Method for coupling ligands to composite materials

Abstract

To provide methods for coupling a ligand to a composite material useful as a chromatographic separation medium.SOLUTION: Provided is a method for coupling a ligand to a functionalized composite material, comprising the steps of: a. providing a functionalized composite material, the functionalized composite material being arranged in a coplanar stack of coextensive sheets, a tubular configuration, or a spiral wound configuration, comprising: i. a support member comprising a plurality of pores extending through the support member; and ii. a macroporous cross-linked gel, the macroporous cross-linked gel comprising a polymer formed from a reaction of one or more polymerizable monomers with one or more cross-linkers, comprising pendant reactive functional groups, being located in the pores of the support member, and having macropores smaller than the pores of the support member; and b. flowing at a first flow rate a first solution substantially through or substantially across the functionalized composite material, the first solution comprising a plurality of first ligands, such that a plurality of covalent bonds forms between the reactive functional groups and the first ligands.SELECTED DRAWING: None

Description

[Background technology]

[0001] Membrane-based water treatment methods were first introduced in the 1970s. Since then, membrane-based separation technologies have been utilized in many other industries. In the pharmaceutical and biotechnology industries, the use of preparative chromatography, direct flow filtration (DFF), and tangential flow filtration (TFF), including microfiltration, ultrafiltration, nanofiltration, and diafiltration, are established methods for separating dissolved molecules or suspended particles. Ultrafiltration (UF) and microfiltration (MF) membranes have become essential for separation and purification in the production of biomolecules. Biomolecular production, regardless of its scale, generally employs one or more steps that utilize filtration. The appeal of these membrane separations lies in several features, including, for example, high separation efficiency and simplicity, requiring only the application of a pressure difference between the feed flow and the permeate. This simple, reliable one-step filtration of a sample into two fractions makes membrane separation a valuable approach to separation and purification.

[0002] Ligands conjugated to fluid-accessible surfaces of composite materials such as membranes are useful in separation and purification methods. However, chemical modification of membranes is more difficult than that of resins. Resins can be easily suspended in solution and therefore can be modified in large reactors where stirring the suspension facilitates the diffusion of reagents into the resin. Membranes are more difficult to modify because they must be supported during the modification process to avoid damage to the membrane structure. This can be achieved in roll-to-roll methods where the membrane physically moves through a trough of the reaction solution when the reaction rate is very fast. Membrane modification in slower reaction chemistry, such as coupling of protein A ligands with activated membranes, requires longer reaction times. Longer reaction times hinder roll-to-roll membrane modification methods, which require extremely slow membrane movement and therefore extremely long processing times.

[0003] A composite material modification step is required in which the reaction solution flows through the support assembly of the composite material over a long period of time. Increasing the ligand coupling to the composite material improves the binding capacity of the affinity medium. The conjugation method should utilize a reaction that is fast, efficient, and easy to control in order to couple the ligand to the composite material.

Summary of the Invention

Means for Solving the Problems

[0004] In one aspect, the present invention is a method for coupling a ligand to a functionalized composite material, wherein the functionalized composite material is arranged in a stack, a tubular configuration, or a spiral winding configuration on the same plane of a sheet having the same spread, comprising: a. Providing a functionalized composite material, comprising: i. A support member comprising a plurality of pores extending therethrough, and ii. A macroporous crosslinked gel comprising a polymer formed from the reaction of one or more polymerizable monomers and one or more crosslinking agents, the macroporous crosslinked gel comprising a plurality of pendant reactive functional groups, the macroporous crosslinked gel being located in the pores of the support member, the macropores of the macroporous crosslinked gel being smaller than the pores of the support member. Including the step of: b. Flowing a first solution at a first flow rate substantially through or substantially across the functionalized composite material, the first solution comprising a plurality of first ligands, whereby a plurality of covalent bonds are formed between the reactive functional groups and the first ligands.

Brief Description of the Drawings

[0005] [Figure 1A] Schematic diagram of an exemplary composite material laminated between layers of an interleaved layer (e.g., a screen) through which fluid flows (tangential flow) substantially across the layer of the functionalized composite material. [Figure 1B]This is a schematic diagram of an exemplary composite material, laminated between layers of interleaf material (e.g., a screen) through which a fluid flows substantially (direct flow) through a layer of functionalized composite material. [Figure 2] This figure shows an exemplary composite material having an interleaf layer with a spiral winding configuration. [Figure 3] This figure shows the dynamic IgG binding capacity of a protein A affinity ligand membrane conjugated using the flow-through method at a flow rate of 10 membrane volumes / min, compared to a membrane conjugated using the batch method. [Figure 4] This illustration shows a composite material layer (i.e., membrane), an interleaf layer (i.e., screen), and a flow divider assembled in a chromatography column. This pattern, containing 10 membranes, was repeated nine more times until 100 membranes were assembled. Then, one additional flow divider was added. [Figure 5A] This figure shows the dynamic IgG binding capacity as a function of membrane position within the stack. [Figure 5B] This figure shows the membrane bundle as a function of the membrane position within the stack. [Figure 6] This illustration shows different locations where the circular portions of the composite material are extracted from the rectangular film sheet after tangential flow coupling with protein A ligands on spiral-shaped winding rolls arranged alternately with the screen. [Modes for carrying out the invention]

[0006] overview The capacity of an affinity medium largely depends on the amount of affinity ligands that can be conjugated to the fluid-accessible surface of a medium, such as a composite material. Conjugation methods that increase ligand coupling to a composite material increase the binding capacity. In some embodiments, these methods utilize a fast, efficient, and easily controllable reaction to functionalize the composite material. In some embodiments, the composite material is an adsorbent macroporous chromatographic membrane. In some embodiments, methods for affinity ligand conjugation involving a direct flow of ligand solution through the membrane (i.e., dead-end flow) or across the membrane (i.e., tangential flow) produce affinity membranes with improved dynamic binding capacity compared to batch or static conjugation methods.

[0007] Chromatographic membranes utilize a high-speed convection mass transport mechanism to facilitate rapid purification or separation operations. However, to maximize the productivity of these operations, the membrane's binding capacity to the target compound must be maximized. In some embodiments, the present invention describes a flow-through or dead-end flow method for conjugating ligands to a membrane containing pendant-reactive functional groups under appropriate conditions of flow rate, buffer pH and concentration, affinity ligand concentration, and exposure time. In some embodiments, the present invention describes a cross-flow or tangential flow method for conjugating ligands to a membrane containing pendant-reactive functional groups under appropriate conditions of flow rate, buffer pH and concentration, affinity ligand concentration, and exposure time. In some embodiments, the method produces conjugated affinity chromatographic membranes with a greater protein binding capacity than that achieved by using a batch non-flow conjugation method with similar buffer conditions and affinity ligands.

[0008] Flow-through and cross-flow conjugation methods can be implemented using instruments that include in-line measurement tools, which consistently provide higher membrane binding capacity, enable real-time observation of reaction progress, and thereby allow for optimization of the reaction process. Higher membrane binding capacity, coupled with rapid binding kinetics, enables rapid and highly productive chromatographic purification operations.

[0009] definition For convenience, prior to any further description of the present invention, the specific terms used herein, in the examples and in the appended claims are set forth herein. These definitions should be read in light of the remainder of this disclosure and understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0010] Various terms are used in describing this invention. Standard technical terms are widely used in filtration, fluid delivery, and general fluid processing technologies.

[0011] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the article. For example, "an element" means one or more elements.

[0012] The terms "comprise" and "comprising" are used in a comprehensive, open sense, meaning that additional elements may be included.

[0013] The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0014] The term "affinity chromatography" refers to a separation method based on specific binding interactions between immobilized ligands and their binding partners. Examples of specific binding interactions include, but are not limited to, antibody / antigen, enzyme / substrate, and enzyme / inhibitor interactions.

[0015] The term "affinity medium" refers to a material containing multiple immobilized ligands. For example, a composite material containing covalently bonded ligands.

[0016] The term "polymer" refers to a large molecule formed by the bonding of repeating units (monomers). The term polymer also encompasses copolymers.

[0017] The term "copolymer" refers to a polymer of at least two different monomers. A copolymer may consist of a crosslinking agent and monomers, provided that the crosslinking agent is a bifunctional monomer.

[0018] The term "functionalized composite material" refers to a macroporous cross-linked gel containing multiple pendant-reactive functional groups located in the pores of a support member.

[0019] The term "pendant-reactive functional group" refers to a functional group that forms one or more covalent bonds with a ligand when the ligand solution is brought into contact with the functional group. Examples of pendant-reactive functional groups that form covalent bonds with ligands containing amine groups include, but are not limited to, epoxides, aldehydes, carboxylic acids, reactive halogens, reactive esters, isocyanates, isothiocyanates, sulfonyl halides, carboniimides, acyl azides, fluorobenzenes, carbonates, N-hydroxysuccinimide esters, imide esters, and fluorophenyl esters. Examples of pendant-reactive functional groups that form covalent bonds with ligands containing thiol groups include, but are not limited to, epoxides, thiols, disulfides, carbon-carbon double bonds, carbon-carbon triple bonds, maleimides, haloacetyls, pyridyl disulfides, thiosulfates, and reactive halogens.

[0020] The term "ligand" refers to a molecule that binds to a specific binding partner. For example, proteins, antibodies, hormones, or drugs bind to specific receptors.

[0021] As used herein, the terms “Protein A” or “PrA” refer to a bacterial protein, protein A derivative, or recombinant protein A derived from Staphylococcus aureus that has the ability to bind with high affinity to mammalian antibodies against class immunoglobulin G (IgG). For example, protein A can be recovered from their natural sources (e.g., Staphylococcus aureus). Protein A can be produced synthetically (e.g., by peptide synthesis or recombinant technology), and its fragments and variants retain the ability to bind to proteins having CH2 / CH3 regions, such as the Fc region. Protein A can be commercially purchased, for example, from Repligen, Pharmacia, EMD Millipore, and Fermatech. The gene for protein A has been cloned and expressed in Escherichia coli, enabling the production of large quantities of recombinant protein A and protein A derivatives.

[0022] In relation to coupling methods, the term "washing solution" refers to a solution that removes the coupling reaction products; that is, a solution that removes any excess polymerizable monomers and any excess ligands.

[0023] In coupling methods, the term "quenched solution" is used to mean a solution containing a reactive compound that covalently bonds with any residual pendant reactive functional group to form a non-reactive group. In other words, the reactive compound converts any residual pendant reactive functional group into a non-reactive group.

[0024] The term "non-reactive group" refers to a group that does not form a covalent bond under the conditions of further coupling reactions and separation methods. For example, exposure of a non-reactive group to a fluid containing a mixture of substances does not result in the formation of a covalent bond between the substances and the non-reactive group.

[0025] The term "buffer" refers to a solution that resists pH changes due to the action of its acid-base conjugate components. Various buffers that can be used in the methods described herein are listed under Buffers. D., ed., A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, Calbiochem Corporation (1975). Different buffers maintain different pH ranges; for example, phosphate buffer is typically used at pH between 6.0 and 8.0, but borate buffer can be used at higher pH levels, and carbonate buffer at lower pH levels. Those skilled in the art will be able to quickly identify the buffer best suited for use depending on the pH to be maintained. Non-limiting examples of buffers that can be used in the methods according to the present invention include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, carbonate, borate, and ammonium buffers, as well as combinations thereof.

[0026] The term "crossflow" in relation to fluid flow and filtration is used to mean a fluid flow or filtration configuration in which the flowing fluid is directed along the surface of a composite material (e.g., a filter material), and a portion of the fluid passing through such a composite material has a "crosswise" velocity component, i.e., a velocity component perpendicular to the direction of the fluid flowing along the surface of such a composite material.

[0027] The terms "tangential flow" or "tangential filtration" are used to mean a fluid flow or filtration method in which the flowing fluid is directed substantially parallel (i.e., tangentially) to the surface of a composite material (e.g., a filter material), and a portion of the fluid passes through such a composite material to provide a permeate. The terms "tangential filtration" and "cross-flow filtration" are often used interchangeably in the art.

[0028] The term "dead end" in relation to fluid flow and filtration is used to mean a fluid flow or filtration configuration through which a flowing fluid is directed, passing through a composite material (e.g., a filter material), and a portion of the fluid passing through such a composite material has a velocity component that is in the direction of the fluid flowing through such a composite material, i.e., parallel to it.

[0029] The terms “direct flow” or “direct filtration” are used to mean a fluid flow or filtration method in which a flowing fluid is directed substantially (i.e., directly) through the surface of a composite material (e.g., a filter material), and the majority of the fluid passes through such a composite material to provide a filtrate. The terms “direct filtration” and “dead-end filtration” are often used interchangeably in the art.

[0030] The term “permeate” is used to mean a portion of the fluid that passes through the filter material and is discharged through a first outlet port of a first filtration device operably connected to such filter material. The term “decantate” is used to mean a portion of the fluid that flows along the surface of the filter material but does not pass through such filter material and is discharged through a second outlet port of a filtration device operably connected to such filter material.

[0031] Cross-flow filtration and tangential filtration are well-known filtration methods. For example, it may be necessary to refer to U.S. Patents No. 5,681,464, No. 6,461,513, No. 6,331,253, No. 6,475,071, No. 5,783,085, and No. 4,790,942, the disclosures of which are incorporated herein by reference. It may also be necessary to refer to "Filter and Filtration Handbook," 4th edition, T. Christopher Dickenson, Elsevier Advanced Technology, 1997, the disclosures of which are incorporated herein by reference.

[0032] As used herein, the “binding-elution mode” refers to an operational approach to chromatography in which buffer conditions are established so that both the target protein and undesirable contaminants bind to the chromatographic support or composite material. Fractionation of the target protein from other components is subsequently achieved by changing the conditions so that the target protein and contaminants are eluted separately. In certain embodiments, the membranes described herein may be used in a “binding-elution mode” characterized by high dynamic binding capacity with high conductivity, high volume throughput, and selectivity. In certain embodiments, the amount of target protein in the eluent is reduced by about 50% to about 99%. In certain embodiments, the eluent is reduced by about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% in aggregates of the target protein.

[0033] As used herein, the term “flow-through mode” refers to an operational approach to chromatography in which buffer conditions are established so that intact target proteins flow through the membrane upon application, while selectively retaining contaminants. In certain embodiments, the membranes described herein can be used in “flow-through mode” in a post-purification step of protein A to remove important contaminants such as DNA, host cell proteins (HCPs), leached protein A, undesirable aggregates, and viruses in a single step.

[0034] The term "average pore size" for a macroporous crosslinked gel can be understood by those skilled in the art as being determined by any preferred method. For example, the average pore size can be estimated by environmentally controlled scanning electron microscopy (ESEM) imaging of the surface. ESEM can be a very simple and useful technique for characterizing microfiltration membranes. Clear and concise images of the membrane can be obtained with respect to the upper layer, cross-section, and lower layer, and the porosity and pore size distribution can be estimated from the images.

[0035] The "volume porosity" of a support member can be determined by a simple calculation. For example, for a polypropylene support member, the external dimensions of the support member are measured and the aggregate volume is calculated [for example, for a flat circular disk: V = πr 2 [h, the volume of the support member if it is solid or non-porous]. The mass of the support member is then determined. Since the density of polypropylene is known or can be determined from Brandrup et al., Polymer Handbook, Chapter VII, Wiley and Sons, New York, 1999, the volumetric porosity is calculated as in the following example.

[0036] Volume porosity = {(Volume of support material in the case of a solid) - [(Mass of support material) / (Density of polypropylene)]} / (Volume of support material in the case of a solid).

[0037] In this calculation, the void volume of the support member is = (volume of the external dimensions of the support member) - [(mass of the support member) / (density of polypropylene)]. For example, the density of polypropylene = 0.91 g / cm³ 3 That is the case.

[0038] The volume porosity ε of the composite material is a value experimentally determined for each composite material. It is calculated in terms of mass. The macroporous crosslinked gel is incorporated into the void volume of the support member. The mass of the incorporated gel is measured after drying to a certain weight. The partial specific volume of the polymer is known or can be determined from Brandrup et al., Polymer Handbook, Chapter VII, Wiley and Sons, New York, 1999. The maximum volume that the gel can occupy is the void volume of the support member (calculated as above). The volume porosity of the gel is then calculated.

[0039] ε = {(Void volume of the support member) - [(Mass of the gel) × (Partial specific volume of the gel polymer)]} / (Void volume of the support member)

[0040] In some embodiments, the ligand is protein A (PrA). PrA capture chromatography is a crucial step in the downstream purification of monoclonal antibodies (mAbs) for biological therapeutics. The PrA ligand selectively binds to the Fc and / or Fab binding domains on the mAb, while simultaneously allowing most impurities (host cell proteins, DNA, residual cell culture medium) to pass through the resin. Typically, the PrA medium is washed after the loading step to remove further impurities, and then the captured mAb is eluted at a low pH. The mAb during elution is significantly purer and also concentrated against the clarified cell culture. However, affinity media containing PrA, for example, are very expensive and must be used for many batches over several years to reduce the cost per batch. Ideally, the affinity media can be used for its maximum number of capture chromatography cycles (approximately 200) for the purification of a single batch of the target substance, e.g., an mAb, significantly reducing the volume of PrA medium required to process a single batch. The PrA medium can then be discarded after the purification of a single batch of mAbs, reducing the costs associated with resin storage. Currently, most PrA media used for the downstream purification of mAbs for biological therapeutics are in resin form. The slow mass transfer of mAbs into porous resin structures requires long loading times with residence times ranging from 2 to 10 minutes. Reducing the residence time during the loading step significantly decreases the dynamic binding capacity of the PrA resin, which is defined as the mass of mAbs loaded onto the chromatographic medium divided by the volume of the chromatographic medium. Longer loading times prevent the resin from being circulated several more times (2-4 times) per batch without extending the PrA capture chromatography step to several days.

[0041] PrA membranes can be loaded with much shorter residence times (e.g., 0.1–1 minute), thus providing an opportunity to rapidly cycle the capture chromatography step. In some embodiments, rapid cycling of the PrA membrane allows for the purification of a much larger amount of mAbs in the same amount of time for the same volume of PrA resin. Thus, over a given period, rapid cycling of a small amount of PrA membrane can capture the same amount of mAbs as a much larger amount of resin that is cycled fewer times. In some embodiments, the PrA membrane offers the possibility of using the entire lifespan of the PrA membrane to process a single batch of mAbs, significantly reducing the initial cost of establishing a downstream mAb purification method and eliminating the costs associated with resin storage.

[0042] In some embodiments, protein A is an affinity ligand. In some embodiments, protein A is a protein, peptide, or recombinant protein comprising a ligand that binds to a monoclonal antibody (e.g., an IgG antibody) and a moiety that can form a covalent bond with a pendant-reactive functional group (e.g., a Cys thiol or a Lys amine). In some embodiments, protein A is a protein, peptide, or recombinant protein comprising a ligand that binds to the Fc domain of an antibody and a moiety that can form a covalent bond with a pendant-reactive functional group. In some embodiments, protein A is a protein, peptide, or recombinant protein comprising a ligand that binds to the Fab domain of an antibody and a moiety that can form a covalent bond with a pendant-reactive functional group. In some embodiments, protein A can form multiple covalent bonds with multiple pendant-reactive functional groups. In some embodiments, protein A forms multiple covalent bonds in a functionalized composite material.

