Affinity chromatography device comprising a fibrillated polymer membrane and concentrator comprising the affinity chromatography device

Abstract

The present disclosure relates to affinity chromatography devices comprising a fibrillated polymer membrane containing inorganic particles having a spherical shape and a particle size distribution of D90 / D10 less than or equal to 3. Blends or combinations of spherical inorganic particles can be used. The nominal particle size of the spherical inorganic particles is about 5 microns to about 20 microns. Affinity ligands can be bonded to the spherical inorganic particles and / or bonded to the fibrillated polymer membrane. Further, the affinity chromatography devices have a hydraulic permeability of about 100 (X 10 ‑12 cm 2 to about 500 (X 10 ‑ 12 cm 2 ) at an operating pressure of no more than 0.3 MPa. In addition, the affinity chromatography devices have a cycle durability of at least 100 cycles at an operating pressure of no more than 0.3 MPa. Also disclosed are collectors containing a plurality of the affinity chromatography devices in parallel configuration and multi-collectors in parallel configuration.

Description

Technical Field

[0001] This disclosure generally relates to affinity chromatography, and more specifically to an affinity chromatography apparatus comprising a fibrillated polymer membrane containing a blend of spherical particles having a D90 / D10 size distribution less than or equal to 3, and capable of separating target molecules from aqueous mixtures. Also disclosed are manifolds comprising multiple affinity chromatography apparatuses and manifolds with parallel configurations. Background Technology

[0002] Chromatographic methods are commonly used to separate and / or purify molecules of interest, such as proteins, nucleic acids, and polysaccharides, from mixtures. Affinity chromatography specifically involves passing a mixture through a matrix containing a specific ligand (i.e., a specific binding partner) that binds to the molecule of interest. Once exposed to the ligand, the molecule of interest binds to the matrix and is thus retained from the mixture. Affinity chromatography offers several advantages over other types of chromatography. For example, it provides a purification method that can separate a target protein from a mixture of other biomolecules in a single step with high yields.

[0003] Despite the many advantages of current affinity chromatography devices, there is a need in the art for a chromatography device that can be used for shorter residence times than conventional devices, while providing the same or better binding capacity as current products, and that can be reused. Invention Overview

[0005] In one aspect ("Aspect 1"), an affinity chromatography apparatus includes a fluid inlet, a fluid outlet fluidly connected to the fluid inlet, a protofibrous polymer membrane located between the fluid inlet and the fluid outlet (and containing inorganic particles having a spherical shape and a nominal particle size of about 5 micrometers to about 20 micrometers), and a shell member surrounding the fluid inlet, the fluid outlet, and the protofibrous polymer membrane, wherein the particle size distribution has a D90 / D10 of less than or equal to 3, and wherein at least one of the protofibrous polymer membrane and the inorganic particles is covalently bonded to an affinity ligand that reversibly binds to a target molecule.

[0006] According to another aspect of aspect 1 ("Aspect 2"), the target molecule is a protein, antibody, viral vector, or combination thereof.

[0007] According to another aspect of aspect 1 or 2 ("Aspect 3"), it includes approximately 100 (X 10) -12 cm 2 ) to approximately 500 (X 10 -12 cm 2 Hydraulic penetration rate.

[0008] According to another aspect of any of aspects 1 to 3 ("Aspect 4"), the spherical inorganic particles are selected from silica, zeolite, hydroxyapatite, metal oxides and combinations thereof.

[0009] According to another aspect of any of aspects 1 to 4 ("Aspect 5"), the fibrillated polymer membrane includes expanded polytetrafluoroethylene membrane, expanded modified polytetrafluoroethylene membrane, expanded polytetrafluoroethylene copolymer membrane or expanded polyethylene membrane.

[0010] According to another aspect of any of aspects 1 to 5 ("Aspect 6"), the fibrillated polymer membrane is an expanded polytetrafluoroethylene membrane.

[0011] According to another aspect of any of aspects 1 to 6 ("Aspect 7"), the affinity ligand is selected from protein A, protein G, protein L, human Fc receptor protein, antibody, polysaccharide and combination thereof.

[0012] According to another aspect of any of aspects 1 to 7 ("Aspect 8"), the inorganic particles include at least a first inorganic particle having a spherical shape and a first nominal particle size and a second inorganic particle having a spherical shape and a second nominal particle size, the first nominal particle size and the second nominal particle size being different from each other.

[0013] According to another aspect of any of aspects 1 to 8 ("Aspect 9"), the nominal particle size is selected from about 5 micrometers, about 10 micrometers, about 15 micrometers, about 20 micrometers, and combinations thereof.

[0014] According to another aspect of any of aspects 1 to 9 ("Aspect 10"), the inorganic particles having a spherical shape include a blend of 10-micrometer spherical particles and 20-micrometer spherical particles, and the blend is 10:90 to 90:10.

[0015] According to another aspect of any of aspects 1 to 9 ("Aspect 11"), the inorganic particles having a spherical shape include a blend of spherical particles of 5 micrometers and spherical particles of 10 micrometers, and the blend is 10:90 to 90:10.

[0016] According to another aspect of any of aspects 1 to 9 ("Aspect 12"), the inorganic particles having a spherical shape include a blend of spherical particles of 5 micrometers and spherical particles of 20 micrometers, and the blend is 10:90 to 90:10.

[0017] According to another aspect of any of aspects 1 to 12 ("Aspect 13"), it includes a dynamic binding capacity (DBC) of at least 35 mg / ml at a residence time of 20 seconds.

[0018] According to another aspect of any of aspects 1 to 13 ("Aspect 14"), it includes cyclic durability of at least 100 cycles at an operating pressure of less than 0.3 MPa.

[0019] According to another aspect of any of aspects 1 to 14 ("Aspect 15"), the fibrillated polymer membrane has a wound structure.

[0020] According to another aspect of any of aspects 1 to 15 ("Aspect 16"), the fibrillated polymer membrane has a stacked structure.

[0021] According to another aspect of any of aspects 1 to 16 ("Aspect 17"), the fibrillated polymer membrane has a wound structure, a stacked structure, and a combination thereof.

[0022] According to another aspect of any of aspects 1 to 17 ("Aspect 18"), the inner intermediate material surrounds the outer surface of the core, and the protofibrous polymer membrane surrounds the inner intermediate material.

[0023] According to another aspect of any of aspects 1 to 17 ("Aspect 18"), wherein the outer intermediate material surrounds the protofibrous polymer membrane.

[0024] According to another aspect of any of aspects 1 to 16 ("Aspect 17"), the inner intermediate material and the outer intermediate material are selected from porous fluoropolymer membranes, porous non-fluoropolymer membranes, porous nonwoven materials and porous woven materials.

[0025] According to another aspect of any of aspects 18 to 20 ("Aspect 21"), at least one of the inner layer intermediate material and the outer layer intermediate material is a polypropylene nonwoven material.

[0026] According to another aspect ("Aspect 22"), articles such as those in any of Aspects 18 to 21 are used to separate target molecules from a fluid stream.

[0027] According to another aspect ("Aspect 23"), an aggregator includes at least two affinity chromatography devices arranged in parallel configurations as described in any of Aspects 1 to 21.

[0028] According to another aspect of aspect 23 ("Aspect 24"), the collector is enclosed in a housing.

[0029] In one aspect (“Aspect 25”), an article comprises a first collector and a second collector constructed in parallel, wherein each of the first collector and the second collector comprises at least two affinity chromatography devices as described in any of Aspects 1 to 21.

[0030] According to another aspect of aspect 25 (“Aspect 26”), the first collector and the second collector are enclosed in a housing.

