Affinity chromatography apparatus comprising a fibrillated polymer membrane and a manifold comprising the same

Abstract

The present disclosure relates to an affinity chromatography apparatus comprising a fibrillated polymer membrane comprising inorganic particles having a spherical shape and a particle size distribution in which D90 / D10 is 3 or less. A blend or combination of spherical inorganic particles may be utilized. The nominal particle size of the spherical inorganic particles is about 5 microns to about 20 microns. An affinity ligand may be bound to the spherical inorganic particles and / or the fibrillated polymer membrane. Additionally, the affinity chromatography apparatus has a permeability of about 100 (×10⁻¹² cm²) to about 500 (×10⁻¹² cm²). Additionally, the affinity chromatography apparatus has a cycling endurance of at least 100 cycles without exceeding an operating pressure of 0.3 MPa. A manifold comprising a multiple affinity chromatography apparatus in a parallel configuration and a multiple manifold in a parallel configuration are also disclosed.

Description

Technology Field

[0001] The present disclosure generally relates to affinity chromatography, and more specifically, to an affinity chromatography apparatus comprising a fibrillated polymer membrane capable of separating a target molecule from an aqueous mixture, the mixture comprising spherical particles having a particle size distribution of D90 / D10 of 3 or less. A manifold comprising a plurality of affinity chromatography apparatuses and a manifold in a parallel configuration are also disclosed. Background Technology

[0002] Chromatographic methods are generally used to separate and / or purify molecules of interest, such as proteins, nucleic acids, and polysaccharides, from mixtures. Affinity chromatography involves passing a mixture over a matrix that has ligands specific to the bound molecule of interest (i.e., specific binding partners). Upon contact with the ligands, the molecule of interest binds to the matrix and is separated from the mixture. Affinity chromatography offers specific advantages over other types of chromatography. For example, affinity chromatography provides a purification method capable of separating a target protein from a mixture of the target protein and other biomolecules in a single step with high yield.

[0003] Despite the advantages of current affinity chromatography devices, there is a demand in the industry for a reusable chromatography device that can be used with a shorter residence time than conventional devices, provides the same or better binding capacity as current products, and is reusable. means of solving the problem

[0004] In one embodiment ("Embodiment 1"), the affinity chromatography apparatus comprises a fluid inlet, a fluid outlet connected to the fluid inlet in a fluid manner, a fibrillated 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 microns to about 20 microns, and a housing surrounding the fluid inlet, the fluid outlet and the fibrillated polymer membrane, wherein the particle size distribution is D90 / D10 of 3 or less, and at least one of the fibrillated polymer membrane and the inorganic particles has an affinity ligand covalently bonded to reversibly bind to a target molecule.

[0005] According to another embodiment ("Embodiment 2") in addition to Embodiment 1, the target molecule is a protein, antibody, viral vector, and a combination thereof.

[0006] According to another modality ("modality 3") in addition to modality 1 or 2, approximately 100 (×10 -12 ㎠) to about 500 (×10 -12 It has a permeability of cm².

[0007] In addition to any one of embodiments 1 to 3, according to another embodiment ("Embodiment 4"), the inorganic particles having a spherical shape are selected from silica, zeolite, hydroxyapatite, metal oxides, and combinations thereof.

[0008] According to another embodiment ("Embodiment 5") in addition to any one of embodiments 1 to 4, the fibrillated polymer membrane comprises a foamed polytetrafluoroethylene membrane, a foamed modified polytetrafluoroethylene membrane, a foamed tetrafluoroethylene copolymer membrane, or a foamed polyethylene membrane.

[0009] According to another embodiment ("Embodiment 6") in addition to any one of embodiments 1 to 5, the fibrillated polymer membrane is a foamed polytetrafluoroethylene membrane.

[0010] In addition to any one of embodiments 1 to 6, according to another embodiment ("Embodiment 7"), the affinity ligand is selected from protein A, protein G, protein L, human Fc receptor protein, antibody, polysaccharide, and combinations thereof.

[0011] According to another embodiment ("Embodiment 8") in addition to any one of embodiments 1 to 7, 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, wherein the first and second nominal particle sizes are different from each other.

[0012] According to another embodiment ("Embodiment 9") in addition to any one of embodiments 1 to 8, the nominal particle size is selected from about 5 microns, about 10 microns, about 15 microns, about 20 microns, and combinations thereof.

[0013] According to another embodiment ("Embodiment 10") in addition to any one of embodiments 1 to 9, the inorganic particles having a spherical shape comprise a blend of 10-micron spherical particles and 20-micron spherical particles, and the blend is 10:90 to 90:10.

[0014] According to another embodiment ("Embodiment 11") in addition to any one of embodiments 1 to 9, the inorganic particles having a spherical shape comprise a blend of 5-micron spherical particles and 10-micron spherical particles, and the blend is 10:90 to 90:10.

[0015] According to another embodiment ("Embodiment 12") in addition to any one of embodiments 1 to 9, the inorganic particles having a spherical shape comprise a blend of 5-micron spherical particles and 20-micron spherical particles, and the blend is 10:90 to 90:10.

[0016] According to another embodiment ("Embodiment 13") in addition to any one of embodiments 1 to 12, it has a dynamic binding dose (DBC) of at least 35 mg / ml at a retention time of 20 seconds.

[0017] According to another embodiment ("Embodiment 14") in addition to any one of embodiments 1 to 13, it has a cycle endurance of at least 100 cycles at an operating pressure of less than 0.3 MPa.

[0018] According to another embodiment ("Embodiment 15") in addition to any one of embodiments 1 to 14, the fibrillated polymer film has a wound configuration.

[0019] According to another embodiment ("Embodiment 16") in addition to any one of embodiments 1 to 15, the fibrillated polymer film has a laminated structure.

[0020] According to another embodiment ("Embodiment 17") in addition to any one of embodiments 1 to 16, the fibrillated polymer film has a wound configuration, a laminated configuration and a combination thereof.

[0021] According to another embodiment ("Embodiment 18") in addition to any one of embodiments 1 to 17, an internal intermediate material is positioned perimeter-wise on the outer surface of the core, and the fibrillated polymer film is positioned perimeter-wise on the internal intermediate material.

[0022] According to another embodiment ("Embodiment 19") in addition to Embodiment 18, an external intermediate material is positioned perimeter-wise on a fibrillated polymer film.

[0023] According to another embodiment ("Embodiment 20") in addition to embodiment 18 or 19, the inner intermediate material and the outer intermediate material are selected from porous fluoropolymer film, porous non-fluoropolymer film, porous nonwoven fabric and porous fabric.

[0024] According to another embodiment ("Embodiment 21") in addition to any one of embodiments 18 to 20, at least one of the inner intermediate material and the outer intermediate material is a polypropylene nonwoven.

[0025] According to another embodiment ("Embodiment 22"), any one of embodiments 18 to 21 is used to separate target molecules from a fluid stream.

[0026] In one embodiment ("Embodiment 23"), the manifold comprises at least two affinity chromatography devices of any one of embodiments 1 to 21 arranged in a parallel configuration.

[0027] According to another embodiment ("Embodiment 24") in addition to Embodiment 23, the manifold is embedded within a housing.

[0028] In one embodiment ("Embodiment 25"), the article comprises a first manifold and a second manifold in parallel configuration, and each of the first manifold and the second manifold comprises a plurality of affinity chromatography devices of any one of embodiments 1 to 21.

[0029] According to another embodiment ("Embodiment 26") in addition to embodiment 25, the first manifold and the second manifold are embedded within a housing.

[0030] In one embodiment ("Embodiment 27"), the article comprises a centrally positioned core, a fibrillated polymer membrane wound around the core and containing spherical inorganic particles having a spherical shape and a nominal particle size of about 5 microns to about 20 microns, a housing member surrounding both the core and the fibrillated polymer membrane, a first end cap located at a first end of the housing member, and a second end cap located at a second end of the housing member, wherein the particle size distribution is D90 / D10 of less than 3, and at least one of the fibrillated polymer membrane and the spherical inorganic particles covalently bonded to an affinity ligand that reversibly binds to a target molecule.

