Method for coupling a ligand to a composite material

Abstract

Methods for coupling ligands to composite materials are disclosed. A covalent bond is formed between the functionalized composite material and the ligand as the ligand solution flows through or across the composite material. The composite material can be used as a chromatographic separation medium.

Description

[0001] This application is a divisional application of the patent application for invention with the international application number PCT / EP2020 / 082787, filed on November 20, 2020, the Chinese national application number 202080092416.4, and the invention title "Method for Coupling Ligands to Composite Materials". Background Art

[0002] Membrane-based water treatment methods were first introduced in the 1970s. Since then, membrane-based separation technologies have been used in many other industries. In the pharmaceutical and biotech industries, preparative chromatography, direct flow filtration (DFF), and tangential flow filtration (TFF) (including microfiltration, ultrafiltration, nanofiltration, and diafiltration) are well-established methods for separating dissolved molecules or suspended particles. For the separation and purification in biomolecule manufacturing, ultrafiltration (UF) membranes and microfiltration (MF) membranes have become essential. Biomolecule manufacturing, regardless of its scale, typically employs one or more steps using filtration. The attraction of these membrane separations lies in several features, including, for example, high separation capacity and simplicity, requiring only a pressure difference to be applied between the feed stream and the permeate. This simple and reliable one-step filtration of a sample into two parts makes membrane separation a valuable method for separation and purification.

[0003] Ligands conjugated to the fluid-accessible surface of composite materials, such as membranes, can be used in separation and purification methods. However, chemical modification of membranes is more challenging than that of resins. Resins can be easily suspended in solution, and thus resins can be modified in large reactors (where reagent diffusion into the resin is facilitated by stirring the suspension). Modifying membranes is more challenging because they must be supported during the modification process to avoid damaging the membrane structure. This can be achieved in a roll-to-roll process, where the membrane moves physically through a bath of reaction solution when the reaction kinetics are very fast. Modifying membranes with slower reaction chemistries, such as the conjugation of protein A ligands to activated membranes, requires longer reaction times. The longer reaction times make the roll-to-roll membrane modification method unworkable, which requires extremely slow movement of the membrane and thus extremely long processing times.

[0004] There is a need for composite material modification methods in which the reaction solution flows through the support assembly of the composite material over a long period of time. Increasing the coupling of ligands to the composite material increases the binding capacity of the affinity medium. The conjugation method should make full use of fast, efficient, and easily controllable reactions to couple ligands to the composite material. Summary of the Invention

[0005] In one aspect, the present invention relates to a method for coupling a ligand to a functionalized composite material, wherein the functionalized composite material is arranged in a coplanar stack of coextensive sheets, a tubular configuration, or a helically wound configuration, the method comprising the steps of:

[0006] a. Providing a functionalized composite material comprising:

[0007] i. A support member comprising a plurality of pores extending therethrough; and

[0008] ii. A macroporous crosslinked gel, wherein the macroporous crosslinked gel comprises a polymer formed by the reaction of one or more polymerizable monomers with 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; and

[0009] b. Flowing a first solution substantially through or substantially across the functionalized composite material at a first flow rate, wherein the first solution comprises a plurality of first ligands such that a plurality of covalent bonds are formed between the reactive functional groups and the first ligands. Description of the Drawings

[0010] Figure 1A A schematic diagram depicting an exemplary composite material stacked between interlayers (e.g., a screen), wherein fluid substantially flows across the layers of the functionalized composite material (tangential flow).

[0011] Figure 1B A schematic diagram depicting an exemplary composite material stacked between interlayers (e.g., a screen), wherein fluid substantially flows through the layers of the functionalized composite material (direct flow).

[0012] Figure 2 An exemplary composite material with interlayers in a helically wound configuration is shown.

[0013] Figure 3 The IgG dynamic binding capacity of a protein A affinity ligand membrane conjugated using a flow through method is shown compared to a membrane conjugated using a batch method at a flow rate of 10 membrane volumes per minute.

[0014] Figure 4 A sketch illustrating a composite material layer (i.e., a membrane), an interlayer (i.e., a screen), and a flow distribution layer assembled in a chromatography column. This pattern containing 10 membranes is repeated 9 more times until 100 membranes are assembled. Then, an additional flow distribution layer is added.

[0015] Figure 5A Shows the IgG dynamic binding capacity varying with the membrane position within the stack.

[0016] Figure 5B Shows the membrane flux varying with the membrane position within the stack.

[0017] Figure 6 Is a sketch illustrating different positions, where after tangential flow coupling of the protein A ligand on a spiral wound roll sandwiched with a screen, a circular section of the composite material is taken out from a rectangular membrane sheet. Detailed Description

[0018] Overview

[0019] The capacity of the affinity medium depends to a large extent on the amount of affinity ligand that can be conjugated to the fluid-accessible surface of the medium, such as the composite material. Conjugation methods that increase the coupling of the ligand to the composite material will increase the binding capacity. In some embodiments, these methods make full use of rapid, efficient, and easily controllable reactions to functionalize the composite material. In some embodiments, the composite material is an adsorptive macroporous chromatography membrane. In some embodiments, methods for affinity ligand conjugation that involve flowing the ligand solution directly through the membrane (i.e., dead-end flow) or across the membrane (i.e., tangential flow) result in affinity membranes with improved dynamic binding capacity compared to batch or static conjugation methods.

[0020] Chromatography membranes utilize rapid convective mass transfer mechanisms to facilitate rapid purification or separation operations. However, to maximize the productivity of these operations, the binding capacity of the membrane for the target compound must be maximized. In some embodiments, the present invention describes flowthrough or dead-end flow methods for conjugating ligands to membranes 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 crossflow or tangential flow methods for conjugating ligands to membranes 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 methods produce conjugated affinity chromatography membranes having a protein binding capacity greater than that achieved by using batch, non-flowing conjugation methods with similar buffer conditions and affinity ligands.

[0021] Flowthrough conjugation methods and crossflow conjugation methods result in consistently higher membrane binding capacities and can be carried out on equipment containing in-line measurement tools that allow real-time observation of the reaction progress and thereby optimization of the reaction process. The higher membrane binding capacity combined with rapid binding kinetics enables rapid, high-productivity chromatographic purification operations.

[0022] Definition

[0023] For convenience, before further describing the present invention, certain terms employed in the specification, examples, and appended claims are collected herein. These definitions should be construed in light of the remainder of the disclosure and will be understood by those of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0024] In describing the present invention, various terms are used in the specification. Standard terms are widely used in the fields of filtration, fluid conveyance, and general fluid handling.

[0025] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. For example, "an element" refers to one element or more than one element.

[0026] The terms "comprise" and "comprising" are used in an inclusive, open - ended sense, which means that additional elements may be included.

[0027] The term "including" is used to mean "including but not limited to". "Including" and "including but not limited to" are used interchangeably.

[0028] The term "affinity chromatography" refers to a separation method based on specific binding interactions between an immobilized ligand and its binding partner. Examples of specific binding interactions include, but are not limited to, antibody / antigen interactions, enzyme / substrate interactions, and enzyme / inhibitor interactions.

[0029] The term "affinity medium" refers to a material that contains multiple immobilized ligands. For example, a composite material containing covalently - bound ligands.

[0030] The term "polymer" refers to a macromolecule formed by the combination of repeating units (monomers). The term polymer also includes copolymers.

[0031] The term "copolymer" refers to a polymer of at least two or more different monomers. If the cross - linker is a bifunctional monomer, the copolymer can consist of the cross - linker and monomers.

[0032] 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.

[0033] The term "side-hanging reactive functional group" refers to a functional group that will form one or more covalent bonds with a ligand when the ligand solution comes into contact with the functional group. Examples of side-hanging reactive functional groups that will 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, imidates, and fluorophenyl esters. Examples of side-hanging reactive functional groups that will 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.

[0034] The term "ligand" refers to a molecule that binds to a specific binding partner. For example, a protein, antibody, hormone, or drug binds to a specific receptor.

[0035] As used herein, the term "protein A" or "PrA" refers to a bacterial protein, protein A derivative, or recombinant protein A from Staphylococcus aureus that has the ability to bind mammalian antibodies of the immunoglobulin G (IgG) class with high affinity. For example, protein A can be recovered from its natural source (e.g., Staphylococcus aureus). Protein A can be produced synthetically (e.g., by peptide synthesis or by recombinant technology), and its fragments and variants retain the ability to bind proteins having a CH2 / CH3 region (such as the Fc region). Protein A can be purchased commercially (e.g., from Repligen, Pharmacia, EMD Millipore, and Fermatech). The gene for protein A has been cloned and expressed in Escherichia coli, allowing for the production of large amounts of recombinant protein A and protein A derivatives.

[0036] The term "washing solution" in relation to the coupling method refers to a solution that will carry away the coupling reactants. That is, a solution that will remove any excess polymerizable monomer and any excess ligand.

[0037] The term "quenching solution" in relation to the coupling method is used to mean a solution containing a reactive compound that will covalently bond with any remaining side-hanging reactive functional groups to form non-reactive groups. That is, the reactive compound will convert any remaining side-hanging reactive functional groups into non-reactive groups.

[0038] 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 will not result in the formation of a covalent bond between the substance and the non-reactive group.

[0039] The term "buffer" refers to a solution that resists changes in pH through the action of its acid-base conjugate components. The various buffers that can be used in the methods described herein are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975). Different buffers maintain different pH ranges. For example, phosphate buffers are commonly used for pH values between 6.0 and 8.0, while for higher pH values, borate buffers can be used, and for lower pH values, carbonate buffers can be used. One of ordinary skill in the art will be able to readily identify the appropriate buffer to be used based on the pH to be maintained. Non-limiting examples of buffers that can be used in the methods according to the invention include MES buffer, MOPS buffer, MOPSO buffer, Tris buffer, HEPES buffer, phosphate buffer, acetate buffer, citrate buffer, succinate buffer, carbonate buffer, borate buffer, and ammonium salt buffer, and combinations thereof.

[0040] The term "crossflow" as related 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 filtration medium), and a portion of the fluid passing through such composite material has a velocity component that is "cross-wise" (i.e., perpendicular to the direction of the fluid flowing along the surface of such composite material).

[0041] The term "tangential flow" or "tangential filtration" is used to mean a fluid flow or filtration method in which the flowing fluid is directed substantially parallel (i.e., tangent) to the surface of a composite material (e.g., a filtration medium), and a portion of the fluid passes through such composite material to provide a permeate. The terms "tangential filtration" and "crossflow filtration" are often used interchangeably in the art.

[0042] The term "dead end" as related to fluid flow and filtration is used to mean a fluid flow or filtration configuration in which the flowing fluid is directed through a composite material (e.g., a filtration medium), and a portion of the fluid passing through such composite material has a velocity component that is through-going (i.e., parallel to the direction of the fluid flowing through such composite material).

[0043] The term "direct current" or "direct filtration" is used to mean a fluid flow or filtration method in which the flowing fluid is directed substantially through (i.e., up to) the surface of a composite material (e.g., a filtration medium), and most of the fluid passes through such composite material to provide a filtrate. The terms "direct filtration" and "dead-end filtration" are often used interchangeably in the art.

[0044] The term "permeate" is used to mean the portion of a fluid that passes through a filtration medium and exits through a first outlet port in a filtration device, the first outlet port being operably connected to such filtration medium. The term "decantate" is used to mean the portion of a fluid that flows along the surface of a filtration medium but does not pass through such filtration medium and exits through a second outlet port in the filtration device, the second outlet port being operably connected to such filtration medium.

[0045] Crossflow filtration and tangential filtration are well-known filtration methods. Reference can be made, for example, to U.S. Patent Nos. 5,681,464, 6,461,513, 6,331,253, 6,475,071, 5,783,085, 4,790,942 (the disclosures of which are incorporated herein by reference). Reference can also be made to "Filter and Filtration Handbook", 4th Ed., T. Christopher Dickenson, Elsevier Advanced Technology, 1997 (the disclosure of which is incorporated herein by reference).