[0043] In some embodiments, protein A comprises multiple domains. In some embodiments, protein A comprises one, two, three, four, five, six, seven or more domains. In some embodiments, the protein A domains are identical to one another. In some embodiments, the protein A domains are distinct from one another. In some embodiments, protein A is resistant to degradation.

[0044] In some embodiments, protein A is immobilized on a solid support material. In some embodiments, protein A is covalently bonded to a composite material. In some embodiments, protein A refers to an affinity chromatography resin or column containing a chromatography solid support matrix to which protein A is covalently bonded.

[0045] Chemical modification of composite materials is more difficult than that of resins. For example, roll-to-roll modification methods for films with slow reaction chemistry require extremely slow movement of the film through the reaction solution and extremely long processing times that are incompatible with large-scale modification.

[0046] In some embodiments, composite materials such as membranes can be modified using the ligand coupling methods disclosed herein.

[0047] In one aspect, the present invention is a method for coupling ligands to a functionalized composite material, a. A step of providing a functionalized composite material, i. A support member comprising a plurality of pores extending through it, and ii. A macroporous crosslinked gel, wherein the macroporous crosslinked gel comprises a polymer formed from the reaction of one or more polymerizable monomers and one or more crosslinking agents, the macroporous crosslinked gel comprises a plurality of pendant-reactive functional groups, the macroporous crosslinked gel is located in the pores of the support member, and the macropores of the macroporous crosslinked gel are smaller than the pores of the support member. Steps including, b. A step of flowing a first solution at a first flow rate substantially through or substantially across a functionalized composite material, wherein the first solution comprises a plurality of first ligands, thereby forming a plurality of covalent bonds between the reactive functional group and the first ligands. Steps, including, The present invention relates to a method for arranging functionalized composite materials in a stack of sheets having the same extent on the same plane, in a tubular configuration, or in a spiral winding configuration.

[0048] Exemplary functionalized composite materials Gel composition In some embodiments, the crosslinked gel may be formed by an in-situ reaction between one or more polymerizable monomers and one or more crosslinking agents. In certain embodiments, the gel may be formed by a reaction between one or more crosslinkable polymers and one or more crosslinking agents.

[0049] In some embodiments, the crosslinked polymer is macroporous. Porosity within the polymer can be promoted during polymerization by the degree of crosslinking, solvent exclusion of polymer chains during polymer network formation, or several combinations of both. In some embodiments, high concentrations of crosslinking agent produce a macroporous crosslinked gel. In some embodiments, porosity is influenced by varying the degree of polymer-(diluent + monomer) interaction, the amount of crosslinking agent, the amount of diluent, the initiator concentration, and the polymerization temperature.

[0050] The degree of crosslinking in a polymer can be adjusted by adjusting the monomer ratio. The chain length of the polymer in the polymer network, and therefore the degree of crosslinking, can also be controlled by using specific monomers that impart specific physicochemical properties to the final polymer and film. These "adjusted" monomers can influence the interaction between the polymer chains and the solvent system. Furthermore, the hydrophilic / hydrophobic properties of these monomers can affect the final aqueous swelling properties of the resulting gel and the hydrophilic / hydrophobic surface properties of the polymer network.

[0051] To minimize the formation of non-porous composite materials, the solvent system and monomers are selected to ensure that there is sufficient driving force to remove the growing polymer chains from the solution at specific points, thereby forming macropores. Specifically, a mixture of solvent and non-solvent is adjusted to provide a suitable reaction system that can initially dissolve all of the reactants but acts as a poor solvent for the crosslinked polymer chains as they grow above a certain molecular weight. A solvent system with too high a proportion of the poor solvent (for the polymer chains) can lead to rapid precipitation of the growing polymer chains, reducing porosity. The size of the macropores generally depends on the properties and concentration of the crosslinking agent, the properties of one or more solvents on which the gel is formed, the amount of any polymerization initiator or catalyst, and, if present, the properties and concentration of the pologen. In certain embodiments, the composite material may have a narrow pore size distribution.

[0052] In some embodiments, the macroporous crosslinked gel is formed as a result of phase separation during free radical crosslinking polymerization of polymerizable monomers in the presence of an inert diluent. In some embodiments, the reaction system includes a polymer network, a soluble polymer, and low molecular weight compounds (monomers and diluents) for forming the macroporous crosslinked polymer. In some embodiments, the macroporous crosslinked gel swells only slightly in the solvent.

[0053] Generally, many highly porous and non-rigid polymer materials are relatively weak and cannot withstand the pressures generated during typical membrane separation processes (e.g., liquid chromatography). Therefore, in certain embodiments, to produce mechanically suitable membranes, composite materials comprising both a porous substrate (such as a woven substrate made of chemically inert polypropylene) and a porous crosslinked polymer are produced by directly synthesizing the polymer within the substrate pores.

[0054] In certain embodiments, when examined using an environmentally controlled scanning electron microscope (ESEM), the composite material showed a well-connected gel network embedded within the substrate fibers.

[0055] In some embodiments, the formation of high polymer density regions, also called polymer chain bundling or lateral aggregation, leaves macropores between the high polymer density regions. In some embodiments, the macroporous crosslinked gel has a non-uniform appearance.

[0056] In certain embodiments, the composite materials used as films in the present invention are described in U.S. Patent Nos. 7,316,919, 8,206,958, 8,187,880, 8,211,682, 8,652,849, 8,192,971, 8,206,982, 8,367,809, 8,383,782, 8,133,840, 9,962,691 and 10,357,766, as well as U.S. Patent Applications Nos. 14 / 190,650, 16 / 055,786 and 16 / 516,500, all of which are incorporated herein by reference.

[0057] In certain embodiments, the present invention provides a functionalized composite material, i. A support member comprising a plurality of pores extending through it, and ii. A method disclosed herein for a macroporous crosslinked gel, wherein the macroporous crosslinked gel comprises a polymer formed from the reaction of one or more polymerizable monomers and one or more crosslinking agents, the macroporous crosslinked gel comprises a plurality of pendant-reactive functional groups, the macroporous crosslinked gel is located in the pores of the support member, and the macropores of the macroporous crosslinked gel are smaller than the pores of the support member.

[0058] In certain embodiments, the present invention relates to any one of the methods disclosed herein, wherein a macroporous crosslinked gel of a composite material has macropores with an average diameter of about 5 nm to about 10,000 nm. In certain embodiments, the macroporous crosslinked gel has macropores with an average diameter between about 10 nm and about 3,000 nm. In certain embodiments, the macroporous crosslinked gel has macropores with an average diameter between about 25 nm and about 1,500 nm. In certain embodiments, the macroporous crosslinked gel has macropores with an average diameter between about 50 nm and about 1,000 nm. In certain embodiments, the macroporous crosslinked gel has macropores with an average diameter of about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm, or about 700 nm.

[0059] In certain embodiments, the diameter of the macropores is estimated by one of the techniques described herein. In certain embodiments, the diameter of the macropores is calculated by capillary flow porometry. Since only the maximum porosity is an intrinsic property of a given material, it is appropriate to define macroporosity with respect to the maximum porosity.

[0060] In certain embodiments, the present invention relates to any one of the methods disclosed herein, wherein the macroporous crosslinked gel of the composite material is a neutral hydrogel, a charged hydrogel, a polymer electrolyte gel, a hydrophobic gel, a neutral gel, or a gel containing functional groups. In certain embodiments, the present invention relates to any one of the methods disclosed herein in which a macroporous crosslinked gel of a composite material is a neutral or charged hydrogel, the neutral or charged hydrogel being selected from the group consisting of crosslinked poly(vinyl alcohol), poly(acrylamide), poly(isopropylacrylamide), poly(vinylpyrrolidone), poly(hydroxymethyl acrylate), poly(ethylene oxide), copolymers of acrylic acid or methacrylic acid with acrylamide, isopropylacrylamide or vinylpyrrolidone, copolymers of acrylamide-2-methyl-1-propanesulfonic acid with acrylamide, isopropylacrylamide or vinylpyrrolidone, copolymers of (3-acrylamide-propyl)trimethylammonium chloride with acrylamide, isopropylacrylamide or N-vinylpyrrolidone, and copolymers of diallyldimethylammonium chloride with acrylamide, isopropylacrylamide or vinylpyrrolidone. In certain embodiments, the present invention relates to any one of the methods disclosed herein in which the macroporous crosslinked gel of a composite material is a polymer electrolyte gel, the polymer electrolyte gel being selected from the group consisting of crosslinked poly(acrylamide-2-methyl-1-propanesulfonic acid) and its salts, poly(acrylic acid) and its salts, poly(methacrylic acid) and its salts, poly(styrenesulfonic acid) and its salts, poly(vinylsulfonic acid) and its salts, poly(alginic acid) and its salts, poly[(3-acrylamidepropyl)trimethylammonium] salt, poly(diallyldimethylammonium) salt, poly(4-vinyl-N-methylpyridinium) salt, poly(vinylbenzyl-N-trimethylammonium) salt, and poly(ethyleneimine) and its salts.In certain embodiments, the present invention relates to any one of the methods disclosed herein, wherein the macroporous crosslinked gel of the composite material is a hydrophobic gel, the hydrophobic gel being selected from the group consisting of ethyl acrylate, n-butyl acrylate, propyl acrylate, octyl acrylate, dodecyl acrylate, octadecyl acrylamide, stearyl acrylate, and styrene crosslinked polymers or copolymers. In certain embodiments, the present invention relates to any one of the methods disclosed herein, wherein the macroporous crosslinked gel of the composite material is a neutral gel, the neutral gel being selected from the group consisting of acrylamide, N,N-dimethylacrylamide, N-methacryloylacrylamide, N-methyl-N-vinylacetamide, and N-vinylpyrrolidone crosslinked polymers or copolymers.

[0061] In certain embodiments, a crosslinked composite material (e.g., a film) is further grafted with chemical functional groups or molecular species to provide a functionalized composite material. In certain embodiments, a crosslinked polymer may be functionalized by post-polymerization modification to form a functionalized composite material. In certain embodiments, a functionalized composite material containing a functionalized crosslinked polymer may be coupled to ligands by post-polymerization modification. In this two-step method, excess pendant-reactive functional groups, e.g., excess thiol or alkene groups generated during thiol-alkene polymerization, are modified in a separate grafting step. By controlling the supply ratio of monomers and crosslinkers, the final polymer may have excess pendant-reactive functional groups. The pendant-reactive functional groups of the functionalized composite material can then be used in coupling reactions, such as click reactions, to further modify the chemical properties or functionalities of the final polymer. In certain embodiments, the crosslinked polymer in the composite material contains residual reactive groups, called pendant-reactive functional groups, such as thiols or unsaturated carbon-carbon bonds, which can be used to attach various ligands via coupling reactions. In certain embodiments, this approach is useful for creating polymer composite materials (e.g., membranes) containing various ligands useful for chromatography. For example, chromatographic separation of biomolecules (e.g., proteins). For example, this approach can be used to introduce ion-exchange functional groups (e.g., carboxylates, sulfonates, quaternary ammonium compounds, amines), hydrophobic interaction moieties (such as octyl groups using 1-octanthiol or 1-octene), and biomolecules for bioaffinity chromatography (such as cysteine-protein A for monoclonal antibody purification) into composite materials (e.g., membranes).

[0062] In some embodiments, the composite material exhibits high selectivity and high flow rate, low back pressure, is inexpensive, and enables long column life, short process time, and overall operational flexibility.

[0063] In certain embodiments, the present invention relates to any one of the aforementioned composite materials, wherein the composite material is a film.

[0064] In certain embodiments, the present invention relates to any one of the aforementioned composite materials, wherein the composite material has a water contact angle of about 50° to about 120°.

[0065] Porous support member In some embodiments, the support member has a void volume, and the void volume of the support member is substantially filled with a macroporous crosslinked gel. In some embodiments, the porous support member has a volume porosity of about 40% to about 90%. In some embodiments, the porous support member has a volume porosity of about 50% to about 80%. In some embodiments, the porous support member has a volume porosity of about 50%, about 60%, about 70%, or about 80%.

[0066] In certain embodiments, the porous support is flat.

[0067] In certain embodiments, the porous support is disc-shaped.

[0068] Many porous substrates or membranes can be used as support members. In some embodiments, the porous support member is made of a polymer material. In certain embodiments, the support member may be a polyolefin, which is available at low cost. In certain embodiments, the polyolefin may be poly(ethylene), poly(propylene), or poly(vinylidene difluoride). Examples include expanded polyolefin membranes prepared by thermally induced phase separation (TIPS) or non-solvent-induced phase separation. In certain embodiments, the support member may be made from a natural polymer such as cellulose or its derivatives. In certain embodiments, preferred supports include polyethersulfone membranes, poly(tetrafluoroethylene) membranes, nylon membranes, cellulose ester membranes, glass fibers, or filter paper. In some embodiments, the support member includes a polymer material selected from the group consisting of polysulfone, polyethersulfone, polyphenylene oxide, polycarbonate, polyester, cellulose, and cellulose derivatives.

[0069] In certain embodiments, the porous support is composed of a fibrous material, such as a woven or nonwoven fabric, or a polyolefin such as polypropylene. Such fibrous woven or nonwoven support members can have larger pore sizes than TIPS support members, sometimes up to approximately 75 μm. The larger pores of the support member allow for the formation of composite materials with larger macropores than macroporous gels. Non-polymer support members, such as ceramic-based supports, can also be used. Porous support members can take on a variety of shapes and sizes.

[0070] In some embodiments, the support member is in the form of a membrane.

[0071] In some embodiments, the support member has a thickness of approximately 10 to approximately 2000 μm, approximately 10 to approximately 1000 μm, or approximately 10 to approximately 500 μm. In some embodiments, the support member has a thickness of approximately 30 μm to approximately 300 μm. In some embodiments, the thickness of the support member is approximately 30 μm, approximately 50 μm, approximately 100 μm, approximately 150 μm, approximately 200 μm, approximately 250 μm, or approximately 300 μm.

[0072] In some embodiments, the pores of the support member have an average pore diameter of about 0.1 μm to about 50 μm. In some embodiments, the pores of the support member have an average pore diameter of about 0.1 μm to about 25 μm. In some embodiments, the pores of the support member have an average pore diameter of about 0.5 μm to about 15 μm. In some embodiments, the pores of the support member have an average pore diameter of about 0.5 μm, about 1 μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, or about 15 μm.

[0073] In other embodiments, multiple porous support units can be combined, for example, by lamination. In one embodiment, a stack of porous support films, for example, 2 to 10 films, can be assembled before a gel is formed in the voids of the porous support. In another embodiment, a single support member unit is used to form a composite material film, which is then laminated before use.

[0074] Relationship between gel and support member The gel may be fixed within the support member. The term “fixed” is intended to mean that the gel is retained within the pores of the support member, but this term is not necessarily limited to meaning that the gel is chemically bonded to the pores of the support member. The gel can be retained by the physical constraints imposed on it, by interlocking and entangling with the structural elements of the support member, without actually being chemically grafted into the support member, although in some embodiments the gel may be grafted onto the surface of the pores of the support member.

[0075] In certain embodiments, the crosslinked gel is macroporous. In these examples, the macropores of the gel must be smaller than the pores of the support member, since the macropores are present in the gel occupying the pores of the support member. Therefore, the flow and separation characteristics of the composite material depend on the characteristics of the gel, but are largely independent of the characteristics of the porous support member, provided that the size of the pores present in the support member is larger than the size of the macropores of the gel. The porosity of the composite material can be controlled, when used, by filling the support member with a gel whose porosity is partially or completely defined by the properties and amounts of monomers or polymers, crosslinkers, reaction solvents, and pologens. The properties of the composite material are determined, if not completely, in part by the properties of the gel. The net result is that the present invention provides control over the macropore size, permeability, and surface area of ​​the composite material.

[0076] If present, the number of macropores in the composite material is not determined by the number of pores in the support material. The number of macropores in the composite material can be much greater than the number of pores in the support member, because the macropores are smaller than the pores in the support member. As described above, the influence of the pore size of the support material on the pore size of the macroporous gel is generally negligible. There are exceptions when there is a large difference in pore size and pore size distribution between the support member and the support member, and a macroporous gel with very small pore sizes and a narrow range of pore size distribution is required. In these cases, large variations in the pore size distribution of the support member are weakly reflected in the pore size distribution of the macroporous gel. In certain embodiments, a support member with a somewhat narrower pore size range may be used in these situations.

[0077] In certain embodiments, the present invention relates to one of the aforementioned composite materials, wherein the composite material is relatively nontoxic.

[0078] Preparation of composite materials In certain embodiments, the composite materials of the present invention may be prepared by a single-step method. In certain embodiments, these methods may use water or other environmentally friendly solvents as reaction solvents. In certain embodiments, the method may be rapid and therefore result in a simple and / or rapid manufacturing process. In certain embodiments, the preparation of the composite materials may be inexpensive.

[0079] In certain embodiments, the composite material may be prepared by mixing one or more monomers, one or more crosslinking agents, one or more initiators, and optionally one or more pologens in one or more suitable solvents. In certain embodiments, the resulting mixture may be homogeneous. In certain embodiments, the mixture may be heterogeneous. In certain embodiments, the mixture may then be introduced into a suitable porous support, where a gel-forming reaction may occur.

[0080] In certain embodiments, pologens may be added to the reactant mixture, and pologens can be broadly described as pore-forming additives. In certain embodiments, pologens may be selected from the group consisting of thermodynamically poor solvents and extractable polymers (e.g., poly(ethylene glycol)), surfactants, and salts.

[0081] In some embodiments, the gel-forming reaction must be initiated. In certain embodiments, the gel-forming reaction may be initiated by any known method, for example, by thermal activation or exposure to UV irradiation. In certain embodiments, the reaction may be initiated by UV irradiation in the presence of a photoinitiator. In certain embodiments, the photoinitiator may be selected from the group consisting of 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure 2959), 4,4'-azobis(4-cyanovaleric acid) (ACVA), 2,2-dimethoxy-2-phenylacetophenone (DMPA), benzophenone, benzoin and benzoin ethers, such as benzoin ethyl ether and benzoin methyl ether, dialkoxyacetophenone, hydroxyalkylphenone, and α-hydroxymethylbenzoin sulfonic acid esters. Thermal activation may require the addition of a thermal initiator. In certain embodiments, the thermal initiator may be selected from the group consisting of 1,1'-azobis(cyclohexanecarbonilate) (VAZO(R) catalyst 88), azobis(isobutyronitrile) (AIBN), potassium persulfate, ammonium persulfate, and benzoyl peroxide.