[0031] In one aspect (“Aspect 27”), an article of article includes a core located at a center, a protofibrous polymer membrane (containing spherical inorganic particles having a spherical shape and a nominal particle size of about 5 micrometers to about 20 micrometers) wound around the core, a shell member surrounding the core and the protofibrous polymer membrane, a first end cap disposed at a first end of the shell member, and a second end cap disposed at a second end of the shell member, wherein the particle size distribution has a D90 / D10 of less than 3, and wherein at least one of the protofibrous polymer membrane and the spherical inorganic particles is covalently bonded to an affinity ligand, the affinity ligand reversibly binding to a target molecule.

[0032] According to another aspect of aspect 27 ("Aspect 28"), wherein the target molecule is a protein, antibody, viral vector, or combination thereof.

[0033] According to another aspect of aspect 27 or 28 ("Aspect 29"), it includes approximately 100 (x 10) -12 cm 2 ) to approximately 500 (X10) -12 cm 2 Hydraulic penetration rate.

[0034] According to another aspect of any of aspects 27 to 29 ("Aspect 30"), wherein the spherical inorganic particles are selected from silica, zeolite, hydroxyapatite, metal oxides and combinations thereof.

[0035] According to another aspect of any of aspects 27 to 30 ("Aspect 31"), the fibrillated polymer membrane comprises at least one of expanded polytetrafluoroethylene membrane, expanded modified polytetrafluoroethylene membrane, expanded polytetrafluoroethylene copolymer membrane or expanded polyethylene membrane.

[0036] According to another aspect of any of aspects 27 to 31 ("Aspect 32"), the fibrillated polymer membrane is an expanded polytetrafluoroethylene membrane.

[0037] According to another aspect of any of aspects 27 to 32 ("Aspect 33"), the affinity ligand is selected from protein A, protein G, protein L, human Fc receptor protein, antibody, polysaccharide and combination thereof.

[0038] According to another aspect of any of aspects 27 to 33 ("Aspect 34"), the inorganic particles include at least a first inorganic particle having a spherical shape and a first nominal particle size and a second inorganic particle having a spherical shape and a second nominal particle size, and wherein the first nominal particle size and the second nominal particle size are different from each other.

[0039] According to another aspect of any of aspects 37 to 34 ("Aspect 35"), the nominal particle size is selected from about 5 micrometers, about 10 micrometers, about 15 micrometers, about 20 micrometers, and combinations thereof.

[0040] According to another aspect of any of aspects 27 to 34 ("Aspect 36"), the inorganic particles having a spherical shape include a blend of 10-micrometer spherical particles and 20-micrometer spherical particles, and the blend is 10:90 to 90:10.

[0041] According to another aspect of any of aspects 27 to 34 ("Aspect 37"), the inorganic particles having a spherical shape include a blend of 5-micrometer spherical particles and 10-micrometer spherical particles, and the blend is 10:90 to 90:10.

[0042] According to another aspect of any of aspects 27 to 34 ("Aspect 38"), the inorganic particles having a spherical shape comprise a blend of 5-micrometer spherical particles and 20-micrometer spherical particles, and the blend is 10:90 to 90:10.

[0043] According to another aspect of any of aspects 27 to 38 ("Aspect 39"), it includes a dynamic binding capacity (DBC) of at least 35 mg / ml at a residence time of 20 seconds.

[0044] According to another aspect of any of aspects 27 to 39 ("Aspect 40"), it includes cycle durability of at least 100 cycles and operating pressure of less than 0.3 MPa.

[0045] According to another aspect of any of aspects 27 to 40 ("Aspect 41"), the inner intermediate material surrounds the outer surface of the core, and the protofibrous polymer membrane surrounds the inner intermediate material.

[0046] According to another aspect of any of aspects 27 to 41 ("Aspect 42"), it includes an outer intermediate material surrounding the original fibrous polymer membrane.

[0047] According to another aspect of aspect 42 ("Aspect 43"), the inner intermediate material and the outer intermediate material are selected from porous fluoropolymer membranes, porous non-fluoropolymer membranes, porous nonwoven materials and porous woven materials.

[0048] According to another aspect of aspect 43 ("Aspect 44"), at least one of the inner layer intermediate material and the outer layer intermediate material is a polypropylene nonwoven material.

[0049] According to another aspect ("Aspect 45"), an aggregator includes at least two affinity chromatography devices as described in any one of Aspects 27 to 44 arranged in parallel configuration.

[0050] According to another aspect of aspect 45 ("Aspect 46"), the collector is enclosed in a housing.

[0051] According to one aspect ("Aspect 47"), an article comprises a first collector and a second collector constructed in parallel, wherein each of the first collector and the second collector comprises at least two affinity chromatography devices as described in any one of Aspects 27 to 44.

[0052] According to another aspect of aspect 47 (“Aspect 48”), the first collector and the second collector are enclosed in a housing.

[0053] In one aspect (Aspect 49), an affinity chromatography apparatus includes a housing, an inlet for fluid inflow into a housing component, first and second fluid distributors (located at opposite ends of the housing), an outlet for fluid outflow from the housing, and a stacked membrane assembly disposed within the housing between the fluid inlet and the fluid outlet, the stacked membrane assembly comprising: a two- or multi-layer stacked protofibrous polymer membrane, the protofibrous polymer membrane comprising a blend of inorganic particles having a spherical shape and a nominal particle size of about 5 micrometers to about 20 micrometers, the particle size distribution having a D90 / D10 ratio of less than 3, and at least one of the protofibrous polymer membrane and the spherical inorganic particles being covalently bonded to an affinity ligand, the affinity ligand being reversibly bound to a target molecule.

[0054] According to another aspect of aspect 49 ("Aspect 50"), it includes first and second fluid distributors located at opposite ends of the housing member.

[0055] According to another aspect of aspect 49 or 50 ("Aspect 51"), wherein the target molecule is a protein, antibody, viral vector, or combination thereof.

[0056] According to another aspect of any of aspects 49 to 51 ("Aspect 52"), it includes approximately 100 (x 10)-12 cm 2 ) to approximately 500 (X 10 -12 cm 2 Hydraulic penetration rate.

[0057] According to another aspect of any of aspects 49 to 52 ("Aspect 53"), wherein the spherical inorganic particles are selected from silicon dioxide, zeolite, hydroxyapatite, metal oxides and combinations thereof.

[0058] According to another aspect of any of aspects 49 to 53 ("Aspect 54"), the fibrillated polymer membrane includes expanded polytetrafluoroethylene membrane, expanded modified polytetrafluoroethylene membrane, expanded polytetrafluoroethylene copolymer membrane or expanded polyethylene membrane.

[0059] According to another aspect of any of aspects 49 to 54 ("Aspect 55"), the fibrillated polymer membrane is an expanded polytetrafluoroethylene membrane.

[0060] According to another aspect of any of aspects 49 to 55 ("Aspect 56"), the affinity ligand is selected from protein A, protein G, protein L, human Fc receptor protein, antibody, polysaccharide and combination thereof.

[0061] According to another aspect of any of aspects 49 to 56 ("Aspect 57"), the inorganic particles include at least a first inorganic particle having a spherical shape and a first nominal particle size and a second inorganic particle having a spherical shape and a second nominal particle size, wherein the first nominal particle size and the second nominal particle size are different from each other.

[0062] According to another aspect of any of aspects 49 to 57 ("Aspect 58"), the nominal particle size is selected from about 5 micrometers, about 10 micrometers, about 15 micrometers, about 20 micrometers, and combinations thereof.

[0063] According to another aspect of any of aspects 49 to 58 ("Aspect 59"), the inorganic particles having a spherical shape comprise a blend of 10-micrometer spherical particles and 20-micrometer spherical particles, and the blend is 90:10 to 10:90.

[0064] According to another aspect of any of aspects 49 to 58 ("Aspect 60"), the inorganic particles having a spherical shape comprise a blend of 5-micrometer spherical particles and 10-micrometer spherical particles, and the blend is 10:90 to 90:10.

[0065] According to another aspect of any of aspects 49 to 58 ("Aspect 61"), the inorganic particles having a spherical shape comprise a blend of 5-micrometer spherical particles and 20-micrometer spherical particles, and the blend is 10:90 to 90:10.