[0046] As used herein, "bind-and-elute mode" refers to an operating method for chromatography in which buffer conditions are established such that both a target protein and undesired contaminants bind to a chromatography support or composite material. The fractionation of the target protein from other components is then achieved by changing the conditions such that the target protein and the contaminants are eluted separately. In certain embodiments, the membranes described herein can be used in "bind-and-elute mode", characterized by high conductivity, high volumetric flux, and high dynamic binding capacity at selectivity. In certain embodiments, the amount of the target protein in the eluate is reduced by about 50% to about 99%. In certain embodiments, the amount of the target protein aggregates in the eluate is reduced by about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.

[0047] As used herein, the term "flow-through mode" refers to an operating method for chromatography in which buffer conditions are established such that the intact target protein flows through the membrane after application, while contaminants are selectively retained. In certain embodiments, the membranes described herein can be used in "flow-through mode" during the process following Protein A purification to remove key contaminants (such as DNA, host cell protein (HCP), leached Protein A, unwanted aggregates, and viruses) in a single step.

[0048] The term "average pore size" of the macroporous crosslinked gel can be understood by those of ordinary skill in the art to be determined by any suitable method. For example, the average pore size can be estimated from environmental scanning electron microscopy (ESEM) images of the surface. ESEM can be a very simple and useful technique for characterizing microfiltration membranes. Clear and concise pictures of the membrane can be obtained from the top, cross-section, and bottom perspectives; the porosity and pore size distribution can be estimated from the pictures.

[0049] The "volume porosity" of the support member is determined by a simple calculation. For example, for a support member made of polypropylene, the external dimensions of the support member are measured, and the total volume [e.g., for a flat disc: V = πr 2 h, the volume of the support member (if it were solid or not porous)] is calculated. Then, the mass of the support member is determined. Since the density of polypropylene is known or can be determined from the Polymer Handbook, edited by Brandrup et al., Chapter VII, Wiley and Sons, New York, 1999, the volume porosity is calculated as follows:

[0050] Volume porosity = { (the volume of the support member (if solid)) - [ (the mass of the support member)

[0051] / (the density of polypropylene) ]} / (the volume of the support member (if solid)).

[0052] In this calculation, the void volume of the support member = (the volume of the external dimensions of the support member) - [ (the mass of the support member) / (the density of polypropylene) ]. For example, the density of polypropylene = 0.91 g / cm 3 .

[0053] The volume porosity ε of the composite material is an experimentally determined value for each composite material. It is calculated from the mass. The macroporous crosslinked gel is incorporated into the void volume of the support member. After drying to constant weight, the mass of the incorporated gel is measured. The differential specific volume of the polymer is known or can be determined from the Polymer Handbook, edited by Brandrup et al., 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 described above). Calculate the volume porosity of the gel:

[0054] ε = { (void volume of the support member) - [(gel mass) × (differential specific

[0055] volume of the gel polymer)]} / (void volume of the support member)

[0056] In some embodiments, the ligand is Protein A (PrA). PrA capture chromatography is an important step in the downstream purification of biotherapeutic monoclonal antibodies (mAbs). The PrA ligand selectively binds to the Fc and / or Fab binding domains on the mAb while allowing most impurities (host cell proteins, DNA, residual cell culture medium) to flow through the resin. The PrA medium is typically washed after the loading step to remove additional impurities and then the captured mAb is eluted at low pH. The purity of the mAb in the eluate is significantly greater and it has also been concentrated relative to the clarified cell culture. However, affinity media (e.g., those with PrA) are very expensive and must be used for many batches over several years to reduce the cost per batch. Ideally, the affinity medium can be used for the maximum number of capture chromatography cycles (∼200) for which it is used to purify a single batch of the target substance (e.g., mAb), which would significantly reduce the volume of PrA medium required to process a single batch. The PrA medium can then be disposed of after purifying a single batch of mAb, thereby eliminating the costs associated with storing the resin. Currently, most PrA media used in the downstream purification of biotherapeutic mAbs are in resin form. The slow mass transfer of the mAb into the porous resin structure requires long loading times, with residence times ranging from 2 min to 10 min. Reducing the residence time during the loading step significantly reduces the dynamic binding capacity of the PrA resin, which is defined as the mass of mAb loaded onto the chromatography medium divided by the volume of the chromatography medium. Longer loading times prevent the resin from being cycled more than a few times (2 - 4 times) per batch without extending the PrA capture chromatography step to several days.

[0057] PrA membranes can be loaded at much shorter residence times (e.g., 0.1 - 1 min), and thus provide the opportunity to rapidly cycle the capture chromatography step. In some embodiments, the rapid cycling of the PrA membrane allows for the purification of a much greater amount of mAb in the same amount of time, relative to the same volume of PrA resin. Thus, in a given time period, the rapid cycling of a small volume PrA membrane can be used to capture the same amount of mAb as a much larger volume of resin that is cycled fewer times. In some embodiments, the PrA membrane offers the potential to process a single batch of mAb over the full lifetime of the PrA membrane, thereby significantly reducing the upfront costs associated with establishing an mAb downstream purification process and eliminating the costs associated with storing the resin.

[0058] In some embodiments, Protein A is an affinity ligand. In some embodiments, Protein A is a protein, peptide, or recombinant protein that comprises 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., the thiol of Cys or the amine of Lys). In some embodiments, Protein A is a protein, peptide, or recombinant protein that comprises 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 that comprises 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 with a functionalized composite material.

[0059] In some embodiments, Protein A comprises multiple domains. In some embodiments, Protein A comprises 1, 2, 3, 4, 5, 6, 7, or more domains. In some embodiments, the Protein A domains are identical to each other. In some embodiments, the Protein A domains are different from each other. In some embodiments, Protein A is degradation-resistant.

[0060] 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 is suitable for use in an affinity chromatography resin or column that comprises a chromatographic solid support matrix covalently linked to Protein A.

[0061] Chemical modification of composite materials is more challenging than that of resins. For example, a roll-to-roll modification method for a membrane with a slow reaction chemistry would require the membrane to move very slowly through the reaction solution and a very long processing time, which is incompatible with large-scale modification.

[0062] In some embodiments, the ligand coupling methods disclosed herein can be used to modify composite materials such as membranes.

[0063] In one aspect, the present invention relates to a method for coupling ligands to a functionalized composite material, the method comprising the steps of:

[0064] a. Providing a functionalized composite material comprising:

[0065] i. A support member comprising a plurality of pores extending through the support member; and

[0066] ii. A macroporous crosslinked gel, wherein the macroporous crosslinked gel comprises a polymer formed by the reaction of one or more polymerizable monomers with 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; and

[0067] b. Flowing a first solution substantially through or substantially across the functionalized composite material at a first flow rate, wherein the first solution comprises a plurality of first ligands such that a plurality of covalent bonds are formed between the reactive functional groups and the first ligands;

[0068] wherein the functionalized composite material is arranged in a coplanar stack of coextensive sheets, a tubular configuration, or a helically wound configuration.

[0069] Exemplary functionalized composite materials

[0070] Composition of the gel

[0071] In some embodiments, the crosslinked gel can be formed by the in-situ reaction of one or more polymerizable monomers with one or more crosslinking agents. In certain embodiments, the gel can be formed by the reaction of one or more crosslinkable polymers with one or more crosslinking agents.

[0072] In some embodiments, the crosslinked polymer is macroporous. The porosity within the polymer can be increased during polymerization by the degree of crosslinking, solvent exclusion of polymer chains during polymer network formation, or some combination of both. In some embodiments, an increased concentration of crosslinking agent results in a macroporous crosslinked gel. In some embodiments, the porosity is affected by the degree of alteration of polymer-(diluent + monomer) interactions, the amount of crosslinking agent, the amount of diluent, the initiator concentration, and the polymerization temperature.

[0073] The degree of crosslinking in the polymer can be adjusted by tuning the monomer ratio. The chain length of the polymers in the polymer network and thus the degree of crosslinking can also be controlled by using specific monomers that impart specific physicochemical properties to the final polymer and the membrane. These "tuning" monomers can affect the interaction of the polymer chains with the solvent system. In addition, the hydrophilicity / hydrophobicity of these monomers affects the final water swelling properties of the resulting gel and the hydrophilic / hydrophobic surface properties of the polymer network.

[0074] To minimize the formation of composites without pores, the solvent system and monomers are chosen to ensure that there is sufficient driving force to exclude the growing polymer chains from the solution at a certain point, thereby forming macropores. Specifically, a mixture of a solvent and a nonsolvent is adjusted to provide a suitable reaction system that can initially dissolve all the reactants but acts as a poor solvent for the crosslinked polymer chains when the crosslinked polymer chains grow to a molecular weight greater than a certain value. A solvent system with too high a proportion of poor solvent (for the polymer chains) will cause rapid precipitation of the growing polymer chains, which reduces the porosity. The size of the macropores generally depends on the nature and concentration of the crosslinking agent, the nature of one or more solvents in which the gel is formed, the amount of any polymerization initiator or catalyst, and the nature and concentration of the porogen (if present). In some embodiments, the composite material can have a narrow pore size distribution.

[0075] In some embodiments, macroporous crosslinked gels are formed due to phase separation during free radical crosslinking polymerization of polymerizable monomers in the presence of an inert diluent. In some embodiments, the reaction system comprises a polymer network, a soluble polymer, and low molecular weight compounds (monomers and diluents) to form a macroporous crosslinked polymer. In some embodiments, the macroporous crosslinked gel swells only slightly in the solvent.

[0076] Typically, many highly porous and non-rigid polymeric materials are relatively weak and cannot withstand the pressures generated during typical membrane separation processes (e.g., liquid chromatography). Thus, to prepare a mechanically suitable membrane, in some embodiments, a composite material comprising both a porous substrate (such as a fabric substrate made of chemically inert polypropylene) and a porous crosslinked polymer is produced by directly synthesizing the polymer within the pores of the substrate.

[0077] In some embodiments, when examined using environmental scanning electron microscopy (ESEM), the composite material shows a well-connected gel network incorporated within the substrate fibers.

[0078] In some embodiments, the formation of high polymer density regions (also known as bundling or lateral aggregation of polymer chains) leaves macropores between the high polymer density regions. In some embodiments, the macroporous crosslinked gel has a heterogeneous appearance.

[0079] In certain embodiments, the composite materials used as membranes 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; 10,357,766; and U.S. Patent Application Serial Nos. 14 / 190,650, 16 / 055,786, and 16 / 516,500, all of which are hereby incorporated by reference in their entirety.

[0080] In certain embodiments, the present invention relates to any of the methods disclosed herein, wherein the functionalized composite material comprises:

[0081] i. A support member comprising a plurality of pores extending therethrough; and

[0082] ii. A macroporous crosslinked gel, wherein the macroporous crosslinked gel comprises a polymer formed by the reaction of one or more polymerizable monomers with 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.

[0083] In certain embodiments, the present invention relates to any of the methods disclosed herein, wherein the macroporous crosslinked gel of the composite material has macropores with an average diameter of from 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.

[0084] 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 a characteristic property of a given material, it is appropriate to define the macroporosity relative to the maximum porosity.

