between the inlet, vent, chamber, and media surface.

[0061] As used herein, for convenience, upper housing 12 is a relative term, and in one embodiment, the upper housing is the housing portion having both the inlet 16 to chamber 24 and the vent 20. In a similar manner, for convenience, the lower housing is a relative term. The first housing portion may be used in place of the upper housing, and the second housing portion may be used in place of the lower housing. Throughout this specification, where the term "upper" appears, "first" may be substituted, and where the term "lower" appears, "second" may be substituted.

[0062] like Figure 5 、 Figure 6 and Figure 7 As shown, a compression extension 46 extends from the lower surface 44 of the upper disc 36 of the upper housing 12. In some embodiments, the compression extension is a protruding annular structure. For media perimeter geometries other than circular, such as square or hexagonal, the compression extension will assume the same corresponding shape as the media perimeter. The compression extension 46 cooperates with the boss 60 supporting the media 22 to compress the edge or perimeter of the media to a distance X as shown. This seals the edge or perimeter of the media, preventing fluid from leaking from the chamber 24 and around the edge or perimeter of the media to the outlet 18. Sufficient compression is necessary to prevent leakage; however, if the media is over-compressed, excessive media area is lost due to compression, and the performance of a laboratory device can deviate significantly from that of a large-scale or production device using the same media. Therefore, the height of the compression extension protruding from the lower surface 44, combined with the tightness with which the upper and lower housings are pressed together during ultrasonic welding, controls the distance X and the resulting edge compression of the media disc 22. During actual ultrasonic welding, the weld energy is set to control the relative edge compression of the media. While the housing is depicted with a compression extension 46 extending from the lower surface of the upper housing in conjunction with a boss 60 on the lower housing, the two components can be switched and the compression extension 46 can extend from the lower housing, while the boss 60 can reside on the upper housing. Also in an alternative embodiment, the boss 60 can protrude from the surrounding surface of the housing interior. For example, the two extensions can be used to compress the perimeter of the media to seal it.

[0063] Projecting from the lower surface 44 of the upper shell 12 is an interlocking weld extension 48 that is welded to the lower shell 14. In some embodiments, the interlocking weld extension is also a protruding ring with a chamfered tip 49 for use during the ultrasonic welding process. The interlocking weld extension is located outboard of the compression extension, at a greater distance from the longitudinal axis of the shells. The interlocking weld extension 48 initially abuts a step 51 in an optional groove 53 in the lower shell, as shown. Figure 7As shown. The center portion of the shell has a height Y' before welding, and the compressed extension has a height X'. Due to the bevel and the step, the final height of the shell Y can change as the amount of ultrasonic energy applied to the shell during welding increases. This then affects the final compressed edge dimension X. As more energy is applied during the ultrasonic welding process, the step height 51 decreases more and the chamfered tip 49 slides deeper into the groove 53. Figure 7 and Figure 6 For comparison, even if the parts are completely welded to one another, the final assembly height Y of the capsule can change, which in turn changes the edge compression distance X. Applying more energy reduces the final assembly height Y by allowing the chamfered tip 53 and interlocking weld extension 49 to slide deeper into the groove 53, resulting in more edge compression of the media and a reduced height X. The opposite occurs for less applied welding energy, resulting in a greater final assembly height Y and less edge compression, along with a larger dimension X. While the housing is depicted as having an interlocking weld extension 48 extending from the lower surface 44 of the upper housing 12 and a step 51 located on the lower housing 14, the interlocking weld extension 48 can extend from the lower housing 14, and the step 51 can reside on the upper housing. The cross-sectional profile of the interlocking weld extension and the shape of its perimeter can be adjusted for different housing geometries. The annular shape is suitable for circular media 22, as shown.

[0064] In one embodiment, a circular media stack is used, and the compression extension and interlocking weld extension are protruding rings, as described below. A first protruding ring 46, used for the compression extension to compress the media around the edge or perimeter, and a second protruding ring 48, used for the interlocking weld extension, extend from the lower surface 44 of the upper disc 36, with the second protruding ring positioned inboard of the outer diameter 50 of the upper disc 36. The longitudinal length of the first protruding ring is selected to compress and seal the media around the edge or perimeter. The longitudinal length of the second protruding ring is selected to align with and coordinate with features on the ultrasonically welded lower housing, while allowing dimension X to vary within a certain height range while still maintaining a media seal around the edge or perimeter. Media with significant thickness variations may require different longitudinal length dimensions for the protruding rings to avoid over-compression of the media, thereby degrading performance or failing to seal the media around the edge to prevent bypass. As shown, the first and second protruding rings have sidewalls that taper with a thicker base and a narrower tip. Other cross-sectional geometries may be utilized. The first sidewall 52 of the first protruding ring 46 forms a portion of the sidewall of the chamber 24 below the inlet and vent.

[0065] As in Figure 5As best seen in FIG, 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 groove 53 is formed between the two rings, into which the interlocking weld extension is positioned for ultrasonic welding attachment to the lower housing. 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 longitudinal ribs 58 spaced along the perimeter to provide an enhanced grip when holding the housing 10. The inner sidewalls of the third and fourth protruding rings are angled to match the taper of the second protruding ring on the upper housing for nesting the two housing portions. Nesting the interlocking weld extension 48 between the third and fourth protruding rings 52, 54 provides greater structural integrity to the welded housing, allowing the assembled housing to better resist lateral forces against the housing without breaking the ultrasonic weld. Additionally, the inner surface of the third protruding ring 52 serves as a guide and centering device for positioning the circular media 22 onto the boss 60 when the components are assembled. Figure 7 Best shown.