[0082] In certain embodiments, the gel-forming reaction may be initiated by UV irradiation. In certain embodiments, a photoinitiator may be added to the reactants of the gel-forming reaction, and the support member containing the mixture of monomer, crosslinker and photoinitiator may be exposed to UV rays with wavelengths of about 250 nm to about 400 nm for a period of several seconds to several hours. In certain embodiments, the support member containing the mixture of monomer, crosslinker and photoinitiator may be exposed to UV rays of about 350 nm for a period of several seconds to several hours. In certain embodiments, the support member containing the mixture of monomer, crosslinker and photoinitiator may be exposed to UV rays of about 350 nm for about 10 minutes. In certain embodiments, polymerization may be initiated using visible wavelength light. In certain embodiments, the support member must have a low absorbance at the wavelength used so that energy can be transferred through the support member.

[0083] In certain embodiments, the rate at which polymerization takes place can affect the size of the macropores obtained in the macroporous gel. In certain embodiments, as the concentration of the crosslinking agent in the gel rises to a sufficient level, the components of the gel begin to aggregate, creating regions of high polymer density and regions containing little or no polymer, the latter of which are referred to herein as "macropores." This mechanism is affected by the polymerization rate.

[0084] In certain embodiments, once the composite materials are prepared, they may be washed with a variety of solvents to remove any unreacted components and any polymers or oligomers that are not fixed within the support. In certain embodiments, suitable solvents for washing the composite materials include water, acidic (e.g., HCl) or basic (e.g., NaOH) aqueous solutions, salt aqueous solutions (e.g., NaCl), acetone, methanol, ethanol, propanol, and DMF.

[0085] Exemplary methods for coupling ligands to functionalized composite materials A method for coupling ligands to a functionalized composite material is provided herein by flowing a first solution containing ligands across or through the functionalized composite material.

[0086] In one aspect, the present invention is a method for coupling ligands to a functionalized composite material, a. A step of providing a functionalized composite material, i. A support member comprising a plurality of pores extending through it, and ii. A macroporous crosslinked gel, wherein the macroporous crosslinked gel comprises a polymer formed from the reaction of one or more polymerizable monomers and one or more crosslinking agents, the macroporous crosslinked gel comprises a plurality of pendant-reactive functional groups, the macroporous crosslinked gel is located in the pores of the support member, and the macropores of the macroporous crosslinked gel are smaller than the pores of the support member. Steps including, b. A step of flowing a first solution at a first flow rate substantially through or substantially across a composite material, wherein the first solution comprises a plurality of first ligands, thereby forming a plurality of covalent bonds between the reactive functional group and the first ligands, The present invention relates to a method for arranging functionalized composite materials in a stack of sheets having the same extent on the same plane, in a tubular configuration, or in a spiral winding configuration.

[0087] In certain embodiments, the present invention relates to providing one of the aforementioned functionalized composite materials.

[0088] In certain embodiments, the present invention relates to one of the aforementioned methods, wherein the first solution flows substantially across the functionalized composite material. In some embodiments, the fluid channel is tangential to the surface of the functionalized composite material (Figure 1A). In some embodiments, tangential flow provides a lower pressure drop and / or allows simultaneous coupling of a stack consisting of many layers of the composite material.

[0089] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein a first solution flows substantially through a functionalized composite material. In some embodiments, the fluid channel flows directly through the functionalized composite material (Figure 1B). In some embodiments, the direct flow increases the pressure drop as the number of layers of the composite material increases. In some embodiments, the pressure drop limits the number of layers in the stack. In some embodiments, one or more layers of interleaf act to distribute the flow. In some embodiments, the direct flow increases the mass transfer rate of ligands into the porous composite material structure.

[0090] In certain embodiments, the crosslinked macroporous gel is further grafted with chemical functional groups or molecular species to provide a functionalized composite material. In certain embodiments, the crosslinked polymer may be functionalized by post-polymerization modification to form a functionalized composite material. In this two-step method, excess pendant-reactive functional groups, for example, excess thiol or alkene groups generated during thiol-alkene polymerization, are modified in a separate grafting step. By controlling the supply ratio of monomers and crosslinkers, the final polymer can have excess pendant-reactive functional groups.

[0091] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the pendant reactive functional group is selected from the group consisting of aldehydes, amines, carbon-carbon double bonds, carbon-carbon triple bonds, epoxides, hydroxyls, thiols, anhydrides, azides, reactive halogens, acid chlorides, and mixtures thereof.

[0092] In some embodiments of the method, the pendant-reactive functional group is selected from the group consisting of carbon-carbon double bonds, carbon-carbon triple bonds, and thiols. In some embodiments, the pendant-reactive functional group is derived from a molecule containing a thiol functional group or a molecule containing an unsaturated carbon-carbon bond. In some embodiments, the pendant-reactive functional group is derived from a molecule containing a thiol functional group, and the molecule containing a thiol functional group is derived from 3-mercaptopropionic acid, 1-mercaptosuccinic acid, polypeptides containing cysteine ​​residues, proteins containing cysteine ​​residues, recombinant proteins containing cysteine ​​residues, bacterial immunoglobulin-binding proteins containing cysteine ​​residues, recombinant fusion proteins containing cysteine ​​residues, cysteamine, 1-thiohexitol, poly(ethylene glycol)2-mercaptoethyl ether acetate, poly(ethylene glycol)methyl ether thiol, 1-thioglycerol, 2-naphthol The following are selected from the group consisting of talentchiol, biphenyl-4-thiol, 3-amino-1,2,4-triazole-5-thiol, 5-(trifluoromethyl)pyridine-2-thiol, 1-[2-(dimethylamino)ethyl]-1H-tetrazole-5-thiol, 1-propanthol, 1-butanethiol, 1-pentanethiol, 1-hexanethiol, 1-octanthiol, 8-amino-1-octanthiol hydrochloride, 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro-1-octanthiol, 8-mercapto-1-octanol, and γ-Glu-Cys.

[0093] In some embodiments, the molecule containing a thiol functional group is selected from the group consisting of polypeptides containing cysteine ​​residues, proteins containing cysteine ​​residues, recombinant proteins containing cysteine ​​residues, bacterial immunoglobulin-binding proteins containing cysteine ​​residues, and recombinant fusion proteins containing cysteine ​​residues. In some embodiments, the molecule containing a thiol functional group is a protein containing cysteine ​​residues.

[0094] In some embodiments, the pendant-reactive functional group is derived from a molecule containing an unsaturated carbon-carbon bond, and the molecule containing an unsaturated carbon-carbon bond is selected from the group consisting of 1-octene, 1-hexine, 4-bromo-1-butene, allyldiphenylphosphine, allylamine, allyl alcohol, 3,4-dihydroxy-1-butene, 7-octen-1,2-diol, 3-allyloxy-1,2-propanediol, 3-butenoic acid, 3,4-dehydro-L-proline, vinyl laurate, 1-vinyl-2-pyrrolidinone, vinyl cinnamate, acylamide, or acrylate.

[0095] In some embodiments, the pendant-reactive functional group is selected from the group consisting of acid chlorides, acyl azides, aldehydes, amines, anhydrides, azides, carbonates, carbon-carbon double bonds, carbon-carbon triple bonds, carboniimides, carboxylic acids, disulfides, epoxides, fluorobenzenes, fluorophenyl esters, haloacetyls, hydroxyls, imide esters, isocyanates, isothiocyanates, maleimides, N-hydroxysuccinimide esters, pyridyl disulfide, reactive esters, reactive halogens, sulfonyl halogens, thiols, and thiosulfates. In some embodiments, the pendant-reactive functional group is selected from the group consisting of aldehydes, amines, epoxides, hydroxyls, anhydrides, azides, reactive halogens, and acid chlorides. In some embodiments, the pendant-reactive functional group is selected from the group consisting of aldehydes, amines, epoxides, and hydroxyls. In some embodiments, the pendant-reactive functional group is selected from the group consisting of epoxides, aldehydes, carboxylic acids, reactive halogens, reactive esters, isocyanates, isothiocyanates, sulfonyl halides, carbodiimides, acyl azides, fluorobenzenes, carbonates, N-hydroxysuccinimide esters, imide esters, and fluorophenyl esters. In some embodiments, the pendant-reactive functional group is selected from the group consisting of epoxides, aldehydes, carboxylic acids, reactive halogens, reactive esters, isocyanates, isothiocyanates, sulfonyl halides, carboniimides, acyl azides, fluorobenzenes, carbonates, N-hydroxysuccinimide esters, imide esters, and fluorophenyl esters, and reacts with the amine group. In some embodiments, the pendant-reactive functional group is selected from the group consisting of epoxides, thiols, disulfides, carbon-carbon double bonds, carbon-carbon triple bonds, maleimides, haloacetyls, pyridyl disulfides, thiosulfates, and reactive halogens.In some embodiments, the pendant reactive functional group is selected from the group consisting of epoxides, thiols, disulfides, carbon-carbon double bonds, carbon-carbon triple bonds, maleimides, haloacetyls, pyridyl disulfides, thiosulfates, and reactive halogens.

[0096] In some embodiments, one or more monomers containing a pendant-reactive functional group are selected from the group consisting of glycidyl methacrylate, acrylamide oxime, acrylic anhydride, azelaic anhydride, maleic anhydride, hydrazide, acryloyl chloride, 2-bromoethyl methacrylate, and vinyl methyl ketone.

[0097] In some embodiments, the pendant-reactive functional group is an aldehyde. In some embodiments, one or more monomers containing the pendant-reactive functional group are vinyl methyl ketones.

[0098] In some embodiments, the pendant-reactive functional group is an amine.

[0099] In some embodiments, the pendant-reactive functional group is an epoxide. In some embodiments, one or more monomers containing the pendant-reactive functional group are glycidyl methacrylates.

[0100] In some embodiments, the pendant-reactive functional group is hydroxyl.

[0101] In some embodiments, the first ligand comprises a first functional group. In some embodiments, the first ligand further comprises at least one graft-terminated group, the first functional group being selected from the group consisting of cationic, anionic, hydrophobic, hydrophilic, thiophilic, hydrogen bond donor, hydrogen bond acceptor, π-π bond donor, π-π bond acceptor, metal chelater, biological molecule, and biological ion. In some embodiments, the first functional group is selected from the group consisting of cationic, anionic, hydrophobic, hydrophilic, thiophilic, hydrogen bond donor, hydrogen bond acceptor, π-π bond donor, and π-π bond acceptor.

[0102] In some embodiments, individual functionalities are included by incorporating functional monomers. In some embodiments, the relative amounts of each functional group can be easily and quickly adjusted for optimal performance characteristics.

[0103] In some embodiments, the molecule comprises a first functional group, and the molecule is 2-(diethylamino)ethyl methacrylate, 2-aminoethyl methacrylate, 2-carboxyethyl acrylate, 2-(methylthio)ethyl methacrylate, acrylamide, N-acryloxysuccinimide, butyl acrylate or methacrylate, N,N-diethylacrylamide, N,N-dimethylacrylamide, 2-(N,N-dimethylamino)ethyl acrylate or methacrylate, N-[3-(N,N-dimethylamino)propyl]methacrylamide, N,N-dimethylacrylamide, ethyl acrylate or methacrylate, 2-ethylhexyl methacrylate, hydroxypropyl methacrylate, glycidyl acrylate or methacrylate, ethylene glycol phenyl ether methacrylate, methacrylamide, methacrylic anhydride, propyl acrylate or methacrylate. Selected from the group consisting of acrylate, N-isopropylacrylamide, styrene, 4-vinylpyridine, vinylsulfonic acid, N-vinyl-2-pyrrolidinenon (VP), acrylamide-2-methyl-1-propanesulfonic acid, styrenesulfonic acid, alginic acid, (3-acrylamidopropyl)trimethylammonium halide, diallyldimethylammonium halide, 4-vinyl-N-methylpyridinium halide, vinylbenzyl-N-trimethylammonium halide, methacryloxyethyltrimethylammonium halide, 3-sulfopropyl methacrylate, 2-(2-methoxy)ethyl acrylate or methacrylate, hydroxyethylacrylamide, N-(3-methoxypropylacrylamide), N-[tris(hydroxymethyl)methyl]acrylamide, N-phenylacrylamide, N-tert-butylacrylamide, or diacetoneacrylamide.

[0104] In some embodiments, the first functional group is a metal chelate functional group. In some embodiments, the first functional group includes a metal chelate functional group selected from the group consisting of octadentate, hexadentate, tetradentate, tridentate, and bidentate iminodicarboxylic acids, iminodiacetic acids, and salts of iminodiacetic acids.

[0105] In some embodiments, the metal chelate functional group complexes with multiple metal ions. In some embodiments, the metal chelate functional group is selected from the group consisting of iminodicarboxylic acids, iminodiacetic acid, and salts of iminodiacetic acid complexed with multiple metal ions selected from the group consisting of transition metal ions, lanthanide ions, poor metal ions, and alkaline earth metal ions. In some embodiments, the metal chelate functional group is selected from the group consisting of iminodicarboxylic acids, iminodiacetic acid, and salts of iminodiacetic acid complexed with multiple metal ions selected from the group consisting of nickel, zirconium, lanthanum, cerium, manganese, titanium, cobalt, iron, copper, zinc, silver, gallium, platinum, palladium, lead, mercury, cadmium, and gold. In some embodiments, the metal chelate functional group is iminodiacetic acid or a salt of iminodiacetic acid complexed with multiple metal ions, where the metal ions are nickel or zirconium.

[0106] In some embodiments, the first functional group is a biological molecule or biological ion. In some embodiments, the first functional group includes a biological molecule or biological ion functional group selected from the group consisting of albumin, lysozyme, virus, cell, human and animal gamma globulin, immunoglobulin of both human and animal origin, recombinant or naturally occurring proteins including synthetic or naturally occurring polypeptides, interleukin-2 and its receptor, enzyme, monoclonal antibody, antigen, lectin, bacterial immunoglobulin-binding protein, trypsin and its inhibitor, cytochrome C, myoglobulin, recombinant human interleukin, recombinant fusion protein, protein A, protein G, protein L, peptide H, nucleic acid-derived products, synthetic or naturally occurring DNA, and synthetic or naturally occurring RNA. In some embodiments, the first functional group comprises a biological molecule or biological ionic functional group selected from the group consisting of human and animal γ-globulins, immunoglobulins of both human and animal origin, recombinant or naturally occurring proteins including synthetic or naturally occurring polypeptides, monoclonal antibodies, bacterial immunoglobulin-binding proteins, recombinant fusion proteins, protein A, protein G, and protein L. In certain embodiments, the first functional group comprises a biological molecule or biological ionic functional group selected from the group consisting of polypeptides, proteins, recombinant proteins, bacterial immunoglobulin-binding proteins, recombinant fusion proteins, protein A, protein G, protein L, and peptide H.

[0107] In some embodiments, the first functional group comprises protein A. In some embodiments, the first functional group comprises protein A derivatives and recombinant protein A selected from the group consisting of polypeptides containing cysteine ​​residues, proteins containing cysteine ​​residues, recombinant proteins containing cysteine ​​residues, bacterial immunoglobulin-binding proteins containing cysteine ​​residues, and recombinant fusion proteins containing cysteine ​​residues. In some embodiments, the first functional group comprises protein A selected from the group consisting of proteins, peptides, or recombinant proteins that include a ligand that binds to a monoclonal antibody (e.g., an IgG antibody) and a moiety that can form a covalent bond with a pendant-reactive functional group. In some embodiments, the first functional group comprises protein A selected from the group consisting of proteins, peptides, or recombinant proteins that include a ligand that binds to the Fc domain of an antibody and a moiety that can form a covalent bond with a pendant-reactive functional group. In some embodiments, the first functional group comprises protein A selected from the group consisting of proteins, peptides, or recombinant proteins that include a ligand that binds to the Fab domain of an antibody and a moiety that can form a covalent bond with a pendant-reactive functional group.

[0108] In some embodiments, the first ligand comprises a first functional group and at least one graft-end group selected from the group consisting of aldehydes, amines, carbon-carbon double bonds, carbon-carbon triple bonds, epoxides, hydroxyls, thiols, and mixtures thereof. In some embodiments, at least one graft-end group is an aldehyde. In some embodiments, at least one graft-end group is an amine. In some embodiments, at least one graft-end group is a carbon-carbon double bond or a carbon-carbon triple bond. In some embodiments, at least one graft-end group is an epoxide. In some embodiments, at least one graft-end group is a hydroxyl. In some embodiments, at least one graft-end group is a thiol.

[0109] In certain embodiments, thiol-engrafts are an attractive option for attaching biomolecules to crosslinked polymers of membranes. The reaction is fast, can be carried out efficiently in aqueous media, works well at room temperature, and can be photoinitiated using relatively long wavelength light (365 nm), thus having very limited impact on the bioactivity of proteins. In addition, it can allow for controlled biomolecule attachment, which can be advantageous in terms of preserving the bioactivity and 3D structure of the biomolecules.

[0110] In certain embodiments, any biomolecule having a free thiol functional group can be immobilized on the composite material described herein. This can be very useful for creating bioaffinity membranes for bioseparation or biocatalytic membranes (by immobilizing enzymes). In certain embodiments, the composite material can be functionalized with oligonucleotide probes for DNA detection.

[0111] In certain embodiments, the present invention is c. The present invention relates to any one of the above methods, further comprising the step of removing excess first ligand by flowing a first cleaning solution at a second flow rate substantially through or substantially across the composite material.

[0112] In certain embodiments, the present invention is d. A step of flowing a quenched solution at a third flow rate substantially through or substantially across a composite material, wherein the quenched solution contains a reactive compound that converts any residual pendant reactive functional groups into non-reactive groups, e. The present invention relates to any one of the above methods, further comprising the step of optionally flowing a second cleaning solution at a fourth flow rate substantially through or substantially across the composite material to remove any residual reactive compounds.

[0113] In some embodiments, the first solution from step b. is recycled through or across the composite material.

[0114] In some embodiments, the quenched solution from step d. is recirculated through or across the composite material.

[0115] In certain embodiments, the present invention is c. Optionally, the step of flowing the first cleaning solution at a second flow rate substantially through or substantially across the composite material to remove excess first ligand, d. A step of flowing a second solution at a third flow rate substantially through or substantially across a functionalized composite material, wherein the second solution comprises a plurality of second ligands comprising at least three reactive groups, the second ligands being a crosslinking agent and optionally a polymerizable monomer comprising at least two pendant reactive functional groups, thereby forming a plurality of covalent bonds between the reactive functional groups and the second ligands. This relates to any one of the aforementioned methods, further including the above.

[0116] In some embodiments, the second ligand is added in at least two separate additions. In some embodiments, the polymerizable monomer containing at least two pendant-reactive functional groups is added in at least two separate additions.