[0031] According to another embodiment ("Embodiment 28") in addition to Embodiment 27, the target molecule is a protein, antibody, viral vector, and combinations thereof.

[0032] According to another modality ("modality 29") in addition to modality 27 or 28, approximately 100 (×10 -12 cm 2 ) to about 500(×10 -12 cm 2 It has a pitching rate of ).

[0033] According to another embodiment ("Embodiment 30") in addition to any one of embodiments 27 to 29, the inorganic particles having a spherical shape are selected from silica, zeolite, hydroxyapatite, metal oxides and combinations thereof.

[0034] According to another embodiment ("Embodiment 31") in addition to any one of embodiments 27 to 30, the fibrillated polymer membrane comprises a foamed polytetrafluoroethylene membrane, a foamed modified polytetrafluoroethylene membrane, a foamed tetrafluoroethylene copolymer membrane, or a foamed polyethylene membrane.

[0035] According to another embodiment ("Embodiment 32") in addition to any one of embodiments 27 to 31, the fibrillated polymer membrane is a foamed polytetrafluoroethylene membrane.

[0036] In addition to any one of embodiments 27 to 32, according to another embodiment ("Embodiment 33"), the affinity ligand is selected from protein A, protein G, protein L, human Fc receptor protein, antibody, polysaccharide, and combinations thereof.

[0037] According to another embodiment ("Embodiment 34") in addition to any one of embodiments 27 to 33, 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, wherein the first and second nominal particle sizes are different from each other.

[0038] According to another embodiment ("Embodiment 35") in addition to any one of embodiments 27 to 34, the nominal particle size is selected from about 5 microns, about 10 microns, about 15 microns, about 20 microns, and combinations thereof.

[0039] According to another embodiment ("Embodiment 36") in addition to any one of embodiments 27 to 34, the inorganic particles having a spherical shape comprise a blend of 10-micron spherical particles and 20-micron spherical particles, and the blend is 10:90 to 90:10.

[0040] According to another embodiment ("Embodiment 37") in addition to any one of embodiments 27 to 34, the inorganic particles having a spherical shape comprise a blend of 5-micron spherical particles and 10-micron spherical particles, and the blend is 10:90 to 90:10.

[0041] According to another embodiment ("Embodiment 38") in addition to any one of embodiments 27 to 34, the inorganic particles having a spherical shape comprise a blend of 5-micron spherical particles and 20-micron spherical particles, and the blend is 10:90 to 90:10.

[0042] According to another embodiment ("Embodiment 39") in addition to any one of embodiments 27 to 38, it has a dynamic binding dose (DBC) of at least 35 mg / ml at a retention time of 20 seconds.

[0043] According to another embodiment ("Embodiment 40") in addition to any one of embodiments 27 to 39, it has a cycle endurance of at least 100 cycles at an operating pressure of less than 0.3 MPa.

[0044] According to another embodiment ("Embodiment 41") in addition to any one of embodiments 27 to 40, an internal intermediate material is positioned perimeter-wise on the outer surface of the core, and the fibrillated polymer film is positioned perimeter-wise on the internal intermediate material.

[0045] According to another embodiment ("Embodiment 42") in addition to any one of embodiments 27 to 41, an external intermediate material is provided that is positioned perimeter-wise on the fibrillated polymer film.

[0046] According to another embodiment ("Embodiment 43") in addition to Embodiment 42, the inner intermediate material and the outer intermediate material are selected from porous fluoropolymer film, porous non-fluoropolymer film, porous nonwoven fabric and porous fabric.

[0047] According to another embodiment ("Embodiment 44") in addition to Embodiment 44, at least one of the inner intermediate material and the outer intermediate material is a polypropylene nonwoven fabric.

[0048] According to one embodiment ("Embodiment 45"), the manifold comprises at least two affinity chromatography devices of any one of embodiments 27 to 44 arranged in a parallel configuration.

[0049] According to another embodiment ("Embodiment 46"), the manifold is embedded within a housing.

[0050] According to one embodiment ("Embodiment 47"), the device comprises a first manifold and a second manifold in a parallel configuration, and each of the first manifold and the second manifold comprises at least two affinity chromatography devices of any one of embodiments 27 to 44.

[0051] According to another embodiment ("Embodiment 48") in addition to Embodiment 47, the first manifold and the second manifold are embedded within a housing.

[0052] In one embodiment ("Embodiment 49"), the affinity chromatography apparatus comprises a housing, an inlet allowing fluid flow into the housing and first and second fluid distributors located at both ends of the housing, an outlet allowing fluid flow out of the housing, and a stacked membrane assembly disposed between the fluid inlet and the fluid outlet within the housing, wherein the stacked membrane assembly comprises two or more fibrillated polymer membranes of a stacked configuration—the fibrillated polymer membranes contain within a blend of inorganic particles having a spherical shape and a nominal particle size of about 5 microns to about 20 microns—the particle size distribution has a D90 / D10 of less than 3, and at least one of the fibrillated polymer membranes and the spherical inorganic particles covalently bonded to an affinity ligand that reversibly binds to a target molecule.

[0053] According to another embodiment ("Embodiment 50") in addition to embodiment 49, it includes first and second flow distributors located at both ends of the housing member.

[0054] According to another embodiment ("Embodiment 51") in addition to embodiment 49 or 50, the target molecule is a protein, antibody, viral vector, and a combination thereof.

[0055] According to another mode ("mode 52") in addition to any one of modes 49 to 51, approximately 100 (×10 -12 ㎠) to about 500 (×10 -12 It has a permeability of cm².

[0056] According to another embodiment ("Embodiment 53") in addition to any one of embodiments 49 to 52, the inorganic particles having a spherical shape are selected from silica, zeolite, hydroxyapatite, metal oxides and combinations thereof.

[0057] According to another embodiment ("Embodiment 54") in addition to any one of embodiments 49 to 53, the fibrillated polymer membrane comprises a foamed polytetrafluoroethylene membrane, a foamed modified polytetrafluoroethylene membrane, a foamed tetrafluoroethylene copolymer membrane, or a foamed polyethylene membrane.

[0058] According to another embodiment ("Embodiment 55") in addition to any one of embodiments 49 to 54, the fibrillated polymer membrane is a foamed polytetrafluoroethylene membrane.

[0059] In addition to any one of embodiments 49 to 55, according to another embodiment ("Embodiment 56"), the affinity ligand is selected from protein A, protein G, protein L, human Fc receptor protein, antibody, polysaccharide, and combinations thereof.

[0060] According to another embodiment ("Embodiment 57") in addition to any one of embodiments 49 to 56, 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, wherein the first and second nominal particle sizes are different from each other.

[0061] According to another embodiment ("Embodiment 58") in addition to any one of embodiments 49 to 57, the nominal particle size is selected from about 5 microns, about 10 microns, about 15 microns, about 20 microns, and combinations thereof.

[0062] According to another embodiment ("Embodiment 59") in addition to any one of embodiments 49 to 58, the inorganic particles having a spherical shape comprise a blend of 10-micron spherical particles and 20-micron spherical particles, and the blend is 90:10 to 10:90.

[0063] According to another embodiment ("Embodiment 60") in addition to any one of embodiments 49 to 58, the inorganic particles having a spherical shape comprise a blend of 5-micron spherical particles and 10-micron spherical particles, and the blend is 10:90 to 90:10.

[0064] According to another embodiment ("Embodiment 61") in addition to any one of embodiments 49 to 58, the inorganic particles having a spherical shape comprise a blend of 5-micron spherical particles and 20-micron spherical particles, and the blend is 10:90 to 90:10.