[0066] According to another aspect of any of aspects 49 to 61 ("Aspect 62"), it includes a dynamic binding capacity (DBC) of at least 35 mg / ml at a residence time of 20 seconds.

[0067] According to another aspect of any of aspects 49 to 62 ("Aspect 63"), it includes cycle durability of at least 100 cycles and operating pressure of less than 0.3 MPa.

[0068] According to another aspect of any of aspects 49 to 63 ("Aspect 64"), at least one first intermediate material is located on a first side of the stacked membrane assembly and a second intermediate material is located on a second side of the stacked membrane assembly, the second side being opposite to the first side.

[0069] According to another aspect of aspect 64 ("Aspect 65"), the first intermediate material and the second intermediate material are selected from porous fluoropolymer membranes, porous non-fluoropolymer membranes, porous nonwoven materials and porous woven materials.

[0070] According to another aspect of aspect 65 ("Aspect 66"), at least one of the first intermediate material and the second intermediate material is a polypropylene nonwoven material.

[0071] In one aspect ("Aspect 67"), an aggregator comprises at least two affinity chromatography devices arranged in parallel configurations as described in any one of Aspects 49 to 66.

[0072] According to another aspect of aspect 67 (“Aspect 68”), the collector is enclosed in a housing.

[0073] According to one aspect ("Aspect 69"), an article comprises a first collector and a second collector constructed in parallel, wherein each of the first collector and the second collector comprises at least two affinity chromatography devices as described in any one of aspects 49 to 66.

[0074] According to another aspect of aspect 69 ("Aspect 70"), the first collector and the second collector are enclosed in a housing.

[0075] In another aspect ("Aspect 71"), a diagnostic device includes a protofibrotic polymer membrane (containing inorganic particles having a spherical shape and a nominal particle size of about 5 micrometers to about 20 micrometers), wherein the particle size distribution has a D90 / D10 of less than or equal to 3 and wherein at least one of the protofibrotic polymer membrane and the inorganic particles is covalently bonded to its ligand, the ligand being reversibly bonded to a target molecule in a biological fluid.

[0076] According to another aspect of aspect 71 ("Aspect 72"), it includes a fluid inlet and a fluid outlet fluidly connected to the fluid inlet.

[0077] According to another aspect of aspect 71 or 72 ("Aspect 73"), it includes a housing component surrounding the fluid inlet, the fluid outlet, and the original fibrous polymer membrane.

[0078] Brief description of the attached figures

[0079] The accompanying drawings, which are incorporated in and form part of this specification, are used to aid in a further understanding of this disclosure and illustrate embodiments thereof, and together with the specification serve to explain the principles of this disclosure.

[0080] Figure 1 This is an exploded view of a chromatographic apparatus according to at least one embodiment, the apparatus including a wound membrane assembly including a protofibrous polymer membrane containing spherical inorganic polymer particles. Figure 2 This is a cross-sectional view of a chromatographic apparatus according to at least one embodiment, depicting the outer flow channel and the inner flow channel according to at least one embodiment; Figure 3 This is a schematic cross-sectional view of another helical winding, vertical (normal) flow chromatography apparatus according to at least one embodiment.

[0081] Figure 4 This is an exploded view of a chromatographic apparatus containing a stacked membrane assembly according to at least one embodiment, the assembly including a fibrillated polymer membrane containing spherical inorganic particles.

[0082] Figure 5 This is a diagram illustrating the relationship between the dynamic binding capacity (DBC) and liquid permeability of the affinity chromatography apparatus described in Examples 1 and 2; Figure 6 This is a diagram illustrating the relationship between devices S and T with spirally wound membranes according to at least one embodiment and affinity chromatography devices C to R with stacked membranes.

[0083] Figure 7 It is a schematic diagram of the front view of an aggregator containing two parallel chromatographic devices.

[0084] Figure 8 yes Figure 7 A schematic diagram of the top view of the collector; Figure 9 It is a schematic perspective view of an collector containing four parallel chromatographic devices; Figure 10 yes Figure 9 A schematic diagram of the top view of the collector; Figure 11 This is a schematic diagram of the front view of two parallel-constructed collectors; Figure 12 yes Figure 11 A schematic diagram of the top view of the collector construction; Figure 13 This is a diagram illustrating a representative purification cycle of the dual collectors V and W described in Example 3; Figure 14 This is a diagram illustrating a representative purification cycle of the dual collector X described in Example 3; and Figure 15 This is a diagram illustrating a representative purification cycle of the four-collector described in Example 4. Invention Details

[0086] Those skilled in the art will readily understand that various aspects of this disclosure can be implemented by any number of methods and apparatuses configured to perform the desired functions. It should also be noted that the accompanying drawings are not necessarily drawn to scale but may be enlarged to illustrate various aspects of this disclosure, and in this respect, these drawings should not be considered limiting. It should be understood that, as used herein, the term "on..." means one element (such as a polymer film) directly on another element, or there may be intermediate elements present.

[0087] It should be understood that the terms "spherical particles," "spherical inorganic particles," and "inorganic particles with a spherical shape" are used interchangeably herein. Furthermore, the term "spiral-wound membrane assembly" refers to both a single fibrillated polymer membrane and a fibrillated polymer membrane with an intermediate nonwoven material. Similarly, the term "stacked membrane assembly" refers to both a single fibrillated polymer membrane and a fibrillated polymer membrane with one or more intermediate materials. Additionally, the terms "affinity chromatography apparatus" and "chromatographic apparatus" are used interchangeably herein.

[0088] This disclosure relates to an affinity chromatography apparatus that separates target molecules from an aqueous mixture containing the target molecules. Target molecules include, but are not limited to, proteins, antibodies, viral vectors, and combinations thereof. In some embodiments, this disclosure relates to a diagnostic apparatus for isolating a target disease from a biological sample. The chromatographic apparatus and diagnostic apparatus include a protofibroblastic polymer membrane containing a blend of inorganic particles having a spherical shape and a particle size distribution with a D90 / D10 ratio less than or equal to 3. In some embodiments, blends or compositions of spherical inorganic particles of various sizes are used. The nominal particle size of the spherical inorganic particles is from about 5 micrometers to about 20 micrometers. Affinity ligands may be bonded to the spherical inorganic particles and / or to the protofibroblastic polymer membrane. Furthermore, at a residence time of 20 seconds, the chromatographic apparatus has a dynamic binding capacity (DBC) greater than 40 mg / ml at 10% breakthrough. Additionally, the affinity chromatography apparatus has cycle durability of at least 100 cycles at an operating pressure not exceeding 0.3 MPa. It should be understood that the term "about" as used in this document means + / - 10% of the specified unit of measurement.

[0089] look Figure 1 and Figure 2The document describes a wound chromatography apparatus 100. In forming the chromatography apparatus 100, at least one layer of fibrillated polymer membrane containing spherical inorganic particles is wound around a cylindrical core 150. As used herein, fibrillation refers to the inclusion of fibrils in a polymer membrane, for example, a membrane with a microstructure characterized by nodes interconnected by fibrils, where voids are spaces between the nodes and fibrils. In some embodiments, at least one inner intermediate material 200 may be circumferentially positioned (e.g., wind around) around the core 150 to achieve a desired width or predetermined amount. A fibrillated polymer membrane 210 containing spherical inorganic particles is then wound around the core 150 around the inner intermediate material 200 to achieve the desired width or predetermined amount, and an outer circumferentially positioned (e.g., wind around) of at least one outer intermediate material 220 is wound around the fibrillated polymer membrane 210 to achieve the desired width or predetermined amount. In this document, the combination of inner intermediate material 200, fibrillated polymer membrane 210, and outer intermediate material 220 will be referred to as a "wound membrane assembly". In some embodiments, a "wound membrane assembly" may include a fibrillated polymer membrane and one or more inner intermediate materials, a fibrillated polymer membrane and one or more outer intermediate materials, and any combination of polymer and intermediate materials wound around a core. The cylindrical core 150 may have a hollow or solid interior. In either case, the core 150 includes a solid outer wall such that an aqueous mixture flowing through the chromatography apparatus 100 flows within an internal channel formed by one or more inner intermediate materials, which will be discussed in detail below. Using a hollow core 150 can reduce the amount of material used to form the core 150, reduce the weight of the apparatus 100, and lower manufacturing costs.