[0066] A circular boss 60 is located inside the base of the fourth protruding ring at its innermost sidewall. This boss supports the periphery of the circular media disc 22 placed in the center of the lower housing. The fourth protruding ring generally guides the media into position and centers the media on the support boss 60. The distance X from the boss's upper surface 62 to the tip 64 (compression extension) of the first protruding ring 46 is selected to affect the necessary compression of the media to provide a fluid-tight seal along the edge or periphery of the media disc. By controlling this distance during the ultrasonic welding process, the performance of the laboratory device can be optimally matched to that of a large-scale device or production device. An optional circular dished recess 66 is located below the circular boss and acts as a funnel to guide the filtered fluid to the outlet. A cylindrical protrusion 32, parallel to and concentric with the housing longitudinal axis 26, extends from the lower surface 68 of the lower disc 56. The cylindrical protrusion has a tapered internal bore 38 to accommodate the Luer lock taper and is in fluid communication with the dished recess 66. The tapered internal bore 38 of the outlet is in fluid communication with a cylindrical passage 42 leading to the dished recess, thereby allowing fluid to flow from the dished recess through the cylindrical passage and out of the housing through the tapered bore.

[0067] A unique feature of the housing design is the combination of media compression and the discussed edge or perimeter sealing of the media within the device. Typically, chromatographic separation devices have O-rings or gaskets to seal and compress the media. One of the features of this design is the combination of media compression between the upper and lower housings via the tip of the first protruding ring and the circular boss, as shown in FIG. Figure 5This provides a straightforward way to control media compression and accommodate thickness variations in the chromatographic separation media, as the housing sections are ultrasonically welded under a fixed load, and the resulting weld height can vary as the thickness of the media stack changes. This design of the device assembly, combined with ultrasonic welding of the upper and lower housing sections, ensures consistent edge effects despite variations in media stack thickness and provides a predictable approach to device capacity and pressure drop.

[0068] Typical laboratory-grade chromatographic separation devices on the market today are manufactured by assembling a two-piece housing (inlet housing portion, outlet housing portion) with the internal media disposed between the housing portions, compressing the entire assembled housing to a fixed height, and then using an overmolding process. The overmolding process involves compressing the two housing portions to a final specified height and then ejecting molten plastic onto the exterior of the housing assembly to form a fluid-tight housing that maintains the predetermined compressed assembly height before the molten plastic is applied. During this process, several tons of force can be applied to the chromatographic separation media, resulting in a protruding and large compression edge or peripheral area. This large compression edge or peripheral area reduces the performance of the chromatographic separation device as discussed above. During the overmolding process, the high compressive force on the assembled housing portions is necessary to contain the molten plastic and prevent burrs. A predetermined mold height is selected to prevent burrs. Therefore, thicker media stacks experience greater compression than thinner media stacks, resulting in significant performance changes in small-volume chromatographic separation devices. This method of manufacturing a chromatographic separation device is not very flexible in the sense that multiple molds for the overmolding process would be required to ensure that the compression of the chromatographic separation media stack at the edges or perimeter is the same for various media thicknesses.

[0069] On the other hand, ultrasonic bonding can involve compressing the assembly of the inlet housing portion, the outlet housing portion, and the internal chromatographic separation media stack to a specified force rather than a fixed height. When the predetermined force is reached, the ultrasonic welding process begins, and the vibration energy applied to the energy director causes local melting and bonding. In this method, the edges of the chromatographic separation media are only subjected to a force of several pounds (many orders of magnitude less than the force observed during the overmolding method). Ultrasonic bonding results in a smaller compressed edge or peripheral area, and the actual amount of compression can be controlled by the energy applied during the ultrasonic welding process, which changes the final height of the assembled housing. In addition, because the start of welding is triggered by the force set point, normal variations in media thickness will not significantly affect the size of the compressed area affected at the edge or periphery. Thicker media stacks will have a higher shell height, while thinner media stacks will have a shorter shell height. The welding process is self-compensating for variations in media thickness. Therefore, the present design provides a simple way to ensure consistent assembly performance.

[0070] This method of manufacturing chromatographic separation devices is highly versatile and can accommodate chromatographic separation media of varying thicknesses. Large variations in media stack thickness for different product types may require different longitudinal lengths for the compression extensions and interlocking weld extensions, but nominal variations in manufacturing tolerances are easily manageable, and chromatographic separation devices manufactured using this method will have more consistent performance.

[0071] Now see Figure 6 , another embodiment of the chromatographic separation device is shown in the figure. A baffle 70 extends from the top plate of the chamber, which redirects the inlet flow to a direction more parallel to the longitudinal axis of the housing, as shown by the arrow. In one embodiment, the baffle is a fifth protruding ring having a sidewall 72 that is roughly parallel to the longitudinal axis 26 and extends a sufficient distance in the longitudinal direction to redirect the inlet flow as described, but is short enough to prevent interference between the swelling medium and the baffle feature. The outer diameter of the fifth protruding ring is small enough to fit between the cylindrical channel 42 of the inlet and the vent, wherein the fifth protruding ring nominally starts from the inside of the position where these channels intersect with the chamber 24, as shown in the figure. In some embodiments, the height of the fifth protruding ring in the longitudinal direction is the same order of magnitude as the diameter of the Luer connector, such as between about 3 mm and about 6 mm.