[0065] According to another embodiment ("Embodiment 62") in addition to any one of embodiments 49 to 61, it has a dynamic binding dose (DBC) of at least 35 mg / ml at a retention time of 20 seconds.

[0066] According to another embodiment ("Embodiment 63") in addition to any one of embodiments 49 to 62, it has a cycle endurance of at least 100 cycles at an operating pressure of less than 0.3 MPa.

[0067] According to another embodiment ("Embodiment 64") in addition to any one of embodiments 49 to 63, at least one first intermediate material is located on a first surface of the laminated membrane assembly, and a second intermediate material is located on a second surface of the laminated membrane assembly—opposite to the first surface.

[0068] According to another embodiment ("Embodiment 65") in addition to Embodiment 64, the first intermediate material and the second intermediate material are selected from porous fluoropolymer film, porous non-fluoropolymer film, porous nonwoven fabric and porous fabric.

[0069] According to another embodiment ("Embodiment 66") in addition to Embodiment 65, at least one of the first intermediate material and the second intermediate material is a polypropylene nonwoven fabric.

[0070] In one embodiment ("Embodiment 67"), the manifold comprises at least two affinity chromatography devices of any one of embodiments 49 to 66 arranged in a parallel configuration.

[0071] According to another embodiment ("Embodiment 68") in addition to Embodiment 67, the manifold is embedded within a housing.

[0072] In one embodiment ("Embodiment 69"), the device comprises a first manifold and a second manifold in a parallel configuration, and each of the first manifold and the second manifold comprises at least two affinity chromatography devices of any one of embodiments 49 to 66.

[0073] According to another embodiment ("Embodiment 70") in addition to embodiment 69, the first manifold and the second manifold are embedded within a housing.

[0074] In another embodiment ("Embodiment 71"), the diagnostic article comprises a fibrillated polymer membrane containing inorganic particles having a spherical shape and a nominal particle size of about 5 microns to about 20 microns, the particle size distribution having a D90 / D10 of 3 or less, and at least one of the fibrillated polymer membrane and the inorganic particles covalently bonds a ligand that reversibly binds to a target substance in a biological fluid.

[0075] According to another embodiment ("Embodiment 72") in addition to Embodiment 71, it includes a fluid inlet and a fluid outlet connected to the fluid inlet in a fluid-communication manner.

[0076] According to another embodiment ("Embodiment 73") in addition to embodiment 71 or 72, the fluid inlet, the fluid outlet, and the housing member surrounding the fibrillated polymer membrane are included. Brief explanation of the drawing

[0077] The attached drawings are included to provide a detailed understanding of the present disclosure and are incorporated into the specification to form part of the specification, illustrate embodiments, and explain the principles of the present disclosure together with the description. FIG. 1 is an exploded view of a chromatography apparatus comprising a wound membrane assembly including a fibrillated polymer membrane including spherical inorganic polymer particles according to at least one embodiment; FIG. 2 is a cross-sectional view of a chromatography apparatus showing an external flow channel and an internal flow channel according to at least one embodiment; FIG. 3 is a schematic cross-sectional view of another spirally wound normal flow chromatography apparatus according to at least one embodiment; FIG. 4 is an exploded view of a chromatography apparatus comprising a laminated membrane assembly including a fibrillated polymer membrane including spherical inorganic particles according to at least one embodiment; FIG. 5 is a graphic diagram depicting the relationship between dynamic binding force (DBC) and liquid permeability from the affinity chromatography device described in Examples 1 and 2; FIG. 6 is a graphic drawing depicting the relationship between a device (S and T) having a spirally wound film and an affinity chromatography device (C to R) having a stacked film according to at least one embodiment; FIG. 7 is a schematic front view of a manifold comprising two chromatography devices arranged in a parallel configuration; FIG. 8 is a schematic plan view of the manifold of FIG. 7; FIG. 9 is a schematic perspective view of a manifold comprising four chromatography devices arranged in a parallel configuration; FIG. 10 is a schematic plan view of the manifold of FIG. 9; FIG. 11 is a schematic front view of two manifolds in a parallel configuration; FIG. 12 is a schematic plan view of the manifold configuration of FIG. 11; FIG. 13 is a graphic diagram of a typical purification cycle for the dual manifold (V and W) described in Example 3; FIG. 14 is a graphic diagram of a typical purification cycle for the dual manifold (X) described in Example 3; Figure 15 is a graphic diagram of a typical purification cycle for the quadruple manifold described in Example 4. Specific details for implementing the invention

[0078] Those skilled in the art will readily recognize that various aspects of the present disclosure may be realized by any number of methods and devices configured to perform the intended functions. Furthermore, it should be noted that the accompanying drawings mentioned herein are not necessarily made in proportion and may be exaggerated to illustrate various aspects of the present disclosure, and in this respect, the drawings should not be interpreted as limiting. As used herein, the term “on” should be understood to mean that an element such as a polymer film exists immediately above another element, or that an intermediate element may exist.

[0079] It should be recognized that the expressions “spherical particle,” “spherical inorganic particle,” and “inorganic particle having a spherical shape” may be used interchangeably in this specification. Additionally, the term “spiral wound membrane assembly” is intended to include both fibrillated polymer membranes alone and fibrillated polymer membranes having an intermediate nonwoven. Additionally, the term “laminated membrane assembly” is intended to include both fibrillated polymer membranes alone and fibrillated polymer membranes having one or more intermediate material(s). Furthermore, the terms “affinity chromatography device” and “chromatography device” may be used interchangeably in this specification.

[0080] The present disclosure relates to an affinity chromatography apparatus for separating a target molecule from an aqueous mixture containing the target molecule. The target molecule includes, but is not limited to, proteins, antibodies, viral vectors, and combinations thereof. In some embodiments, the present disclosure relates to a diagnostic apparatus for separating a target disease from a biological sample. The chromatography apparatus and the diagnostic apparatus comprise a fibrillated polymer membrane comprising a mixture of inorganic particles having a spherical shape and a particle size distribution having a D90 / D10 of 3 or less. In some embodiments, a mixture or combination of spherical inorganic particles of various sizes is utilized. The nominal particle size of the spherical inorganic particles is about 5 microns to about 20 microns. An affinity ligand may be bound to the spherical inorganic particles and / or the fibrillated polymer membrane. Additionally, the chromatography apparatus has a dynamic binding capacity (DBC) of greater than 40 mg / ml at a 10% breakthrough at a retention time of 20 seconds. In addition, the affinity chromatography device has a cycling endurance of at least 100 cycles without exceeding an operating pressure of 0.3 MPa. It should be understood that the term “about” as used herein means + / - 10% of the specified unit of measurement.

[0081] Referring to FIGS. 1 and 2, a wound chromatography apparatus (100) is illustrated. When forming the chromatography apparatus (100), at least one fibrillated polymer membrane containing spherical inorganic particles wraps around a cylindrical core (150). As used herein, fibrillation refers to the inclusion of fibrils in a polymer membrane, such as a membrane having a microstructure characterized by nodes interconnected by fibrils such that a void is formed in the space between the nodes and the fibrils. In some embodiments, at least one type of internal intermediate material (200) may be placed perimeterly with respect to the core (150) in a desired width or a predetermined amount (e.g., wound). Subsequently, a fibrillated polymer membrane (210) containing spherical inorganic particles is wound over an inner intermediate material (200) around the core (150) in a desired width or a predetermined amount, and an outer layer of at least one type of outer intermediate material (220) may be placed perimeterly (e.g., wound) over the fibrillated polymer membrane (210) in a desired width or a predetermined amount. Here, the combination of the inner intermediate material (200), the fibrillated polymer membrane (210), and the outer intermediate material (220) will be referred to as a "wound membrane assembly." In some embodiments, the "wound membrane assembly" may include a fibrillated polymer membrane and inner intermediate material(s), a fibrillated polymer membrane and outer intermediate material(s), and any combination of polymers and / or an intermediate material wrapping the polymer and the core. The cylindrical core (150) may have a hollow or solid interior. In any case, the core (150) includes a solid outer wall to allow the aqueous mixture flowing through the chromatography device (100) to flow within an internal flow channel formed of an internal intermediate material(s), which is described in detail below. The use of a hollow core (150) reduces the amount of material used to form the core (150), reduces the weight of the device (100), and reduces manufacturing costs.