[0090] The membrane module 110 and the central core 150 may be located within the housing 50. In some embodiments, the housing 50 is cylindrical. Figure 1 and Figure 2In the embodiments described herein, the outer intermediate material 220 forms the outer flow channel 130, and the inner intermediate material 200 forms the inner flow channel 140. It should be understood that the intermediate materials 200 and 220 in the embodiments described herein may be different or the same. Furthermore, two or more intermediate materials may be used to form one or both of the outer flow channel 130 and the inner flow channel 140. In use, an aqueous mixture flows into the inlet 80 located within the inlet cap 60, whereby the mixture flows through the distributor cap 65 and is directed to the outer flow channel 130 formed by the outer intermediate material 220. The distributor cap 65 directs the aqueous mixture 90 degrees from the feed direction to the outer flow channel 130 (i.e., the intermediate material 220). This redirection promotes a more uniform flow of the aqueous mixture into the outer flow channel 130. The outer intermediate layer 220 forming the outer flow channel 130 is located between the housing 50 and the wound polymer film 210. The distributor cap 65 may be polyolefin or coated with polyolefin. It should be understood that the aqueous mixture flows along the outer channel gap 165 and connects to the inner channel 130.

[0091] The aqueous mixture flows through the outer channel 130 (i.e., the outer intermediate material 220) in a vertical direction (e.g., vertical flow) across the wound polymer membrane 210. As the aqueous mixture flows vertically through the wound polymer membrane 210 from the outer channel 130 (i.e., the outer intermediate material 220), the affinity ligands reversibly bind to the target protein, thereby effectively removing it from the aqueous mixture. The aqueous mixture then enters the inner channel 140 (i.e., the inner intermediate material 200) located between the solid outer wall of the central core 150 and the wound polymer membrane 210.

[0092] The aqueous mixture is then redirected at the bottom of the inner flow channel 140 by the outlet cap 75. The aqueous mixture then exits the chromatographic apparatus 100 through the outlet 85 located within the outlet cap 75. It should be understood that the diameter and / or height of the central core 150 (and / or the width and / or height of the protofibrotic polymer membrane and / or intermediate material) can be adjusted to achieve a larger volume without negatively impacting the performance of the apparatus. Furthermore, the target protein can be removed from the affinity ligand, for example, by passing a liquid with a lower pH through the chromatographic apparatus, as is known to those skilled in the art.

[0093] There are no particular limitations on intermediate materials 200, 220, and 40, provided that the aqueous mixture can flow through them. Some non-limiting examples of suitable intermediate materials include, but are not limited to, porous fluoropolymer membranes or porous non-fluoropolymer membranes (e.g., porous polypropylene or other porous polyolefin membranes), porous nonwoven materials, or porous woven materials. In some embodiments, the wound membrane assembly includes an integrated inlet end cap 60 at one end of the core 150 and an integrated outlet end cap 75 at the other end of the core 150 to form an integrated, reusable chromatography device.

[0094] There is no particular limitation on the total number of fibrillated polymer membrane layers present in a wound membrane assembly, and it depends on the desired end use and / or the desired mass transport flow rate within the membrane assembly. A wound membrane assembly may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 (or more) polymer membrane layers. It should be understood that hundreds or even thousands of polymer membrane layers may be present in a stacked membrane assembly. Furthermore, the fibrillated polymer membrane present in the wound membrane assembly may have a single-layer thickness of about 1 micrometer to about 10,000 micrometers, about 100 micrometers to about 5,000 micrometers, about 500 micrometers to about 3,000 micrometers, or about 650 micrometers to about 1,000 micrometers. As used herein, the term "thickness" refers to the direction of the fibrillated polymer membrane perpendicular to its length-area.

[0095] Figure 3 The function of the chromatographic apparatus 300 described in the text is the same as... Figure 2 The chromatographic apparatus 100 described herein is substantially similar. For example, an aqueous mixture is introduced into the chromatographic apparatus 300 through an inlet 80 disposed within an inlet cap 60 in the direction of arrow 62. The aqueous mixture is guided from the feed direction by a dispenser cap 65. For ease of description, the aqueous mixture may flow toward the outer channel 130 (which may be formed of an outer intermediate material 220) in the direction described by arrow 55. The aqueous mixture flows along the outer channel gap 165 and connects to the inner channel 140, in which it flows in the direction of arrow 30. The aqueous mixture flows through the wound polymer membrane 210 in a direction perpendicular to arrow 70 (e.g., vertical flow) from the inner channel 130 to the inner channel 140. As the aqueous mixture passes through the wound polymer membrane 210, affinity ligands reversibly bind to the target molecule. It should be understood that the inner channel 140 may be formed of an inner intermediate material 200.

[0096] The aqueous mixture, free of the target molecule, flows along the inner flow channel 140 in the direction described by arrow 40. At the bottom of the inner flow channel 140, the aqueous mixture is redirected, flowing towards the central portion of the chromatography apparatus 300 as described by arrow 52. The aqueous mixture, free of the target molecule, flows out of the chromatography apparatus 300 through the outlet 85 within the outlet end cap 75 in the direction of arrow 45.

[0097] In other implementations, such as Figure 4As described, the chromatographic apparatus 200 includes a fibrillated polymer membrane configured such that individual discs 240 are stacked together to form a stacked membrane assembly 220. The fibrillated polymer membranes 240 can be formed into a stacked configuration by simply stacking the fibrillated polymer membrane discs 240 onto each other. Alternatively, the fibrillated polymer membrane discs 240 can be stacked and then laminated together using heat and / or pressure or other conventional methods. It should be understood that, for ease of interpretation, the stacked membrane assembly 240 described herein refers to fibrillated polymer membrane discs. Fibrillated polymer membranes formed in one or more geometries and / or one or more non-geometric shapes are considered to be within the scope of this disclosure.

[0098] The chromatography apparatus 200 includes at least one upper intermediate material 260 disposed on top of the stacked membrane assembly 220 and at least one lower intermediate material 280 disposed below the stacked membrane assembly 220. The upper and lower intermediate materials 260 and 280 can be the same or different. Similar to the wound membrane assembly discussed above, there are no particular limitations on the intermediate materials 260 and 280 used to form the stacked membrane assembly 220, as long as the aqueous mixture can flow through. Non-limiting examples of suitable intermediate materials include, but are not limited to, porous fluoropolymer membranes or porous non-fluoropolymer membranes (e.g., porous polypropylene or other porous polyolefin membranes), porous nonwoven materials, or porous woven materials.

[0099] The stacked membrane assembly 220 can be housed within a housing 250, which has an inlet cap 265 and an outlet cap 275 at opposite ends. In some embodiments, the housing 250 is cylindrical, although any geometry capable of accommodating the stacked membrane assembly and achieving the desired dynamic binding capacity is considered to be within the scope of this disclosure. In some embodiments, the intermediate materials 260, 280, the housing 250, the inlet cap 265, and the outlet cap 275 may be formed of a thermoplastic polymer (such as polypropylene, polyethylene, or other polyolefins). Alternatively, one or both of the intermediate materials 260, 280 may be formed of inorganic or metallic materials, provided that the porous intermediate materials 260, 280 do not impede the operation of the chromatographic apparatus.