[0072] Depending on the surface tension and wetting properties of the chromatographic separation medium, a baffle 70 may be required. The baffle helps direct the inlet flow of buffer or solute along the chamber sidewalls and onto the compressed edge or periphery of the medium. Since the permeability of the medium in this area may be lower, the buffer or solute tends to redistribute toward the center of the medium disk to pass through the medium. This phenomenon is another way to counteract the tunneling effect described above.

[0073] The cylindrical protrusions forming the inlet, outlet, and vent can be sized to fit a tapered Luer lock connector. To facilitate the Luer lock connector, the outer surface of these cylindrical protrusions 32 can have two opposing transverse tabs 80 extending from the outer circular diameter of the cylindrical protrusion and positioned near the distal end of the cylindrical protrusion. The tabs engage with threads in the male Luer lock connector. Optionally, other fluid connectors, such as hose barbs, can also be used to guide fluid into and out of the chromatographic separation device.

[0074] The chromatographic separation device is preferably injection molded from a suitable material. Advantageously, the material is easily ultrasonically welded so that the upper and lower housings can be joined in a fluid-tight manner. Suitable materials for the housing include thermoplastics such as acetal (POM), acrylic (PMMA), acrylonitrile butadiene styrene (ABS), polycarbonate (PC), polyethylene (LD / HDPE), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polypropylene (PP), and the like.

[0075] Alternatively, other means of fastening the upper housing to the lower housing may be used, such as, for example, a liquid-tight threaded connection as used on common water pipes. The upper and lower housings may be made of a suitable material for threaded connection, such as plastic or metal.

[0076] Alternatively, the housing can be 3D printed using a three-dimensional printer. In this case, the housing can be bonded together using an adhesive such as an epoxy or acrylic resin to form a liquid-tight seal.

[0077] Spacer ring

[0078] like Figure 9 、 Figure 10 、 Figure 11 、 Figure 13 、 Figure 14 and Figure 15 As shown, in some embodiments, one or more spacer rings 86 may optionally be used in the chromatographic separation device 8. The function of the spacer rings is to provide an air gap between the leading and trailing media layers in the direction of fluid flow in the chromatographic separation device. Without being bound by theory, it is believed that the air gap allows liquid to disperse more quickly to the edges of the media, thereby helping to prevent tunneling through the center of the media. The air gap allows the fluid to flow directly to the edge of the media, rather than relying on capillary action to move the fluid to the edge of the media.

[0079] Certain media, such as functionalized nonwovens, can swell when in contact with liquids, and this swelling action, combined with the edge compression seals, can lead to undesirable tunneling. By spacing the layers only slightly apart, air gaps can be provided that allow liquid exiting one media layer to flow laterally before entering the next media layer.

[0080] Therefore, the optimal height of the spacer rings depends on the expected media swelling that may occur. Although air gaps and spacer ring heights as small as 0.001 inches can be used with non-swelling media layers, generally speaking, larger air gaps and spacer ring heights are used with functionalized nonwovens that swell more easily.

[0081] In some embodiments, the height of the spacer ring in the longitudinal direction between the dielectric layers may be equal to or greater than 0.001, 0.005, 0.010, 0.020, or 0.030 inches. The maximum height of the spacer ring is typically limited by the overall length of the housing and the height of the internal chamber in which the dielectric layers forming the dielectric stack may be placed. Too high a height of the spacer ring reduces the thickness of the dielectric layers that can be placed into the chromatographic separation device. Typically, the spacer ring will have a height of less than or equal to 1.0, 0.90, 0.80, 0.60, or 0.50 inches in the longitudinal direction between the dielectric layers. Ranges between these heights are within the scope of the present invention. Particularly suitable spacer rings have a height of between 0.030 and 0.050 inches in the longitudinal direction between the dielectric layers.

[0082] The spacer ring can be constructed similar to a washer, bushing, or short tube having an outer diameter, inner diameter, center hole or opening, and height. The outer diameter is typically sized to be the same as the outer compression seal diameter of the media. This typically corresponds to the outer diameter of the compression extension 46, such as Figure 5 and Figure 9 shown.

[0083] The inner diameter can be smaller than the inner compression seal diameter of the media; however, this will reduce capacity and increase the compression edge effect in the media edge because more media surface will be covered. Therefore, the inner diameter is approximately the same as or slightly smaller than the inner diameter of the media compression extension 46, as shown in FIG. Figure 5 and Figure 9 shown.

[0084] In many cases, the width of the spacer ring matches the width of the compression extension 46, such as Figure 9 This provides sufficient sealing area without canceling edge effects or causing loss of device capacity.

[0085] The appropriate dimensions of the outer diameter, inner diameter, and width of the spacer ring can be determined based on the specific housing design. As the diameter of the housing becomes larger or smaller for various sizes of the chromatographic separation device, the diameter of the spacer ring is also adjusted accordingly. In one embodiment, the outer diameter of the spacer ring is about 1.1 inches, the inner diameter is about 0.9 inches, and the width is 0.1 inches.