[0082] The membrane assembly (110) and the central core (150) may be located within the housing (50). In some embodiments, the housing (50) is cylindrical. In the embodiments illustrated in FIGS. 1 and 2, the outer intermediate material(s) (220) form the outer flow channel (130), and the inner intermediate material(s) (200) form the inner flow channel (140). It should be noted that the intermediate material(s) (200, 220) in the embodiments described herein may be the same or different from each other. Additionally, two or more types of intermediate materials may be used to form one or both of the outer flow channel (130) and the inner flow channel (140). When used, an aqueous mixture is introduced into an inlet (80) located within the inlet cap (60), where the mixture flows over the distributor cap (65) and is guided into the outer flow channel (130) formed by the outer intermediate material(s) (220). The distributor cap (65) directs the aqueous mixture into the outer flow channel (130) (i.e., intermediate material(s) (220)) at a 90-degree angle from the supply direction. This change in direction encourages the aqueous mixture to flow more uniformly 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 membrane (210). The distributor cap (65) may be made of polyolefin or coated with polyolefin. It should be noted that the aqueous mixture flows along the outer channel gap (165) and connects to the inner flow channel (130).

[0083] The aqueous mixture flows across the wound polymer membrane (210) in a normal direction (e.g., normal flow) through the outer flow channel (130) (i.e., outer intermediate material(s) (220)). As the aqueous mixture passes through the wound polymer membrane (210) in a normal flow from the outer flow channel (130) (i.e., outer intermediate material(s) (220)), the affinity ligand reversibly binds to the target protein and effectively removes it from the aqueous mixture. The aqueous mixture then enters the inner flow channel (140) (i.e., inner intermediate material(s) (200)) located between the solid outer wall of the central core (150) and the wound polymer membrane (210).

[0084] Then, the aqueous mixture is diverted from the bottom of the internal flow channel (140) by the outflow cap (75). Then, the aqueous mixture is discharged from the chromatography device (100) through the outlet (85) located within the outflow cap (75). It should be noted that the diameter and / or height of the central core (150) (and / or the width and / or height of the fibrillated polymer membrane and / or intermediate material) can be adjusted to achieve a larger volume without negatively affecting the performance of the device. Additionally, the target protein can be removed from the affinity ligand by passing a fluid having a lower pH through the chromatography device, for example, as is known to those skilled in the art.

[0085] The intermediate material(s) (200, 220, and 40) are not particularly limited as long as an aqueous mixture can pass through them. Some non-limiting examples of suitable intermediate materials include, but are not limited to, porous fluoropolymer films or porous nonfluoropolymer films (e.g., porous polypropylene or other porous polyolefin films), porous nonwovens or porous wovens. In some embodiments, the winding membrane assembly forms an integrated reusable chromatography device comprising an inlet end cap (60) integrated at one end of the core (150) and an outlet end cap (75) integrated at the opposite end of the core (150).

[0086] The total number of fibrillated polymer membrane layers present within a wound membrane assembly is not specifically limited and depends on the desired end use and / or the desired mass flow through the membrane assembly. A wound membrane assembly may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 (or more) total polymer membrane layers. It should be noted that a stacked membrane assembly may contain hundreds or thousands of polymer membrane layers. Additionally, the fibrillated polymer film present in the wound film assembly may have a single layer thickness of about 1 micron to about 10,000 microns, about 100 microns to about 5,000 microns, about 500 microns to about 3,000 microns, or about 650 microns to about 1,000 microns. As used herein, the term “thickness” refers to the direction of the fibrillated polymer film perpendicular to the length region of the fibrillated polymer film.

[0087] The function of the chromatography device (300) illustrated in FIG. 3 is substantially similar to that of the chromatography device (100) illustrated in FIG. 2. For example, an aqueous mixture is introduced into the chromatography device (300) through an inlet (80) located within an inlet cap (60) in the direction of arrow (62). The aqueous mixture is guided from the supply direction by a distributor cap (65). For convenience of explanation, the aqueous mixture may flow toward an external flow channel (130) (which may be formed of external intermediate material(s)) in the direction indicated by arrow (55). The aqueous mixture flows along the external channel gap (165) and connects to an internal flow channel (140) to flow in the direction of arrow (30). The aqueous mixture flows across the wound polymer membrane (210) in a normal direction (e.g., normal flow) from the internal flow channel (130) to the internal flow channel (140) as indicated by arrow (70). As the aqueous mixture passes through the wound polymer membrane (210), the affinity ligand reversibly binds to the target molecule. It should be noted that the internal flow channel (140) can be formed from the internal intermediate material(s) (200).

[0088] The aqueous mixture without target molecules flows along the internal flow channel (140) in the direction indicated by the arrow (40). The aqueous mixture is redirected from the bottom of the internal flow channel (140) toward the central part of the chromatography device (300) as indicated by the arrow (52). The aqueous mixture without target molecules is discharged from the chromatography device (300) through the outlet (85) located within the outlet end cap (75) in the direction indicated by the arrow (45).

[0089] In other embodiments as generally illustrated in FIG. 4, the chromatography apparatus (200) comprises fibrillated polymer membranes composed of individual discs (240) stacked together to form a stacked membrane assembly (220). The fibrillated polymer membranes (240) may be arranged in a stacked configuration simply by overlapping the fibrillated polymer membrane discs (240) with one another. Alternatively, the fibrillated polymer membrane discs (240) may be stacked together using heat and / or pressure or other conventional methods after stacking. It should be understood that the stacked membrane assembly (240) described herein relates to fibrillated polymer membrane discs for convenience of description. Fibrillated polymer membranes formed in geometric shape(s) and / or non-geometric shape(s) are considered to be within the scope of the present disclosure.

[0090] The chromatography device (200) comprises at least one upper intermediate material (260) positioned above the laminated membrane assembly and at least one lower intermediate material (280) positioned below the laminated membrane assembly (220). The upper and lower intermediate material(s) (260, 280) may each be the same or different. Similar to the wound membrane assembly described above, the intermediate material(s) (260, 280) used to form the laminated membrane assembly (220) are not particularly limited as long as an aqueous mixture can pass through them. Non-limiting examples of suitable intermediate materials include, but are not limited to, porous fluoropolymer films or porous nonfluoropolymer films (e.g., porous polypropylene or other porous polyolefin films), porous nonwovens or porous fabrics.

[0091] A laminated membrane assembly (220) may be disposed within a housing (250), and an inlet cap (265) and an outlet end cap (275) may be disposed at both ends of the housing (250). In some embodiments, the housing (250) is cylindrical, but any shape capable of accommodating the laminated membrane assembly and achieving the desired dynamic coupling capacity is considered to be within the scope of the present disclosure. In some embodiments, the intermediate material(s) (260, 280), the housing (250), the inlet cap (265), and the outlet cap (275) may be formed from a thermoplastic polymer such as polypropylene, polyethylene, or other polyolefins. Alternatively, one or both of the intermediate materials (260, 280) may be formed from an inorganic material or a metal material, provided that the porous intermediate material (260, 280) does not interfere with the operation of the chromatography device.