[0085] In certain embodiments, the present invention relates to any of the methods disclosed herein, wherein the macroporous crosslinked gel of the composite material is a neutral hydrogel, a charged hydrogel, a polyelectrolyte gel, a hydrophobic gel, a neutral gel, or a gel containing functional groups. In certain embodiments, the present invention relates to any of the methods disclosed herein, wherein the macroporous crosslinked gel of the composite material is a neutral hydrogel or a charged hydrogel; and the neutral hydrogel or charged hydrogel is selected from crosslinked poly(vinyl alcohol), poly(acrylamide), poly(isopropylacrylamide), poly(vinylpyrrolidone), poly(hydroxyethyl methacrylate), 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-acrylamidopropyl)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 of the methods disclosed herein, wherein the macroporous crosslinked gel of the composite material is a polyelectrolyte gel; and the polyelectrolyte gel is selected from crosslinked poly(acrylamido-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(algic acid) and its salts, poly[(3-acrylamidopropyl)trimethylammonium] salts, poly(diallyldimethylammonium) salts, poly(4-vinyl-N-methylpyridinium) salts, poly(vinylbenzyl-N-trimethylammonium) salts, and poly(ethyleneimine) and its salts. In certain embodiments, the present invention relates to any of the methods disclosed herein, wherein the macroporous crosslinked gel of the composite material is a hydrophobic gel; and the hydrophobic gel is selected from crosslinked polymers or copolymers of ethyl acrylate, n-butyl acrylate, propyl acrylate, octyl acrylate, dodecyl acrylate, octadecyl acrylamide, octadecyl acrylate, and styrene. In certain embodiments, the present invention relates to any of the methods disclosed herein, wherein the macroporous crosslinked gel of the composite material is a neutral gel; and the neutral gel is selected from crosslinked polymers or copolymers of acrylamide, N,N-dimethylacrylamide, N-methylacryloyl acrylamide, N-methyl-N-vinylacetamide, and N-vinylpyrrolidone.

[0086] In certain embodiments, the crosslinked composite material (e.g., a membrane) is further grafted with chemical functional groups or molecular species to provide a functionalized composite material. In certain embodiments, the crosslinked polymer can be functionalized by post-polymerization modification to form a functionalized composite material. In certain embodiments, the functionalized composite material comprising the functionalized crosslinked polymer can be coupled to a ligand by post-polymerization modification. In this two-step process, the excess pendant reactive functional groups are modified during a separate grafting step, e.g., the excess thiol or alkene groups generated during thiol-ene polymerization. By controlling the feed ratio of the monomer to the crosslinker, the final polymer can have an excess of pendant reactive functional groups. The pendant reactive functional groups of the functionalized composite material can then be used in a coupling reaction (such as a click reaction) to further modify the final polymer chemistry or functional groups. In certain embodiments, the crosslinked polymer in the composite material contains residual reactive groups (referred to as pendant reactive functional groups, such as thiols or unsaturated carbon-carbon bonds), and the reactive functional groups can be used to link various ligands via a coupling reaction. In certain embodiments, this method can be used to prepare polymer composite materials (e.g., membranes) containing various ligands that can be used in chromatography. For example, chromatographic separation of biomolecules (e.g., proteins). For example, this method can be used to introduce ion-exchange functional groups (e.g., carboxylates, sulfonates, quaternary ammonium salts, amines), hydrophobic interaction moieties (such as octyl groups by using 1-octanethiol or 1-octene), and biomolecules for bioaffinity chromatography (such as cysteine-protein A for monoclonal antibody purification) into the composite material (e.g., a membrane).

[0087] In some embodiments, the composite material exhibits high selectivity, high flow rate, low backpressure, is inexpensive and allows for long column life, short processing times, and overall operational flexibility.

[0088] In certain embodiments, the present invention relates to any of the foregoing composite materials, wherein the composite material is a membrane.

[0089] In certain embodiments, the present invention relates to any of the foregoing composite materials, wherein the water contact angle of the composite material is from about 50° to about 120°.

[0090] Porous support member

[0091] 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 volume porosity of the porous support member is from about 40% to about 90%. In some embodiments, the volume porosity of the porous support member is from about 50% to about 80%. In some embodiments, the volume porosity of the porous support member is about 50%, about 60%, about 70%, or about 80%.

[0092] In certain embodiments, the porous support is flat.

[0093] In certain embodiments, the porous support is disc-shaped.

[0094] Many porous substrates or membranes can be used as the support member. In some embodiments, the porous support member is made of a polymeric material. In certain embodiments, the support can be a polyolefin that can be obtained at low cost. In certain embodiments, the polyolefin can be poly(ethylene), poly(propylene), or poly(vinylidene fluoride). Extended polyolefin membranes prepared by thermally induced phase separation (TIPS) or nonsolvent induced phase separation are mentioned. In certain embodiments, the support member can be made of a natural polymer such as cellulose or its derivatives. In certain embodiments, suitable supports include polyethersulfone membranes, poly(tetrafluoroethylene) membranes, nylon membranes, cellulose ester membranes, glass fibers, or filter papers. In some embodiments, the support member comprises a polymeric material selected from polysulfone, polyethersulfone, polyphenylene oxide, polycarbonate, polyester, cellulose, and cellulose derivatives.

[0095] In certain embodiments, the porous support is composed of a woven fibrous material or a non-woven fibrous material (such as a polyolefin, such as polypropylene). Such fibrous woven or non-woven support members can have larger pore sizes than TIPS support members, up to about 75 μm in some cases. The larger pores in the support member allow for the formation of composites with larger macropores in the macroporous gel. Non-polymeric support members (such as ceramic-based supports) can also be used. The porous support member can assume various shapes and sizes.

[0096] In some embodiments, the support member is in the form of a membrane.

[0097] In some embodiments, the thickness of the support member is from about 10 μm to about 2000 μm, from about 10 μm to about 1000 μm, or from about 10 μm to about 500 μm. In some embodiments, the thickness of the support member is from about 30 μm to about 300 μm. In some embodiments, the thickness of the support member is about 30 μm, about 50 μm, about 100 μm, about 150 μm, about 200 μm, about 250 μm, or about 300 μm.

[0098] In some embodiments, the average pore diameter of the pores of the support member is from about 0.1 μm to about 50 μm. In some embodiments, the average pore diameter of the pores of the support member is from about 0.1 μm to about 25 μm. In some embodiments, the average pore diameter of the pores of the support member is from about 0.5 μm to about 15 μm. In some embodiments, the average pore diameter of the pores of the support member is 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.

[0099] In other embodiments, multiple porous support units can be combined (e.g., by stacking). In one embodiment, a stack of porous support membranes (e.g., 2 to 10 membranes) can be assembled prior to forming a gel within the voids of the porous support. In another embodiment, individual support member units are used to form a composite membrane, which is then stacked prior to use.

[0100] Relationship between the gel and the support member

[0101] The gel can be anchored within the support member. The term "anchored" is intended to mean that the gel remains within the pores of the support member, but the term is not necessarily limited to meaning that the gel is chemically bound to the pores of the support member. Although in some embodiments the gel can be grafted to the surface of the pores of the support member, the gel can be held by physical constraints imposed thereon by entanglement and tangling with the structural elements of the support member without actually being chemically grafted to the support member.

[0102] In certain embodiments, the crosslinked gel is macroporous. In these cases, because macropores are present in the gel that occupies the pores of the support member, the macropores of the gel must be smaller than the pores of the support member. Thus, the flow and separation characteristics of the composite material depend on the properties of the gel, but are to a large extent independent of the properties of the porous support member, provided that the size of the pores present in the support member is greater than the size of the macropores of the gel. The porosity of the composite material can be adjusted by filling the support member with a gel, the porosity of which depends in part or in whole on the nature and amount of the monomer or polymer, crosslinking agent, reaction solvent, and pore former (if used). The properties of the composite material are determined in part (if not entirely) by the properties of the gel. The end result is that the present invention provides control over the macropore size, permeability, and surface area of the composite material.

[0103] When present, the number of macropores in the composite material does not depend on the number of pores in the support material. The number of macropores in the composite material can be much larger 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. Exceptions are found in cases where there are large differences in the pore size and pore size distribution of the support member and in cases where macroporous gels with very small pore sizes and narrow pore size distribution ranges are sought. 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, support members with a slightly narrower pore size range can be used in these cases.

[0104] In certain embodiments, the present invention relates to any of the foregoing composite materials, wherein the composite material is relatively non-toxic.

[0105] Preparation of the composite material

[0106] In certain embodiments, the composite materials of the present invention can be prepared by a one-step process. In certain embodiments, these methods can use water or other environmentally friendly solvents as reaction solvents. In certain embodiments, the method can be rapid and can thus result in a simple and / or rapid manufacturing process. In certain embodiments, the preparation of the composite material can be inexpensive.

[0107] In certain embodiments, the composite material can be prepared by mixing one or more monomers, one or more crosslinking agents, one or more initiators, and optionally one or more pore-forming agents in one or more suitable solvents. In certain embodiments, the resulting mixture can be homogeneous. In certain embodiments, the mixture can be heterogeneous. In certain embodiments, the mixture can then be introduced into a suitable porous support, in which the gel formation reaction can occur.

[0108] In certain embodiments, a pore-forming agent can be added to the reactant mixture, where the pore-forming agent can be broadly described as a pore-forming additive. In certain embodiments, the pore-forming agent can be selected from solvents with poor thermodynamics and extractable polymers (e.g., poly(ethylene glycol)), surfactants, and salts.

[0109] In some embodiments, the gel-forming reaction must be initiated. In certain embodiments, the gel-forming reaction can be initiated by any known method (e.g., by thermal activation or exposure to UV radiation). In certain embodiments, the reaction can be initiated by UV radiation in the presence of a photoinitiator. In certain embodiments, the photoinitiator can be selected from 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 sulfonate. Thermal activation may require the addition of a thermal initiator. In certain embodiments, the thermal initiator can be selected from 1,1'-azobis(cyclohexanecarbonitrile) ( Catalyst 88), azobis(isobutyronitrile) (AIBN), potassium persulfate, ammonium persulfate, and benzoyl peroxide.

[0110] In certain embodiments, the gel-forming reaction can be initiated by UV radiation. In certain embodiments, a photoinitiator can be added to the reactants of the gel-forming reaction, and a support member containing a mixture of monomer, crosslinker, and photoinitiator can be exposed to UV radiation having a wavelength of from about 250 nm to about 400 nm for a period of seconds to hours. In certain embodiments, the support member containing the mixture of monomer, crosslinker, and photoinitiator can be exposed to UV radiation at about 350 nm for a period of seconds to hours. In certain embodiments, the support member containing the mixture of monomer, crosslinker, and photoinitiator can be exposed to UV radiation at about 350 nm for about 10 minutes. In certain embodiments, light of visible wavelength can be used to initiate polymerization. In certain embodiments, the support member must have low absorbance at the wavelength used such that energy can be transmitted through the support member.

[0111] In certain embodiments, the rate of polymerization may affect the size of the macropores obtained in the macroporous gel. In certain embodiments, when the crosslinker concentration in the gel increases to a sufficient concentration, the components of the gel begin to aggregate to produce regions of high polymer density and regions with little or no polymer, the latter regions being referred to herein as "macropores". This mechanism is affected by the rate of polymerization.

[0112] In certain embodiments, once the composites are prepared, they can be washed with various solvents to remove any unreacted components and any polymers or oligomers that are not anchored in the support. In certain embodiments, solvents suitable for washing the composites include water, acidic (e.g., HCl) or alkaline (e.g., NaOH) aqueous solutions, aqueous salt solutions (e.g., NaCl), acetone, methanol, ethanol, propanol, and DMF.

[0113] Exemplary method for coupling a ligand to the functionalized composite material

[0114] Provided herein are methods of coupling a ligand to a functionalized composite material by flowing a first solution comprising the ligand across or through the functionalized composite material.

[0115] In one aspect, the present invention relates to a method for coupling a ligand to a functionalized composite material, the method comprising the steps of:

[0116] a. providing a functionalized composite material, the functionalized composite material comprising:

[0117] i. a support member comprising a plurality of holes extending through the support member; and

[0118] ii. a macroporous cross-linked gel, wherein the macroporous cross-linked gel comprises a polymer formed by the reaction of one or more polymerizable monomers and one or more cross-linking agents; the macroporous cross-linked gel comprises a plurality of pendant reactive functional groups; the macroporous cross-linked gel is located in the pores of the support member; and the macropores of the macroporous cross-linked gel are smaller than the pores of the support member; and

[0119] b. flowing a first solution substantially through or substantially across the functionalized composite material at a first flow rate, wherein the first solution comprises a plurality of first ligands such that a plurality of covalent bonds are formed between the reactive functional groups and the first ligands;

[0120] wherein the functionalized composite material is arranged in a coplanar stack of coextensive sheets, a tubular configuration, or a spirally wound configuration.