[0086] The number of spacer rings can vary and is typically one less than the number of media layers in the chromatographic separation device. Thus, a two-layer device may have one spacer ring between the two media layers in the media stack, while a three-layer device may have one spacer ring between the first and second media layers in the media stack, and one spacer ring between the second and third media layers. Due to the swelling of this layer, it is generally more important to separate the functionalized nonwoven layer from the next layer, while if the overall height of the media stack becomes an issue to fit within the chosen housing, then separating the functionalized membrane layer from the next layer is less important (because the functionalized membrane layer and the next layer generally do not swell).

[0087] The spacer ring can be made of a variety of different materials. The choice of material generally depends on the reactivity of the fluid to be processed to the material. The same materials that are suitable for the molded housing are also suitable for the spacer ring. Generally speaking, the material selected for the housing will also be used for the spacer ring. Suitable materials for molding the spacer ring include thermoplastics, such as acetal (POM), acrylics (PMMA), acrylonitrile butadiene styrene (ABS), polycarbonate (PC), polyethylene (LD / HDPE), polyphenylene oxide (PPO), polyphenylene sulfide (PPS) and polypropylene (PP). These thermoplastic materials are easy to injection mold and can be used to make spacer rings of appropriate sizes.

[0088] In many embodiments, the housing has a compression extension 46 and a boss 60 between which the media stack is compressed to form an edge seal. In some embodiments, the distal portions of these surfaces that contact the media are relatively flat or planar, such as Figure 5 In other embodiments, the distal portions of these surfaces that contact the medium include compression protrusions. Figure 8 As shown, the distal end of the compression extension 46 of the upper housing has a longer length near the interior chamber 24 and a shorter length toward the exterior of the housing. Similarly, the circular boss 60 in the lower housing also has a compression protrusion extending from its surface. Although not shown, the spacer ring 86 may have a compression protrusion located on one or both of these surfaces, rather than having a smooth upper surface and a smooth lower surface as shown.

[0089] The function of the hold-down projections is to bite into the media to prevent it from sliding out of the edge compression zone. Preferably, the hold-down projections are positioned so that the edge or perimeter of the media is compressed more in the area closer to the interior chamber 24 and less in the area further from the interior chamber. This creates a slightly less compressed area of the media toward the exterior of the housing, which would have to be further compressed and pulled under the hold-down projections during any swelling of the media, making this less likely to occur.

[0090] The compression protrusion can be a continuous ring, ridge, step, or other feature that rises from the distal surface of the compression extension, boss, or spacer ring. Alternatively, the compression protrusion can be a discontinuous surface consisting of short segments or protrusions that compress or bite into the surface of the media. The compression protrusion has been found to effectively enhance the seal of the media, particularly the functionalized nonwoven layer, which tends to swell and pull out of the edge compression area of the housing during use of the chromatographic separation device.

[0091] Sealing layer

[0092] like Figure 12 As shown, sealing layer 88 is positioned as the last layer through which fluid flows from the inlet to the outlet in the at least two-layer media stack within the chromatographic separation device. This sealing layer contacts the housing, and at least a portion of its edge or perimeter forms a compressive seal within the chromatographic separation device. Because it is the last compressive seal of the layer (the lowest layer in the media stack) through which fluid can pass below or above its outlet from the housing, it is the most important layer in the chromatographic separation device for sealing against the housing.

[0093] The sealing layer is a "non-functionalized" layer as defined herein. It has been found that a functionalized layer as defined herein does not achieve a good compression seal with the housing and, when the layer is the last layer in the media stack used for sealing, the LRV of the chromatographic separation device is less than a similarly constructed chromatographic separation device having the same media stack layer configuration but with the addition of a sealing layer of non-functionalized media as the last layer in the media stack in contact with the housing.

[0094] The sealing layer is a non-functionalized porous medium having a relatively smooth surface and sufficient rigidity due to its thickness to support the functionalized layer above it and prevent it from sagging and sliding out of the sealed edge under pressure due to the pressure differential generated by the flow through the medium layer during use. The sealing layer can be a film or a nonwoven layer. In a preferred embodiment, the sealing layer is a film because the sealing layer generally has a smoother surface roughness. The sealing layer can include a multi-component material having a ply of a film and another medium such as a scrim layer. Alternatively, the sealing layer can include two or more plies of a film or other medium.

[0095] When the sealing layer is a membrane, it can be formed by any suitable film-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 can be a supported membrane, which means that the supported membrane is cast on a porous support layer, such as a nonwoven layer, a spunbond layer, a fabric layer, a scrim, etc. Alternatively, the membrane can be an unsupported membrane, which means that it is formed by a film-forming material without the help of a support layer. The membrane can be a symmetrical membrane, which means that the average pore size at any position between the two outer major surfaces of the membrane is substantially the same. Alternatively, the membrane can be an asymmetric membrane or a gradient membrane, which means that the average pore size in the region near one major surface of the membrane is significantly larger than the average pore size in the region near the relative major surface of the membrane. Further, the membrane can be a multi-region membrane, which means that the membrane includes a full thickness region between its main outer surfaces, and the average pore size of the full thickness region is different from the average pore size of another full thickness region between the main outer surfaces of the membrane. In some embodiments, the sealing layer comprises a supported nylon-6,6 membrane as described in US Pat. No. 6,264,044, entitled “Reinforced, three zone microporous membrane,” issued Jul. 24, 2001, which is hereby incorporated by reference in its entirety.