[0092] The fibrillated polymer membrane (240) within the laminated membrane assembly (220) may be attached to the housing (250) on the inner wall of the housing (250) by any conventional process (e.g., melt sealing or the use of a sealant) that prevents flow between the periphery of the fibrillated polymer membrane (240) and the housing (250). The inlet cap (265) and the outlet cap (275) may be sealed to the housing (250) by a similar or identical process. The inlet cap and the outlet cap (265, 275) each include an inlet (280) and an outlet (285), which are intended to allow the flow of an aqueous mixture 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). When in use, the aqueous mixture flows sequentially through an intermediate material (260), a fibrillated polymer membrane (240) forming a laminated membrane assembly (220), and intermediate material(s) (280). As the aqueous mixture passes through the chromatography device (200), the affinity ligand reversibly binds to the target molecule and effectively removes it from the aqueous mixture. The target molecule may be removed from the affinity ligand by passing a fluid having a lower pH through the device, for example, as is known to those skilled in the art.

[0093] The total number of fibrillated polymer membranes present within the laminated membrane assembly is not specifically limited and depends on the desired end use and / or the desired mass flow through the membrane assembly. The laminated membrane assembly may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 (or more) total polymer membranes. It should be recognized that hundreds or thousands of polymer membranes may be present in the laminated membrane assembly. Additionally, the fibrillated polymer membranes present in the laminated membrane assembly may have a thickness of about 1 micron to about 10,000 microns, about 100 microns to about 5,000 microns, about 500 microns to about 3,000 microns, or about 650 microns to about 1,000 microns. As used herein, the term “thickness” refers to the direction of the fibrillated polymer film perpendicular to the length region of the fibrillated polymer film.

[0094] In both the wound membrane assembly and the laminated membrane assembly, the fibrillated polymer membrane comprises spherical inorganic particles, or particles having a spherical configuration. As used herein, the term “spherical” means that the inorganic particles have a circular or nearly circular shape in which the distance from the center of the inorganic particle to the outer edge of the particle at any point is equal or nearly equal. In some embodiments, the spherical inorganic particles have a particle size distribution in which D90 / D10 is 3 or less, 2.5 or less, 2 or less, 1.5 or less, or 1 or less. The spherical inorganic particles have a nominal particle size that may be about 5 microns, about 10 microns, about 15 microns, about 20 microns, and combinations and mixtures thereof. In some embodiments, the spherical inorganic particles are polydisperse particles.

[0095] In some embodiments, the fibrillated polymer membrane comprises spherical inorganic particles of two or more nominal particle sizes and / or two or more types within the fibrillated polymer membrane. The fibrillated polymer membrane may comprise about 10 mass% fibrillated polymer membrane to about 90 mass% spherical inorganic particles, about 15 mass% fibrillated polymer membrane to about 85 mass% spherical inorganic particles, about 20 mass% fibrillated polymer membrane to about 80 mass% spherical inorganic particles, about 30 mass% fibrillated polymer membrane to about 70 mass% spherical inorganic particles, about 35 mass% fibrillated polymer membrane to about 65 mass% spherical inorganic particles, about 40 mass% fibrillated polymer membrane to about 60 mass% spherical inorganic particles, about 45 mass% fibrillated polymer membrane to about 55 mass% spherical inorganic particles, or about 50 mass% fibrillated polymer membrane to about 50 mass% spherical inorganic particles. Non-limiting examples of suitable inorganic particles include silica, zeolites, hydroxyapatite, metal oxides, and combinations thereof. Additionally, the inorganic particles may be solid or porous. Furthermore, the affinity chromatography apparatus described herein is approximately 100 (×10⁻⁶). -12 ㎠) to about 500 (×10 -12 cm²), approximately 150 (×10⁻⁶ -12 ㎠) to about 500 (×10 -12 cm²), approximately 200 (×10⁻⁶ -12 ㎠) to about 500 (×10 -12 cm²), approximately 250 (×10⁻⁶ -12 ㎠) to about 500 (×10 -12 cm²), approximately 200 (×10⁻⁶ -12 ㎠) to about 450 (×10 -12 cm²), approximately 200 (×10⁻⁶ -12 ㎠) to about 400 (×10 -12 cm²), approximately 250 (×10⁻⁶ -12 ㎠) to about 400 (×10 -12 cm²), or about 300 (×10⁻⁶ -12 ㎠) to about 400 (×10 -12It has a hydraulic permeability of cm².

[0096] In at least one embodiment, the fibrillated polymer membrane comprises a blend of spherical inorganic particles having different nominal particle sizes. For example, the fibrillated polymer membrane may comprise a 90:10 blend of a first nominal particle size (e.g., 5 microns) and a second nominal particle size (e.g., 20 microns) of the same or different spherical inorganic particles. The blend of spherical inorganic particles within the fibrillated polymer membrane may comprise any blend, such as, 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 inorganic particles having a spherical shape comprise 10-micron spherical particles and 20-micron spherical particles in a blend of 10:90 to 90:10. In another embodiment, the inorganic particles having a spherical shape comprise 5-micron spherical particles and 10-micron spherical particles in a blend of 10:90 to 90:10. In yet another embodiment, the inorganic particles having a spherical shape comprise 5-micron spherical particles and 20-micron spherical particles in a blend of 10:90 to 90:10.

[0097] In some embodiments, the affinity ligand is covalently bonded to a spherical inorganic particle. In other embodiments, the affinity ligand is covalently bonded to a fibrillated polymer membrane. In other embodiments, the affinity ligand may be bound to both the polymer membrane and the spherical inorganic particle(s). 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, for example, to the Fc region of an antibody, an antibody moiety, an Fc fusion protein, or an antibody / drug conjugate. In other embodiments, the affinity ligand is a polysaccharide that reversibly binds to an antibody, protein L, or a protein or protein moiety having specificity. Exemplary affinity ligands used in an affinity chromatography device include, but are not limited to, protein A, protein G, protein L, human Fc receptor protein, antibodies that specifically bind to other proteins, and heparin. The affinity ligand may be a natural product, a recombinant, or a synthetic product. In another embodiment, the affinity ligand is a metal-affinity ligand that reversibly binds to a His-Tagged protein. In another embodiment, the affinity ligand may be an antibody or a polysaccharide that reversibly binds to a viral vector having specificity.

[0098] In at least one embodiment, the fluoropolymer membrane is a polytetrafluoroethylene (PTFE) membrane or an expanded polytetrafluoroethylene (ePTFE) membrane. An expanded polytetrafluoroethylene (ePTFE) membrane prepared according to the methods described in U.S. Patent No. 7,306,729 (Bacino et al.), U.S. Patent No. 3,953,566 (Gore), U.S. Patent No. 5,476,589 (Bacino), or U.S. Patent No. 5,183,545 (Branca et al.) may be used in the present invention. Additionally, the fluoropolymer membrane may be made hydrophilic (e.g., water-wettable) by using methods known in the art, such as, for example but not limited to, the method disclosed in U.S. Patent No. 4,113,912 granted to Okita et al. A coating that effectively binds to a ligand such as that described in U.S. Patent No. 5,897,955, U.S. Patent No. 5,914,182, or U.S. Patent No. 8,591,932, granted to Drumheller, can be applied to a polymer membrane.

[0099] The fluoropolymer membrane may also include a polymer material comprising a functional tetrafluoroethylene (TFE) copolymer membrane, wherein the functional TFE copolymer material comprises a functional copolymer of TFE and PSVE (perfluorosulfonyl vinyl ether) or, but is not limited to, TFE having other suitable functional monomers such as vinylidene fluoride (VDF), vinyl acetate or vinyl alcohol.

[0100] Throughout this application, the term “PTFE” is used in this application for convenience and should be understood to mean not only polytetrafluoroethylene but also expanded PTFE, expanded modified PTFE, and expanded copolymers of PTFE as described in U.S. Patent No. 5,708,044 (Branca), U.S. Patent No. 6,541,589 (Baillie), U.S. Patent No. 7,531,611 (Sabol et al.), U.S. Patent No. 8,637,144 (Ford), and U.S. Patent No. 9,139,669 (Xu et al.).