[0117] In some embodiments, the second ligand comprises a second functional group. In some embodiments, the second ligand further comprises at least one graft-terminated group, the second functional group being selected from the group consisting of cationic, anionic, hydrophobic, hydrophilic, thiophilic, hydrogen bond donor, hydrogen bond acceptor, π-π bond donor, π-π bond acceptor, metal chelater, biological molecule, and biological ion. In some embodiments, the second functional group is selected from the group consisting of cationic, anionic, hydrophobic, hydrophilic, thiophilic, hydrogen bond donor, hydrogen bond acceptor, π-π bond donor, and π-π bond acceptor.

[0118] In some embodiments, the second ligand comprises a biological molecule or biological ion having at least one graft-terminant group selected from the group consisting of amine, hydroxyl, and thiol functional groups. In some embodiments, the biological molecule or biological ion is selected from the group consisting of albumin, lysozyme, viruses, cells, human and animal gamma globulins, immunoglobulins of both human and animal origin, recombinant or naturally occurring proteins including synthetic or naturally occurring polypeptides, interleukin-2 and its receptors, enzymes, monoclonal antibodies, antigens, lectins, bacterial immunoglobulin-binding proteins, trypsin and its inhibitors, cytochrome C, myoglobulin, recombinant human interleukin, recombinant fusion proteins, protein A, protein G, protein L, peptide H, nucleic acid-derived products, synthetic or naturally occurring DNA, and synthetic or naturally occurring RNA. In some embodiments, the biological molecule or biological ion is selected from the group consisting of human and animal-derived γ-globulin, immunoglobulins of both human and animal origin, recombinant or naturally occurring proteins including synthetic or naturally occurring polypeptides, monoclonal antibodies, antigens, bacterial immunoglobulin-binding proteins, recombinant fusion proteins, protein A, protein G, protein L, and peptide H. In some embodiments, the second ligand is a polypeptide, protein, recombinant protein, bacterial immunoglobulin-binding protein, recombinant fusion protein, protein A, protein G, protein L, and peptide H.

[0119] In some embodiments, the first ligand and the second ligand are the same. In some embodiments, the first ligand and the second ligand are different.

[0120] In some embodiments, the polymerizable monomer containing at least two pendant-reactive functional groups is poly(ethylene glycol) divinyl ether.

[0121] In certain embodiments, the present invention is e. The present invention relates to any one of the above methods, further comprising the step of flowing a second cleaning solution at a fourth flow rate substantially through or substantially across the composite material to remove any excess of the second ligand and optionally any excess of the polymerizable monomer.

[0122] In certain embodiments, the present invention is f. A step of flowing a quenched solution at a fifth flow rate substantially through or substantially across a composite material, wherein the quenched solution contains a reactive compound that converts any residual pendant reactive functional groups into non-reactive groups. g. Optionally, a step of flowing a third cleaning solution at a sixth flow rate substantially through or substantially across the composite material to remove any residual reactive compounds, This relates to any one of the aforementioned methods, further including the above.

[0123] In some embodiments, the present invention is a method for coupling ligands to a functionalized composite material, a. A step of providing a functionalized composite material, i. A support member comprising a plurality of pores extending through it, and ii. A macroporous crosslinked gel, wherein the macroporous crosslinked gel comprises a polymer formed from the reaction of one or more polymerizable monomers and one or more crosslinking agents, the macroporous crosslinked gel comprises a plurality of pendant-reactive functional groups, the macroporous crosslinked gel is located in the pores of the support member, and the macropores of the macroporous crosslinked gel are smaller than the pores of the support member. Steps including, b. A step of flowing a first solution at a first flow rate substantially through or substantially across the functionalized composite material, wherein the first solution comprises a plurality of first ligands, thereby forming a plurality of covalent bonds between the reactive functional group and the first ligands. c. Optionally, the step of removing excess first ligand by flowing a first cleaning solution at a second flow rate substantially through or substantially across the composite material, d. A step of flowing a quenching solution at a third flow rate substantially through or substantially across the composite material, wherein the quenching solution comprises a reactive compound that converts any residual pendant reactive functional group into a non-reactive group, e. optionally, the step of flowing a second cleaning solution at a fourth flow rate substantially through or substantially across the composite material to remove any residual reactive compounds, The present invention relates to a method for arranging functionalized composite materials in a stack of sheets having the same extent on the same plane, in a tubular configuration, or in a spiral winding configuration.

[0124] In some embodiments, the present invention is a method for coupling ligands to a functionalized composite material, a. A step of providing a functionalized composite material, i. A support member comprising a plurality of pores extending through it, and ii. A macroporous crosslinked gel, wherein the macroporous crosslinked gel comprises a polymer formed from the reaction of one or more polymerizable monomers and one or more crosslinking agents, the macroporous crosslinked gel comprises a plurality of pendant-reactive functional groups, the macroporous crosslinked gel is located in the pores of the support member, and the macropores of the macroporous crosslinked gel are smaller than the pores of the support member. Steps including, b. A step of flowing a first solution at a first flow rate substantially through or substantially across the functionalized composite material, wherein the first solution comprises a plurality of first ligands, thereby forming a plurality of covalent bonds between the reactive functional group and the first ligands. c. Optionally, the step of removing excess first ligand by flowing a first cleaning solution at a second flow rate substantially through or substantially across the composite material, d. A step of flowing a second solution at a third flow rate substantially through or substantially across the functionalized composite material, wherein the second solution comprises a plurality of second ligands comprising at least three reactive groups, the second ligands being a crosslinking agent and optionally a polymerizable monomer comprising at least two pendant reactive functional groups, thereby forming a plurality of covalent bonds between the reactive functional groups and the second ligands. e. Optionally, the step of flowing a second cleaning solution at a fourth flow rate substantially through or substantially across the composite material to remove any excess of the second ligand and optionally any excess of polymerizable monomer, f. A step of flowing a quenching solution at a fifth flow rate substantially through or substantially across the composite material, wherein the quenching solution comprises a reactive compound that converts any residual pendant reactive functional group into a non-reactive group. g. Optionally, a step of flowing a third cleaning solution at a sixth flow rate substantially through or substantially across the composite material to remove any residual reactive compounds, It further includes, The present invention relates to a method for arranging functionalized composite materials in a stack of sheets having the same extent on the same plane, in a tubular configuration, or in a spiral winding configuration.

[0125] In some embodiments, the functionalized composite material is a wet film.

[0126] In some embodiments, the wetted membrane is placed in a holder attached to the chromatography system. In some embodiments, various solutions and fluids (e.g., a first solution, a first washing solution, a second solution, a quenching solution, a second washing solution, and a third washing solution) are pumped through the membrane holder.

[0127] In certain embodiments, the present invention relates to one of the aforementioned methods, wherein the functionalized composite material is arranged in a coplanar stack of sheets having the same extent, in a tubular configuration, or in a spiral winding configuration.

[0128] Membrane stack In some embodiments, the composite material is arranged in substantially coplanar stacks of sheets having substantially the same extent. In some embodiments, the composite material has 2 to 300 separate support members. In some embodiments, the composite material has 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 35, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300 separate support members. In some embodiments, the composite material has 5 to 200 separate support members. In some embodiments, the composite material has 5 to 100 separate support members.

[0129] In some embodiments, when composite materials are arranged in substantially coplanar stacks of sheets having substantially the same extent, one or more distinct support members are separated by one or more interleaf layers. In some embodiments, the present invention provides for layers of composite material and interleaf (i.e., (composite material-interleaf) x or (interleaf-composite material) xThe present invention relates to one of the aforementioned methods, which is an alternating layer of ). In some embodiments, the composite material is layered with 1 to 250 distinct interleaf layers. In some embodiments, the composite material has 1 to 100 distinct interleaf layers. In some embodiments, the composite material has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 It has 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100 separate interleaf layers. In some embodiments, the composite material has 1 to 50 separate interleaf layers. In some embodiments, the composite material has 1 to 25 separate interleaf layers.

[0130] In some embodiments, the interleaf is a flow-dividing layer. In some embodiments, the interleaf layer extends beyond the edge of the composite material layer. In some embodiments, the interleaf layer also provides a lower pressure drop across the stack, as it allows flow through the membrane while also allowing the solution to flow around the membrane.

[0131] In some embodiments, when the composite material is arranged in a substantially coplanar stack of sheets having substantially the same extent, one or more separate support members, and optionally one or more interleaf layers, are separated by one or more flow dividers. In some embodiments, the one or more flow dividers allow for uniform coupling within the stack. In some embodiments, the one or more flow dividers allow for more uniform coupling of ligands to the composite material throughout the stack compared to coupling without flow dividers. In some embodiments, the composite material has 1 to 250 separate flow dividers. In some embodiments, the composite material has 1 to 100 separate flow dividers. In the first embodiment, the composite material is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 It has 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100 separate diversion layers. In some embodiments, the composite material has 1 to 50 separate diversion layers. In some embodiments, the composite material has 1 to 25 separate diversion layers.

[0132] In some embodiments, one or more diversion layers are non-porous sheets. In some embodiments, one or more diversion layers are non-porous sheets containing pores. In some embodiments, the diversion layers are periodically distributed within the membrane stack. In some embodiments, the diversion layers consist of a composite material layer and an interleaf layer (i.e., ((composite material-interleaf) x (Diversion) y or (interleaf-composite material) x (Diversion) y It is periodically distributed within a membrane stack containing ).

[0133] In some embodiments, the present invention relates to any one of the aforementioned methods, wherein the support member comprises a polymer material selected from the group consisting of polysulfone, polyethersulfone, polyphenylene oxide, polycarbonate, polyester, cellulose, and cellulose derivatives.

[0134] In some embodiments, the composite material is arranged in a tubular configuration.

[0135] Spiral winding configuration In some embodiments, the composite material is arranged in a substantially spiral winding configuration. In some embodiments, the substantially spiral winding configuration includes a composite material forming a layer wound around an inner core. In some embodiments, the substantially spiral winding configuration includes a composite material and an interleaving wound around the inner core.

[0136] Interleaving of spiral winding configuration In some embodiments, the present invention relates to any one of the aforementioned methods, wherein the layers of the composite material and the interleaving are alternating layers of the composite material and the interleaving (i.e., (composite material - interleaving) x or (interleaving - composite material) x ). In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the layers of the composite material and the interleaving are arranged as (interleaving - first composite material - second composite material) x or (first composite material - second composite material - interleaving) x . In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the layers of the composite material and the interleaving are arranged in a combination of the aforementioned arrangements. In certain embodiments, the first composite material and the second composite material are the same.

[0137] In some embodiments, the composite material includes about 3 to about 50 layers of composite material wound around an inner core.

[0138] In some embodiments, the present invention relates to one of the aforementioned methods (e.g., a film stack or a spiral winding configuration) in which the composite material is in contact with one or more interleaf layers. In some embodiments, the interleaf layers provide some mechanical support to the composite material.

[0139] In some embodiments, the interleaf helps to reduce back pressure.

[0140] In some embodiments, the present invention relates to one of the aforementioned methods (e.g., a membrane stack or a spiral winding configuration) in which one or more interleaf layers are selected from the group consisting of screen, mesh, polypropylene, polyethylene, paper, and cellulose. In some embodiments, the interleaf is a screen or nonwoven material. In some embodiments, the interleaf is a mesh. In some embodiments, the interleaf is polypropylene or polyethylene. In some embodiments, the interleaf is nonwoven polypropylene. In some embodiments, the interleaf is paper. In certain embodiments, the interleaf is cellulose.

[0141] In certain embodiments, the interleaf is a mesh. In certain embodiments, the mesh interleaf is an extruded net. In certain embodiments, the mesh interleaf is a mesh of approximately 0.45 mm. In certain embodiments, the mesh interleaf is a two-plane thermoplastic net. In certain embodiments, the mesh interleaf is substantially the same as Naltex (a specific two-plane thermoplastic net) manufactured by DelStar Technologies, Inc.

[0142] In certain embodiments, the interleaf is spunbond polypropylene. In certain embodiments, the interleaf is approximately 0.70 ounces / yd 2 ~Approximately 0.95 ounces / yd 2 The basis weight is spunbond polypropylene. In certain embodiments, the interleaf is approximately 0.70 ounces / yd2 Approximately 0.75 ounces / yd 2 Approximately 0.80 ounces / yd 2 Approximately 0.85 ounces / yd 2 Approximately 0.90 ounces / yd 2 Or approximately 0.95 ounces / yd 2 It is a spunbond polypropylene with a basis weight of approximately 0.86 ounces / yd 2 This is spunbond polypropylene with a basis weight.

[0143] In certain embodiments, the interleaf has a thickness of approximately 50 μm to approximately 300 μm. In certain embodiments, the interleaf has a thickness of approximately 50 μm, approximately 100 μm, approximately 150 μm, approximately 200 μm, approximately 250 μm, or approximately 300 μm.

[0144] In certain embodiments, the interleaf has a volume porosity of about 50% to about 99%. In certain embodiments, the interleaf has a volume porosity of about 70% to about 95%. In certain embodiments, the interleaf has a volume porosity of about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In certain embodiments, the interleaf has a volume porosity of about 80% to about 90%. In certain embodiments, the interleaf is substantially compressible.

[0145] In some embodiments, one or more interleaf layers are in contact with one or more diversion layers.

[0146] Inner core with spiral winding configuration In some embodiments, the inner core is made of plastic. In some embodiments, the inner core is made of polypropylene or polysulfone.

[0147] In some embodiments, the inner core is a cylinder. In certain embodiments, the inner core is a cylinder capped or sealed at both ends.

[0148] In certain embodiments, the inner core is a cylindrical tube. In certain embodiments, the inner core is a perforated cylindrical tube. In certain embodiments, the inner core is a perforated cylindrical tube capped or sealed at one end.

[0149] In some embodiments, the inner core is a screen wrapped around a cylinder. In certain embodiments, the screen provides a path through which a fluid can flow.

[0150] In some embodiments, the present invention relates to any one of the aforementioned methods, wherein the composite material is a film.

[0151] In some embodiments, the present invention relates to any one of the aforementioned methods, wherein the crosslinked gel is a neutral hydrogel, a charged hydrogel, a polymer electrolyte gel, a hydrophobic gel, a neutral gel, or a gel containing functional groups.

[0152] In some embodiments, the present invention relates to any one of the aforementioned methods, wherein the macroporous crosslinked gel comprises macropores having an average size of 10 nm to 3000 nm.

[0153] In some embodiments, the present invention relates to any one of the aforementioned methods, wherein the pores of the support member have an average pore diameter of about 0.1 μm to about 50 μm.

[0154] In some embodiments, the present invention relates to one of the aforementioned methods, wherein the fluid channel passes through a substantially composite material.

[0155] In some embodiments, the present invention relates to one of the aforementioned methods, wherein the fluid channel substantially crosses the composite material.

[0156] Exemplary method for preparing composite materials In certain embodiments, the present invention relates to one of the aforementioned methods in which the ratio of pendant-reactive functional groups to graft-terminal groups in the monomer mixture is about 1:10 to about 2:1, for example, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, or about 2:1. In some embodiments, the alkyne group is equivalent to two alkene groups.

[0157] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first monomer is present in the monomer mixture in an amount of about 5% to about 25% by weight of the monomer mixture. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first monomer is present in the monomer mixture in an amount of about 5% to about 20% by weight of the monomer mixture.

[0158] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the second monomer is present in the monomer mixture in an amount of about 0.1% to about 20% by weight of the monomer mixture.

[0159] In certain embodiments, the present invention relates to any one of the methods described above, wherein the first crosslinking agent is present in the monomer mixture in an amount of about 1% to about 20% by weight of the monomer mixture.

[0160] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the photoinitiator is present in the monomer mixture in an amount of about 0.1% to about 2% by weight of the monomer mixture.

[0161] In certain embodiments, the present invention relates to a photoinitiator that is benzoin or benzoin ether, benzophenone, dialkoxyacetophenone, 2,2-dimethoxy-2-phenylacetophenone, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, hydroxyalkylphenone, 1-hydroxycyclohexyl-phenyl-ketone, 4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone, 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy The present invention relates to any one of the aforementioned methods, wherein the material is -2-methyl-1-propan-1-one, 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone, α-hydroxymethylbenzoin sulfonate, 2-hydroxy-2-methylpropiophenone, lithium acylphosphinate, or 2-methyl-1-[4-(methylthio)phenyl]-2-(4-morpholinyl)-1-propanone, 4,4'-azobis(4-cyanovaleric acid) (ACVA), or a mixture thereof.

[0162] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first solvent comprises N,N'-dimethylacetamide (DMAc), (±)-1,3-butanediol (budiol), di(propylene glycol)methyl ether acetate (DPMA), water, di(propylene glycol)dimethyl ether (DPM), di(propylene glycol)propyl ether (DPGPE), di(propylene glycol)methyl ether (DPGME), tri(propylene glycol)butyl ether (TPGBE), 3-methyl-1,3-butanediol, 3,3-dimethyl-1,2-butanediol, 3-methoxy-1-butanol, dimethyl sulfoxide (DMSO), ethylene glycol, di(ethylene glycol), tri(ethylene glycol), tetra(ethylene glycol), hexylene glycol, sodium dodecyl sulfate, or N,N-dimethylformamide (DMF), or a mixture thereof.

[0163] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein N,N'-dimethylacetamide (DMAc) is present in the monomer mixture in an amount of about 0% to about 70% by weight of the monomer mixture. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein N,N'-dimethylacetamide (DMAc) is present in the monomer mixture in an amount of about 0% to about 50% by weight of the monomer mixture. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein N,N'-dimethylacetamide (DMAc) is present in the monomer mixture in an amount of about 0% to about 70% by weight of the total solvent. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein N,N'-dimethylacetamide (DMAc) is present in the monomer mixture in an amount of about 0% to about 50% by weight of the total solvent.

[0164] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein (±)-1,3-butanediol (budiol) is present in the monomer mixture in an amount of about 0% to about 50% by weight of the monomer mixture. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein (±)-1,3-butanediol (budiol) is present in the monomer mixture in an amount of about 0% to about 50% by weight of the total solvent.

[0165] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein di(propylene glycol) methyl ether acetate (DPMA) is present in the monomer mixture in an amount of about 0% to about 60% by weight of the monomer mixture. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein di(propylene glycol) methyl ether acetate (DPMA) is present in the monomer mixture in an amount of about 0% to about 60% by weight of the total solvent.

[0166] In certain embodiments, the present invention relates to any one of the aforementioned methods in which water is present in the monomer mixture in an amount of about 0% to about 50% by weight of the monomer mixture. In certain embodiments, the present invention relates to any one of the aforementioned methods in which water is present in the monomer mixture in an amount of about 0% to about 30% by weight of the monomer mixture. In certain embodiments, the present invention relates to any one of the aforementioned methods in which water is present in the monomer mixture in an amount of about 0% to about 30% by weight of the total solvent.

[0167] In certain embodiments, the present invention relates to one of the aforementioned methods, wherein the covered support member is irradiated at approximately 350 nm.

[0168] In certain embodiments, the present invention relates to one of the aforementioned methods, wherein the duration is approximately 1 minute, approximately 5 minutes, approximately 10 minutes, approximately 15 minutes, approximately 20 minutes, approximately 30 minutes, approximately 45 minutes, or approximately 1 hour.