[0096] In one embodiment, the sealing layer is a nylon-6,6 membrane with an average pore size of 0.8 microns. The thickness of the sealing layer is in the range of 17 mils to 20 mils. Other materials suitable for the sealing layer include nylon-6,6 membrane with an average pore size of 0.8 microns and a thickness of 8.5 mils to 10.0 mils; nylon-6,6 membrane with an average pore size of 0.65 microns and a thickness of 6.0 mils to 7.0 mils; nylon-6,6 membrane with an average pore size of 0.2 microns and a thickness of 6.0 mils to 7.0 mils; nylon-6,6 membrane with a pore size in the range of 0.2 microns to 1.2 microns and a thickness of 13.0 mils to 15.4 mils.

[0097] In various embodiments of the present invention, the film used for the sealing layer may have a minimum pore size within the film of 0.1 to 5.0 microns, or 0.1 to 3.0 microns, or 0.2 to 1.2 microns and a thickness of 6.0 to 20.0 mils.

[0098] As will be seen in the examples, the following materials were found not to increase the LRV of the chromatographic separation device and did not function as a suitable sealing layer: a spunbond nonwoven having an average thickness of 9 mils. A polyethersulfone membrane having an average pore size of 0.2 microns and a thickness of 100 to 120 microns and the above-mentioned spunbond nonwoven were both ultrasonically bonded to the outlet housing of the laboratory device to provide a rigid support structure.

[0099] Without being bound by theory, it is believed that the sealing layer provides a consistent seal because it cannot undergo morphological changes like the functionalized layer when in contact with the challenge solution. Moreover, when a membrane is used as a sealing layer, the medium generally does not allow any significant tangential flow, thereby improving sealing performance. Typically, the sealing layer can be formed from the same medium used to prepare the functionalized layer, but without any coating or grafting treatment to functionalize it. Therefore, it can have a relatively similar thickness and pore distribution to that of the functionalized layer in the chromatographic separation device. In a preferred embodiment, a functionalized membrane is used as one of the chromatographic separation medium layers, and the same precursor membrane before functionalization is used as the sealing layer.

[0100] When using spacer ring, sealing layer or both, the chromatographic separation device that medium volume or membrane volume is about 0.08mL is evaluated.Use mass melt flow rate (MFR) is the polypropylene random copolymer injection molding upper housing and lower housing and spacer ring (height is 0.050 inch) of 9.0g / 10min.Selected chromatographic separation medium has three kinds of main components: anion exchange nonwoven, anion exchange membrane and sealing layer.The anion exchange nonwoven layer is made up of the polypropylene nonwoven of the quaternary ammonium functional polymer that four plies have covalent attachment.The anion exchange membrane layer is made up of the high porosity polyamide membrane of the guanidinium functional polymer that three plies have covalent attachment.In some embodiments, be the polyamide non-functionalized membrane of the sealing layer in the housing capsule of assembling afterwards.

[0101] Example, Figures 8 to 15

[0102] To assemble the chromatographic separation device, the media components were punched to obtain a 1.0625 inch diameter disc. The disc was placed in the lower housing 14 on the inside of the protruding ring 54. A spacer ring 86 was added between the functionalized membrane and the nonwoven layer. The upper housing was positioned on top of the media so that the protruding ring 48 of the upper housing slid between the protruding rings 52 and 54 of the lower housing. The assembly was inverted and placed in a nest or fixture so that the outer surface 68 of the lower housing 14 could contact the ultrasonic horn. To weld the parts, a Branson 20kHz ultrasonic welder (2000xdt model), a black intensifier, and a horn with a gain of 2.5x were used. An air pressure of 80psi, a descent rate of 10%, an amplitude of 80%, a welding time of 2 seconds, and a starting welding trigger force of 200lbf were set as fixed parameters. The welding energy varied from 200 joules to 600 joules to obtain a sample with a consistent compression level, specifically depending on the number of spacer rings and sealing layers. The housing assembly with the chromatographic separation media is placed in the nest directly below the horn, with the housing longitudinal axis 26 aligned with the axis of the ultrasonic horn. When the welding process begins, the horn compresses the housing and media assembly downward on the lower housing until a force of 200 lbf is reached. At this point, the shear director is in compression. The horn begins to vibrate, delivering a set amount of energy to the plastic director and causing localized melting and bonding. After the weld duration, the horn retracts, leaving the welded chromatographic separation device in the nest.

[0103] For the polyethersulfone (PES) sample, as will be seen, the PES membrane and thin spunbond nonwoven layer were placed in the lower housing 14 inside the protruding ring 54 so that the spunbond nonwoven layer was in contact with the lower housing 14. The lower housing was positioned in the lower nest so that the PES membrane could be in contact with the ultrasonic horn. To weld the membrane and support nonwoven layer, a Branson 20kHz ultrasonic welder (2000xdt model) was used, with a gold intensifier and a horn with a gain of 1.5x. An air pressure of 10 psi, a descent rate of 10%, an amplitude of 80%, a weld time of 0.05 seconds, and a start weld trigger force of 10 lbf were set as fixed parameters. It was found that a weld energy of 100 J provided a good, uniform weld without damaging the membrane.