[0121] In certain embodiments, the present invention is directed to providing any of the aforementioned functionalized composite materials.

[0122] In certain embodiments, the present invention relates to any of the aforementioned methods, wherein the first solution flows substantially across the functionalized composite material. In some embodiments, the fluid flow path is tangential to the surface of the functionalized composite material ( Figure 1A ). In some embodiments, tangential flow provides lower pressure drop and / or allows stacks consisting of multiple layers of composite materials to be coupled simultaneously.

[0123] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the first solution substantially flows through the functionalized composite material. In some embodiments, the fluid flow path passes directly through the functionalized composite material ( Figure 1B ). In some embodiments, as the number of composite material layers increases, the direct current provides an increased pressure drop. In some embodiments, the pressure drop limits the number of layers in the stack. In some embodiments, one or more interlayers are used to distribute the flow rate. In some embodiments, the direct current increases the mass transfer rate of the ligand into the porous composite material structure.

[0124] 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 can be functionalized by post-polymerization modification to form a functionalized composite material. In this two-step method, the excess pendant reactive functional groups are modified during a separate grafting step, for example, the excess thiol or olefin groups generated during thiol-ene polymerization. By controlling the feed ratio of the monomer to the crosslinker, the final polymer can have an excess of pendant reactive functional groups.

[0125] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the pendant reactive functional groups are selected from aldehydes, amines, carbon-carbon double bonds, carbon-carbon triple bonds, epoxides, hydroxyl groups, thiols, acid anhydrides, azides, reactive halogens, acyl chlorides, and mixtures thereof.

[0126] In some embodiments of the method, the pendant reactive functional groups are selected from carbon-carbon double bonds, carbon-carbon triple bonds, and thiols. In some embodiments, the pendant reactive functional groups are 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 groups are derived from a molecule containing a thiol functional group; and the molecule containing a thiol functional group is selected from 3-mercaptopropionic acid, 1-mercaptosuccinic acid, 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, a recombinant fusion protein containing a cysteine residue, cysteamine, 1-thiohexitol, poly(ethylene glycol) 2-mercaptoethyl ether acetate, poly(ethylene glycol) methyl ether thiol, 1-thioglycerol, 2-naphthalenethiol, 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-propanethiol, 1-butanethiol, 1-pentanethiol, 1-hexanethiol, 1-octanethiol, 8-amino-1-octanethiol hydrochloride, 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro-1-octanethiol, 8-mercapto-1-octanol, and γ-Glu-Cys.

[0127] In some embodiments, the molecule containing a thiol functional group is selected from 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.

[0128] 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 1-octene, 1-hexyne, 4-bromo-1-butene, allyldiphenylphosphine, allylamine, allyl alcohol, 3,4-dihydroxy-1-butene, 7-octene-1,2-diol, 3-allyloxy-1,2-propanediol, 3-butenoic acid, 3,4-dehydro-L-proline, vinyl laurate, 1-vinyl-2-pyrrolidone, vinyl cinnamate, amide, or acrylate.

[0129] In some embodiments, the pendant reactive functional group is selected from acyl chloride, acyl azide, aldehyde, amine, acid anhydride, azide, carbonate, carbon-carbon double bond, carbon-carbon triple bond, carbodiimide, carboxylic acid, disulfide, epoxide, fluorobenzene, fluorophenyl ester, haloacetyl, hydroxyl, imidate, isocyanate, isothiocyanate, maleimide, N-hydroxysuccinimide ester, pyridyl disulfide, reactive ester, reactive halogen, sulfonyl halide, thiol, and thiosulfate. In some embodiments, the pendant reactive functional group is selected from aldehyde, amine, epoxide, hydroxyl, acid anhydride, azide, reactive halogen, and acyl chloride. In some embodiments, the pendant reactive functional group is selected from aldehyde, amine, epoxide, and hydroxyl. In some embodiments, the pendant reactive functional group is selected from epoxide, aldehyde, carboxylic acid, reactive halogen, reactive ester, isocyanate, isothiocyanate, sulfonyl halide, carbodiimide, acyl azide, fluorobenzene, carbonate, N-hydroxysuccinimide ester, imidate, and fluorophenyl ester. In some embodiments, the pendant reactive functional group is selected from epoxide, aldehyde, carboxylic acid, reactive halogen, reactive ester, isocyanate, isothiocyanate, sulfonyl halide, carbodiimide, acyl azide, fluorobenzene, carbonate, N-hydroxysuccinimide ester, imidate, and fluorophenyl ester, and reacts with an amino group. In some embodiments, the pendant reactive functional group is selected from epoxide, thiol, disulfide, carbon-carbon double bond, carbon-carbon triple bond, maleimide, haloacetyl, pyridyl disulfide, thiosulfate, and reactive halogen. In some embodiments, the pendant reactive functional group is selected from epoxide, thiol, disulfide, carbon-carbon double bond, carbon-carbon triple bond, maleimide, haloacetyl, pyridyl disulfide, thiosulfate, and reactive halogen.

[0130] In some embodiments, one or more monomers comprising pendant reactive functional groups are selected from glycidyl methacrylate, acrylamide oxime, acrylic anhydride, azelaic anhydride, maleic anhydride, hydrazide, acryloyl chloride, 2-bromoethyl methacrylate, and vinyl methyl ketone.

[0131] In some embodiments, the pendant reactive functional group is an aldehyde. In some embodiments, one or more monomers comprising pendant reactive functional groups is vinyl methyl ketone.

[0132] In some embodiments, the pendant reactive functional group is an amine.

[0133] In some embodiments, the pendant reactive functional group is an epoxide. In some embodiments, one or more monomers comprising pendant reactive functional groups is glycidyl methacrylate.

[0134] In some embodiments, the pendant reactive functional group is a hydroxyl group.

[0135] In some embodiments, the first ligand comprises a first functionality. In some embodiments, the first ligand further comprises at least one grafting end group; and the first functionality is selected from cationic functional groups, anionic functional groups, hydrophobic functional groups, hydrophilic functional groups, thiophilic functional groups, hydrogen bond donating functional groups, hydrogen bond accepting functional groups, π-π bond donating functional groups, π-π bond accepting functional groups, metal chelating functional groups, biomolecules, and bioions. In some embodiments, the first functionality is selected from cationic functional groups, anionic functional groups, hydrophobic functional groups, hydrophilic functional groups, thiophilic functional groups, hydrogen bond donating functional groups, hydrogen bond accepting functional groups, π-π bond donating functional groups, and π-π bond accepting functional groups.

[0136] In some embodiments, individual functional groups are included by incorporating functional monomers. In some embodiments, the relative amounts of each functional group can be readily and easily adjusted to obtain optimal performance characteristics.

[0137] In some embodiments, the molecule comprises a first functional group, and the molecule is selected from 2-(diethylamino)ethyl methacrylate, 2-aminoethyl methacrylate, 2-carboxyethyl acrylate, 2-(methylthio)ethyl methacrylate, acrylamide, N-acryloxysuccinimide, butyl acrylate or butyl methacrylate, N,N-diethylacrylamide, N,N-dimethylacrylamide, 2-(N,N-dimethylamino)ethyl acrylate or 2-(N,N-dimethylamino)ethyl methacrylate, N-[3-(N,N-dimethylamino)propyl]methacrylamide, N,N-dimethylacrylamide, ethyl acrylate or ethyl methacrylate, 2-ethylhexyl methacrylate, hydroxypropyl methacrylate, glycidyl acrylate or glycidyl methacrylate, ethylene glycol phenyl ether methacrylate, methacrylamide, methacrylic anhydride, propyl acrylate or propyl methacrylate, N-isopropylacrylamide, styrene, 4-vinylpyridine, vinylsulfonic acid, N-vinyl-2-pyrrolidone (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, methacryloyloxyethyltrimethylammonium halide, 3-sulfopropyl methacrylate, 2-(2-methoxy)ethyl acrylate or 2-(2-methoxy)ethyl methacrylate, hydroxyethylacrylamide, N-(3-methoxypropylacrylamide), N-[tris(hydroxymethyl)methyl]acrylamide, N-phenylacrylamide, N-tert-butylacrylamide or diacetoneacrylamide.

[0138] In some embodiments, the first functional group is a metal chelating functional group. In some embodiments, the first functional group comprises a metal chelating functional group selected from: an octadentate functional group, a hexadentate functional group, a tetradentate functional group, a tridentate functional group, a bidentate functional group, iminodicarboxylic acid, iminodiacetic acid, and salts of iminodiacetic acid.

[0139] In some embodiments, the metal chelating functional group complexes with multiple metal ions. In some embodiments, the metal chelating functional group is selected from iminodicarboxylic acid, iminodiacetic acid, and salts of iminodiacetic acid that complex with multiple metal ions selected from transition metal ions, lanthanide ions, poor metal ions, and alkaline earth metal ions. In some embodiments, the metal chelating functional group is selected from iminodicarboxylic acid, iminodiacetic acid, and salts of iminodiacetic acid that complex with multiple metal ions selected from nickel, zirconium, lanthanum, cerium, manganese, titanium, cobalt, iron, copper, zinc, silver, gallium, platinum, palladium, lead, mercury, cadmium, and gold. In some embodiments, the metal chelating functional group is iminodiacetic acid or a salt of iminodiacetic acid that complexes with multiple metal ions, where the metal ions are nickel or zirconium.

[0140] In some embodiments, the first functional group is a biomolecule or a bioion. In some embodiments, the first functional group comprises a biomolecule or bioion functional group selected from the group consisting of: albumin, lysozyme, virus, cell, gamma-globulin from human and animal sources, immunoglobulin from human and animal sources, recombinant or natural source proteins including, synthetic or natural source polypeptides, interleukin-2 and its receptor, enzyme, monoclonal antibody, antigen, lectin, bacterial immunoglobulin-binding protein, trypsin and its inhibitor, cytochrome C, myosin, recombinant human interleukin, recombinant fusion protein, protein A, protein G, protein L, peptide H, nucleic acid derivatives, synthetic or natural source DNA, and synthetic or natural source RNA. In some embodiments, the first functional group comprises a biomolecule or bioion functional group selected from the group consisting of: gamma-globulin from human and animal sources, immunoglobulin from human and animal sources, recombinant or natural source proteins including, synthetic or natural source polypeptides, monoclonal antibody, bacterial immunoglobulin-binding protein, recombinant fusion protein, protein A, protein G, and protein L. In certain embodiments, the first functional group comprises a biomolecule or bioion functional group selected from the group consisting of: polypeptide, protein, recombinant protein, bacterial immunoglobulin-binding protein, recombinant fusion protein, protein A, protein G, protein L, and peptide H.

[0141] In some embodiments, the first functional group comprises protein A. In some embodiments, the first functional group comprises protein A, and the protein A includes 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, recombinant fusion proteins containing cysteine residues. In some embodiments, the first functional group comprises protein A, and the protein A is selected from proteins, peptides, or recombinant proteins that comprise a ligand that binds to a monoclonal antibody (e.g., 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, and the protein A is selected from proteins, peptides, or recombinant proteins that comprise 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, and the protein A is selected from proteins, peptides, or recombinant proteins that comprise 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.

[0142] In some embodiments, the first ligand comprises a first functional group and at least one graft end group, and the graft end group is selected from 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.