[0169] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the composite material includes macropores.

[0170] In certain embodiments, the present invention relates to one of the aforementioned methods, wherein the average pore diameter of macropores is smaller than the average pore diameter of pores.

[0171] Determining pore size SEM and ESEEM As described above, in certain embodiments, the cross-linked gel is a macroporous cross-linked gel. The average diameter of macropores in a macroporous cross-linked gel can be estimated by one of many methods. One method that can be used is scanning electron microscopy (SEM). SEM is generally an established method for determining pore size and porosity, and in particular for characterizing membranes. See the book "Basic Principles of Membrane Technology" ((C)1996) ("Mulder") by Marcel Mulder, particularly Chapter IV. Mulder provides an overview of methods for characterizing membranes. For porous membranes, the first method mentioned is electron microscopy. SEM is a very simple and useful technique for characterizing microfiltration membranes. Clear and concise images of the membrane can be obtained with respect to the upper layer, cross-section, and lower layer. In addition, porosity and pore size distribution can be estimated from the images.

[0172] Environmental scanning electron microscopy (ESEM) is a technique that enables non-destructive imaging of wet samples by allowing a gaseous environment within the sample chamber. The environmental secondary detector (ESD) requires a gas background to function and operates at approximately 3 Torre to 20 Torre. These pressure constraints limit the ability to vary the humidity within the sample chamber. For example, at 10 Torre, the relative humidity at a given temperature is as follows:

[0173] [Table 1]

[0174] This is a useful guide for relative humidity in the sample chamber at different temperatures. In certain embodiments, the relative humidity in the sample chamber during imaging is about 1% to about 99%. In certain embodiments, the relative humidity in the sample chamber during imaging is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In certain embodiments, the relative humidity in the sample chamber during imaging is about 45%.

[0175] In certain embodiments, the microscope has nanometer resolution and a magnification of up to approximately 100,000X.

[0176] In certain embodiments, the temperature in the sample chamber during imaging is approximately 1°C to approximately 95°C. In certain embodiments, the temperature in the sample chamber during imaging is approximately 2°C, approximately 3°C, approximately 4°C, approximately 5°C, approximately 6°C, approximately 7°C, approximately 8°C, approximately 9°C, approximately 10°C, approximately 12°C, approximately 14°C, approximately 16°C, approximately 18°C, approximately 20°C, approximately 25°C, approximately 30°C, approximately 35°C, approximately 40°C, approximately 45°C, approximately 50°C, approximately 55°C, approximately 60°C, approximately 65°C, approximately 70°C, approximately 75°C, approximately 80°C, or approximately 85°C. In certain embodiments, the temperature in the sample chamber during imaging is approximately 5°C.

[0177] In certain embodiments, the pressure in the sample chamber during imaging is approximately 0.5 Torr to approximately 20 Torr. In certain embodiments, the pressure in the sample chamber during imaging is approximately 4 Torr, approximately 6 Torr, approximately 8 Torr, approximately 10 Torr, approximately 12 Torr, approximately 14 Torr, approximately 16 Torr, approximately 18 Torr, or approximately 20 Torr. In certain embodiments, the pressure in the sample chamber during imaging is approximately 3 Torr.

[0178] In certain embodiments, the working distance from the electron beam source to the sample is approximately 6 mm to 15 mm. In certain embodiments, the working distance from the electron beam source to the sample is approximately 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, or 15 mm. In certain embodiments, the working distance from the electron beam source to the sample is approximately 10 mm.

[0179] In certain embodiments, the voltage is approximately 1kV to approximately 30kV. In certain embodiments, the voltage is approximately 2kV, approximately 4kV, approximately 6kV, approximately 8kV, approximately 10kV, approximately 12kV, approximately 14kV, approximately 16kV, approximately 18kV, approximately 20kV, approximately 22kV, approximately 24kV, approximately 26kV, approximately 28kV, or approximately 30kV. In certain embodiments, the voltage is approximately 20kV.

[0180] In certain embodiments, the average pore diameter can be measured by estimating the pore diameter of a representative sample of images taken from the top or bottom of the composite material. Those skilled in the art can recognize and understand the various experimental variables associated with obtaining ESEM images of a wetted film and design the experiment accordingly.

[0181] Capillary flow spectroscopy Capillary flow porometry is an analytical technique used to measure the pore size of porous materials. In this technique, a wetting solution is used to fill the pores of a test sample, and the pressure of a non-reactive gas is used to replace the liquid from the pores. The gas pressure and flow rate through the sample are accurately measured, and the pore diameter is determined using the following formula: The gas pressure required to remove the liquid from the pores is related to the pore size by the following formula: D = 4 × γ × cosθ / P D=pore diameter γ=liquid surface tension θ=liquid contact angle P = Gas differential pressure

[0182] This equation shows that the pressure required to replace the liquid in a wet sample is inversely proportional to the pore size. Because this technique involves liquid flow from the pores of the test sample under pressure, it is useful for characterizing "through-pores" (interconnected pores that allow fluid flow from one side of the sample to the other). Other pore types (closed pores and blind pores) cannot be detected by this method.

[0183] Capillary flow porometry detects the presence of pores when gas begins to flow through them. This only occurs when the gas pressure is high enough to displace the liquid from the narrowest part of the pore. Therefore, the pore diameter calculated using this method is the diameter of the narrowest part of the pore, and each pore is detected as a single pore of this narrowest diameter. The maximum pore diameter (called the bubble point) is determined by the minimum gas pressure required to initiate flow through the wet sample, and the average pore diameter is calculated from the average flow pressure. In addition, this technique can be used to determine both the narrowed pore diameter range and the pore size distribution.

[0184] This method can be performed on a small membrane sample (e.g., approximately 2.5 cm in diameter) immersed in a test fluid (e.g., water, buffer solution, alcohol). The applied gas pressure can be selected from a range of approximately 0 to approximately 500 psi.

[0185] Other methods for determining pore size Mulder describes other methods for characterizing the average pore size of porous membranes, including atomic force microscopy (AFM) (p. 164), transmittance calculation (p. 169), gas adsorption-desorption (p. 173), thermoporometry (p. 176), perporometry (p. 179), and liquid displacement (p. 181). Mulder and the references cited herein are incorporated herein by reference.

[0186] Exemplary use of composite materials In certain embodiments, the present invention relates to a method for a fluid to pass through one of the aforementioned crosslinked gels of composite materials. Good selectivity can be obtained by adjusting the conditions for bonding or fractionation.

[0187] In certain embodiments, the present invention is f. A method further comprising the step of flowing a first fluid containing a substance at a fifth flow rate substantially through or substantially across a composite material, thereby adsorbing or absorbing a portion of the substance onto the composite material.

[0188] In certain embodiments, the present invention is The present invention relates to a method comprising the step of adsorbing or absorbing a portion of a substance onto a composite material by flowing a first fluid containing the substance at a seventh flow rate substantially through or substantially across the composite material.

[0189] In certain embodiments, the first fluid further comprises fragmentation antibodies, agglutinating antibodies, host cell proteins, polynucleotides, endotoxins, or viruses. In some embodiments, the first fluid is a suspension of cells or a suspension of aggregates.

[0190] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the fluid channel for the first fluid substantially passes through the macropores of a composite material.

[0191] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the fluid channel for the first fluid is substantially perpendicular to the macropores of the composite material.

[0192] In certain embodiments, the present invention relates to one of the aforementioned methods, wherein substantially all of the substance is adsorbed or absorbed onto the composite material after a first fluid has flowed substantially through or substantially across the composite material.

[0193] In certain embodiments, the present invention is g. The present invention relates to any one of the above methods, further comprising the step of bringing a second fluid into contact with a substance adsorbed or absorbed on the composite material at a sixth flow rate, thereby releasing a portion of the substance from the composite material.

[0194] In certain embodiments, the present invention is i. The present invention relates to any one of the above methods, further comprising the step of bringing a second fluid into contact with a substance adsorbed or absorbed on the composite material at an eighth flow rate, thereby releasing a portion of the substance from the composite material.

[0195] In some embodiments, the present invention is a method for coupling ligands to a functionalized composite material, a. A step of providing a functionalized composite material, i. A support member comprising a plurality of pores extending through it, and ii. A macroporous crosslinked gel, wherein the macroporous crosslinked gel comprises a polymer formed from the reaction of one or more polymerizable monomers and one or more crosslinking agents, the macroporous crosslinked gel comprises a plurality of pendant-reactive functional groups, the macroporous crosslinked gel is located in the pores of the support member, and the macropores of the macroporous crosslinked gel are smaller than the pores of the support member. Steps including, b. A step of flowing a first solution at a first flow rate substantially through or substantially across the functionalized composite material, wherein the first solution comprises a plurality of first ligands, thereby forming a plurality of covalent bonds between the reactive functional group and the first ligands. c. Optionally, the step of flowing a first cleaning solution at a second flow rate substantially through or substantially across the composite material to remove excess first ligands, d. A step of flowing a quenching solution at a third flow rate substantially through or substantially across the composite material, wherein the quenching solution comprises a reactive compound that converts any residual pendant reactive functional group into a non-reactive group, e. Optionally, the step of flowing a second cleaning solution at a fourth flow rate substantially through or substantially across the composite material to remove any residual reactive compounds, f. A step of adsorbing or absorbing a portion of the substance onto the composite material by flowing a first fluid containing the substance at a fifth flow rate substantially through or substantially across the composite material, g. The process includes bringing a second fluid into contact with the substance adsorbed or absorbed on the composite material at a sixth flow rate, thereby releasing a portion of the substance from the composite material. The present invention relates to a method for arranging functionalized composite materials in a stack of sheets having the same extent on the same plane, in a tubular configuration, or in a spiral winding configuration.

[0196] In some embodiments, the present invention is a method for coupling ligands to a functionalized composite material, a. A step of providing a functionalized composite material, i. A support member comprising a plurality of pores extending through it, and ii. A macroporous crosslinked gel, wherein the macroporous crosslinked gel comprises a polymer formed from the reaction of one or more polymerizable monomers and one or more crosslinking agents, the macroporous crosslinked gel comprises a plurality of pendant-reactive functional groups, the macroporous crosslinked gel is located in the pores of the support member, and the macropores of the macroporous crosslinked gel are smaller than the pores of the support member, step, b. A step of flowing a first solution at a first flow rate substantially through or substantially across the functionalized composite material, wherein the first solution comprises a plurality of first ligands, thereby forming a plurality of covalent bonds between the reactive functional group and the first ligands. c. Optionally, the step of flowing a first cleaning solution at a second flow rate substantially through or substantially across the composite material to remove excess first ligands, d. A step of flowing a second solution at a third flow rate substantially through or substantially across the functionalized composite material, wherein the second solution comprises a plurality of second ligands comprising at least three reactive groups, the second ligands being a crosslinking agent and optionally a polymerizable monomer comprising at least two pendant reactive functional groups, thereby forming a plurality of covalent bonds between the reactive functional groups and the second ligands. e. Optionally, the step of flowing a second cleaning solution at a fourth flow rate substantially through or substantially across the composite material to remove any excess of the second ligand and optionally any excess of polymerizable monomer, f. A step of flowing a quenching solution at a fifth flow rate substantially through or substantially across the composite material, wherein the quenching solution comprises a reactive compound that converts any residual pendant reactive functional group into a non-reactive group, g. Optionally, a step of flowing a third cleaning solution at a sixth flow rate substantially through or substantially across the composite material to remove any residual reactive compounds, h. A step of adsorbing or absorbing a portion of a substance onto the composite material by flowing a first fluid containing the substance at a seventh flow rate substantially through or substantially across the composite material, i. The process includes bringing a second fluid into contact with the substance adsorbed or absorbed on the composite material at an eighth flow rate, thereby releasing a portion of the substance from the composite material, The present invention relates to a method for arranging functionalized composite materials in a stack of sheets having the same extent on the same plane, in a tubular configuration, or in a spiral winding configuration.

[0197] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the fluid channel for the second fluid substantially passes through the macropores of the composite material.

[0198] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the fluid channel for the second fluid is substantially perpendicular to the macropores of the composite material.

[0199] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the substance is a biological molecule, a biological ion, a virus, or a viral particle.

[0200] In certain embodiments, the present invention relates to a method for separating biological molecules or biological ions, such as proteins or immunoglobulins, from a solution. In certain embodiments, the present invention relates to a method for purifying biological molecules or biological ions, such as proteins or immunoglobulins. In certain embodiments, the present invention relates to a method for purifying proteins or monoclonal antibodies with high selectivity. In certain embodiments, the present invention relates to a method for preserving the tertiary or quaternary structure of a biological molecule or biological ion, which may be important for retaining its biological activity.

[0201] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the substance is a biological molecule or biological ion selected from the group consisting of albumin, lysozyme, viruses, cells, human and animal gamma globulins, human and animal immunoglobulins, hIgG, recombinant and naturally occurring proteins, synthetic and naturally occurring polypeptides, interleukin-2 and its receptors, enzymes, monoclonal antibodies, trypsin and its inhibitors, cytochrome C, myoglobin, myoglobulins, α-chymotrypsinogen, recombinant human interleukins, recombinant fusion proteins, nucleic acid-derived products, synthetic and naturally occurring DNA, and synthetic and naturally occurring RNA.

[0202] In some embodiments, the present invention relates to substances such as albumin, lysozyme, viruses, cells, human and animal gamma globulins, human and animal immunoglobulins, hIgG, immunoglobulin M, recombinant and naturally occurring proteins (e.g., recombinant human growth hormone, recombinant human insulin, recombinant follicle-stimulating hormone, recombinant factor VII (antihemophilic factor), recombinant human erythropoietin, recombinant granulocyte colony-stimulating factor, recombinant alpha-galactosidase a, recombinant idulonidase, recombinant galsulfase, recombinant dorunase alpha, recombinant tissue plasminogen activator, recombinant human interferol The present invention relates to any one of the aforementioned methods, wherein the biological molecule or biological ion is selected from the group consisting of (recombinant insulin-like growth factor 1 and recombinant asparaginase), synthetic and naturally occurring polypeptides, interleukin-2 and its receptor, enzymes, monoclonal antibodies, trypsin and its inhibitors, cytochrome C, myoglobin, myoglobulin, α-chymotrypsinogen, recombinant human interleukin, recombinant fusion proteins, factor VIII, factor IX, antithrombin III, alpha-I-antitrypsin, nucleic acid-derived products, synthetic and naturally occurring DNA, and synthetic and naturally occurring RNA.

[0203] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the biological molecule or biological ion is lysozyme, hIgG, myoglobin, human serum albumin, soy trypsin inhibitor, transferase, enolase, ovalbumin, ribonuclease, egg trypsin inhibitor, cytochrome c, annexin V, or α-chymotrypsinogen.

[0204] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the substance is a biological molecule, or the biological ion is selected from the group consisting of human and animal γ-globulins, immunoglobulins of both human and animal origin, recombinant or naturally occurring proteins including synthetic or naturally occurring polypeptides, monoclonal antibodies, antigens, bacterial immunoglobulin-binding proteins, recombinant fusion proteins, protein A, protein G, protein L, and peptide H.

[0205] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the substance is a biological molecule, or the biological ion is selected from the group consisting of polypeptides, proteins, recombinant proteins, bacterial immunoglobulin-binding proteins, recombinant fusion proteins, protein A, protein G, protein L, and peptide H.

[0206] In certain embodiments, the present invention relates to a method for recovering antibody fragments from associated variants, impurities, or contaminants. In certain embodiments, the separation or purification of biological molecules or biological ions may substantially occur in a crosslinked gel. In certain embodiments, if the crosslinked gel has macropores, the separation or purification of biological molecules or biological ions may substantially occur in the macropores of the crosslinked gel.

[0207] In certain embodiments, the present invention relates to a method for reversible adsorption of a substance. In certain embodiments, the adsorbed substance can be released by altering the liquid flowing through the gel. In certain embodiments, the uptake and release of the substance can be controlled by variations in the composition of the crosslinked gel.

[0208] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first fluid is a clarified cell culture supernatant.

[0209] In certain embodiments, the present invention relates to a method by which a substance can be applied to a composite material from a buffer solution. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first fluid is a buffer solution. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the concentration of the buffer solution in the first fluid is about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 0.1 M, about 0.11 M, about 0.12 M, about 0.13 M, about 0.14 M, about 0.15 M, about 0.16 M, about 0.17 M, about 0.18 M, about 0.19 M, or about 0.2 M.

[0210] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the pH of the first fluid is about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, or about 9.

[0211] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first fluid comprises sodium phosphate.

[0212] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first fluid contains a salt. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the concentration of the salt in the first fluid is about 50 mM, about 60 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 0.1 M, about 0.11 M, about 0.12 M, about 0.13 M, about 0.14 M, about 0.15 M, about 0.16 M, about 0.17 M, about 0.18 M, about 0.19 M, about 0.2 M, about 0.25 M, or about 0.3 M. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the salt is sodium chloride.

[0213] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the substance is the bonding partner. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the composite material comprises a coupled ligand, and the substance is the bonding partner of the ligand.

[0214] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the concentration of the substance in the first fluid is about 0.01 mg / mL to about 1,000 mg / mL. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the concentration of the substance in the first fluid is about 0.2 mg / mL to about 10 mg / mL. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the concentration of the substance in the first fluid is about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / L, about 1 mg / mL, about 1.2 mg / mL, about 1.4 mg / mL, about 1.6 mg / mL, about 1.8 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about mg / mL, or about 10 mg / mL.

[0215] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth flow rates are each independently selected from about 1 membrane volume (MV) / min to about 75 MV / min. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth flow rates are each independently selected from about 3 membrane volumes (MV) / min to about 70 MV / min. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth flow rates are each independently selected from about 5 MV / min to about 50 MV / min. In a particular embodiment, the present invention provides a first flow rate, a second flow rate, a third flow rate, a fourth flow rate, a fifth flow rate, a sixth flow rate, a seventh flow rate, and an eighth flow rate, each independently, at approximately 5 MV / min, approximately 6 MV / min, approximately 7 MV / min, approximately 8 MV / min, approximately 9 MV / min, approximately 10 MV / min, approximately 11 MV / min, approximately 12 MV / min, approximately 13 MV / min, approximately 14 MV / min, approximately 15 MV / min, approximately 16 MV / min, approximately 17 MV / min, approximately 18 MV / min, approximately 19 MV / min, approximately 20 MV / min, approximately 20 MV / min, approximately 21 MV / min, approximately 22 MV / min, approximately 23 MV / min, approximately 24 MV / min, and approximately 25 MV / min. This relates to one of the aforementioned methods selected from the group consisting of approximately 26 MV / min, approximately 27 MV / min, approximately 28 MV / min, approximately 29 MV / min, approximately 30 MV / min, approximately 30 MV / min, approximately 31 MV / min, approximately 32 MV / min, approximately 33 MV / min, approximately 34 MV / min, approximately 35 MV / min, approximately 36 MV / min, approximately 37 MV / min, approximately 38 MV / min, approximately 39 MV / min, approximately 40 MV / min, approximately 40 MV / min, approximately 41 MV / min, approximately 42 MV / min, approximately 43 MV / min, approximately 44 MV / min, approximately 45 MV / min, approximately 46 MV / min, approximately 47 MV / min, approximately 48 MV / min, approximately 49 MV / min, and approximately 50 MV / min. In certain embodiments, the present invention relates to one of the aforementioned methods, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth flow rates are each independently selected from approximately 10 MV / min to approximately 20 MV / min.