[0104] All chromatography separation device samples were autoclaved at 121°C for 30 minutes using a pre-vacuum cycle. After autoclaving, the samples were allowed to cool completely at room temperature before testing.

[0105] To evaluate the virus removal performance of the chromatographic separation device, a Cole Parmer MasterFlex peristaltic pump was set up with MasterFlex tubing and sterilized with 0.5M sodium hydroxide (NaOH) for at least 30 minutes. After sterilization, the sterilized chromatographic separation device was connected to the peristaltic pump. Chloride (Cl) was measured in the sterilized sample as described above according to U.S. Patent Application Serial No. 67 / 783,319. - )DBC. The chromatographic separation device was then rinsed with 15 mL of 50 mM Tris-HCl buffer solution with a pH of 8 and a conductivity of 20 mS / cm. Next, 15 mL of the viral challenge solution was perfused through the chromatographic separation device and the eluate was collected. The input and eluate solutions were plated and the LRV was calculated using Equations 2 and 3.

[0106] At GE Healthcare Life Sciences Pure System The BSA DBC was tested on a chromatographic separation device using a Pure System (GE Healthcare Life Sciences). Coaxial UV monitoring at 280 nm was used to detect protein penetration. Chloride (Cl) was first measured in sterilized samples as described above according to U.S. Patent Application Serial No. 67 / 783,319. - )DBC. Then, 25 mM Tris 50 mM NaCl buffer with a pH of 8 was used at a rate of 1.94 (mL / min) / cm 2 The chromatographic separation device was rinsed with the medium. The chromatographic separation device was then challenged with a solution of approximately 1 mg / mL BSA in the aforementioned buffer until 10% breakthrough occurred. To determine the endpoint of the test, the absorbance of the BSA challenge solution at 280 nm was measured and the 10% breakthrough value was calculated. The dynamic binding capacity (DBC) of the chromatographic separation device at 10% breakthrough was calculated using Equation 1.

[0107] Now see Figures 8 to 15 , various combinations of the above-mentioned medium layers, spacer rings and sealing membranes are shown in different chromatographic separation devices.

[0108] comparison

[0109] Figure 8 is a chromatographic separation device having a media stack in the direction from the inlet to the outlet, the media stack comprising a functionalized nonwoven layer 82, a functionalized nonwoven layer 82, and a functionalized membrane layer 84. No spacer ring or sealing membrane was used, which represents a control device of the following construction. It was found that the Cl of this device - DBC is 4.07Cl - / cm 2 , BSA DBC is 14.73 mg / cm2 And the LRV is 3.83.

[0110] Spacer ring

[0111] Figure 9 The device is a chromatographic separation device having a media stack from the inlet to the outlet, the media stack including a functionalized nonwoven layer 82, a functionalized nonwoven layer 82, a spacer ring 86 and a functionalized membrane layer 84. The Cl of the device was found to be - DBC is 5.74Cl - / cm 2 , BSA DBC is 19.42 mg / cm 2 And the LRV is 4.28.

[0112] Figure 10 The device is a chromatographic separation device having a media stack from the inlet to the outlet, the media stack including a functionalized nonwoven layer 82, a spacer ring 86, a functionalized nonwoven layer 82 and a functionalized membrane layer 84. The Cl of the device was found to be - DBC is 5.28Cl - / cm 2 , BSA DBC is 18.04 mg / cm 2 And the LRV is 4.22.

[0113] Figure 11 The device is a chromatographic separation device having a media stack from the inlet to the outlet, the media stack including a functionalized nonwoven layer 82, a first spacer ring 86, a functionalized nonwoven layer 82, a second spacer ring 86 and a functionalized membrane layer 84. The Cl of the device was found to be - DBC is 6.21Cl - / cm 2 , BSA DBC is 20.23 mg / cm 2 And the LRV is 4.47.

[0114] Sealing layer

[0115] Figure 12 The device is a chromatographic separation device having a media stack from the inlet to the outlet, the media stack including a functionalized nonwoven layer 82, a functionalized nonwoven layer 82, a functionalized membrane layer 84 and a sealing layer 88. The Cl of the device was found to be - DBC is 3.27Cl - / cm 2 , BSA DBC is 13.34 mg / cm 2 And the LRV is 6.74.

[0116] Sealing layer and spacer ring

[0117] Figure 13 The device is a chromatographic separation device having a media stack from the inlet to the outlet, the media stack including a functionalized nonwoven layer 82, a functionalized nonwoven layer 82, a spacer ring 86, a functionalized membrane layer 84 and a sealing layer 88. The Cl of the device was found to be - DBC is 5.42Cl - / cm 2 , BSA DBC is 19.79 mg / cm 2 And the LRV is 7.62.

[0118] Figure 14 The device is a chromatographic separation device having a media stack from the inlet to the outlet, the media stack including a functionalized nonwoven layer 82, a spacer ring 86, a functionalized nonwoven layer 82, a functionalized membrane layer 84 and a sealing layer 88. The Cl of the device was found to be - DBC is 4.48Cl - / cm 2 , BSA DBC is 17.22 mg / cm 2 And the LRV is 7.00.