[0101] In addition, the fibrillated polymer membrane may be, for example, a fibrillated polyolefin membrane (e.g., a polyethylene membrane).

[0102] The intermediate material(s) may be a fluoropolymer film or a non-fluoropolymer film (e.g., polyethylene, expanded polyethylene, or other polyolefin films). Additionally, the intermediate film may be porous. In some embodiments, the intermediate film is a thermoplastic or thermosetting polymer film.

[0103] Advantageously, the chromatography apparatus can be used multiple times. Additionally, the chromatography apparatus can be reused by being cleaned with a cleaning solution (e.g., sodium hydroxide, phosphoric acid, citric acid, ethanol, etc.) after each separation process or after multiple separation processes.

[0104] The affinity chromatography apparatus described herein has a dynamic binding capacity (DBC) greater than 35 mg / ml at a residence time of 20 seconds. Additionally, the affinity chromatography apparatus has a cycling endurance of at least 100 cycles without exceeding an operating pressure of 0.3 MPa. Furthermore, the chromatography apparatus can be used multiple times without losing significant dynamic binding capacity. In particular, the chromatography apparatus can be reused after being cleaned with a cleaning solution (e.g., sodium hydroxide) after each separation process. Although embodiments of a wound membrane assembly (110) and a 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 of the type of fibrillated polymer membrane, the type of spherical inorganic particle, the size of the spherical inorganic particle, and the orientation of the fibrillated polymer membrane(s) within the membrane assembly (110, 220), are within the scope of the present disclosure. In addition, some or all of the fibrillated polymer membranes may differ in composition, thickness, permeability, etc.

[0105] The chromatography apparatus and its components described herein may be manufactured using various processes. In some embodiments, injection molding may be used to manufacture the chromatography components provided herein. Other suitable processes may include, but are not limited to, extrusion, compression molding, solvent casting, and combinations thereof. Embodiments using two fibrillated polymer membranes that are co-foamed to create a composite membrane assembly are also considered to be within the scope of this disclosure. Such a composite membrane assembly may comprise two (or more) layers of fibrillated polymer membranes that can be co-extruded or incorporated together.

[0106] In some embodiments, the affinity chromatography devices described herein are utilized in a dual manifold (750) having affinity chromatography devices (700, 701) arranged in a parallel configuration as generally illustrated in FIG. 7. It should be recognized that the affinity chromatography devices illustrated in FIG. 7–12 may include a stacked membrane assembly, a wound membrane assembly, or a combination thereof. As illustrated, an aqueous mixture flows into a distribution element (740). The distribution element (740) is not particularly limited, as long as the distribution element (740) divides the flow of the aqueous mixture into at least two inlet tubes (760, 761). The divided aqueous mixture within the inlet tubes (760, 761) flows into the chromatography devices (700, 701), where the target molecule is captured. The aqueous solution (i.e., the aqueous mixture excluding the target molecule captured by the affinity ligand) is discharged from the chromatography device (700, 701) through the respective outflow tubes (780, 781). The aqueous solution in the outflow tubes (780, 781) is combined in the distribution element (790) and reconstituted into a single aqueous solution. A plan view of a dual manifold (750) comprising two parallel affinity chromatography devices (700, 701) is shown in FIG. 8. In some embodiments, the manifold (750) is located within a housing (720).

[0107] FIGS. 7 and 8 show two chromatography devices (700, 701) for illustrative purposes only, and it should be understood that multiple (i.e., three or more) chromatography devices described herein may be utilized in a parallel configuration manifold, provided that there is a similar distribution of flow and permeability among the chromatography devices. Such similarity across the affinity chromatography devices allows for scalability of device volume and performance. The affinity chromatography devices used in the manifold (e.g., double, quadruple, etc.) may be identical or different from one another. Additionally, the affinity chromatography devices may be merged into a parallel configuration system without any modification or addition to the manifold (750).

[0108] An example of a manifold comprising a plurality of chromatographic devices as described in this specification is the quadruple manifold shown in FIG. 9. A top view of this quadruple manifold (950) is shown in FIG. 10. Similar to the double manifold shown in FIG. 7 and FIG. 8, the aqueous mixture flows into a distribution element (940). The distribution element (940) divides the flow of the aqueous mixture into flows into inlet tubes (960, 961, 962 and 963). It should be noted that in FIG. 9, 962 and 963 are hidden behind 961 and 962, respectively. The divided aqueous mixtures in the inlet tubes (960, 961, 962, and 963 (962 and 963 not shown)) each flow into the chromatography device (900, 901, 902, and 903 (902 and 903 (not shown))) where the target molecule is captured. The aqueous solution is discharged from each of the chromatography devices (900, 901, 902, and 903 (not shown)) through the outlet tubes (980, 981, 982, and 983) respectively (note that 982 and 983 are hidden behind 980 and 981). The aqueous solutions (982 and 983 (not shown)) within the outlet tubes (980, 981, 982, and 983) are combined at the distribution element (990) and reconstituted into a single aqueous solution. In some embodiments, quadruple The manifold (950) can be located within the housing (920).

[0109] FIG. 11 illustrates a device (1000) comprising two manifolds (1150, 1151) in a parallel configuration. A plan view of the device (1000) is shown in FIG. 12. Each manifold (1150, 1151) comprises four affinity chromatography devices, among which two affinity chromatography devices (1100, 1101 and 1102, 1103) of each manifold (1150, 1151) are shown in FIG. 11. The ability to utilize at least two manifolds in parallel has the advantage of increasing volumetric capacity while utilizing the chromatography devices described herein. That is, the device (1000) does not need to move to a larger volumetric chromatography device. Additionally, as shown in FIG. 11, arranging the manifolds in parallel reduces the risk of excessive pressure being applied to the device (1000).

[0110] When in use, the aqueous mixture flows into the distribution element (1140). The distribution element (1140) divides the flow of the aqueous mixture into two distribution tubes (1141, 1142). The aqueous mixture divided in the distribution tubes (1141, 1142) is further divided into inlet tubes by the distribution element (1145, 1146). In the embodiment illustrated in FIG. 11, among the four inlet tubes of the manifold (1150), inlet tubes (1160, 1161) are illustrated. Similarly, among the four inlet tubes of the manifold (1151), inlet tubes (1162, 1163) are illustrated. It should be noted that the remaining inlet tubes (1164, 1165, 1166, 1167) are hidden behind the inlet tubes (1160, 1161, 1162, 1163) in FIG. 11. The aqueous mixture in the inlet tubes of the first and second manifolds (1150, 1151) flows into an affinity chromatography device. It should be noted that an affinity chromatography device (1100, 1101) is illustrated in manifold (1150), and an affinity chromatography device (1102, 1103) is illustrated in manifold (1151). The remaining chromatography devices (1104, 1105, 1106, 1107) are hidden behind the affinity chromatography devices (1100, 1101, 1102, 1103) in FIG. 11. Target molecules are captured in the affinity chromatography devices.

[0111] The aqueous solution is discharged from each chromatography device through the discharge tubes. Fig. 11 shows the discharge tubes (1180, 1181) of the manifold (1150) and the discharge tubes (1182, 1183) of the manifold (1151). It should be noted that the remaining discharge tubes are hidden behind the discharge tubes (1180, 1181, 1182, 1183) in Fig. 11. The aqueous solution in the discharge tubes is combined at the distribution element (1170) and the distribution element (1171). Then, the combined solution flows from the distribution element (1170, 1170) to the collection tubes (1110, 1111). The aqueous solution in the collection tubes (1110, 1111) is combined at the distribution element (1190) and reconstituted into a single aqueous solution. The first manifold (1150) and the second manifold (1151) can be embedded within the housing (1120).