[0100] The fibrillated polymer membrane 240 in the stacked membrane assembly 220 can be adhered to the inner wall of the housing 250 by any conventional method (e.g., melt sealing or using a sealant) to prevent flow between the periphery of the fibrillated polymer membrane 240 and the housing 250. The inlet cap 265 and outlet cap 275 can be sealed to the housing 250 by a similar or identical process. The inlet cap and outlet caps 265, 275 each include an inlet 280 and an outlet 285 to allow the aqueous mixture to flow through the affinity chromatography device 200. Specifically, the inlet cap 265 allows fluid flow of the aqueous mixture into the housing 250, and the outlet cap 285 allows fluid flow of the aqueous mixture out of the housing 250. In use, the aqueous mixture flows sequentially through intermediate material 260, through the fibrillated polymer membrane 240 forming the stacked membrane assembly 220, and through intermediate material 280. As the aqueous mixture passes through the chromatography device 200, affinity ligands reversibly bind to the target molecule, thereby effectively removing it from the aqueous mixture. The target molecule can be removed from the affinity ligand, for example, by passing a liquid with a lower pH through the device, as is known to those skilled in the art.

[0101] There is no particular limitation on the total number of fibrillated polymer membranes present in a stacked membrane module, depending on the desired end use and / or the desired mass transport flow rate within the membrane module. A stacked membrane module may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 (or more) layers of polymer membranes. It should be understood that there may be hundreds or even thousands of polymer membrane layers in a stacked membrane module. Furthermore, the fibrillated polymer membranes present in the stacked membrane module may have a thickness of about 1 micrometer to about 10,000 micrometers, about 100 micrometers to about 5,000 micrometers, about 500 micrometers to about 3,000 micrometers, or about 650 micrometers to about 1,000 micrometers. As used herein, the term "thickness" refers to the direction of the fibrillated polymer membrane perpendicular to its length-area.

[0102] Both the fibrillated polymer membranes in wound membrane assemblies and stacked membrane assemblies contain spherical inorganic particles, or particles with a spherical structure. As used herein, the term "spherical" refers to an inorganic particle having a circular or nearly circular shape, wherein the distance from the center of the inorganic particle to any point on the outer edge of the particle is the same or nearly the same. In some embodiments, the spherical inorganic particles have a particle size distribution of D90 / D10 less than or equal to 3, less than or equal to 2.5, less than or equal to 2, less than or equal to 1.5, or less than or equal to 1. The nominal particle size of the spherical inorganic particles can be about 5 micrometers, about 10 micrometers, about 15 micrometers, about 20 micrometers, and combinations and blends thereof. In some embodiments, the spherical inorganic particles are polydisperse.

[0103] In some embodiments, the fibrillated polymer membrane includes more than one nominal particle size and / or more than one type of spherical inorganic particles. The fibrillated polymer membrane may comprise about 10% by mass of the fibrillated polymer membrane to about 90% by mass of spherical inorganic particles, about 15% by mass of the fibrillated polymer membrane to about 85% by mass of spherical inorganic particles, about 20% by mass of the fibrillated polymer membrane to about 80% by mass of spherical inorganic particles, about 30% by mass of the fibrillated polymer membrane to about 70% by mass of spherical inorganic particles, about 35% by mass of the fibrillated polymer membrane to about 65% by mass of spherical inorganic particles, about 40% by mass of the fibrillated polymer membrane to about 60% by mass of spherical inorganic particles, about 45% by mass of the fibrillated polymer membrane to about 55% by mass of spherical inorganic particles, or about 50% by mass of the fibrillated polymer membrane to about 50% by mass of spherical inorganic particles. Non-limiting examples of suitable inorganic particles include silica, zeolites, hydroxyapatite, metal oxides, and combinations thereof. Furthermore, the inorganic particles can be solid or porous. Additionally, the hydraulic permeability of the affinity chromatography apparatus described herein is approximately 100 (X 10⁻⁶). -12 cm 2 ) to approximately 500 (X 10 -12 cm 2 ), approximately 150 (X 10) -12 cm 2 ) to approximately 500 (X 10 -12 cm 2 ), approximately 200 (X 10) -12 cm 2 ) to approximately 500 (X 10 -12 cm 2 ), approximately 250 (X 10) -12 cm 2 ) to approximately 500 (X 10 -12 cm 2 ), approximately 200 (X 10) -12 cm 2 ) to approximately 450 (X 10 -12 cm 2 ), approximately 200 (X 10) -12 cm 2 ) to approximately 400 (X 10 -12 cm 2 ), approximately 250 (X 10) -12 cm 2 ) to approximately 400 (X 10 -12 cm 2 ), or approximately 300 (X 10 -12 cm 2 ) to approximately 400 (X 10-12 cm 2 ).

[0104] In at least one embodiment, the protofibrotic polymer membrane comprises a blend of spherical inorganic particles with different nominal particle sizes. For example, the protofibrotic polymer membrane may comprise a 90:10 mixture of the same or different spherical inorganic particles with a first nominal particle size (e.g., 5 micrometers) and a second nominal particle size (e.g., 20 micrometers). The blend of spherical inorganic particles in the protofibrotic polymer membrane can be any blend, for example, a blend of 10:90 to 90:10, a blend of 30:70 to 70:30, a blend of 60:40 to 40:60, a blend of 25:75 to 75:25, a blend of 20:80 to 80:20, or a blend of 50:50. In one embodiment, the spherical inorganic particles comprise spherical particles of 10 micrometers and spherical particles of 20 micrometers, blended in a ratio of 10:90 to 90:10. In another embodiment, the spherical inorganic particles include 5-micrometer spherical particles and 10-micrometer spherical particles, blended in a ratio of 10:90 to 90:10. In another embodiment, the spherical inorganic particles include 5-micrometer spherical particles and 20-micrometer spherical particles, blended in a ratio of 10:90 to 90:10.

[0105] In some embodiments, the affinity ligand is covalently bonded to spherical inorganic particles. In another embodiment, the affinity ligand is covalently bonded to a protofibrotic polymer membrane. In yet another embodiment, the affinity ligand may bind to both the polymer membrane and the spherical inorganic particles. The affinity ligand may be a protein, antibody, or polysaccharide that reversibly binds to a target molecule. In one embodiment, the affinity ligand is a protein that reversibly binds to, for example, the Fc region of an antibody, an antibody fragment, an Fc fusion protein, or an antibody / drug conjugate. In another embodiment, the affinity ligand is an antibody, L protein, or polysaccharide that reversibly binds to a protein or protein fragment it is specific to. Exemplary affinity ligands for affinity chromatography apparatus include, but are not limited to, protein A, protein G, protein L, human Fc receptor proteins, antibodies that specifically bind to other proteins, and heparin. The affinity ligand may be natural, recombinant, or synthetic. In another embodiment, the affinity ligand is a metal affinity ligand that reversibly binds to a histidine-tagged protein. In another embodiment, the affinity ligand may be an antibody or polysaccharide that reversibly binds to the viral vector to which it is specific.

[0106] In at least one embodiment, the fluoropolymer membrane is a polytetrafluoroethylene (PTFE) membrane or an expanded polytetrafluoroethylene (ePTFE) membrane. Expanded polytetrafluoroethylene (ePTFE) membranes prepared according to the methods described in U.S. Patent No. 7,306,729 to Bacino et al., U.S. Patent No. 3,953,566 to Gore, U.S. Patent No. 5,476,589 to Bacino, or U.S. Patent No. 5,183,545 to Branca et al. are applicable herein. Furthermore, the fluoropolymer membrane can be made hydrophilic (e.g., water-wetting) by methods known in the art, such as, but not limited to, U.S. Patent No. 4,113,912 to Okita et al., methods disclosed in coatings that can effectively bind to ligands, such as applying the coating to the polymer membrane as described in U.S. Patent Nos. 5,897,955, 5,914,182, or 8,591,932 to Drumheller.