[0119] Figure 15 The device is a chromatographic separation device having a media stack from the inlet to the outlet, the media stack comprising a functionalized nonwoven layer 82, a first spacer ring 86, a functionalized nonwoven layer 82, a second spacer ring 86, a functionalized membrane layer 84 and a sealing layer 88. - DBC is 5.82Cl - / cm 2 , BSA DBC is 19.93 mg / cm 2 And the LRV is 7.74.

[0120] Table-1

[0121]

[0122] Will Figure 8 The control and Figures 9 to 11 By comparison, it can be seen that adding one or more spacer rings without a sealing layer increases the dynamic binding capacity by 22.5% to 52.6%. See Table 3. This is a significant increase in performance without adding more functionalized media! Figure 8 The control and Figure 12 In comparison, the addition of the sealing layer increased the LRV by 75.8%. This significantly improved the virus clearance of the chromatographic separation device. Figure 8 For the control, two spacer rings were used in combination with the sealing layer. Figure 15 The CL-DBC of the chromatographic separation device increased by 43.0% and the LRV increased by 102%.

[0123] Additional sealing layer example

[0124] In order to demonstrate the effect of the sealing layer on the virus removal performance of the chromatographic separation device, samples with different sealing layers were prepared. The LRV values of the various embodiments discussed herein are summarized in Table-2.

[0125] Table-2

[0126]

[0127]

[0128] Figure 15 A configuration is illustrated in which a sealing layer 88 is used as the last layer adjacent to the outlet of a chromatographic separation device and is in compression sealing contact with the housing to prevent bypass of the sealing layer. As fluid moves from the inlet to the outlet, the chromatographic separation device comprises two layers of functionalized nonwoven layer 82, a 50-mil thick spacer ring 86, two layers of a second functionalized nonwoven layer 82, another 50-mil thick spacer ring 86, three layers of functionalized membrane layer 84, and sealing layer 88.

[0129] Figure 11 A configuration is illustrated in which a functionalized membrane is used as the last layer adjacent to the outlet of a chromatographic separation device and is in compression sealing contact with the housing to prevent bypass. As the fluid moves from the inlet to the outlet, the chromatographic separation device has two layers of functionalized nonwoven layer 82, a 50 mil thick spacer ring 86, two layers of a second functionalized nonwoven layer 82, another 50 mil thick spacer ring 86, and three layers of functionalized membrane layer 84. Figure 15 Examples of the same material and material weight.

[0130] See Table 2, Figure 15 The log reduction value (LRV) of the construct is at least 6.49, while Figure 11The LRV of the media stack in the chromatographic separation device was only 4.47. When the functionalized membrane is used as the last layer of the chromatographic separation device and is in sealing contact with the housing, viruses are able to more easily leak through the lower surface of the media in compressive contact with the housing, bypass the functionalized layer and enter the outlet, thereby reducing the LRV of the device. On the other hand, when a sealing layer is used in this position directly below the functionalized layer, an improved compression seal is formed and the LRV is increased by an astonishing 45.2%. These performance improvements are achieved without adding any additional functionalized materials to the device. In various embodiments, the LRV of the media stack in the chromatographic separation device using the sealing layer can be increased by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100% compared to the same media stack in the control chromatographic separation device without the sealing layer.

[0131] Additional Spacer Ring Discussion

[0132] Figure 9 、 Figure 10 、 Figure 11 、 Figure 13 、 Figure 14 and Figure 15 Various configurations are illustrated in which at least one spacer ring is placed between two layers in a chromatographic separation device. In some embodiments, only a single spacer ring is used, and in other embodiments, two spacer rings are used. As fluid moves from an inlet to an outlet, each chromatographic separation device in the chromatographic separation device has a two-ply functionalized nonwoven layer, a two-ply second functionalized nonwoven layer, and a three-ply functionalized membrane layer. As shown, one or more spacer rings are placed between the layers.

[0133] Figure 8 A control chromatographic separation device is shown, which has two sheets of functionalized nonwoven layer, two sheets of second functionalized nonwoven layer and three sheets of functionalized membrane layer as the fluid moves from the inlet to the outlet. Figure 9 、 Figure 10 and Figure 11 Examples of the same material and material weight. Figure 12 A chromatographic separation device is shown having a nylon 6,6 membrane with an average pore size of 0.8 micrometers as a sealing layer. The thickness of the sealing layer is in the range of 17 mils to 20 mils. Figure 13 、 Figure 14 and Figure 15 Various configurations are exemplified in which, in addition to a bonding sealing layer, at least one spacer ring is placed between two layers in a chromatographic separation device.

[0134] Table-3

[0135]

[0136] See Table 3, Figure 8 The BSA dynamic binding capacity of the control was 14.73 (mg / cm2), and the chloride capacity was 4.07 (mL). Figure 9 、 Figure 10 and Figure 11 The various spacer ring configurations in the two tests increased capacity by a range of 22.5% to 52.6%. Figure 13 、 Figure 14 and Figure 15 The capacity of various spacer ring configurations with sealing layers in the two tests increased by 10.1% to 43.2%. Figure 8 The control chromatographic separation device in FIG2 has no additional material added to the layers, which significantly increases capacity. As seen above, the media stack in the chromatographic separation device using one or more spacer rings can increase DBC by at least 10%, 20%, 30%, 40%, or 50% when tested for Cl-DBC or BSA DBC compared to the same media stack in the control chromatographic separation device without the spacer rings.