[0143] In certain embodiments, thiol-ene grafting is an attractive choice for cross-linking polymers for attaching biomolecules to membranes. The reaction is fast, can be performed effectively in aqueous media, performs well at room temperature, and can be phototriggered using relatively long wavelength light (365 nm), which has a very limited effect on protein bioactivity. In addition, it can allow controlled biomolecule attachment, which may be advantageous in terms of maintaining the bioactivity and 3D structure of the biomolecule.

[0144] In certain embodiments, any biomolecule with a free thiol functional group can be immobilized on the composite material described herein. This can be very useful for preparing bioaffinity membranes or biocatalytic membranes (by immobilizing one or more enzymes) for bioseparation. In certain embodiments, the composite material can be functionalized with oligonucleotide probes for DNA detection.

[0145] In certain embodiments, the present invention relates to any one of the aforementioned methods, further comprising the following steps:

[0146] c. flowing the first wash solution substantially through or substantially across the composite material at a second flow rate to remove excess first ligand.

[0147] In certain embodiments, the present invention relates to any one of the aforementioned methods, further comprising the following steps:

[0148] d. flowing a quench solution substantially through or across the composite material at a third flow rate, wherein the quench solution comprises a reactive compound to convert any remaining pendant reactive functional groups to non-reactive groups; and

[0149] e. Optionally, flowing a second wash solution substantially through or across the composite material at a fourth flow rate to remove any remaining reactive compounds.

[0150] In some embodiments, the first solution of step b. is recirculated through or across the composite material.

[0151] In some embodiments, the quenching solution of step d. is recycled through or across the composite material.

[0152] In certain embodiments, the present invention relates to any of the foregoing methods, which further comprise the steps of:

[0153] c. Optionally, flowing a first washing solution substantially through or substantially across the composite material at a second flow rate to remove any excess of the first ligand;

[0154] d. Flowing a second solution substantially through or substantially across the functionalized composite material at a third flow rate, wherein the second solution comprises a plurality of second ligands each comprising at least three reactive groups 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, wherein the second ligand is a crosslinker.

[0155] In some embodiments, the second ligand is added in at least two portions. In some embodiments, the polymerizable monomer comprising at least two pendant reactive functional groups is added in at least two portions.

[0156] In some embodiments, the second ligand comprises a second functional group. In some embodiments, the second ligand further comprises at least one grafting end group; and the second functional group is selected from cationic functional groups, anionic functional groups, hydrophobic functional groups, hydrophilic functional groups, thiophilic functional groups, hydrogen bond donating functional groups, hydrogen bond accepting functional groups, π-π bond donating functional groups, π-π bond accepting functional groups, metal chelating functional groups, biomolecules, and bioions. In some embodiments, the second functional group is selected from cationic functional groups, anionic functional groups, hydrophobic functional groups, hydrophilic functional groups, thiophilic functional groups, hydrogen bond donating functional groups, hydrogen bond accepting functional groups, π-π bond donating functional groups, and π-π bond accepting functional groups.

[0157] In some embodiments, the second ligand comprises a biomolecule or bioion, the biomolecule or bioion comprising at least one grafted end group selected from an amine, a hydroxyl and a thiol functional group. In some embodiments, the biomolecule or bioion is selected from the group consisting of albumin, lysozyme, virus, cell, γ-globulin of human and animal origin, immunoglobulin of human and animal origin, protein of recombinant or natural origin including, polypeptides of synthetic or natural origin, interleukin-2 and its receptor, enzyme, monoclonal antibody, antigen, lectin, bacterial immunoglobulin-binding protein, trypsin and its inhibitor, cytochrome C, myosin, recombinant human interleukin, recombinant fusion protein, protein A, protein G, protein L, peptide H, nucleic acid derivatives, DNA of synthetic or natural origin, and RNA of synthetic or natural origin. In some embodiments, the biomolecule or bioion is selected from the group consisting of: gamma-globulins of human and animal origin, immunoglobulins of human and animal origin, proteins of recombinant or natural origin, including polypeptides of synthetic or natural origin, 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, a protein, a recombinant protein, a bacterial immunoglobulin-binding protein, a recombinant fusion protein, protein A, protein G, protein L, and peptide H.

[0158] 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.

[0159] In some embodiments, the polymerizable monomer comprising at least two pendant reactive functional groups is poly(ethylene glycol) divinyl ether.

[0160] In certain embodiments, the present invention relates to any one of the aforementioned methods, further comprising the following steps:

[0161] e. flowing a second wash solution substantially through or substantially across the composite material at a fourth flow rate to remove any excess second ligand and optionally any excess polymerizable monomer.

[0162] In certain embodiments, the present invention relates to any one of the aforementioned methods, further comprising the following steps:

[0163] f. flowing a quench solution substantially through or substantially across the composite material at a fifth flow rate, wherein the quench solution comprises a reactive compound to convert any remaining pendant reactive functional groups to non-reactive groups; and

[0164] g. Optionally, flowing a third wash solution substantially through or substantially across the composite material at a sixth flow rate to remove any remaining reactive compounds.

[0165] In some embodiments, the present invention relates to a method for conjugating a ligand to a functionalized composite material, the method comprising the steps of:

[0166] a. Providing a functionalized composite material comprising:

[0167] i. A support member comprising a plurality of pores extending through the support member;

[0168] And

[0169] ii. A macroporous crosslinked gel, wherein the macroporous crosslinked gel comprises a polymer formed by the reaction of one or more polymerizable monomers with 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;

[0170] b. Flowing a first solution substantially through or substantially across the functionalized composite material at a first flow rate, wherein the first solution comprises a plurality of first ligands such that a plurality of covalent bonds are formed between the reactive functional groups and the first ligands;

[0171] c. Optionally, flowing a first wash solution substantially through or substantially across the composite material at a second flow rate to remove any excess first ligands;

[0172] d. Flowing a quenching solution substantially through or substantially across the composite material at a third flow rate, wherein the quenching solution comprises a reactive compound to convert any remaining pendant reactive functional groups into non-reactive groups; and

[0173] e. Optionally, flowing a second wash solution substantially through or substantially across the composite material at a fourth flow rate to remove any remaining reactive compound;

[0174] wherein the functionalized composite material is arranged in a coplanar stack of coextensive sheets, a tubular configuration, or a helically wound configuration.

[0175] In some embodiments, the present invention relates to a method for conjugating a ligand to a functionalized composite material, the method comprising the steps of:

[0176] a. Providing a functionalized composite material comprising:

[0177] i. A support member comprising a plurality of pores extending through the support member;

[0178] And

[0179] ii. A macroporous crosslinked gel, wherein the macroporous crosslinked gel comprises a polymer formed by the reaction of one or more polymerizable monomers with 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;

[0180] b. Flowing a first solution through or across the functionalized composite material substantially at a first flow rate, wherein the first solution comprises a plurality of first ligands such that a plurality of covalent bonds are formed between the reactive functional groups and the first ligands;

[0181] c. Optionally, flowing a first washing solution through or across the composite material substantially at a second flow rate to remove any excess first ligands;

[0182] d. Flowing a second solution through or across the functionalized composite material substantially at a third flow rate, wherein the second solution comprises a plurality of second ligands comprising at least three reactive groups 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, wherein the second ligand is a crosslinking agent;

[0183] e. Optionally, flowing a second washing solution through or across the composite material substantially at a fourth flow rate to remove any excess second ligands and any excess polymerizable monomer that may be present;

[0184] f. Flowing a quenching solution through or across the composite material substantially at a fifth flow rate, wherein the quenching solution comprises a reactive compound to convert any remaining pendant reactive functional groups into non-reactive groups; and

[0185] g. Optionally, flowing a third washing solution through or across the composite material substantially at a sixth flow rate to remove any remaining reactive compound;

[0186] wherein the functionalized composite material is arranged in a coplanar stack of coextensive sheets, a tubular configuration, or a helically wound configuration.

[0187] In some embodiments, the functionalized composite material is a wetted membrane.

[0188] In some embodiments, the wetted membrane is placed in a holder attached to a chromatography system. In some embodiments, various solutions and fluids (e.g., the first solution, the first washing solution, the second solution, the quenching solution, the second washing solution, and the third washing solution) are pumped through the membrane holder.

[0189] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the functionalized composite material is arranged in a coplanar stack of coextensive sheets, a tubular configuration, or a helical wound configuration.

[0190] Film stack

[0191] In some embodiments, the composite material is arranged in a substantially coplanar stack of substantially coextensive sheets. In some embodiments, the composite material has from 2 to 300 individual 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 individual support members. In some embodiments, the composite material has from 5 to 200 individual support members. In some embodiments, the composite material has from 5 to 100 individual support members.

[0192] In some embodiments, when the composite material is arranged in a substantially coplanar stack of substantially coextensive sheets, one or more individual support members are separated by one or more interlayers. In some embodiments, the present invention relates to any of the foregoing methods, wherein the composite material layer and the interlayer are alternating layers of the composite material and the interlayer (i.e., (composite material - interlayer) x or (interlayer - composite material) x)。In some embodiments, the composite material is stratified by 1 to 250 individual interlayers. In some embodiments, the composite material has 1 to 100 individual interlayers. 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, 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 individual interlayers. In some embodiments, the composite material has 1 to 50 individual interlayers. In some embodiments, the composite material has 1 to 25 individual interlayers.

[0193] In some embodiments, the interlayer is a flow distribution layer. In some embodiments, the interlayer extends beyond the edge of the composite material layer. In some embodiments, the interlayer allows flow through the membrane while also providing a lower pressure drop across the stack because the solution can also flow around the membrane.

[0194] In some embodiments, when the composite material is arranged as a substantially coplanar stack of substantially coextensive sheets, one or more individual support members and any optional one or more interlayers present are separated by one or more flow distribution layers. In some embodiments, the one or more flow distribution layers permit uniform coupling within the stack. In some embodiments, the one or more flow distribution layers permit ligands to couple more uniformly to the composite material throughout the stack as compared to coupling without a flow distribution layer. In some embodiments, the composite material has from 1 to 250 individual flow distribution layers. In some embodiments, the composite material has from 1 to 100 individual flow distribution 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, 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 individual flow distribution layers. In some embodiments, the composite material has from 1 to 50 individual flow distribution layers. In some embodiments, the composite material has from 1 to 25 individual flow distribution layers.

[0195] In some embodiments, the one or more flow distribution layers are non-porous sheets. In some embodiments, the one or more flow distribution layers are non-porous sheets containing holes. In some embodiments, the flow distribution layers are periodically distributed within the membrane stack. In some embodiments, the flow distribution layers are periodically distributed within a membrane stack comprising composite material layers and interlayers (i.e., (composite material - interlayer) x (flow distribution) y or (interlayer - composite material) x (flow distribution) y ).

[0196] In some embodiments, the invention relates to any of the foregoing methods, wherein the support member comprises a polymeric material selected from polysulfone, polyethersulfone, polyphenylene oxide, polycarbonate, polyester, cellulose, and cellulose derivatives.

[0197] In some embodiments, the composite material is arranged in a tubular configuration.

[0198] Spiral wound configuration

[0199] In some embodiments, the composite material is arranged in a substantially spiral wound configuration. In some embodiments, the substantially spiral wound configuration comprises layers of composite material wound around a core. In some embodiments, the substantially spiral wound configuration comprises composite material and interlayers wound around a core.

[0200] Interlayers of the spiral wound configuration

[0201] In some embodiments, the present invention relates to any of the foregoing methods, wherein the composite material layers and the interlayers are alternating layers of composite material and interlayers (i.e., (composite material - interlayer) x or (interlayer - composite material) x ). In certain embodiments, the present invention relates to any of the above methods, wherein the composite material layers and the interlayers are arranged as (interlayer - first composite material - second composite material) x or (first composite material - second composite material - interlayer) x . In certain embodiments, the present invention relates to any of the above methods, wherein the composite material layers and the interlayers are arranged as a combination of the foregoing arrangements. In certain embodiments, the first composite material and the second composite material are the same.

[0202] In some embodiments, the composite material comprises from about 3 to about 50 composite material layers around the core.