[0107] The heat-treated fibrillated fluoropolymer membrane may also include polymer materials containing functionalized tetrafluoroethylene (TFE) copolymer membranes, wherein the functionalized TFE copolymer materials include functionalized copolymers of TFE and PSVE (perfluorosulfonyl vinyl ether), or TFE with another suitable functional monomer, such as, but not limited to, vinylidene fluoride (VDF), vinyl acetate, or vinyl alcohol.

[0108] It should be understood that throughout this application, the term "PTFE" is used for convenience and means not only polytetrafluoroethylene, but also expanded PTFE, expanded modified PTFE, and expanded copolymers of PTFE, such as those described in U.S. Patent No. 5,708,044 to Branca, U.S. Patent No. 6,541,589 to Baillie, U.S. Patent No. 7,531,611 to Sabol et al., U.S. Patent No. 8,637,144 to Ford, and U.S. Patent No. 9,139,669 to Xu et al.

[0109] Alternatively, the fibrillated polymer membrane can be, for example, a fibrillable polyolefin membrane (e.g., a polyethylene membrane).

[0110] The intermediate material can be a fluoropolymer film or a non-fluoropolymer film (e.g., polyethylene, expanded polyethylene, or other polyolefin films). Furthermore, the intermediate film can be porous. In some embodiments, the intermediate film is a thermoplastic or thermosetting polymer film.

[0111] Advantageously, the chromatographic apparatus can be used multiple times. Furthermore, after each separation process or multiple separation processes, the chromatographic apparatus can be cleaned with a cleaning solution (such as sodium hydroxide, phosphoric acid, citric acid, ethanol, etc.) and reused.

[0112] The affinity chromatography apparatus described herein exhibits a dynamic binding capacity (DBC) greater than 35 mg / ml at a residence time of 20 seconds. Furthermore, the affinity chromatography apparatus demonstrates cycle durability of at least 100 cycles at an operating pressure not exceeding 0.3 MPa. Moreover, the apparatus can be used multiple times without losing substantial dynamic binding capacity. Specifically, after each separation process, the apparatus can be cleaned with a cleaning solution (such as sodium hydroxide) and reused. Although embodiments of the wound membrane assembly 110 and the stacked membrane assembly 220 are described herein, it should be understood that any number of fibrillated polymer membranes, as well as any and all combinations thereof, including the type of fibrillated polymer membranes, the type and size of the spherical inorganic particles, and the orientation of the fibrillated polymer membranes within membrane assemblies 110 and 220, are within the scope of this disclosure. Additionally, some or all of the fibrillated polymer membranes may differ from each other in composition, thickness, permeability, etc.

[0113] The chromatographic apparatus and its components described herein can be manufactured using various methods. In some embodiments, injection molding can be used to manufacture the chromatographic components provided herein. Other suitable methods may include, but are not limited to, extrusion, compression molding, solvent casting, and combinations thereof. Embodiments employing two fibrillated polymer membranes co-expanded to produce composite membrane modules are also considered to be within the scope of this disclosure. Such composite membrane modules may comprise two (or more) fibrillated polymer membranes that may be co-extruded or integrated together.

[0114] In some implementations, for example Figure 7 As roughly described herein, the affinity chromatography apparatus described herein utilizes a dual collector 750 of affinity chromatography apparatuses 700 and 701 arranged in parallel configuration. It should be understood that... Figure 7-12 The affinity chromatography apparatus described herein may include stacked membrane assemblies, wound membrane assemblies, or combinations thereof. As shown, an aqueous mixture flows into a distribution element 740. The distribution element 740 is not particularly limited, as long as it distributes the fluid of the aqueous mixture into at least two inlet pipes 760, 761. The split aqueous mixture in inlet pipes 760, 761 flows into chromatography apparatus 700, 701, where the target molecules are captured. The aqueous solution (i.e., the aqueous mixture excluding the target molecules captured by the affinity ligands) flows out of chromatography apparatus 700, 701 through outlet pipes 780, 781, respectively. The aqueous solutions in outlet pipes 780, 781 are combined in distribution element 790 and then recombined into a single aqueous solution. Figure 8 The image depicts a top view of a dual collector 750 comprising two parallel affinity chromatography devices 700, 701. In some embodiments, the collector 750 is located within a housing 720.

[0115] It should be understood that Figure 7 and Figure 8 The two chromatographic devices 700 and 701 shown are for illustrative purposes only. Multiple (i.e., three or more) chromatographic devices of the parallel configuration described herein can be used in an collector, provided there is similar fluid distribution and permeability between the devices. This similarity between affinity chromatographic devices allows for scalability in device size and performance. The affinity chromatographic devices used in collectors (such as dual collectors, quad collectors, etc.) can be identical or different from each other. Furthermore, affinity chromatographic devices can be used in parallel configuration systems without requiring any changes or additions to collector 750.

[0116] Figure 9 An example of an aggregator that includes multiple chromatographic devices as described herein is a four-aggregator. Figure 10 The image shows a top view of the four-assembly unit 950. (Compared to...) Figure 7 and Figure 8 Similar to the dual collector described herein, the aqueous mixture flows to the distribution element 940. The distribution element 940 divides the fluid of the aqueous mixture into inlet pipes 960, 961, 962, and 963. It should be noted that... Figure 9 962 and 963 are concealed behind 961 and 962, respectively. The split aqueous mixtures in inlet pipes 960, 961, 962, and 963 (962 and 963 not shown) flow into chromatographic apparatuses 900, 901, 902, and 903 (902 and 903 not shown), respectively, where target molecules are captured. The aqueous solutions exit each of the respective chromatographic apparatuses 900, 901, 902, and 903 through outlet pipes 980, 981, 982, and 983 (not shown) (note that 982 and 983 are concealed behind 980 and 981). The aqueous solutions in outlet pipes 980, 981, 982, and 983 (982 and 983 not shown) are combined in dispensing element 990 and then recombined into a single aqueous solution. In some embodiments, a quadruple collector 950 may be located within housing 920.

[0117] Figure 11 An apparatus 1000 is described, comprising two parallel constructs, collectors 1150 and 1151. Figure 12 The diagram depicts a top view of apparatus 1000. Collectors 1150 and 1151 each include four affinity chromatography devices, wherein... Figure 11 The diagram depicts two affinity chromatography units 1100, 1101 and 1102, 1103 for each of the collectors 1150, 1151. The ability to utilize at least two parallel collectors advantageously allows for increased volumetric capacity while utilizing the chromatographic apparatus described herein. In other words, apparatus 1000 eliminates the need to switch to a larger volumetric chromatography apparatus. Furthermore, for example... Figure 11The described parallel placement of the collector reduces concerns about overpressurization of the device 1000.

[0118] In use, the aqueous mixture flows to distribution element 1140. Distribution element 1140 divides the aqueous mixture into two distribution pipes 1141 and 1142. The aqueous mixture diverted in distribution pipes 1141 and 1142 is then further divided into inlet pipes by distribution elements 1145 and 1146. Figure 11 In the depicted embodiment, inlet pipes 1160 and 1161 are depicted among the four inlet pipes of collector 1150. Similarly, inlet pipes 1162 and 1163 are depicted among the four inlet pipes of collector 1151. It should be understood that in Figure 11 In this configuration, the remaining inlet pipes 1164, 1165, 1166, and 1167 are concealed behind inlet pipes 1160, 1161, 1162, and 1163. The aqueous mixture in the inlet pipes of the first and second collectors 1150 and 1151 flows into the affinity chromatography apparatus. It should be understood that affinity chromatography apparatuses 1100 and 1101 are depicted in collector 1150, and affinity chromatography apparatuses 1102 and 1103 are depicted in collector 1151. Figure 11 The remaining chromatographic devices 1104, 1105, 1106, and 1107 are hidden behind affinity chromatographic devices 1100, 1101, 1102, and 1103. The target molecule is captured in the affinity chromatographic devices.