[0216] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth flow rates are each independently selected from about 0.5 mL / min to about 50 L / min. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth flow rates are each independently selected from about 0.5 mL / min to about 25 L / min. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth flow rates are each independently selected from about 0.5 mL / min to about 10 L / min. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth flow rates are each independently selected from about 0.5 mL / min to about 1 L / min. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth flow rates are each independently selected from about 0.5 mL / min to about 0.5 L / min. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth flow rates are each independently selected from about 0.5 mL / min to about 100 mL / min. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth flow rates are each independently selected from about 0.5 mL / min to about 10 mL / min. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth flow rates are each independently selected from about 0.5 mL / min to about 2 mL / min.In certain embodiments, the present invention relates to one of the aforementioned methods, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth flow rates are each independently selected from the group consisting of 0.5 mL / min, about 0.6 mL / min, about 0.7 mL / min, about 0.8 mL / min, about 0.9 mL / min, about 1 mL / min, about 1.1 mL / min, about 1.2 mL / min, about 1.3 mL / min, about 1.4 mL / min, about 1.5 mL / min, about 1.6 mL / min, about 1.7 mL / min, and about 1.8 mL / min.

[0217] In certain embodiments, the present invention relates to a method for eluting a substance using a saline aqueous solution of various concentrations and pH levels. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the second fluid is a buffer solution. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the second fluid comprises glycine-HCl or sodium citrate. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the second fluid comprises glycine-HCl or sodium citrate at a concentration of about 5 mM to about 2 M. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the second fluid comprises glycine-HCl or sodium citrate at a concentration of about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 125 mM, about 150 mM, about 200 mM, about 300 mM, or about 400 mM.

[0218] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the pH of the second fluid is about 2 to about 8. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the pH of the second fluid is about 2, about 2.2, about 2.4, about 2.6, about 2.8, about 3, about 3.2, about 3.4, about 3.6, about 3.8, about 4, about 4.2, about 4.4, about 4.6, about 4.8, about 5, about 5.2, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, and about 8.0.

[0219] In certain embodiments, the present invention relates to a method for exhibiting high binding capacity. In some embodiments, the binding capacity of the composite material was higher when using a flow-through conjugation method compared to when using a batch or dead-end conjugation method. In certain embodiments, the present invention provides a binding capacity of approximately 1 mg / mL at 10% breakthrough. 膜 , about 2mg / mL 膜 , about 3mg / mL 膜 , about 4mg / mL 膜 , about 5mg / mL 膜 , about 6mg / mL 膜 , about 7mg / mL 膜 , about 8mg / mL 膜 Approximately 9 mg / mL 膜 , about 10mg / mL 膜 , about 12mg / mL 膜 , about 14mg / mL 膜 , about 16mg / mL 膜 , about 18mg / mL 膜 , about 20mg / mL 膜 , about 30mg / mL 膜 , about 40mg / mL 膜 , about 50mg / mL 膜 , about 60mg / mL 膜 , about 70mg / mL 膜 , about 80mg / mL 膜 , about 90mg / mL 膜 , about 100mg / mL, about 110mg / mL 膜, about 120mg / mL 膜 , about 130mg / mL 膜 , about 140mg / mL 膜 , about 150mg / mL 膜 , about 160mg / mL 膜 , about 170mg / mL 膜 , about 180mg / mL 膜 , about 190mg / mL 膜 , about 200mg / mL 膜 , about 210mg / mL 膜 , about 220mg / mL 膜 , about 230mg / mL 膜 , about 240mg / mL 膜 , about 250mg / mL 膜 , about 260mg / mL 膜 , about 270mg / mL 膜 , about 280mg / mL 膜 , about 290mg / mL 膜 , about 300mg / mL 膜 , about 320mg / mL 膜 , about 340mg / mL 膜 mg / mL 膜 , about 360mg / mL 膜 , about 380mg / mL 膜 Or approximately 400 mg / mL 膜 Regarding a method for indicating the binding capacity. [Examples]

[0220] The following examples are provided for illustrative purposes only. However, it should be understood that the specific details given in each example are selected for illustrative purposes and should not be construed as limiting the scope of this disclosure. In general, unless otherwise noted, experiments were conducted under similar conditions.

[0221] [Example 1 - General Materials and Methods] protein r-protein A-cys was obtained from Biomedal SL (Seville, Spain). Polyclonal immunoglobulin IgG was obtained from Equitech-BioInc. (Carville, Texas, USA).

[0222] Membrane preparation Protocol A The crosslinking agent and monomers (except for the thiol-functionalized monomer added 10 minutes before casting) were added to the solvent mixture along with the photoinitiator (IRGACURE 2959), and the mixture was stirred for a sufficient amount of time to dissolve all components. A pre-weighed 7-inch x 8-inch porous support substrate sheet (nonwoven polypropylene mesh) was placed on a polyethylene sheet, and then approximately 15 g of the polymer solution was poured onto the substrate sheet. Subsequently, the impregnated substrate was covered with another polyethylene sheet. The sheets were gently pressed by hand in a circular motion to remove excess solution and trapped air bubbles. The polymerization process was initiated by irradiating the polymer solution / substrate sandwiched between polyethylene sheets with UV light (approximately 350 nm) for 10 minutes in a closed chamber. The resulting film was then removed from between the polyethylene sheets and subjected to a thorough washing cycle (2-3 times) with immersion in purified (RO) water for 20-30 minutes while stirring. The clean film was dried by hanging it freely in room temperature air for approximately 16 hours.

[0223] Protocol B The membranes were fabricated by polymerizing acrylate and / or acrylamide monomers with a crosslinking agent in a UV-initiated reaction within a support mesh material. By introducing suitable functional polymerizable groups into the gel polymerization solution, various functional membranes containing protein binding groups (e.g., ion exchange, hydrophobic interactions, and hydrophilic interactions) can be produced in a single polymerization step. The wet-washed membranes may also be subjected to additional heat treatment steps to adjust their performance and properties.

[0224] For example, epoxy-containing membranes can function as reactive media platforms that can be converted into biocompatible membranes by covalently attaching various ligands, such as protein A, to their surface. Other ligands can also be conjugated to other target biomolecules or entities such as viruses.

[0225] Mass increase, wetting and permeability of composite films The weight of the dry membrane was measured and used to calculate the mass increase. Membrane wetting was also determined by dispensing a 50 μL droplet of distilled water onto the membrane surface and measuring the time required for the droplet to be absorbed into the membrane. To estimate membrane permeability, the flux of each membrane was determined using RO water (or acetate buffer pH 5) and a membrane sample with a diameter of 7.7 cm, using an applied pressure of 100 kPa.

[0226] To estimate membrane permeability, the flux of RO water (or 132 mM acetate buffer pH 5) as the mobile phase through each membrane was determined. The membranes were pre-immersed in the test solution for at least 10 minutes before testing, rinsed with approximately 300 mL of the test solution, and then the amount of test solution passing through a circular membrane coupon with a diameter of 7.7 cm (actual usable diameter 7.3 cm) under an applied pressure of 100 kPa was determined. Flux is defined as the amount of liquid per unit area per unit time (kg / m³). 2 It is expressed as h).

[0227] Imaging of porous structures To probe the gel structure and porosity, wet films were imaged using an environmentally controlled scanning electron microscope (ESEM). Small coupons (approximately 7 x 5 mm) were moistened by immersion in distilled water for 10-15 minutes and then examined using an ESEM instrument (FEI QuantaFEG 250 ESEM). The samples were placed on a cooling stage and the temperature was adjusted to 5°C, and the images were examined at low pressure levels (4.5-5.5 Torre) and relative humidity of 50-55%.

[0228] To probe the dry film structure, a Tescan Vega II LSU scanning electron microscope (SEM) (Tescan, Pennsylvania, USA) was used to image the gold-coated films at voltages set to 10–20 kV.

[0229] Pore ​​size measurement The membrane pore size (diameter) was measured using a CFP-1500-AE capillary flow porometer (Porous Materials Inc., Ithaca, New York) operated by CapWin software (V.6).

[0230] A small disk membrane (2.5 cm in diameter) was immersed in Galwick(R) wetting solution (Porous Materials Inc., surface tension = 15.9 dynes / cm) for 10 minutes, and then it was gently squeezed between two pre-wetted filter paper disks (Whatman 5 - 70 mm) to remove the excess solution. The thickness of the wet membrane was determined using a micrometer. Next, the membrane disk was placed on a 2.5 cm stainless steel mesh support disk. The support disk loaded with the test membrane was placed into the designated holder with the membrane facing up. Then, the metal cover was gently placed on the holder, and the test was conducted within a pressure range of 0 - 200 psi.

[0231] Density of Protein A ligand on the composite membrane To measure the density of Protein A ligand on the coupled membrane, the amount of uncoupled protein remaining after the coupling reaction was determined, subtracted from the total ligand amount to obtain the amount of coupled ligand, and then divided by the membrane volume (mL) to express the density in mg of ligand per mL of membrane.

[0232] To determine the amount of Protein A in the solution, a series of protein solutions in 0.1 M phosphate buffer (pH 7.2) were prepared, the absorbance at 280 nm was measured for each, and a calibration curve for determining the slope was constructed.

[0233] For the selected membrane type, 4 cm × 7 cm coupons were cut, their thicknesses were measured, and the volume was calculated from that. The coupling reaction was carried out as outlined previously, and 20 mg was individually loaded into each membrane coupling reaction. When the UV reaction was complete, the reaction solution was collected in a tube, then 3 - 5 mL of 0.1 M phosphate buffer was added to the reaction bag and used to wash the membrane by shaking for 20 - 25 minutes, and then the resulting solution was added to the collection tube.

[0234] After repeating the washing cycle two more times, the absorbance of the final solution was measured, and the amount of uncoupled protein was calculated using the slope of the calibration curve. The amount of coupled ligand was determined by taking the difference between the total reaction amount and the uncoupled amount.

[0235] Coupling capacitance measurement Biocompatible IgG binding capacity A 25 mm diameter membrane disk was placed in a 25 mm Natrix-StainlessSteel (SS) holder. Equilibration was achieved by passing 20 mL of binding buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4) through the disk (approximately 160-200 bed volumes / min). In the binding step, 0.5 mg / mL of polyclonal IgG from the binding buffer was passed through at a flow rate of 1 mL / min until the UV absorbance of the eluate exceeded 10% of the supply solution. Then, 10-15 mL of buffer was passed through at a flow rate of 2 mL / min to remove unbound protein. In the elution step, bound IgG was eluted by passing 10-14 mL of elution buffer (0.1 M glycine-HCl or 0.1 M sodium citrate, both pH 3) through the disk at a flow rate of 2 mL / min.

[0236] Cation exchange IgG binding capacity A 25 mm membrane disk was placed in a 25 mm Natrix-SS holder, and equilibration was achieved by passing 20 mL of binding buffer (132 mM sodium acetate, pH 5.0) through it. Next, a protein solution (0.5 mg / mL human polyclonal IgG in binding buffer (Equitech-Bio Inc.)) was passed through the eluate until the UV absorbance of the eluate exceeded 10% of the supply solution, and then 10-15 mL of buffer was passed through the cells to wash away unbound proteins. In the elution step, bound IgG was eluted by passing 10 mL of elution buffer (132 mM sodium acetate, 1 M NaCl, pH 5.0, or 50 mM Tris, 0.5 M NaCl, pH 8.5) through it.

[0237] Hydrophobic interaction mode IgG binding capacity A 25mm membrane disk was placed in a 25mm Natrix-SS holder, and equilibration was achieved by passing 20mL of binding buffer (50mM sodium phosphate, 1M ammonium sulfate, pH 6.5) through it. Next, a protein solution (0.5mg / mL human polyclonal IgG in binding buffer (Equitech-BioInc.)) was passed through the eluate until the UV absorbance of the eluate exceeded 10% of the supply solution. Subsequently, 15-20mL of buffer was passed through the cells to wash away unbound proteins. In the elution step, 10mL of elution buffer (50mM sodium phosphate, pH 7.0) was passed through to elute the bound IgG.

[0238] [Example 2 - Exemplary Bulk Coupling Protocol] Conjugation of protein A ligand for clicking alkene membranes To investigate the feasibility of chemically bonding biomolecules (containing thiol functional groups) to alkene membranes via hydrothiolation click reactions, we coupled modified protein A ligands containing cysteine ​​residues to alkene membranes (with different chemical formulas) and examined the bioactivity of the immobilized ligands.

[0239] A 50 mg / mL stock solution was prepared by dissolving lyophilized protein A ligand powder (r-protein A-cys) in PBS (20 mM sodium phosphate, 0.15 M NaCl, pH 7.4). To prepare a coupling solution for each membrane, 0.4 mL of the ligand stock solution was transferred to a small Ziploc® plastic bag (5 × 8 cm), 1.6 mL of 2 M phosphate buffer (pH 7.2) was added, and then 50 μL of initiator (4,4'-azobis(4-cyanovaleric acid), ACVA) in DMAc (150 mg / mL) was added. The reaction solutions were thoroughly mixed. The final reaction solution had a volume of approximately 2.0 mL and contained approximately 20 mg of ligand and approximately 7.5 mg of initiator.

[0240] Alternatively, to avoid the use of DMAc, ACVA was dissolved at a concentration of 5 mg / mL in reaction buffer (2M phosphate, pH 7.2). For low-salt experiments, the initiator was dissolved at a concentration of 7.5 mg / mL in 0.5M phosphate.

[0241] A 4 x 7 cm membrane coupon (pre-moistened in water) was added to a bag containing the coupling reaction product. The bag was shaken for 1 minute, and then irradiated with UV light (approximately 365 nm) for 10 minutes. After irradiation, the coupling solution was decanted, and then 15-20 mL of washing buffer (0.1 M phosphate, pH 7.2) was added, and the membrane was placed on a shaker for 10-15 minutes. The washing cycle was repeated three times, and then the membrane was either (i) transferred to 8 mL of trehalose solution (10 wt%), shaken for 10-15 minutes, and dried in an oven (50°C) for 20-30 minutes, or (ii) stored in 0.1 M phosphate buffer.

[0242] For coupling in the presence of additives, ACVA was dissolved in 0.5 M potassium phosphate (pH 7.2) to prepare a solution with a concentration of 7.5 mg / mL. Protein A ligand was dissolved in 20 mM sodium phosphate buffer (pH 7.2) to prepare a stock solution of 50 mg / mL. In each of the three small bags (5 × 8 cm), 0.25 mL of ligand stock solution was mixed with 0.25 mL of initiator solution, and 50 μL of additives were added (cysteamine-HCl in reaction bag B, and 1-mercaptoethanol in reaction bag C).

[0243] After thoroughly mixing the reaction solution, 25 mm diameter membrane discs were placed in each bag, the reaction bags were shaken well, and then irradiated with UV light for 10 minutes. The reaction solution was decanted, and then the membrane coupons were washed three times with 0.1 M sodium phosphate buffer (pH 7.2) and shaken for 10-15 minutes. As outlined above, the composite membrane coupons were stored in buffer (0.1 M sodium phosphate, pH 7.2) and tested for bioaffinity to IgG protein.

[0244] [Example 3 - Flow-through ligand conjugation effect] A sample disc of a wetted membrane containing pendant-reactive functional groups was placed in a stainless steel holder and mounted in an AKTA chromatography system. The affinity ligand solution was pumped into the membrane holder at a specified flow rate for a predetermined time. Subsequently, a washing solution was pumped into the holder, followed by a quenching solution containing a reactive compound that converts residual membrane pendant groups into non-reactive groups. Finally, the washing solution was pumped into the holder. The pump was stopped, the holder was removed from the AKTA system, and the holder was disassembled to extract the conjugated membrane. The human IgG dynamic binding capacity of the conjugated membrane (protein A affinity ligand) was measured at a flow rate of 10 membrane volumes / min and compared to membranes conjugated using a batch non-flow-through method (see Figure 3). When flow-through conjugation was used, the membrane's dynamic binding capacity was consistently observed to be higher.

[0245] [Example 4 - Protein A coupling by flow through a membrane stack without periodic flow dividers] A header containing porous frit was attached to the bottom of a 44mm inner diameter glass chromatography column (VANTAGE(R)L Laboratory Column VL 44x250, catalog number 96440250). A circular membrane with a diameter of 30mm and a thickness of approximately 350 microns (volume 0.25mL) was then placed on the frit inside the column, at an equal distance from the column wall. The membrane had a surface containing epoxide groups. Next, a circular portion of a 44mm diameter polypropylene screen (1:2 twill weave, 500 micron mesh opening) was placed on the column and lowered so that it lay flat on the membrane and in contact with the inner wall of the column. Then, another 30mm diameter membrane was placed in the column and lowered so that it lay flat on the screen and at an equal distance from the column wall. The process of placing a screen on a membrane and then another membrane on a screen was repeated until the column contained 100 membranes (total volume 25mL) layered between 99 screens. Next, the header was added to the top of the column and lowered until the membrane and screen layers were compressed to a height of 9.5 cm.

[0246] Next, a tube was added to the inlet at the bottom of the column and another tube was added to the outlet at the top of the column. The inlet tube was connected through a peristaltic pump, which allowed for a controlled flow of the solution through the column.

[0247] The air in the column and tubing was replaced with PBS buffer consisting of 20 mM sodium phosphate containing 150 mM sodium chloride at pH 7.4. The inlet tube was placed in a glass bottle containing 400 mL of PBS buffer, and the outlet tube was directed towards the waste. The pump was then started, and 200 mL of PBS buffer was flowed through the column at a rate of 50 mL / min for 4 minutes. The pump was stopped, and the column was inverted. The connections of the inlet and outlet tubes to the column were then reversed so that the inlet was again at the bottom of the column and the outlet was at the top of the column. The pump was started, and another 200 mL of PBS buffer was flowed through the column at a rate of 50 mL / min for 4 minutes.

[0248] The pump was stopped, and the inlet tube was transferred to a glass bottle containing 500 mL of coupling buffer consisting of 1.35 M potassium phosphate at pH 9.0. The outlet tube remained pointed towards the waste. The pump was started, and 200 mL of coupling buffer was flowed through the column at a rate of 50 mL / min for 4 minutes. The pump was stopped, and the outlet tube was placed in the same glass bottle as the inlet tube, containing the remaining 300 mL of coupling buffer. In this configuration, where the inlet and outlet tubes are in the same bottle, the solution can be recirculated through the column multiple times. By recirculating the solution through the column, the reaction time can be extended without increasing the required volume of solution.