[0112] In some embodiments, affinity chromatography devices within the manifold(s) and / or the manifold itself (e.g., a double manifold (Fig. 7), a quadruple manifold (Fig. 9)) and / or a manifold in parallel configuration (Fig. 11) may be contained within a housing. For example, there may be a first housing surrounding the chromatography devices within the manifold. Thus, in a double manifold, the first housing surrounds two affinity chromatography devices. In this regard, there may be a second housing surrounding the manifold (and a first housing surrounding the chromatography devices). In other embodiments, only the chromatography devices within the manifold are surrounded by a housing. In other embodiments, the manifold(s) and the affinity chromatography devices may be housed in a single housing (e.g., the chromatography devices contained within the manifold(s) are not separately contained in the housing). The material forming the housing is not particularly limited. Non-limiting examples of suitable materials include, but are not limited to, polyurethane, stainless steel, polypropylene, acrylonitrile butadiene styrene (ABS), polyethylene terephthalate (PET), polyether ether ketone (PEEK), cyclic olefin copolymer (COC), and polyethylene terephthalate glycol (PETG). Additionally, the shape of the housing unit is not limited and may take any shape as long as the shape encapsulates the affinity chromatography device and / or manifold(s).

[0113] In some embodiments, the present disclosure relates to a diagnostic device for removing a target substance from a biological sample. The device comprises a fibrillated polymer membrane comprising inorganic particles having a spherical shape and a nominal particle size of about 5 microns to about 20 microns. The particle size distribution has a D90 / D10 of 3 or less, and at least one of the fibrillated polymer membrane and the inorganic particles covalently bonds a ligand that reversibly binds to a target substance in the biological sample. The device may further comprise a fluid inlet and a fluid outlet connected fluidly to said fluid inlet. Additionally, the device may comprise a housing member surrounding the fluid inlet, the fluid outlet, and the fibrillated polymer membrane.

[0114] Those skilled in the art will readily recognize that various aspects of the present disclosure may be realized by any number of methods and devices configured to perform the intended functions. Furthermore, it should be noted that the accompanying drawings referred to herein are not necessarily made in proportion and may be exaggerated to illustrate various aspects of the present disclosure, and in this regard, the drawings should not be interpreted as restrictive.

[0115] Test method

[0116] Although specific methods and equipment are described below, it should be understood that other methods or equipment deemed appropriate by those skilled in the art may be used as alternatives.

[0117] Method to determine dynamic coupling capacity when breaking through 10%

[0118] The chromatography device was inserted into the flow path of an AKTA™ Pure (Cytiva, Marlborough, Massachusetts) liquid chromatography system, and a single cycle consisting of the following protocol was performed. Table A describes the solutions used, and Table B describes the protocol steps for determining the dynamic binding capacity at 10% breakthrough.

[0119] Table A

[0120]

[0121] Table B

[0122]

[0123] Liquid Permeability Measurement Method

[0124] The liquid permeability of the chromatography apparatus was determined using Darcy's law. Individual apparatuses were characterized for bed cross-sectional area and bed length. Solution A was used as the liquid, and its viscosity was characterized. The pressure drop across the column as a function of liquid flux was measured in the AKTA™ Pure liquid chromatography system.

[0125] Methods for determining particle size and particle size distribution

[0126] Particle size and particle size distribution data were provided by the manufacturer and measured using the Coulter Counter technique.

[0127] Purification method of CHO cell culture

[0128] The chromatography device was inserted into the flow path of the AKTA™ Pure (Cytiva, Marlborough, Massachusetts) liquid chromatography system. Table C describes the solutions used to perform the purification of CHO cell cultures.

[0129] Table C

[0130]

[0131] Method for determining protein purification yield

[0132] The purification yield was first determined by measuring the absorbance of the protein elution pool at a wavelength of 280 nm using a Hitachi U-2900 spectrophotometer. Then, utilizing this absorbance value, the protein concentration of the protein elution pool was calculated using Beer's law described below. The yield was calculated for each elution pool by dividing the total target protein mass (c) of the elution pool by the loaded target protein mass (Co), using the loading volume based on Table L, M, or P and referring to the titer of Culture 1 or Culture 2 in Table C.

[0133] A=εLc

[0134] Here:

[0135] A = absorbance

[0136] ε = extinction coefficient

[0137] L = Sample path length

[0138] c = Total target protein mass upon elution

[0139] c0 = loaded target protein mass

[0140] % yield = (c / c0)×100.

[0141] Method of connecting chromatography devices in parallel

[0142] All affinity chromatography devices described in Tables K and O (described later) were attached in a double manifold configuration including a 2, Y fitting and 1 / 16" PEEK tubing (devices V, W, and X in Table K) or a quadruple manifold configuration including a 6, Y fitting and 1 / 16" PEEK tubing (device Y) or a 6, Y fitting and 1 / 8" FEP tubing (device Z) as described in Table D.

[0143] Table D

[0144]

[0145] example

[0146] Example 1: Stacked film reference particles

[0147] A porous polytetrafluoroethylene (ePTFE) membrane was obtained having 15 mass% ePTFE and 85 mass% porous silica particles with a nominal particle size of 10 microns. Additionally, a porous ePTFE membrane was obtained having 15 mass% PTFE and 85 mass% porous silica particles with a nominal particle size of 20 microns. The porous silica particles of the ePTFE membranes listed in Table E were substantially identical in relation to other chemical and physical properties such as chemical composition, particle shape, nominal particle porosity, nominal particle pore dimensions, and nominal particle surface area. Table E lists some of the physical properties of the two porous ePTFE membranes.

[0148] Table E

[0149]

[0150] Porous ePTFE membranes A and B were used to fabricate an affinity chromatography apparatus. A polypropylene flow distributor was attached to one end of a polypropylene cylinder housing. A porous polypropylene intermediate material was placed in the housing. A desired number of ePTFE membrane layers were stacked on top of the polypropylene intermediate material within the housing (see Table F). A second porous polypropylene intermediate material was placed on top of the ePTFE membrane stack. A second polypropylene flow distributor was attached to the end of the cylindrical housing opposite the first polypropylene flow distributor. The chromatography apparatus was sealed through a heating process.

[0151] Then, the intermediate device was processed in such a way that protein A was covalently bonded to the stacked ePTFE membrane. This method represents a typical method to those skilled in the art and is described in detail in U.S. Patent No. 10,525,376 (MacManaway et al.) and U.S. Patent No. 10,526,367 (MacManaway et al.).

[0152] The affinity chromatography apparatus manufactured as described above was tested to evaluate liquid permeability and 20-second residence time dynamic binding capacity using the protocol described in the test method described herein. The performance of each of these affinity chromatography apparatuses is presented in Table F.

[0153] Table F

[0154]

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

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

[0157] Table G

[0158]

[0159] From the items in Table G, a porous polytetrafluoroethylene (ePTFE) membrane was obtained containing 15 mass% PTFE and 85 mass% spherical porous silica particles. These ePTFE membranes had various mass mixtures of nominal particle sizes. Table H lists the mixing ratios of each particle size and the physical properties of the ePTFE membranes.

[0160] Table H

[0161]

[0162] An affinity chromatography device was prepared in the same manner as in Example 1 using porous membranes (ePTFE) E to K. Then, the chromatography device was treated to covalently bind protein A to the membrane in the same manner as in Example 1. The affinity chromatography device was tested to evaluate liquid permeability and dynamic binding ability at a 20-second residence time. The performance of each of these affinity chromatography devices is presented in Table I.

[0163] Table I

[0164]

[0165] The relationship between dynamic coupling capacity and liquid permeability from the devices described in Examples 1 and 2 is presented in Fig. 5 for comparison.

[0166] Example 3: Spiral wound membrane

[0167] The porous ePTFE membranes F and H described in Table H were used to construct a helical winding affinity chromatography apparatus. The length of each PTFE membrane, with a porous polypropylene intermediate material at both ends, was wound around a hollow polypropylene core. The length of the ePTFE membrane was sufficient to achieve the desired number of windings. The edges of the ePTFE membrane were integrated and sealed into a polypropylene dispensing cap at one end and an outflow cap at the other end. The inlet cap and the outer housing were similarly sealed over the assembly, enabling radial flow through the ePTFE membrane layer so that an axially oriented channel flows from the outer radius to the inner radius and exits through the outflow cap.