[0119] Aqueous solutions flow out of each chromatographic unit through outlet pipes. Figure 11 The text describes the outlet pipes 1180 and 1181 of the collector 1150 and the outlet pipes 1182 and 1183 of the collector 1151. It should be understood that the remaining outlet pipes are... Figure 11 The aqueous solution is concealed behind outlet pipes 1180, 1181, 1182, and 1183. The aqueous solution in the outlet pipes is combined in distribution elements 1170 and 1171. The combined solution then flows from distribution elements 1170 and 1171 into collection pipes 1110 and 1111. The aqueous solution in collection pipes 1110 and 1111 is combined in distribution element 1190 and then reassembled into a single aqueous solution. The first collector 1150 and the second collector 1151 can be enclosed within housing 1120.

[0120] In some implementations, the affinity chromatography device within the collector and / or the collector itself (e.g., a dual collector) Figure 7 ), four-collector ( Figure 9 )) and / or parallel constructed collectors ( Figure 11The affinity chromatography apparatus can be contained within a housing. For example, there can be a first housing surrounding the chromatography apparatus within the collector. Thus, in a dual collector, the first housing surrounds both affinity chromatography apparatuses. Relatedly, there can be a second housing surrounding the collector (and a first housing surrounding the chromatography apparatus). In other embodiments, only the chromatography apparatus within the collector is surrounded by the housing. In further embodiments, the collector and affinity chromatography apparatus can be housed within the housing (e.g., the chromatography apparatus contained within the collector is not separately contained within the housing). There are no particular limitations on the material forming the housing. Non-limiting examples of suitable materials include, but are not limited to, polyurethane, stainless steel, polypropylene, acrylonitrile-butadiene-styrene (ABS), polyethylene terephthalate (PET), polyetheretherketone (PEEK), cyclic olefin copolymers (COC), and polyethylene terephthalate (PETG). Furthermore, the shape of the housing unit is not limited and can take any form, as long as it can encapsulate the affinity chromatography apparatus and / or the collector.

[0121] In some embodiments, this disclosure relates to a diagnostic device for removing a target substance from a biological sample. The device includes a protofibroblastic polymer membrane containing spherical inorganic particles having a spherical shape and a nominal particle size of about 5 micrometers to about 20 micrometers. The particle size distribution has a D90 / D10 ratio of less than or equal to 3, and at least one of the protofibroblastic polymer membrane and the inorganic particles is covalently bonded to a ligand that reversibly binds to the target substance in the biological sample. The device may further include a fluid inlet and a fluid outlet in fluid communication with the fluid inlet. Additionally, the device may include a housing member containing the fluid inlet, the fluid outlet, and the protofibroblastic polymer membrane.

[0122] Those skilled in the art will readily understand that various aspects of this disclosure can be implemented by any number of methods and apparatuses configured to perform the desired functions. It should also be noted that the accompanying drawings are not necessarily drawn to scale, but may be enlarged to illustrate various aspects of this disclosure, and in this respect, these drawings should not be considered limiting.

[0123] Test methods

[0124] It should be understood that while certain methods and apparatuses are described below, other methods or apparatuses that are determined to be suitable by those skilled in the art may also be used alternatively.

[0125] Method for determining dynamic binding capacity at 10% penetration

[0126] Insert the chromatographic apparatus into the flow path of the AKTA™ Pure (Cytiva, Marlborough, Massachusetts) liquid chromatography system and perform a single cycle consisting of the following protocols. Table A lists the solutions used; Table B lists the protocol steps for determining the dynamic binding capacity at 10% breakthrough.

[0127] Table A.

[0128] Table B

[0129] Methods for determining liquid permeability

[0130] The liquid permeability of the chromatographic apparatus was determined using Darcy's law. The bed cross-sectional area and bed length of each apparatus were characterized. Solution A was used as the liquid, and its viscosity was characterized. The relationship between the pressure drop across the column and the liquid flow rate was measured on the AKTA™ purified liquid chromatography system.

[0131] Methods for determining particle size and particle size distribution

[0132] Particle size and particle size distribution data are provided by the manufacturer and measured using Coulter counter technology.

[0133] Methods for purifying CHO cell harvest (Cell Harvest)

[0134] Insert the chromatographic apparatus into the flow path of the AKTA™ Pure (Cytiva, Marlborough, Massachusetts) liquid chromatography system. Table C lists the solutions used for the purification of CHO cell harvest.

[0135] Table C

[0136] Methods for determining protein purification yield

[0137] The purification yield can be determined first by measuring the absorbance of the protein elution cell at 280 nm using a Hitachi U-2900 spectrophotometer. Then, using this absorbance value, the protein concentration in the elution cell is calculated using Beer's Law, as follows. The yield for each elution cell is calculated by dividing the total mass (c) of the target protein in the elution cell by the mass (co) of the loaded target protein, referencing the titer for harvest 1 or harvest 2 in Table C, which uses loading amounts based on Tables L, M, or P.

[0138] in

[0139] A = Absorbance

[0140] ε = extinction coefficient

[0141] L = Sample path length

[0142] c =Total target protein content in the eluent

[0143] c 0 =Target protein quantity loaded

[0144] Connection method of parallel chromatography apparatus

[0145] All affinity chromatography devices listed in Tables D, K, and O (see below) are connected in a dual-assembler configuration (containing two Y-fitting fittings and 1 / 16-inch PEEK tubes (devices V, W, and X in Table K)) or in a quad-assembler configuration (containing six Y-fitting fittings and 1 / 16-inch Peek tubes (device Y), or six Y-fitting fittings and 1 / 8-inch FEP tubes (device Z)).

[0146] Table D

[0147] Example

[0148] Example 1: Stacked film reference particles

[0149] A porous polytetrafluoroethylene (ePTFE) membrane was obtained, comprising 15 mass percent ePTFE and 85 mass percent porous silica particles with a nominal particle size of 10 micrometers. Additionally, a porous ePTFE membrane comprising 15 mass percent PTFE and 85 mass percent porous silica particles with a nominal particle size of 20 micrometers was also obtained. The porous silica particles in the ePTFE membranes listed in Table E are substantially identical in other chemical and physical properties, such as chemical composition, particle shape, nominal particle porosity, nominal particle pore size, and nominal particle surface area. Table E lists some physical properties of these two porous ePTFE membranes.

[0150] Table E

[0151] An affinity chromatography apparatus is constructed using porous ePTFE membranes A and B. A polypropylene fluid distributor is fixed to one end of a polypropylene cylindrical shell. A porous polypropylene intermediate material is placed inside the shell. The desired number of ePTFE membrane layers are stacked on top of the polypropylene intermediate material inside the shell (see Table F). A second porous polypropylene intermediate material is placed on top of the ePTFE membrane layers. A second polypropylene fluid distributor is fixed to the end of the cylindrical shell opposite the first polypropylene fluid distributor. The chromatography apparatus is sealed by heating.

[0152] The intermediate device is then processed in a manner that covalently bonds protein A to the stacked ePTFE membrane. This approach represents a typical approach for those skilled in the art and is further described in U.S. Patent Nos. 10,525,376 and 10,526,367 to McManaway et al.

[0153] The affinity chromatography devices manufactured above were tested, and their liquid permeability and dynamic binding capacity at a residence time of twenty (20) seconds were evaluated using the test methods described herein. The performance of each affinity chromatography device is shown in Table F.

[0154] Table F

[0155] Example 2: Stacked films - spherical, controlled size distribution particles

[0156] Spherical particles with the particle size and particle size distribution described in Table G were obtained. The porous silica particles listed in Table G are substantially the same in terms of chemical and physical properties, such as chemical composition, particle shape, nominal particle porosity, nominal particle pore size, and nominal particle surface area.

[0157] Table G

[0158] Porous polytetrafluoroethylene (ePTFE) membranes were obtained with 15% by mass PTFE and 85% by mass spherical porous silica particles from Table G. These ePTFE membranes were mixtures of nominal particle sizes with different masses. Table H lists the proportions of each particle size mixture and the physical properties of the ePTFE membranes.