[0249] To a glass bottle containing the remaining 300 mL of coupling buffer, 82.4 mL of the PrA ligand stock solution at a concentration of 25.8 g / L in water was added. After starting the pump, the PrA solution was recirculated through the column at a flow rate of 50 mL / min for 4 hours. The total volume of the PrA solution recirculated through the column was calculated to be 422.4 mL, which consisted of the addition of the 300 mL of coupling buffer remaining in the glass bottle, 40 mL of coupling buffer remaining in the column / tube, and 82.4 mL of the PrA stock solution. Assuming that an 82.4 mL stock PrA ligand solution containing 2.12 g of PrA ligand was diluted to a volume of 422.4 mL, the PrA solution recirculated in the system was calculated to have a concentration of 5 g / L. The ligand loading onto the membrane was calculated by dividing the total mass of 2.12 g of PrA ligand by the total membrane deposition of 25 mL, resulting in a ligand loading of 85 g / L.

[0250] After 4 hours, the pump was stopped, the outlet tube was directed towards the waste, and the outlet pipe was directed towards the waste. The inlet tube was placed into a glass bottle containing 200 mL of PBS buffer. The pump was started, and the PBS buffer was flowed through the column at a flow rate of 50 mL / min for 4 minutes.

[0251] Next, the pump was stopped, and the inlet tube was placed into a glass bottle containing 800 mL of 1M ethanolamine. The outlet tube remained directed towards the waste. The pump was started, and 200 mL of 1M ethanolamine was flowed through the column and discarded at a flow rate of 50 mL / min for 4 minutes. The pump was stopped, and the outlet tube was directed towards a glass bottle containing the remaining 600 mL of 1M ethanolamine solution. Then, the pump was started, and the remaining 600 mL of 1M ethanolamine solution was recirculated through the column at a flow rate of 50 mL / min for 3 hours.

[0252] The pump was stopped, and the outlet tube was directed towards the waste. The inlet tube was placed into a glass bottle containing 200 mL of PBS buffer. The pump was started, and the PBS buffer was flowed through the column at a flow rate of 50 mL / min for 4 minutes.

[0253] The pump was stopped and the top column header was removed. The membranes were removed and stored in PBS buffer. The flux and IgG dynamic binding capacity of several membranes at different positions in the stack were determined. Position #1 was closest to the column inlet, and position #100 was closest to the column outlet. It was found that there was no significant shift in membrane flux even when the position was varied (Table 1). The membranes at positions 60 and 80 had much lower IgG dynamic binding capacities, suggesting that the flow distribution in the column was not uniform.

[0254] [Table 2]

[0255] [Example 5 - Protein A coupling by flow through a stack of membranes with periodic flow dividers] A header containing porous frit was attached to the bottom of a 44 mm inner diameter glass chromatography column (VANTAGE(R)L Laboratory Column VL 44x250, catalog number 96440250). Next, a flow divider was constructed from a circular, impermeable plastic sheet approximately 0.025 inches thick and 44 mm in diameter, with a 6 mm diameter hole in the center, and lowered flat over the porous frit. Then, two circular portions of a 44 mm diameter polypropylene screen (1:2 twill weave, 500 micron mesh opening) were placed on top of the flow divider. Next, a circular membrane with a diameter of 30 mm and a thickness of approximately 350 microns (volume 0.25 mL) was placed on the screen, equal to the distance from the column wall. The membrane had a surface containing epoxide groups. Another circular portion of polypropylene screen was lowered flat over the membrane. The process of adding membrane layers, followed by polypropylene screen layers, was repeated nine more times until the column had the following composition shown in Figure 4.

[0256] The process of assembling layers in the column as described above was repeated nine more times until the column contained 100 membrane layers (total volume 25 mL), 120 screen layers, and 10 flow divider layers. Then, additional flow divider layers were added to the stack, and a header with porous frit was added to the top of the column. The header was lowered until the membrane, screen, and flow divider layers were compressed to a height of 10.5 cm.

[0257] Next, a tube was added to the inlet at the bottom of the column and another tube was added to the outlet at the top of the column. The inlet tube was connected through a peristaltic pump, which allowed for a controlled flow of the solution through the column.

[0258] The air in the column and tubing was replaced with PBS buffer consisting of 20 mM sodium phosphate containing 150 mM sodium chloride at pH 7.4. The inlet tube was placed in a glass bottle containing 400 mL of PBS buffer, and the outlet tube was directed towards the waste. The pump was then started, and 200 mL of PBS buffer was flowed through the column at a rate of 50 mL / min for 4 minutes. The pump was stopped, and the column was inverted. The connections of the inlet and outlet tubes to the column were then reversed so that the inlet was again at the bottom of the column and the outlet was at the top of the column. The pump was started, and another 200 mL of PBS buffer was flowed through the column at a rate of 50 mL / min for 4 minutes.

[0259] The pump was stopped, and the inlet tube was transferred to a glass bottle containing 500 mL of coupling buffer consisting of 1.35 M potassium phosphate at pH 9.0. The outlet tube remained pointed towards the waste. The pump was started, and 200 mL of coupling buffer was flowed through the column at a rate of 50 mL / min for 4 minutes. The pump was stopped, and the outlet tube was placed in the same glass bottle as the inlet tube, containing the remaining 300 mL of coupling buffer. In this configuration, where the inlet and outlet tubes are in the same bottle, the solution can be recirculated through the column multiple times. By recirculating the solution through the column, the reaction time can be extended without increasing the required volume of solution.

[0260] To the glass vial containing the remaining 300 mL of coupling buffer, 82.4 mL of PrA ligand stock solution at a concentration of 25.8 g / L in water was added. After starting the pump, the PrA solution was recirculated through the column at a flow rate of 50 mL / min for 4 hours. The total volume of PrA solution recirculated through the column was calculated to be 422.4 mL, consisting of the 300 mL of coupling buffer remaining in the glass vial, the 40 mL of coupling buffer remaining in the column / tube, and the addition of 82.4 mL of PrA stock solution. Assuming that the 82.4 mL of stock PrA ligand solution containing 2.12 g of PrA ligand was diluted to a volume of 422.4 mL, the PrA solution recirculated in the system was calculated to have a concentration of 5 g / L. The ligand load on the membrane was calculated by dividing the total mass of 2.12 g of PrA ligand by the total membrane deposition of 25 mL, resulting in a ligand load of 85 g / L.

[0261] After 4 hours, the pump was stopped and the outlet tube was directed towards the waste. The inlet tube was placed in a glass bottle containing 200 mL of PBS buffer. The pump was started and PBS buffer was flowed through the column at a flow rate of 50 mL / min for 4 minutes.

[0262] Next, the pump was stopped and the inlet tube was placed in a glass bottle containing 800 mL of 1 M ethanolamine. The outlet tube remained pointed towards the waste. The pump was started and 200 mL of 1 M ethanolamine was passed through the column at a flow rate of 50 mL / min for 4 minutes for waste. The pump was stopped and the outlet tube was pointed towards a glass bottle containing the remaining 600 mL of 1 M ethanolamine solution. Next, the pump was started and the remaining 600 mL of 1 M ethanolamine solution was recirculated through the column at a flow rate of 50 mL / min for 3 hours.

[0263] The pump was stopped and the outlet tube was directed towards the waste. The inlet tube was placed in a glass bottle containing 200 mL of PBS buffer. The pump was started and PBS buffer was flowed through the column at a flow rate of 50 mL / min for 4 minutes.

[0264] The pump was stopped and the top column header was removed. The membranes were removed and stored in PBS buffer. The flux and IgG dynamic binding capacity of several membranes at different positions in the stack were determined. Position #1 was closest to the column inlet, and position #100 was closest to the column outlet. It was found that there was no significant shift in membrane flux even when the position was varied (Table 2). Furthermore, no significant deviation was observed in the IgG dynamic binding capacity, which suggested that the flow separation was relatively uniform throughout the column. By adding a flow separation layer, uniform coupling between all epoxide membranes and PrA ligands in the stack was provided (Figures 5A and 5B).

[0265] [Table 3]

[0266] [Example 6 - Protein A coupling by tangential flow through a spiral winding roll of a membrane] A rectangular membrane (total membrane volume 53.6 mL) with a width of 24.5 cm, a length of 62.5 cm, and a thickness of 0.035 cm was wound around a cylindrical core with a diameter of 1.6 cm and a length of 25.4 cm, together with a rectangular plastic portion of a polypropylene screen (1:2 twill weave, 500 micron mesh opening) with a width of 25.4 cm. The membrane was centered so that there was a 0.45 cm gap between the edge of the membrane and the edge of the screen. The membrane and screen were wound around the core, with the screen in contact with the core. The membrane and screen were rolled until the entire membrane was completely covered by the screen layer. The screen layer was continued to be wound around the roll until the diameter of the roll was 32 mm. The screen layer was then cut. The roll was then slid into a glass chromatography column with an inner diameter of 32 mm (Vantage(R)L Laboratory Column VL 32×250, catalog number 96320250) so that the edges of the roll were 2.5 cm from both ends of the column. Next, headers containing porous frit were attached to the bottom and top of the column.

[0267] Next, a tube was added to the inlet at the bottom of the column and another tube was added to the outlet at the top of the column. The inlet tube was connected through a peristaltic pump, which allowed for a controlled flow of the solution through the column.

[0268] The air in the column and tubing was replaced with PBS buffer consisting of 20 mM sodium phosphate containing 150 mM sodium chloride at pH 7.4. The inlet tube was placed in a glass bottle containing 400 mL of PBS buffer, and the outlet tube was directed towards the waste. The pump was then started, and 200 mL of PBS buffer was flowed through the column at a rate of 40 mL / min for 5 minutes. The pump was stopped, and the column was inverted. The connections of the inlet and outlet tubes to the column were then reversed so that the inlet was again at the bottom of the column and the outlet was at the top of the column. The pump was started, and another 200 mL of PBS buffer was flowed through the column at a rate of 40 mL / min for 5 minutes.

[0269] The pump was stopped, and the inlet tube was transferred to a glass bottle containing 1064 mL of coupling buffer consisting of 1.35 M potassium phosphate at pH 9.0. The outlet tube remained pointed towards the waste. The pump was started, and 300 mL of coupling buffer was flowed through the column at a rate of 40 mL / min for 7.5 minutes. The pump was stopped, and the outlet tube was placed in the same glass bottle as the inlet tube, containing the remaining 764 mL of coupling buffer. In this configuration, where the inlet and outlet tubes are in the same bottle, the solution can be recirculated through the column multiple times. By recirculating the solution through the column, the reaction time can be extended without increasing the required volume of solution.

[0270] To the glass vial containing the remaining 764 mL of coupling buffer, 205.6 mL of PrA ligand stock solution at a concentration of 25.8 g / L in water was added. After starting the pump, the PrA solution was recirculated through the column at a flow rate of 40 mL / min for 4 hours. The total volume of PrA solution recirculated through the column was calculated to be 1062.6 mL, which consisted of the 764 mL of coupling buffer remaining in the glass vial, the 93 mL of coupling buffer remaining in the column / tube, and the addition of 205.6 mL of PrA stock solution. Assuming that the 205.6 mL of stock PrA ligand solution containing 5.3 g of PrA ligand was diluted to a volume of 1064 mL, the PrA solution recirculated in the system was calculated to have a concentration of 5 g / L. The ligand load on the membrane was calculated by dividing the total mass of 5.3 g of PrA ligand by the total membrane deposition of 53.6 mL, resulting in a ligand load of 99 g / L.

[0271] After 4 hours, the pump was stopped and the outlet tube was directed towards the waste. The inlet tube was placed in a glass bottle containing 200 mL of PBS buffer. The pump was started and PBS buffer was flowed through the column at a flow rate of 40 mL / min for 5 minutes.

[0272] Next, the pump was stopped and the inlet tube was placed in a glass bottle containing 600 mL of 1 M ethanolamine. The outlet tube remained pointed towards the waste. The pump was started and 200 mL of 1 M ethanolamine was passed through the column at a flow rate of 20 mL / min for 10 minutes for waste. The pump was stopped and the outlet tube was pointed towards a glass bottle containing the remaining 400 mL of 1 M ethanolamine solution. Next, the pump was started and the remaining 400 mL of 1 M ethanolamine solution was recirculated through the column at a flow rate of 20 mL / min for 170 minutes.

[0273] The pump was stopped and the outlet tube was directed towards the waste. The inlet tube was placed in a glass bottle containing 400 mL of PBS buffer. The pump was started and PBS buffer was flowed through the column at a flow rate of 40 mL / min for 10 minutes.

[0274] The pump was stopped and the upper and lower column headers were removed. Next, the membrane and screen winding rolls were removed from the column. The membrane was unwound and separated from the screen. Then, a circular portion of the membrane with a diameter of 30 mm was removed from the rectangular membrane sheet. Five circular portions of the membrane were removed from the following locations (Figure 6): 1. Center: Located in the center of the entire rectangular membrane sheet. 2. Core: Located in the center of the edge of the rectangular membrane sheet closest to the core during the coupling reaction. 3. Shell: Located in the center of the edge of the rectangular membrane sheet closest to the column during the coupling reaction. 4. Inlet: Located in the center of the edge of the rectangular membrane sheet closest to the column inlet during the coupling reaction. 5. Exit: Located in the center of the edge of the rectangular membrane sheet closest to the column inlet during the coupling reaction. 6. The five membranes extracted from each section were assembled into five 5-layer membrane chromatography devices with a total accessible membrane volume of 1.0 mL. As shown in Table 3, the pressure drop and IgG dynamic binding capacity of the five chromatography devices were determined at a flow rate of 10 membrane volumes per minute or 10 mL / min. All 1 mL devices were found to have very similar pressure drop and IgG dynamic binding capacity at all five different locations. These results suggest that tangential flow coupling between the epoxide membrane and the PrA ligand can be achieved uniformly throughout the membrane when interleaved with a polypropylene screen.

[0275] [Table 4]

[0276] Embedding by reference All U.S. patents and U.S. patent application publications cited herein are incorporated herein by reference.