[0168] Then, each device was treated to covalently bind Protein A to the membrane in the same manner as in Example 1. Each affinity chromatography device was tested to evaluate liquid permeability and 20-second residence time dynamic binding capacity. The performance of the affinity chromatography devices is presented in Table J. Figure 6 shows the relationship between the devices with helical wound membranes (S and T) and the membrane-stacked device (CR).

[0169] Table J

[0170]

[0171] Example 4: Chromatography Units V, W, and X - Dual Manifold Configuration

[0172] The chromatography units (V, W, and X) were constructed using the fittings and tubing described in Table D in a dual manifold configuration as shown in Fig. 7. For each of the chromatography units (V, W, and X), five sequential purification cycles were performed using Culture 1 from Table C. The chromatography units (V and W) followed the purification conditions summarized in Table L, and the chromatography unit (X) (Cytiva, Marlboro, Massachusetts – HiScreen MabSelect SuRe LX (PN:17547415)) followed the purification conditions summarized in Table M. The purification cycles were performed to evaluate the purification capability and yield performance in the dual manifold configuration. Fig. 13 shows a representative purification cycle for the chromatography units (V and W). Fig. 14 shows a representative purification cycle for the chromatography unit (X). The yield performance of each dual manifold is presented in Table N. While chromatography devices (V and W) displayed a typical chromatogram for the purified CHO cell culture, chromatography device (X) showed evidence of early breakthrough throughout the loading step by absorbances 500-600 mAU higher than devices (V and W) in Fig. 13 in Fig. 14. Chromatography device (X) also showed evidence of a double elution peak, which can be observed in Fig. 14. This double elution peak was not observed in devices (V and W).

[0173] Table K

[0174]

[0175] Table L

[0176]

[0177] Table M

[0178]

[0179] Table N

[0180]

[0181] Example 5: Chromatography Units Y and Z – Stacked vs. Spiral-Wound Quadruple Manifold Configuration

[0182] The chromatography devices (Y and Z) were constructed using the fittings and tubing described in Table D in a quadruple manifold configuration, as confirmed in Fig. 9. A purification cycle using culture 2 from Table C was performed using the quadruple manifold. The purification capacity and yield performance of the chromatography devices (Y and Z) in the quadruple manifold configuration were evaluated according to the purification conditions described in Table P. Fig. 15 shows a representative purification cycle for both chromatography device (Y) and chromatography device (Z). The yield performance of each device is presented in Table Q.

[0183] Table O

[0184]

[0185] Table P

[0186]

[0187] Table Q

[0188]

[0189] The invention of the present application has been described generally and in relation to specific embodiments as described above. It will be apparent to those skilled in the art that various modifications and changes may be made to the embodiments without departing from the scope of the present disclosure. Accordingly, the embodiments are intended to include modifications and changes of the invention where they fall within the scope of the appended claims and their equivalents.

Claims

Claim 1 An article comprising an affinity chromatography device, wherein the affinity chromatography device comprises: a fluid inlet; a fluid outlet connected to the fluid inlet in a fluid manner; a fibrillated polymer membrane positioned between the fluid inlet and the fluid outlet and containing only inorganic particles having a spherical shape and a nominal particle size selected from about 5 microns, about 10 microns, about 15 microns, about 20 microns, and combinations thereof; and a housing member surrounding the fluid inlet, the fluid outlet, and the fibrillated polymer membrane, wherein the spherical shape comprises a circular or nearly circular shape in which the distance from the center of the inorganic particle to the outer edge of the particle at any point is equal or nearly equal, the particle size distribution has a D90 / D10 of 3 or less, the affinity chromatography device has a dynamic binding capacity (DBC) of at least 35 mg / ml at a residence time of 20 seconds, and the affinity chromatography device has 100 (×10 -12 ㎠) to 500(×10 -12 An article having a permeability of (cm²) and having an affinity ligand that reversibly binds to a target molecule selected from proteins, antibodies, viral vectors, and combinations thereof, covalently bonded to at least one of the fibrillated polymer membrane and the inorganic particle. Claim 2 Article 1, wherein the inorganic particles having a spherical shape are selected from silica, zeolite, hydroxyapatite, metal oxides, and combinations thereof. Claim 3 Article 1, wherein the fibrillated polymer membrane comprises an expanded polytetrafluoroethylene membrane, an expanded modified polytetrafluoroethylene membrane, an expanded tetrafluoroethylene copolymer membrane, or an expanded polyethylene membrane. Claim 4 Article 1, wherein the fibrillated polymer membrane is a foamed polytetrafluoroethylene membrane. Claim 5 Article 1, wherein the affinity ligand is selected from protein A, protein G, protein L, human Fc receptor protein, antibody, polysaccharide, and combinations thereof. Claim 6 Article 1, 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, wherein the first and second nominal particle sizes are different. Claim 7 Article 1, wherein the inorganic particles having a spherical shape comprise a blend of 10-micron spherical particles and 20-micron spherical particles, and the blend is 10:90 to 90:

10. Claim 8 Article 1, wherein the inorganic particles having a spherical shape comprise a blend of 5-micron spherical particles and 10-micron spherical particles, and the blend is 10:90 to 90:

10. Claim 9 Article 1, wherein the inorganic particles having a spherical shape comprise a blend of 5-micron spherical particles and 20-micron spherical particles, and the blend is 10:90 to 90:

10. Claim 10 An article according to claim 1 having a cycle durability of at least 100 cycles at an operating pressure of less than 0.3 MPa. Claim 11 Article 1, wherein the fibrillated polymer film has a wound configuration. Claim 12 Article 1, wherein the fibrillated polymer film has a laminated structure. Claim 13 Article 1, wherein the fibrillated polymer film has a wound configuration, a laminated configuration, and a combination thereof. Claim 14 An article according to claim 1, wherein the internal intermediate material is positioned perimeter-wise on the outer surface of the core, and the fibrillated polymer film is positioned perimeter-wise around the internal intermediate material. Claim 15 Article 14, wherein the external intermediate material is positioned perimeter-wise on the fibrillated polymer membrane. Claim 16 Article 15, wherein the internal intermediate material and the external intermediate material are selected from porous fluoropolymer film, porous non-fluoropolymer film, porous nonwoven fabric and porous fabric. Claim 17 Article 16, wherein at least one of the internal intermediate material and the external intermediate material is a polypropylene nonwoven. Claim 18 Article 1, which is used to separate target molecules from a fluid stream. Claim 19 A manifold comprising at least two affinity chromatography devices of claim 1 arranged in a parallel configuration Claim 20 In paragraph 19, the above manifold is a manifold that is embedded within a housing. Claim 21 An apparatus comprising a first manifold and a second manifold in a parallel configuration, wherein each of the first manifold and the second manifold comprises at least two affinity chromatography apparatuses of claim 1. Claim 22 A device according to claim 21, wherein the first manifold and the second manifold are embedded within a housing. Claim 23 Article 1, wherein the affinity ligand is a metalaffinity ligand that reversibly binds to a His-Tagged protein. Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete Claim 44 delete Claim 45 delete Claim 46 delete Claim 47 delete Claim 48 delete Claim 49 delete Claim 50 delete Claim 51 delete Claim 52 delete Claim 53 delete Claim 54 delete Claim 55 delete Claim 56 delete Claim 57 delete Claim 58 delete Claim 59 delete Claim 60 delete Claim 61 delete Claim 62 delete Claim 63 delete Claim 64 delete Claim 65 delete Claim 66 delete Claim 67 delete Claim 68 delete Claim 69 delete Claim 70 delete Claim 71 delete Claim 72 delete Claim 73 delete

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