[0203] In some embodiments, the present invention relates to any of the foregoing methods (e.g., membrane stack or spiral wound configuration), wherein the composite material is in contact with one or more interlayers. In some embodiments, the interlayers provide some mechanical support to the composite material.

[0204] In some embodiments, the interlayers help to reduce back pressure.

[0205] In some embodiments, the present invention relates to any of the foregoing methods (e.g., membrane stack or spiral wound configuration), wherein one or more interlayers are selected from screens, meshes, polypropylene, polyethylene, paper, and cellulose. In some embodiments, the interlayer is a screen or a nonwoven material. In some embodiments, the interlayer is a mesh. In some embodiments, the interlayer is polypropylene or polyethylene. In some embodiments, the interlayer is nonwoven polypropylene. In some embodiments, the interlayer is paper. In certain embodiments, the interlayer is cellulose.

[0206] In certain embodiments, the interlayer is a mesh. In certain embodiments, the mesh interlayer is an extruded mesh. In certain embodiments, the mesh interlayer is a ~0.45-mm mesh. In certain embodiments, the mesh interlayer is a biaxial thermoplastic mesh. In certain embodiments, the mesh interlayer is substantially similar to Naltex from DelStar Technologies, Inc., a specific biaxial thermoplastic mesh.

[0207] In certain embodiments, the interlayer is spunbond polypropylene. In certain embodiments, the interlayer is a spunbond polypropylene having a fabric basis weight of from about 0.70 oz / yd 2 to about 0.95 oz / yd 2 In certain embodiments, the interlayer is a spunbond polypropylene having a fabric basis weight of from about 0.70 oz / yd 2 to about 0.75 oz / yd 2 to about 0.80 oz / yd 2 to about 0.85 oz / yd 2 to about 0.90 oz / yd 2 or about 0.95 oz / yd 2 In certain embodiments, the interlayer is a spunbond polypropylene having a fabric basis weight of about 0.86 oz / yd 2 In certain embodiments, the interlayer is a spunbond polypropylene having a fabric basis weight of about 0.86 oz / yd

[0208] In certain embodiments, the thickness of the interlayer is from about 50 μm to about 300 μm. In certain embodiments, the thickness of the interlayer is about 50 μm, about 100 μm, about 150 μm, about 200 μm, about 250 μm or about 300 μm.

[0209] In certain embodiments, the volume porosity of the interlayer is from about 50% to about 99%. In certain embodiments, the volume porosity of the interlayer is from about 70% to about 95%. In certain embodiments, the volume porosity of the interlayer is about 70%, about 75%, about 80%, about 85%, about 90% or about 95%. In certain embodiments, the volume porosity of the interlayer is from about 80% to about 90%. In certain embodiments, the interlayer is substantially compressible.

[0210] In some embodiments, one or more interlayers are in contact with one or more flow distribution layers.

[0211] The inner core of the spiral wound configuration

[0212] In some embodiments, the inner core is plastic. In some embodiments, the inner core is polypropylene or polysulfone.

[0213] In some embodiments, the inner core is a cylinder. In certain embodiments, the inner core is a cylinder capped or sealed at both of its ends.

[0214] 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 of its ends.

[0215] In some embodiments, the inner core is a screen wound around a cylinder. In certain embodiments, the screen provides a path through which fluid can flow.

[0216] In some embodiments, the present invention relates to any of the foregoing methods, and the composite material is a membrane.

[0217] In some embodiments, the present invention relates to any of the foregoing methods, wherein the crosslinked gel is a neutral hydrogel, a charged hydrogel, a polyelectrolyte gel, a hydrophobic gel, a neutral gel, or a gel containing functional groups.

[0218] In some embodiments, the present invention relates to any of the foregoing methods, wherein the macroporous crosslinked gel contains macropores with an average size of 10 nm to 3000 nm.

[0219] In some embodiments, the present invention relates to any of the foregoing methods, wherein the average pore diameter of the pores of the support member is about 0.1 μm to about 50 μm.

[0220] In some embodiments, the present invention relates to any of the foregoing methods, wherein the fluid flow path substantially passes through the composite material.

[0221] In some embodiments, the present invention relates to any of the foregoing methods, wherein the fluid flow path substantially traverses the composite material.

[0222] Method for preparing an exemplary composite material

[0223] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the ratio of pendant reactive functional groups to grafted end 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, an alkynyl group is equivalent to two alkenyl groups.

[0224] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the first monomer is present in the monomer mixture in an amount of about 5 wt% to about 25 wt% of the monomer mixture. In certain embodiments, the present invention relates to any of the foregoing methods, wherein the first monomer is present in the monomer mixture in an amount of about 5 wt% to about 20 wt% of the monomer mixture.

[0225] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the second monomer is present in the monomer mixture in an amount of about 0.1 wt% to about 20 wt% of the monomer mixture.

[0226] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the first crosslinking agent is present in the monomer mixture in an amount of about 1 wt% to about 20 wt% of the monomer mixture.

[0227] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the photoinitiator is present in the monomer mixture in an amount of about 0.1 wt% to about 2 wt% of the monomer mixture.

[0228] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the photoinitiator is benzoin or a benzoin ether, benzophenone, dialkoxyacetophenone, 2,2-dimethoxy-2-phenylacetophenone, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, hydroxyalkylphenone, 1-hydroxy-cyclohexyl-phenyl-ketone, 4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone, 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-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.

[0229] In certain embodiments, the present invention relates to any one of the foregoing methods, wherein the first solvent comprises N,N'-dimethylacetamide (DMAc), (±)-1,3-butanediol (Budiol), dipropylene glycol methyl ether acetate (DPMA), water, dipropylene glycol dimethyl ether (DPM), dipropylene glycol propyl ether (DPGPE), dipropylene glycol methyl ether (DPGME), tripropylene 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), hexanediol, sodium dodecyl sulfate, or N,N-dimethylformamide (DMF), or a mixture thereof.

[0230] In certain embodiments, the present invention relates to any one of the foregoing methods, wherein N,N'-dimethylacetamide (DMAc) is present in the monomer mixture in an amount of about 0 wt% to about 70 wt% of the monomer mixture. In certain embodiments, the present invention relates to any one of the foregoing methods, wherein N,N'-dimethylacetamide (DMAc) is present in the monomer mixture in an amount of about 0 wt% to about 50 wt% of the monomer mixture. In certain embodiments, the present invention relates to any one of the foregoing methods, wherein N,N'-dimethylacetamide (DMAc) is present in the monomer mixture in an amount of about 0 wt% to about 70 wt% of the total solvent. In certain embodiments, the present invention relates to any one of the foregoing methods, wherein N,N'-dimethylacetamide (DMAc) is present in the monomer mixture in an amount of about 0 wt% to about 50 wt% of the total solvent.

[0231] In certain embodiments, the present invention relates to any one of the foregoing methods, wherein (±)-1,3-butanediol (Budiol) is present in the monomer mixture in an amount of about 0 wt% to about 50 wt% of the monomer mixture. In certain embodiments, the present invention relates to any one of the foregoing methods, wherein (±)-1,3-butanediol (Budiol) is present in the monomer mixture in an amount of about 0 wt% to about 50 wt% of the total solvent.

[0232] In certain embodiments, the present invention relates to any one of the foregoing methods, wherein dipropylene glycol methyl ether acetate (DPMA) is present in the monomer mixture in an amount of about 0 wt% to about 60 wt% of the monomer mixture. In certain embodiments, the present invention relates to any one of the foregoing methods, wherein dipropylene glycol methyl ether acetate (DPMA) is present in the monomer mixture in an amount of about 0 wt% to about 60 wt% of the total solvent.

[0233] In certain embodiments, the present invention relates to any of the foregoing methods, wherein water is present in the monomer mixture in an amount of from about 0 wt% to about 50 wt% of the monomer mixture. In certain embodiments, the present invention relates to any of the foregoing methods, wherein water is present in the monomer mixture in an amount of from about 0 wt% to about 30 wt% of the monomer mixture. In certain embodiments, the present invention relates to any of the foregoing methods, wherein water is present in the monomer mixture in an amount of from about 0 wt% to about 30 wt% of the total solvent.

[0234] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the covered support member is irradiated at about 350 nm.

[0235] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the time period is about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, or about 1 hour.

[0236] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the composite material comprises macropores.

[0237] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the average pore diameter of the macropores is less than the average pore diameter of the pores.

[0238] Pore size determination

[0239] SEM and ESEM

[0240] As described above, in certain embodiments, the crosslinked gel is a macroporous crosslinked gel. The average diameter of the macropores in the macroporous crosslinked gel can be estimated by one of a variety of methods. One method that can be employed is scanning electron microscopy (SEM). SEM is a well-established method commonly used to determine pore size and porosity, particularly for characterizing membranes. See the book Basic Principles of Membrane Technology by Marcel Mulder ( 1996) (particularly Chapter IV). Mulder provides a review 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 pictures of the membrane can be obtained from the top, cross-sectional, and bottom perspectives. In addition, porosity and pore size distribution can be estimated from the images.

[0241] Environmental scanning electron microscopy (ESEM) is a technique that allows non-destructive imaging of wet samples by permitting a gas environment in the sample chamber. An environmental secondary detector (ESD) requires a gas background to operate and operates at approximately 3 Torr to approximately 20 Torr. These pressure constraints limit the ability to vary the humidity in the sample chamber. For example, at 10 Torr, the relative humidity at a specific temperature is as follows:

[0242] Relative humidity (%) at 10 Torr T(℃) Approximately 80 Approximately 16 Approximately 70 Approximately 18 Approximately 60 Approximately 20 Approximately 40 Approximately 24 Approximately 20 Approximately 40 Approximately 10 Approximately 50 Approximately 2 Approximately 70 Approximately 1 Approximately 100

[0243] This is a useful guide to the relative humidity in the sample chamber at different temperatures. In some embodiments, the relative humidity in the sample chamber during imaging is from about 1% to about 99%. In some 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 some embodiments, the relative humidity in the sample chamber during imaging is about 45%.

[0244] In some embodiments, the microscope has nanometer resolution and a magnification of up to about 100,000×.

[0245] In some embodiments, the temperature in the sample chamber during imaging is from about 1 °C to about 95 °C. In some embodiments, the temperature in the sample chamber during imaging is about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, about 10 °C, about 12 °C, about 14 °C, about 16 °C, about 18 °C, about 20 °C, about 25 °C, about 30 °C, about 35 °C, about 40 °C, about 45 °C, about 50 °C, about 55 °C, about 60 °C, about 65 °C, about 70 °C, about 75 °C, about 80 °C or about 85 °C. In some embodiments, the temperature in the sample chamber during imaging is about 5 °C.

[0246] In some embodiments, the pressure in the sample chamber during imaging is from about 0.5 Torr to about 20 Torr. In some embodiments, the pressure in the sample chamber during imaging is about 4 Torr, about 6 Torr, about 8 Torr, about 10 Torr, about 12 Torr, about 14 Torr, about 16 Torr, about 18 Torr or about 20 Torr. In some embodiments, the pressure in the sample chamber during imaging is about 3 Torr.

[0247] In certain embodiments, the working distance from the electron beam source to the sample is from about 6 mm to about 15 mm. In certain embodiments, the working distance from the electron beam source to the sample is about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, or about 15 mm. In certain embodiments, the working distance from the electron beam source to the sample is about 10 mm.

[0248] In certain embodiments, the voltage is from about 1 kV to about 30 kV. In certain embodiments, the voltage is about 2 kV, about 4 kV, about 6 kV, about 8 kV, about 10 kV, about 12 kV, about 14 kV, about 16 kV, about 18 kV, about 20 kV, about 22 kV, about 24 kV, about 26 kV, about 28 kV, or about 30 kV. In certain embodiments, the voltage is about 20 kV.