[0159] Table H

[0160] Affinity chromatography devices were fabricated using porous membranes (ePTFE) E to K in the same manner as in Example 1. The devices were then treated in the same manner as in Example 1 to covalently bind protein A to the membrane. The affinity chromatography devices were tested to evaluate their liquid permeability and dynamic binding capacity at a residence time of twenty (20) seconds. The performance of each affinity chromatography device is shown in Table I.

[0161] Table I

[0162] Figure 5 The relationship between the dynamic binding capacity and liquid permeability of the devices described in Examples 1 and 2 is shown for comparison; Example 3: Spiral Wound Film The spiral-wound affinity chromatography apparatus is constructed using the porous ePTFE membranes F and H described in Table H. Each PTFE membrane, with a porous polypropylene intermediate material at opposite ends, is wound along its length onto a hollow polypropylene core. The length of the ePTFE membrane is sufficient to achieve the desired number of windings. The edges of the ePTFE membrane are integrated and sealed in a polypropylene dispensing cap at one end and an outlet cap at the other. The inlet cap and housing are also sealed to the assembly, thereby allowing radial flow through the ePTFE membrane layer from the outer radius to the inner radius and out through the outlet cap.

[0163] Each device was then processed to covalently bind protein A to the membrane in the same manner as in Example 1. Each affinity chromatography device was tested to evaluate its liquid permeability and dynamic binding capacity at a residence time of twenty (20) seconds. The performance of the affinity chromatography devices is shown in Table J. Figure 6 The relationship between S and T devices with spiral wound films and C to R devices with stacked films is described.

[0164] Table J

[0165] Example 4: Construction of Chromatographic Apparatus V, W and X – Dual Collectors

[0166] Construct the chromatography apparatus V, W, and X as shown in Table D using the fittings and pipes listed. Figure 7 The dual-collector configuration is shown. Five (5) consecutive purification cycles were performed on chromatographic devices V, W, and X using harvest 1 from Table C. Chromatographic devices V and W followed the purification conditions listed in Table L, while chromatographic device X (Cytiva, Marlborough, MA - HiScreenMabSelect SuRe LX (PN: 17547415)) followed the purification conditions listed in Table M. The purification cycles were performed to evaluate the purification capacity and yield performance of the dual-collector configuration. Figure 13 Representative purification cycles for chromatographic apparatuses V and W are described. Figure 14 Representative purification cycles for chromatographic apparatus X are described. Table N shows the yield performance of each dual collector. Chromatographic apparatuses V and W show typical chromatograms of CHO cell harvest purification, while chromatographic apparatus X shows... Figure 14 Evidence of early penetration throughout the entire loading process is shown, and Figure 13 Compared to device V and W, the absorbance of the device in question is 500 to 600 mAU higher. Figure 14 As can be observed, chromatographic device X also showed evidence of dual elution peaks. These dual elution peaks were not observed in devices V and W.

[0167] Table K

[0168] Table L

[0169] Table M

[0170] Table N

[0171] Example 5: Chromatographic apparatus Y and Z – a four-assembly structure of stacked and helically wound elements

[0172] Construct chromatography apparatus Y and Z using the fittings and pipes listed in Table D. Figure 9 The quad-collector configuration is shown. The harvest 2 from Table C was purified using the quad-collector. Chromatographic apparatuses Y and Z followed the purification conditions listed in Table P to evaluate the purification capacity and yield performance of the quad-collector configuration. Figure 15 Representative purification cycles for chromatographic apparatus Y and chromatographic apparatus Z are described, and Table Q describes the yield performance of each apparatus.

[0173] Table O

[0174] Table P

[0175] Table Q

[0176] The invention of this application has been described for brevity and in conjunction with specific embodiments above. It will be apparent to those skilled in the art that various modifications and variations can be made to the embodiments described herein without departing from the spirit and scope of the invention. Therefore, the embodiments are intended to cover these modifications and variations of the invention, provided that such modifications and variations are within the scope of the appended claims and their equivalents.

Claims

1. An article comprising: Includes the following affinity chromatography apparatus: Fluid inlet; The fluid outlet is connected to the fluid inlet. as well as A fibrillated polymer membrane located between a fluid inlet and a fluid outlet, comprising inorganic particles having a spherical shape and a nominal particle size of about 5 to about 20 micrometers; and The shell structure surrounding the fluid inlet, fluid outlet, and the original fibrous polymer membrane. The particle size distribution has a D90 / D10 ratio of less than or equal to 3, and The affinity chromatography device described herein has a dynamic binding capacity (DBC) of at least 35 mg / ml at a residence time of 20 seconds. The affinity chromatography apparatus has a capacity of 100 (X 10). -12 cm 2 ) to 500 (X 10 -12 cm 2 Hydraulic penetration rate In the case of the protofibrotic polymer membrane and the inorganic particles, at least one of them has been covalently bonded to an affinity ligand, which reversibly binds to the target molecule.

2. The article of claim 1, wherein the target molecule is a protein, antibody, viral vector, or combination thereof.

3. The article of any one of claims 1 to 2, wherein the inorganic particles having a spherical shape are selected from silica, zeolite, hydroxyapatite, metal oxides and combinations thereof.

4. The article of any one of claims 1 to 2, wherein the original fibrous polymer film comprises expanded polytetrafluoroethylene film, expanded modified polytetrafluoroethylene film, expanded polytetrafluoroethylene copolymer film or expanded polyethylene film.

5. The article of any one of claims 1 to 2, wherein the original fibrous polymer membrane is an expanded polytetrafluoroethylene membrane.

6. The article of any one of claims 1 to 2, wherein the affinity ligand is selected from protein A, protein G, protein L, human Fc receptor protein, antibody, polysaccharide and combination thereof.

7. The article of any one of claims 1 to 2, wherein the inorganic particles comprise at least a first inorganic particle having a spherical shape and a first nominal particle size, and a second inorganic particle having a spherical shape and a second nominal particle size, the first nominal particle size and the second nominal particle size being different from each other.

8. The article of any one of claims 1 to 2, wherein the nominal particle size is selected from about 5 micrometers, about 10 micrometers, about 15 micrometers, about 20 micrometers, and combinations thereof.

9. The article of any one of claims 1 to 2, wherein the affinity chromatography apparatus has cycle durability of at least 100 cycles at an operating pressure of less than 0.3 MPa.

10. The article of any one of claims 1 to 2, wherein the fibrillated polymer film has a wound structure.

11. The article of any one of claims 1 to 2, wherein the fibrillated polymer film has a stacked structure.

12. The article of any one of claims 1 to 2, wherein the fibrillated polymer film has a wound structure, a stacked structure, or a combination thereof.

13. The article of any one of claims 1 to 2, wherein the inner intermediate material surrounds the outer surface of the core, and wherein the protofibrotic polymer film surrounds the inner intermediate material.

14. The article of claim 13, wherein the outer intermediate material surrounds the original fibrous polymer membrane.

15. The article of claim 13, wherein the inner intermediate material and the outer intermediate material are selected from porous fluoropolymer membranes, porous non-fluoropolymer membranes, porous nonwoven materials, and porous woven materials.

16. The article of claim 13, wherein at least one of the inner layer intermediate material and the outer layer intermediate material is a polypropylene nonwoven material.

17. Use of the article of any one of claims 1 to 16 to separate target molecules from a fluid stream.

18. An assembler comprising at least two articles of any one of claims 1 to 16 arranged in parallel configuration.

19. The collector of claim 18, wherein the collector is enclosed in a housing.

20. An apparatus comprising a first collector and a second collector constructed in parallel, wherein each of the first collector and the second collector comprises at least two articles of any one of claims 1 to 16.

21. The apparatus of claim 20, wherein the first collector and the second collector are enclosed in a housing.

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