Claims

1. A chromatographic separation device comprising: a housing having an inlet and an outlet; at least one functionalized medium layer, the at least one functionalized medium layer being disposed between the inlet and the outlet inside the housing; a non-functionalized sealing layer, the sealing layer being disposed between the inlet and the outlet inside the housing, and serving as the last dielectric layer in the dielectric stack when fluid flows from the inlet to the outlet through the dielectric stack inside the housing; An edge of the sealing layer in contact with the housing, the edge being compressed by the housing to form a compression seal portion, so as to prevent the fluid from leaking to the outlet through the compression seal portion; and The dielectric stack includes the functionalized dielectric layer and the sealing layer.

2. The chromatographic separation device of claim 1, wherein the sealing layer comprises a membrane. 3 . The chromatographic separation device according to claim 2 , wherein the membrane of the sealing layer comprises a precursor having the same material structure as the functionalized medium layer before functionalization. 4 . The chromatographic separation device according to claim 2 , wherein the membrane of the sealing layer comprises a multi-region membrane having varying pore sizes in different regions.

5. The chromatographic separation device according to claim 2, wherein the membrane of the sealing layer comprises a symmetric membrane having a substantially constant pore size. The chromatographic separation device according to claim 2 , wherein the membrane of the sealing layer comprises polyamide. The chromatographic separation device according to claim 6 , wherein the membrane of the sealing layer comprises nylon 6,6.

8. The chromatographic separation device according to claim 2, wherein the membrane of the sealing layer comprises a supported membrane cast on a porous supporting layer.

9. The chromatographic separation device according to claim 2, wherein the membrane of the sealing layer has an average pore size of 0.1 μm to 5.0 μm.

10. The chromatographic separation device of claim 2, wherein the film of the sealing layer has a thickness of 6 mils to 20 mils.

11. The chromatographic separation device of claim 2, wherein the log reduction value (LRV) of the media stack in the chromatographic separation device using the sealing layer is increased by at least 20% compared to the same media stack in a control chromatographic separation device without the sealing layer.

12. The chromatographic separation device of claim 11, wherein the log reduction value (LRV) is increased by at least 70%.

13. A chromatographic separation device comprising: a housing having an inlet and an outlet; at least two dielectric layers disposed between the inlet and the outlet within the housing to form a dielectric stack, at least one of the dielectric layers comprising a functionalized layer; a spacer ring, the spacer ring being disposed between the two dielectric layers and forming an air gap between the two dielectric layers; a non-functionalized sealing layer, the sealing layer being disposed between the inlet and the outlet inside the housing, and serving as the last dielectric layer in the dielectric stack when fluid flows from the inlet to the outlet through the dielectric stack inside the housing; as well as an edge of the sealing layer in contact with the shell; The rim is compressed by the housing to form a compression seal to prevent fluid from leaking past the compression seal to the outlet. 14 . The chromatographic separation device according to claim 13 , wherein the media stack comprises a functionalized nonwoven layer and a functionalized membrane layer, and the spacer ring is disposed between the functionalized nonwoven layer and the functionalized membrane layer.

15. The chromatographic separation device of claim 13, wherein the media stack comprises a functionalized nonwoven layer, another functionalized nonwoven layer, and the spacer ring disposed between the functionalized nonwoven layer and the another functionalized nonwoven layer.

16. The chromatographic separation device of claim 13, wherein the media stack comprises a functionalized nonwoven layer, a first spacer ring, another functionalized nonwoven layer, a second spacer ring, and a functionalized membrane layer.

17. The chromatographic separation device of claim 13, wherein the functionalized layer is a functionalized nonwoven.

18. The chromatographic separation device of claim 13, wherein the spacer ring has a height, and the height is 0.030 inches to 0.050 inches.

19. The chromatographic separation device of claim 13, 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).

20. The chromatographic separation device according to claim 13, wherein the media stack in the chromatographic separation device using the spacer ring is more stable than the same media stack in a control chromatographic separation device without the spacer ring in a test of chloride dynamic binding capacity (CDBC) of the chromatographic separation device. - The dynamic binding capacity (DBC) of bovine serum albumin (BSADBC) or bovine serum albumin (BSADBC) was increased by at least 10%.

21. The chromatographic separation device of claim 13, wherein the sealing layer comprises a membrane.

22. The chromatographic separation device of claim 13, wherein the membrane of the sealing layer comprises polyamide.

23. The chromatographic separation device according to claim 13, wherein the membrane of the sealing layer comprises a supported membrane cast on a porous support layer.

24. The chromatographic separation device according to claim 13, wherein the membrane of the sealing layer has an average pore size of 0.1 μm to 5.0 μm.

25. The chromatographic separation device of claim 13, wherein the film of the sealing layer has a thickness of 6 mils to 20 mils.

26. The chromatographic separation device of claim 13, wherein the log reduction value (LRV) of the media stack in the chromatographic separation device using the sealing layer is increased by at least 20% compared to the same media stack in a control chromatographic separation device without the sealing layer.

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