[0277] Equivalents Those skilled in the art will recognize, or can confirm by routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims. The embodiments of the present invention include the following: [Embodiment 1] A method for coupling ligands to a functionalized composite material, a. A step of providing a functionalized composite material, wherein the functionalized composite material is arranged in a stack, tubular configuration, or spiral winding configuration on the same plane of sheets having the same extent, i. The support member comprising a plurality of pores extending through it, and ii. A macroporous crosslinked gel, wherein the macroporous crosslinked gel comprises a polymer formed from the reaction of one or more polymerizable monomers and one or more crosslinking agents, the macroporous crosslinked gel comprises a plurality of pendant-reactive functional groups, the macroporous crosslinked gel is located in the pores of the support member, and the macropores of the macroporous crosslinked gel are smaller than the pores of the support member. Steps including, b. A step of flowing a first solution at a first flow rate substantially through or substantially across the functionalized composite material, wherein the first solution comprises a plurality of first ligands, thereby forming a plurality of covalent bonds between the reactive functional group and the first ligands. Steps and methods, including [Embodiment 2] The method of Embodiment 1, wherein the pendant reactive functional group is selected from the group consisting of aldehydes, amines, carbon-carbon double bonds, carbon-carbon triple bonds, epoxides, hydroxyls, thiols, anhydrides, azides, reactive halogens, acid chlorides, and mixtures thereof. [Embodiment 3] The method of Embodiment 1, wherein the pendant reactive functional group is selected from the group consisting of carbon-carbon double bonds, carbon-carbon triple bonds, and thiols. [Embodiment 4] The method according to any of Embodiments 1 to 3, wherein the pendant reactive functional group is derived from a molecule containing a thiol functional group or a molecule containing an unsaturated carbon-carbon bond. [Embodiment 5] The pendant-reactive functional group is derived from a molecule containing a thiol functional group, and the molecule containing the thiol functional group is derived from 3-mercaptopropionic acid, 1-mercaptosuccinic acid, polypeptides containing cysteine ​​residues, proteins containing cysteine ​​residues, recombinant proteins containing cysteine ​​residues, bacterial immunoglobulin-binding proteins containing cysteine ​​residues, recombinant fusion proteins containing cysteine ​​residues, cysteamine, 1-thiohexitol, poly(ethylene glycol) 2-mercaptoethyl ether acetate, poly(ethylene glycol) methyl ether thiol, 1-thioglycerol, 2-naphthalenchiol, and The method of Embodiment 4, selected from the group consisting of phenyl-4-thiol, 3-amino-1,2,4-triazole-5-thiol, 5-(trifluoromethyl)pyridine-2-thiol, 1-[2-(dimethylamino)ethyl]-1H-tetrazole-5-thiol, 1-propanthol, 1-butanethiol, 1-pentanethiol, 1-hexanethiol, 1-octanthiol, 8-amino-1-octanthiol hydrochloride, 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro-1-octanthiol, 8-mercapto-1-octanol, and γ-Glu-Cys. [Embodiment 6] The method of Embodiment 5, wherein the molecule containing a thiol functional group is a polypeptide containing a cysteine ​​residue, a protein containing a cysteine ​​residue, a recombinant protein containing a cysteine ​​residue, a bacterial immunoglobulin-binding protein containing a cysteine ​​residue, and a recombinant fusion protein containing a cysteine ​​residue. [Embodiment 7] The method of Embodiment 6, wherein the molecule containing the thiol functional group is a protein containing a cysteine ​​residue. [Embodiment 8] The method of Embodiment 4, wherein the pendant-reactive functional group is derived from a molecule containing an unsaturated carbon-carbon bond, and the molecule containing an unsaturated carbon-carbon bond is selected from the group consisting of 1-octene, 1-hexine, 4-bromo-1-butene, allyldiphenylphosphine, allylamine, allyl alcohol, 3,4-dihydroxy-1-butene, 7-octen-1,2-diol, 3-allyloxy-1,2-propanediol, 3-butenic acid, 3,4-dehydro-L-proline, vinyl laurate, 1-vinyl-2-pyrrolidinone, vinyl cinnamate, acylamide, or acrylate. [Embodiment 9] The method of Embodiment 1, wherein the pendant reactive functional group is selected from the group consisting of aldehydes, amines, epoxides, hydroxyls, anhydrides, azides, reactive halogens, and acid chlorides. [Embodiment 10] The method of Embodiment 9, wherein the one or more monomers containing a pendant-reactive functional group are selected from the group consisting of glycidyl methacrylate, acrylamide oxime, acrylic anhydride, azelaic anhydride, maleic anhydride, hydrazide, acryloyl chloride, 2-bromoethyl methacrylate, and vinyl methyl ketone. [Embodiment 11] The method of Embodiment 9, wherein the pendant-reactive functional group is an amine. [Embodiment 12] The method of Embodiment 9, wherein the pendant reactive functional group is an epoxide. [Embodiment 13] The method of Embodiment 9, wherein the pendant reactive functional group is hydroxyl. [Embodiment 14] The method according to any of Embodiments 1 to 13, wherein the first ligand comprises the first functional group. [Embodiment 15] The method of Embodiment 14, wherein the first ligand further comprises at least one graft-terminated group, and the first functional group is selected from the group consisting of cationic, anionic, hydrophobic, hydrophilic, thiophilic, hydrogen bond donor, hydrogen bond acceptor, π-π bond donor, π-π bond acceptor, metal chelate, biological molecule, and biological ion. [Embodiment 16] The method of Embodiment 15, wherein the first functional group is selected from the group consisting of cationic, anionic, hydrophobic, hydrophilic, thiophyllic, hydrogen bond donor, hydrogen bond acceptor, π-π bond donor, and π-π bond acceptor. [Embodiment 17] The molecule contains a first functional group, and the molecule is 2-(diethylamino)ethyl methacrylate, 2-aminoethyl methacrylate, 2-carboxyethyl acrylate, 2-(methylthio)ethyl methacrylate, acrylamide, N-acryloxysuccinimide, butyl acrylate or methacrylate, N,N-diethylacrylamide, N,N-dimethylacrylamide, 2-(N,N-dimethylamino)ethyl acrylate or methacrylate, N-[3-(N,N-dimethylamino)propyl]methacrylamide, N,N-dimethylacrylamide, ethyl acrylate or methacrylate, 2-ethylhexyl methacrylate, hydroxypropyl methacrylate, glycidyl acrylate or methacrylate, ethylene glycol phenyl ether methacrylate, methacrylamide, methacrylic anhydride, propyl acrylate or methacrylate, N- The method of Embodiment 15, selected from the group consisting of sopropylacrylamide, styrene, 4-vinylpyridine, vinylsulfonic acid, N-vinyl-2-pyrrolidinenon (VP), acrylamido-2-methyl-1-propanesulfonic acid, styrenesulfonic acid, alginic acid, (3-acrylamidopropyl)trimethylammonium halide, diallyldimethylammonium halide, 4-vinyl-N-methylpyridinium halide, vinylbenzyl-N-trimethylammonium halide, methacryloxyethyltrimethylammonium halide, 3-sulfopropyl methacrylate, 2-(2-methoxy)ethyl acrylate or methacrylate, hydroxyethylacrylamide, N-(3-methoxypropylacrylamide), N-[tris(hydroxymethyl)methyl]acrylamide, N-phenylacrylamide, N-tert-butylacrylamide, or diacetoneacrylamide. [Embodiment 18] The method of Embodiment 15, wherein the first functional group is a metal chelate functional group. [Embodiment 19] The method of Embodiment 15, wherein the first functional group comprises a metal chelate functional group selected from the group consisting of octadentate, hexadentate, tetradentate, tridentate, and bidentate iminodicarboxylic acids and iminodiacetic acids. [Embodiment 20] The method of Embodiment 15, wherein the first functional group is a biological molecule or a biological ion. [Embodiment 21] The method of Embodiment 15, wherein the first functional group comprises a biological molecule or biological ionic functional group selected from the group consisting of albumin, lysozyme, virus, cell, human and animal γ-globulin, immunoglobulin of both human and animal origin, recombinant or naturally occurring proteins including synthetic or naturally occurring polypeptides, interleukin-2 and its receptor, enzyme, monoclonal antibody, antigen, lectin, bacterial immunoglobulin-binding protein, trypsin and its inhibitor, cytochrome C, myoglobulin, recombinant human interleukin, recombinant fusion protein, protein A, protein G, protein L, peptide H, nucleic acid-derived products, synthetic or naturally occurring DNA, and synthetic or naturally occurring RNA. [Embodiment 22] The method according to any one of Embodiments 15 to 21, wherein the at least one graft terminal group is selected from the group consisting of aldehydes, amines, carbon-carbon double bonds, carbon-carbon triple bonds, epoxides, hydroxyls, thiols, and mixtures thereof. [Embodiment 23] The method according to any one of embodiments 15 to 21, wherein at least one of the graft terminal groups is an aldehyde. [Embodiment 24] The method according to any one of embodiments 15 to 21, wherein at least one of the graft terminal groups is an amine. [Embodiment 25] The method according to any one of embodiments 15 to 21, wherein the at least one graft terminal group is a carbon-carbon double bond or a carbon-carbon triple bond. [Embodiment 26] The method according to any one of embodiments 15 to 21, wherein at least one of the graft terminal groups is an epoxide. [Embodiment 27] The method according to any one of embodiments 15 to 21, wherein the at least one graft terminal group is a hydroxyl group. [Embodiment 28] The method according to any one of embodiments 15 to 21, wherein the at least one graft terminal group is a thiol. [Embodiment 29] c. Any method of Embodiments 1 to 28, further comprising the step of removing excess first ligand by flowing a first cleaning solution at a second flow rate substantially through or substantially across the composite material. [Embodiment 30] d. A step of flowing a quenching solution at a third flow rate substantially through or substantially across the composite material, wherein the quenching solution comprises a reactive compound that converts any residual pendant reactive functional group into a non-reactive group, e. A method according to any of embodiments 1 to 29, further comprising the step of optionally flowing a second cleaning solution at a fourth flow rate substantially through or substantially across the composite material to remove any residual reactive compounds. [Embodiment 31] A method of any of embodiments 1 to 30, wherein the first solution from step b. is recirculated through or across the composite material. [Embodiment 32] The method of embodiment 30 or 31, wherein the quenched solution from step d. is recirculated through or across the composite material. [Embodiment 33] c. Optionally, the step of flowing a first cleaning solution at a second flow rate substantially through or substantially across the composite material to remove excess first ligands, d. A step of flowing a second solution at a third flow rate substantially through or substantially across the functionalized composite material, wherein the second solution comprises a plurality of second ligands comprising at least three reactive groups, the second ligands being a crosslinking agent and optionally a polymerizable monomer comprising at least two pendant reactive functional groups, thereby forming a plurality of covalent bonds between the reactive functional groups and the second ligands. Any method of embodiments 1 to 28, further including the above. [Embodiment 34] The method of embodiment 33, wherein the second ligand is added in at least two separate steps. [Embodiment 35] The method of Embodiment 33, wherein the polymerizable monomer containing at least two pendant-reactive functional groups is added in at least two separate steps. [Embodiment 36] The method according to any of embodiments 33 to 35, wherein the second ligand includes a second functional group. [Embodiment 37] The method of Embodiment 36, wherein the second ligand further comprises at least one graft-terminated group, and the second functional group is selected from the group consisting of cationic, anionic, hydrophobic, hydrophilic, thiophilic, hydrogen bond donor, hydrogen bond acceptor, π-π bond donor, π-π bond acceptor, metal chelate, biological molecule, and biological ion. [Embodiment 38] The method of Embodiment 37, wherein the second functional group is selected from the group consisting of cationic, anionic, hydrophobic, hydrophilic, thiophilic, hydrogen bond donor, hydrogen bond acceptor, π-π bond donor, and π-π bond acceptor. [Embodiment 39] The method of Embodiment 37, wherein the second ligand comprises a biological molecule or biological ion having at least one graft-terminated group selected from the group consisting of amine, hydroxyl, and thiol functional groups. [Embodiment 40] The method of Embodiment 39, wherein the biological molecule or biological ion is selected from the group consisting of albumin, lysozyme, virus, cell, human and animal γ-globulin, immunoglobulin of both human and animal origin, recombinant or naturally occurring proteins including synthetic or naturally occurring polypeptides, interleukin-2 and its receptor, enzyme, monoclonal antibody, antigen, lectin, bacterial immunoglobulin-binding protein, trypsin and its inhibitor, cytochrome C, myoglobulin, recombinant human interleukin, recombinant fusion protein, protein A, protein G, protein L, peptide H, nucleic acid-derived products, synthetic or naturally occurring DNA, and synthetic or naturally occurring RNA. [Embodiment 41] The method of Embodiment 39, wherein the biological molecule or biological ion is selected from the group consisting of human and animal-derived γ-globulin, immunoglobulin of both human and animal origin, recombinant or naturally occurring proteins including synthetic or naturally occurring polypeptides, monoclonal antibodies, antigens, bacterial immunoglobulin-binding proteins, recombinant fusion proteins, protein A, protein G, protein L, and peptide H. [Embodiment 42] The method according to any one of Embodiments 39 to 41, wherein the second ligand is a polypeptide, a protein, a recombinant protein, a bacterial immunoglobulin-binding protein, a recombinant fusion protein, protein A, protein G, protein L, and peptide H. [Embodiment 43] The method according to any one of embodiments 33 to 42, wherein the polymerizable monomer containing at least two pendant-reactive functional groups is poly(ethylene glycol) divinyl ether. [Embodiment 44] e. Any method of Embodiments 33 to 43, further comprising the step of flowing a second cleaning solution at a fourth flow rate substantially through or substantially across the composite material to remove any excess second ligand and optionally any excess polymerizable monomer. [Embodiment 45] f. A step of flowing a quenching solution at a fifth flow rate substantially through or substantially across the composite material, wherein the quenching solution comprises a reactive compound that converts any residual pendant reactive functional group into a non-reactive group. g. Optionally, a step of flowing a third cleaning solution at a sixth flow rate substantially through or substantially across the composite material to remove any residual reactive compounds, Any method of embodiments 33 to 44, further including the above. [Embodiment 46] A method according to any of embodiments 1 to 45, wherein the composite material is arranged in a substantially coplanar stack of sheets having substantially the same extent. [Embodiment 47] The composite material comprises 2 to 300 separate support members, according to any of embodiments 1 to 46. [Embodiment 48] The composite material is arranged in a tubular configuration according to any of the embodiments 1 to 45. [Embodiment 49] A method according to any of embodiments 1 to 45, wherein the composite material is arranged in a substantially spiral winding configuration. [Embodiment 50] The method according to any one of Embodiments 1 to 49, wherein the support member comprises a polymer material selected from the group consisting of polysulfone, polyethersulfone, polyphenylene oxide, polycarbonate, polyester, cellulose, and cellulose derivatives. [Embodiment 51] The method according to any of Embodiments 1 to 50, wherein the composite material is a film. [Embodiment 52] A method according to any of embodiments 1 to 51, wherein the composite material is in contact with one or more interleaf layers. [Embodiment 53] The method of Embodiment 52, wherein one or more interleaf layers are selected from the group consisting of screen, polypropylene, polyethylene, and paper. [Embodiment 54] The method of embodiment 52 or 53, wherein one or more interleaf layers are in contact with one or more diversion layers. [Embodiment 55] The method according to any one of Embodiments 1 to 54, wherein the crosslinked gel is a neutral hydrogel, a charged hydrogel, a polymer electrolyte gel, a hydrophobic gel, a neutral gel, or a gel containing a functional group. [Embodiment 56] The method according to any one of Embodiments 1 to 55, wherein the macroporous crosslinked gel contains macropores having an average size of 10 nm to 3000 nm. [Embodiment 57] The method according to any of embodiments 1 to 56, wherein the pores of the support member have an average pore diameter of about 0.1 μm to about 50 μm. [Embodiment 58] A method according to any of embodiments 1 to 57, wherein the fluid channel substantially passes through the composite material. [Embodiment 59] A method according to any of embodiments 1 to 58, wherein the fluid channel substantially crosses the composite material. [Embodiment 60] h. Any method of Embodiments 1 to 59, further comprising the step of causing a portion of the substance to be adsorbed or absorbed onto the composite material by flowing a first fluid containing the substance at a seventh flow rate substantially through or substantially across the composite material. [Embodiment 61] The method of Embodiment 60, wherein the first fluid further comprises a fragmentation antibody, an agglutinating antibody, a host cell protein, a polynucleotide, an endotoxin, or a virus. [Embodiment 62] The method of embodiment 60 or 61, wherein the fluid passage of the first fluid substantially passes through the composite material. [Embodiment 63] The method of embodiment 60 or 61, wherein the fluid channel of the first fluid substantially crosses the composite material. [Embodiment 64] A method according to any of embodiments 60 to 63, wherein substantially all of the substance is adsorbed or absorbed onto the composite material after the first fluid has flowed substantially through or substantially across the composite material. [Embodiment 65] i. Any method of embodiments 60 to 64, further comprising the step of bringing a second fluid into contact with a substance adsorbed or absorbed on the composite material at an eighth flow rate, thereby releasing a portion of the substance from the composite material. [Embodiment 66] The method of embodiment 65, wherein the fluid channel of the second fluid substantially passes through the macropores of the composite material. [Embodiment 67] The method of embodiment 65, wherein the fluid channel of the second fluid is substantially perpendicular to the macropores of the composite material. [Embodiment 68] The method according to any one of embodiments 60 to 67, wherein the substance is a biological molecule, a biological ion, a virus, or a viral particle. [Embodiment 69] The method of Embodiment 68, wherein the biological molecule or biological ion is selected from the group consisting of albumin, lysozyme, virus, cell, human and animal gamma globulin, human and animal immunoglobulin, hIgG, recombinant and naturally occurring proteins, synthetic and naturally occurring polypeptides, interleukin-2 and its receptor, enzyme, monoclonal antibody, trypsin and its inhibitor, cytochrome C, myoglobin, myoglobulin, α-chymotrypsinogen, recombinant human interleukin, recombinant fusion protein, nucleic acid-derived products, synthetic and naturally occurring DNA, and synthetic and naturally occurring RNA. [Embodiment 70] The method of Embodiment 69, wherein the biological molecule or biological ion is lysozyme, hIgG, myoglobin, human serum albumin, soybean trypsin inhibitor, transferase, enolase, ovalbumin, ribonuclease, egg trypsin inhibitor, cytochrome c, annexin V, or α-chymotrypsinogen. [Embodiment 71] A method according to any of embodiments 60 to 70, wherein the concentration of the substance in the first fluid is approximately 0.2 mg / mL to approximately 10 mg / mL. [Embodiment 72] The method according to any of Embodiments 1 to 71, wherein the first flow rate is approximately 1 membrane volume (MV) / min to approximately 75 MV / min. [Embodiment 73] The method according to any of embodiments 29 to 72, wherein the second flow rate is approximately 1 MV / min to approximately 75 MV / min. [Embodiment 74] The method according to any of embodiments 30 to 73, wherein the third flow rate is approximately 1 MV / min to approximately 75 MV / min. [Embodiment 75] The method according to any of embodiments 30 to 74, wherein the fourth flow rate is approximately 1 MV / min to approximately 75 MV / min. [Embodiment 76] The method according to any of embodiments 45 to 75, wherein the fifth flow rate is approximately 1 MV / min to approximately 75 MV / min. [Embodiment 77] The method according to any of embodiments 45 to 76, wherein the sixth flow rate is approximately 1 MV / min to approximately 75 MV / min. [Embodiment 78] The method according to any of embodiments 60 to 77, wherein the seventh flow rate is approximately 1 MV / min to approximately 75 MV / min. [Embodiment 79] The method according to any of embodiments 65 to 78, wherein the flow rate of the eighth method is approximately 1 MV / min to approximately 75 MV / min.

Claims

1. A method for coupling ligands to a composite material, a. A step of providing a composite material, wherein the composite material is arranged as a stack of sheets having the same extent on the same plane, or in a spiral winding configuration, and the sheets having the same extent on the same plane are periodically separated by a screen, or the spiral composite material is wound up by a screen, and the composite material is i. A support member comprising a plurality of pores extending through it, and ii. A macroporous crosslinked gel, wherein the macroporous crosslinked gel comprises a polymer formed from the reaction of one or more polymerizable monomers and one or more crosslinking agents, the macroporous crosslinked gel comprises a plurality of pendant-reactive functional groups, the macroporous crosslinked gel is located in the pores of the support member, and the macropores of the macroporous crosslinked gel are smaller than the pores of the support member. Steps including, b. A step of flowing one liquid or several solutions containing affinity ligands through or across the composite material, wherein the one liquid or several solutions react with pendant-reactive functional groups on the surface of the composite material to form a functionalized composite material having affinity ligands bonded to pendant-reactive functional groups on its surface, c. A step of flowing a first solution at a first flow rate through or across the functionalized composite material, wherein the first solution comprises a plurality of first ligands, thereby forming a plurality of bonds between the affinity ligands and the first ligands. A method wherein the pendant reactive functional group is selected from the group consisting of aldehydes, amines, carbon-carbon double bonds, carbon-carbon triple bonds, epoxides, hydroxyls, thiols, anhydrides, azides, reactive halogens, acid chlorides, and mixtures thereof.

2. The method according to claim 1, wherein the pendant reactive functional group is selected from the group consisting of carbon-carbon double bonds, carbon-carbon triple bonds, and thiols.

3. The method according to claim 1, wherein the pendant-reactive functional group is derived from a molecule containing an unsaturated carbon-carbon bond, and the molecule containing an unsaturated carbon-carbon bond is selected from the group consisting of 1-octene, 1-hexine, 4-bromo-1-butene, allyldiphenylphosphine, allylamine, allyl alcohol, 3,4-dihydroxy-1-butene, 7-octen-1,2-diol, 3-allyloxy-1,2-propanediol, 3-butenic acid, 3,4-dehydro-L-proline, vinyl laurate, 1-vinyl-2-pyrrolidinone, vinyl cinnamate, acylamide, or acrylate.

4. The method according to claim 1, wherein the pendant reactive functional group is selected from the group consisting of aldehydes, amines, epoxides, hydroxyls, anhydrides, azides, reactive halogens, and acid chlorides.

5. The method according to claim 4, wherein the one or more monomers containing a pendant-reactive functional group are selected from the group consisting of glycidyl methacrylate, acrylamide oxime, acrylic anhydride, azelaic anhydride, maleic anhydride, hydrazide, acryloyl chloride, 2-bromoethyl methacrylate, and vinyl methyl ketone.

6. The method according to claim 4, wherein the pendant reactive functional group is an amine.

7. The method according to claim 4, wherein the pendant reactive functional group is an epoxide.

8. The method according to claim 4, wherein the pendant reactive functional group is hydroxyl.

9. The method according to any one of claims 1 to 8, wherein the first ligand comprises a first functional group.

10. The method according to claim 9, wherein the first ligand further comprises at least one graft-terminal group, and the first functional group is selected from the group consisting of biological molecules and biological ions.

11. The method according to claim 10, wherein the first functional group comprises a biological molecule or biological ionic functional group selected from the group consisting of albumin, lysozyme, virus, cell, human and animal γ-globulin, immunoglobulin of both human and animal origin, recombinant or naturally occurring proteins including synthetic or naturally occurring polypeptides, interleukin-2 and its receptor, enzyme, monoclonal antibody, antigen, lectin, bacterial immunoglobulin-binding protein, trypsin and its inhibitor, cytochrome C, myoglobulin, recombinant human interleukin, recombinant fusion protein, nucleic acid-derived products, synthetic or naturally occurring DNA, and synthetic or naturally occurring RNA.

12. The method according to claim 1, wherein the stack of sheets having the same extent on the same plane further includes one or more flow dividers.

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