[0249] In certain embodiments, the average pore size can be measured by estimating the pore size in a representative sample of an image from the top or bottom of the composite material. Those of ordinary skill in the art will recognize and acknowledge the various experimental variables associated with obtaining an ESEM image of a wetting film and will be able to design experiments accordingly.

[0250] Capillary flow porometry

[0251] Capillary flow porometry is an analytical technique for measuring one or more pore sizes of a porous material. In this analytical technique, a wetting liquid is used to fill the pores of a test sample, and the pressure of a non-reactive gas is used to displace the liquid from the pores. The gas pressure and flow rate through the sample are accurately measured, and the pore size 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:

[0252] D = 4 × γ × cosθ / P

[0253] D = pore diameter

[0254] γ = liquid surface tension

[0255] θ = liquid contact angle

[0256] P = gas pressure difference.

[0257] This formula shows that the pressure required to displace the liquid from the wetting sample is negatively correlated with the pore size. Since this technique involves the flow of a liquid under pressure from the pores of a test sample, it can be used to characterize "through pores" (interconnected pores that allow fluid to flow from one side of the sample to the other). Other pore types (closed pores and blind pores) cannot be detected by this method.

[0258] When gas begins to flow through the pores, the presence of the pores is detected by capillary flow porometry. This occurs only when the gas pressure is high enough to displace the liquid from the narrowest part of the pore. Thus, the pore diameter calculated using this method is the diameter of the pore at its narrowest part, and each pore is detected as a single pore with that narrow diameter. The maximum pore diameter (referred to as the bubble point) is determined by the lowest gas pressure required to initiate flow through the wet sample, and the average pore diameter is calculated from the average flow pressure. Additionally, both the narrow pore diameter range and the pore size distribution can be determined using this technique.

[0259] This method can be performed on small membrane samples (e.g., about 2.5 cm in diameter) immersed in a test fluid (e.g., water, buffer, alcohol). The range of gas pressure applied can be selected from about 0 psi to about 500 psi.

[0260] Other methods for determining pore size

[0261] Mulder describes other methods for characterizing the average pore size of porous membranes, including atomic force microscopy (AFM) (page 164), permeability calculations (page 169), gas adsorption - desorption (page 173), thermoporometry (page 176), permporometry (page 179), and liquid displacement (page 181). Mulder and the references cited therein are hereby incorporated by reference.

[0262] Exemplary uses of the composite material

[0263] In certain embodiments, the present invention relates to a method in which a fluid passes through the cross - linked gel of any of the above - mentioned composite materials. Good selectivity can be obtained by adjusting the conditions for binding or fractionation.

[0264] In certain embodiments, the present invention relates to a method that further comprises the following steps:

[0265] f. causing a first fluid containing a substance to flow substantially through or substantially across the composite material at a fifth flow rate, thereby adsorbing or absorbing a portion of the substance onto the composite material.

[0266] In certain embodiments, the present invention relates to a method that comprises the following steps:

[0267] h. causing a first fluid containing a substance to flow substantially through or substantially across the composite material at a seventh flow rate, thereby adsorbing or absorbing a portion of the substance onto the composite material.

[0268] In certain embodiments, the first fluid further comprises fragmented antibodies, aggregated antibodies, host cell proteins, polynucleotides, endotoxins, or viruses. In some embodiments, the first fluid is a cell suspension or an aggregate suspension.

[0269] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the fluid flow path of the first fluid substantially passes through the macropores of the composite material.

[0270] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the fluid flow path of the first fluid is substantially perpendicular to the macropores of the composite material.

[0271] In certain embodiments, the present invention relates to any of the foregoing methods, wherein after the first fluid substantially flows through or substantially flows across the composite material, substantially all of the substances are adsorbed or absorbed onto the composite material.

[0272] In certain embodiments, the present invention relates to any of the foregoing methods, and the method further comprises the following steps:

[0273] g. Contacting a second fluid with the substances adsorbed or absorbed onto the composite material at a sixth flow rate, thereby releasing a portion of the substances from the composite material.

[0274] In certain embodiments, the present invention relates to any of the foregoing methods, and the method further comprises the following steps:

[0275] i. Contacting a second fluid with the substances adsorbed or absorbed onto the composite material at an eighth flow rate, thereby releasing a portion of the substances from the composite material.

[0276] In some embodiments, the present invention relates to a method for conjugating a ligand to a functionalized composite material, the method comprising the following steps:

[0277] a. Providing a functionalized composite material, the functionalized composite material comprising:

[0278] i. A support member, the support member comprising a plurality of pores extending through the support member; and

[0279] ii. A macroporous crosslinked gel, wherein the macroporous crosslinked gel comprises a polymer formed by the reaction of one or more polymerizable monomers with 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;

[0280] b. Flowing the first solution substantially through or substantially across the functionalized composite material at a first flow rate, wherein the first solution comprises a plurality of first ligands such that a plurality of covalent bonds are formed between the reactive functional groups and the first ligands;

[0281] c. Optionally, flowing a first washing solution substantially through or substantially across the composite material at a second flow rate to remove any excess first ligands;

[0282] d. Flowing a quenching solution substantially through or substantially across the composite material at a third flow rate, wherein the quenching solution comprises a reactive compound to convert any remaining pendant reactive functional groups into non-reactive groups;

[0283] e. Optionally, flowing a second washing solution substantially through or substantially across the composite material at a fourth flow rate to remove any remaining reactive compounds;

[0284] f. Flowing a first fluid containing a substance substantially through or substantially across the composite material at a fifth flow rate, thereby adsorbing or absorbing a portion of the substance onto the composite material; and

[0285] g. Contacting a second fluid with the substance adsorbed or absorbed onto the composite material at a sixth flow rate, thereby releasing a portion of the substance from the composite material;

[0286] wherein the functionalized composite material is arranged in a coplanar stack of coextensive sheets, a tubular configuration, or a helically wound configuration.

[0287] In some embodiments, the present invention relates to a method for coupling ligands to a functionalized composite material, the method comprising the following steps:

[0288] a. Providing a functionalized composite material, the functionalized composite material comprising:

[0289] i. A support member, the support member comprising a plurality of pores extending through the support member;

[0290] And

[0291] ii. A macroporous crosslinked gel, wherein the macroporous crosslinked gel comprises a polymer formed by the reaction of one or more polymerizable monomers with 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;

[0292] b. Cause the first solution to flow substantially through or substantially across the functionalized composite material at a first flow rate, wherein the first solution comprises a plurality of first ligands such that a plurality of covalent bonds are formed between the reactive functional groups and the first ligands;

[0293] c. Optionally, cause a first washing solution to flow substantially through or substantially across the composite material at a second flow rate to remove any excess first ligands;

[0294] d. Cause a second solution to flow substantially through or substantially across the functionalized composite material at a third flow rate, wherein the second solution comprises a plurality of second ligands each comprising at least three reactive groups and optionally present polymerizable monomers each comprising at least two pendant reactive functional groups, thereby forming a plurality of covalent bonds between the reactive functional groups and the second ligands, wherein the second ligands are crosslinking agents;

[0295] e. Optionally, cause a second washing solution to flow substantially through or substantially across the composite material at a fourth flow rate to remove any excess second ligands and any excess polymerizable monomers that may be present;

[0296] f. Cause a quenching solution to flow substantially through or substantially across the composite material at a fifth flow rate, wherein the quenching solution comprises a reactive compound to convert any remaining pendant reactive functional groups into non-reactive groups; and

[0297] g. Optionally, cause a third washing solution to flow substantially through or substantially across the composite material at a sixth flow rate to remove any remaining reactive compound;

[0298] h. Cause a first fluid containing a substance to flow substantially through or substantially across the composite material at a seventh flow rate, thereby adsorbing or absorbing a portion of the substance onto the composite material; and

[0299] i. Cause a second fluid to contact the substance adsorbed or absorbed onto the composite material at an eighth flow rate, thereby releasing a portion of the substance from the composite material;

[0300] wherein the functionalized composite material is arranged in a coplanar stack of coextensive sheets, a tubular configuration, or a helically wound configuration.

[0301] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the fluid flow path of the second fluid substantially passes through the macropores of the composite material.

[0302] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the fluid flow path of the second fluid is substantially perpendicular to the macropores of the composite material.

[0303] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the substance is a biomolecule, bioion, virus, or virus particle.

[0304] In certain embodiments, the present invention relates to methods for separating biomolecules or bioions (such as proteins or immunoglobulins) from a solution. In certain embodiments, the present invention relates to methods for purifying biomolecules or bioions (such as proteins or immunoglobulins). In certain embodiments, the present invention relates to methods for purifying proteins or monoclonal antibodies with high selectivity. In certain embodiments, the present invention relates to a method in which the biomolecule or bioion retains its tertiary or quaternary structure, which may be important for maintaining biological activity.

[0305] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the substance is a biomolecule or bioion selected from the group consisting of: albumin, lysozyme, virus, cell, γ-globulin from human and animal sources, immunoglobulin from human and animal sources, hIgG, proteins from recombinant and natural sources, polypeptides from synthetic and natural sources, interleukin-2 and its receptor, enzyme, monoclonal antibody, trypsin and its inhibitor, cytochrome C, myoglobin, myosin, α-chymotrypsinogen, recombinant human interleukin, recombinant fusion protein, nucleic acid derivatives, DNA from synthetic and natural sources, and RNA from synthetic and natural sources.

[0306] In some embodiments, the present invention relates to any of the foregoing methods, wherein the substance is a biomolecule or bioion selected from the group consisting of: albumin, lysozyme, virus, cell, γ-globulin from human and animal sources, immunoglobulin from human and animal sources, hIgG, immunoglobulin M, proteins from recombinant and natural sources (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 α-galactosidase a, recombinant iduronidase, recombinant sulfatase, recombinant dornase alfa, recombinant tissue-type plasminogen activator, recombinant human interferon, recombinant insulin-like growth factor 1, and recombinant asparaginase), polypeptides from synthetic and natural sources, interleukin-2 and its receptor, enzyme, monoclonal antibody, trypsin and its inhibitor, cytochrome C, myoglobin, myosin, α-chymotrypsinogen, recombinant human interleukin, recombinant fusion protein, factor VIII, factor IX, antithrombin III, α-1-antitrypsin, nucleic acid derivatives, DNA from synthetic and natural sources, and RNA from synthetic and natural sources.

[0307] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the biomolecule or bioion is lysozyme, hIgG, myoglobin, human serum albumin, soybean trypsin inhibitor, transferase, enolase, ovalbumin, ribonuclease, ovomucoid trypsin inhibitor, cytochrome c, annexin V, or chymotrypsinogen.

[0308] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the substance is a biomolecule or bioion selected from: gamma globulins of human and animal origin, immunoglobulins of human and animal origin, proteins of recombinant or natural origin including, polypeptides of synthetic or natural origin, monoclonal antibodies, antigens, bacterial immunoglobulin-binding proteins, recombinant fusion proteins, protein A, protein G, protein L, and peptide H.

[0309] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the substance is a biomolecule or bioion selected from: polypeptides, proteins, recombinant proteins, bacterial immunoglobulin-binding proteins, recombinant fusion proteins, protein A, protein G, protein L, and peptide H.

[0310] In certain embodiments, the present invention relates to a method for recovering antibody fragments from associated variants, impurities, or contaminants. In certain embodiments, separation or purification of the biomolecule or bioion can occur substantially in a crosslinked gel. In certain embodiments, when the crosslinked gel has large pores, separation or purification of the biomolecule or bioion can occur substantially in the large pores of the crosslinked gel.

[0311] 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 changing the liquid flowing through the gel. In certain embodiments, absorption and release of the substance can be controlled by changes in the composition of the crosslinked gel.

[0312] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the first fluid is a clarified cell culture supernatant.

[0313] In certain embodiments, the present invention relates to a method in which a substance can be applied from a buffer solution to a composite material. In certain embodiments, the present invention relates to any of the foregoing methods, wherein the first fluid is a buffer solution. In certain embodiments, the present invention relates to any of the foregoing 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.

[0314] In certain embodiments, the present invention relates to any of the foregoing 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.

[0315] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the first fluid contains sodium phosphate.

[0316] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the first fluid contains a salt. In certain embodiments, the present invention relates to any of the foregoing 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 of the foregoing methods, wherein the salt is sodium chloride.

[0317] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the substance is a binding partner. In certain embodiments, the present invention relates to any of the foregoing methods, wherein the composite material contains a coupling ligand, and the substance is a binding partner of the ligand.

[0318] In certain embodiments, the present invention relates to any one of the foregoing methods, wherein the concentration of the substance in the first fluid is from about 0.01 mg / mL to about 1000 mg / mL. In certain embodiments, the present invention relates to any one of the foregoing methods, wherein the concentration of the substance in the first fluid is from about 0.2 mg / mL to about 10 mg / mL. In certain embodiments, the present invention relates to any one of the foregoing 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.

[0319] In certain embodiments, the present invention relates to any one of the foregoing methods, wherein the first flow rate, the second flow rate, the third flow rate, the fourth flow rate, the fifth flow rate, the sixth flow rate, the seventh flow rate, and the eighth flow rate 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 foregoing methods, wherein the first flow rate, the second flow rate, the third flow rate, the fourth flow rate, the fifth flow rate, the sixth flow rate, the seventh flow rate, and the eighth flow rate are each independently selected from about 3 membrane volume (MV) / min to about 70 MV / min. In certain embodiments, the present invention relates to any one of the foregoing methods, wherein the first flow rate, the second flow rate, the third flow rate, the fourth flow rate, the fifth flow rate, the sixth flow rate, the seventh flow rate, and the eighth flow rate are each independently selected from about 5 MV / min to about 50 MV / min. In certain embodiments, the present invention relates to any one of the foregoing methods, wherein the first flow rate, the second flow rate, the third flow rate, the fourth flow rate, the fifth flow rate, the sixth flow rate, the seventh flow rate, and the eighth flow rate are each independently selected from about 5 MV / min, about 6 MV / min, about 7 MV / min, about 8 MV / min, about 9 MV / min, about 10 MV / min, about 11 MV / min, about 12 MV / min, about 13 MV / min, about 14 MV / min, about 15 MV / min, about 16 MV / min, about 17 MV / min, about 18 MV / min, about 19 MV / min, about 20 MV / min, about 20 MV / min, about 21 MV / min, about 22 MV / min, about 23 MV / min, about 24 MV / min, about 25 MV / min, about 26 MV / min, about 27 MV / min, about 28 MV / min, about 29 MV / min, about 30 MV / min, about 30 MV / min, about 31 MV / min, about 32 MV / min, about 33 MV / min, about 34 MV / min, about 35 MV / min, about 36 MV / min, about 37 MV / min, about 38 MV / min, about 39 MV / min, about 40 MV / min, about 40 MV / min, about 41 MV / min, about 42 MV / min, about 43 MV / min, about 44 MV / min, about 45 MV / min, about 46 MV / min, about 47 MV / min, about 48 MV / min, about 49 MV / min, and about 50 MV / min. In certain embodiments, the present invention relates to any one of the foregoing methods, wherein the first flow rate, the second flow rate, the third flow rate, the fourth flow rate, the fifth flow rate, the sixth flow rate, the seventh flow rate, and the eighth flow rate are each independently selected from about 10 MV / min to about 20 MV / min.

[0320] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the first flow rate, the second flow rate, the third flow rate, the fourth flow rate, the fifth flow rate, the sixth flow rate, the seventh flow rate, and the eighth flow rate are each independently selected from about 0.5 mL / min to about 50 L / min. In certain embodiments, the present invention relates to any of the foregoing methods, wherein the first flow rate, the second flow rate, the third flow rate, the fourth flow rate, the fifth flow rate, the sixth flow rate, the seventh flow rate, and the eighth flow rate are each independently selected from about 0.5 mL / min to about 25 L / min. In certain embodiments, the present invention relates to any of the foregoing methods, wherein the first flow rate, the second flow rate, the third flow rate, the fourth flow rate, the fifth flow rate, the sixth flow rate, the seventh flow rate, and the eighth flow rate are each independently selected from about 0.5 mL / min to about 10 L / min. In certain embodiments, the present invention relates to any of the foregoing methods, wherein the first flow rate, the second flow rate, the third flow rate, the fourth flow rate, the fifth flow rate, the sixth flow rate, the seventh flow rate, and the eighth flow rate are each independently selected from about 0.5 mL / min to about 1 L / min. In certain embodiments, the present invention relates to any of the foregoing methods, wherein the first flow rate, the second flow rate, the third flow rate, the fourth flow rate, the fifth flow rate, the sixth flow rate, the seventh flow rate, and the eighth flow rate 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 of the foregoing methods, wherein the first flow rate, the second flow rate, the third flow rate, the fourth flow rate, the fifth flow rate, the sixth flow rate, the seventh flow rate, and the eighth flow rate are each independently selected from about 0.5 mL / min to about 100 mL / min. In certain embodiments, the present invention relates to any of the foregoing methods, wherein the first flow rate, the second flow rate, the third flow rate, the fourth flow rate, the fifth flow rate, the sixth flow rate, the seventh flow rate, and the eighth flow rate are each independently selected from about 0.5 mL / min to about 10 mL / min. In certain embodiments, the present invention relates to any of the foregoing methods, wherein the first flow rate, the second flow rate, the third flow rate, the fourth flow rate, the fifth flow rate, the sixth flow rate, the seventh flow rate, and the eighth flow rate are each independently selected from about 0.5 mL / min to about 2 mL / min. In certain embodiments, the present invention relates to any of the foregoing methods, wherein the first flow rate, the second flow rate, the third flow rate, the fourth flow rate, the fifth flow rate, the sixth flow rate, the seventh flow rate, and the eighth flow rate are each independently selected from about 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.

[0321] In certain embodiments, the present invention relates to a method in which a substance can be eluted using aqueous salt solutions of different concentrations and pH. In certain embodiments, the present invention relates to any of the foregoing methods, wherein the second fluid is a buffer. In certain embodiments, the present invention relates to any of the foregoing methods, wherein the second fluid comprises glycine-HCl or sodium citrate. In certain embodiments, the present invention relates to any of the foregoing methods, wherein the second fluid comprises glycine-HCl or sodium citrate at a concentration of from about 5 mM to about 2 M. In certain embodiments, the present invention relates to any of the foregoing methods, wherein the second fluid comprises 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 of glycine-HCl or sodium citrate.

[0322] In certain embodiments, the present invention relates to any of the foregoing methods, wherein the second fluid has a pH of from about 2 to about 8. In certain embodiments, the present invention relates to any of the foregoing methods, wherein the second fluid has a pH of 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.

[0323] In certain embodiments, the present invention relates to methods that exhibit high binding capacity. In some embodiments, the binding capacity of the composite material is higher when using a flow-through conjugation method compared to using a batch or dead-end conjugation method. In certain embodiments, the present invention relates to exhibiting about 1 mg / mL at 10% breakthrough 膜 , about 2 mg / mL 膜 , about 3 mg / mL 膜 , about 4 mg / mL 膜 , about 5 mg / mL 膜 , about 6 mg / mL 膜 , about 7 mg / mL 膜 , about 8 mg / mL 膜 , about 9 mg / mL 膜 , about 10 mg / mL 膜 , about 12 mg / mL膜 , about 14 mg / mL 膜 , about 16 mg / mL 膜 , about 18 mg / mL 膜 , about 20 mg / mL 膜 , about 30 mg / mL 膜 , about 40 mg / mL 膜 , about 50 mg / mL 膜 , about 60 mg / mL 膜 , about 70 mg / mL 膜 , about 80 mg / mL 膜 , about 90 mg / mL 膜 , about 100 mg / mL 膜 , about 110 mg / mL 膜 , about 120 mg / mL 膜 , about 130 mg / mL 膜 , about 140 mg / mL 膜 , about 150 mg / mL 膜 , about 160 mg / mL 膜 , about 170 mg / mL 膜 , about 180 mg / mL 膜 , about 190 mg / mL 膜 , about 200 mg / mL 膜 , about 210 mg / mL 膜 , about 220 mg / mL 膜 , about 230 mg / mL 膜 , about 240 mg / mL 膜 , about 250 mg / mL 膜 , about 260 mg / mL 膜 , about 270 mg / mL 膜 , about 280 mg / mL 膜 , about 290 mg / mL 膜 , about 300 mg / mL 膜 , about 320 mg / mL 膜 , about 340 mg / mL 膜 mg / mL 膜 , about 360 mg / mL 膜 , about 380 mg / mL 膜 or about 400 mg / mL 膜 for the binding capacity method.

[0324] Examples

[0325] The following examples are provided by way of illustration. However, it will be understood that the specific details given in each example have been chosen for illustrative purposes and should not be construed as limiting the scope of the disclosure. Generally, experiments were conducted under similar conditions unless otherwise indicated.

[0326] Example 1 - General materials and methods

[0327] Protein

[0328] Recombinant Protein A-cys (rProtein A-cys) was obtained from Biomedal S.L (Seville, Spain). Polyclonal immune γ-globulin IgG was obtained from Equitech-Bio Inc. (Kerrville, TX, USA).

[0329] Membrane Preparation

[0330] Protocol A

[0331] One or more crosslinkers and monomers (except for thiol-functionalized monomers, which are added 10 min prior to casting) were added to a solvent mixture together with a photoinitiator (IRGACURE 2959), and the mixture was stirred for a sufficient time to dissolve all components. A pre-weighed 7”×8” porous support substrate sheet (non-woven polypropylene mesh) was placed on a polyethylene sheet, and then ~15 g of the polymer solution was poured onto the substrate sheet. Subsequently, the impregnated substrate was covered with an additional polyethylene sheet. The sheets were gently pressed by hand in a circular motion to remove excess solution and any trapped air bubbles. The polymerization process was initiated by irradiating the polymer solution / substrate sandwiched between the polyethylene sheets with UV light (~350 nm) in a closed chamber for 10 min. Then, the resulting membrane was removed from between the polyethylene sheets and subjected to a thorough washing cycle, which involved a 20 - 30 minute soaking period (2 - 3 times) in purified (RO) water with agitation. The clean membrane was dried by freely hanging in air at room temperature for ~16 hours.

[0332] Protocol B

[0333] Membranes were prepared by polymerizing acrylate and / or acrylamide monomers within a support mesh material with a crosslinker in a UV-initiated reaction. Various functional membranes containing protein-binding groups (e.g., ion exchange, hydrophobic interaction, and hydrophilic interaction) can be produced in a single polymerization step by introducing suitable functional polymerizable groups into the gel polymerization solution. The wet-washed membranes can also be subjected to an additional heat treatment step to adjust their performance and properties.

[0334] For example, an epoxy group-containing film can be used as a reaction medium platform, which can be converted into a bioaffinity film by covalently anchoring various ligands (such as Protein A) to the surface. Other ligands can also be conjugated to target other biomolecules or entities (such as viruses).

[0335] Mass gain, wetting, and permeability of composite membranes

[0336] The weight of the dried film is measured and used to calculate the mass gain. The wetting of the film is also determined by dispensing 50 μL of distilled water droplets on the film surface and measuring the time required for the droplets to be absorbed into the film. To estimate the film permeability, the flux of each film is measured using RO water (or acetate buffer pH 5) and a 7.7-cm diameter film sample, with a 100 kPa applied pressure.

[0337] To estimate the film permeability, the flux of RO water (or 132 mM acetate buffer pH 5) as the mobile phase through each film is determined. The film is pre-soaked in the test fluid for at least 10 minutes before testing, rinsed with ~300 mL of the test liquid, and then the amount of the test liquid passing through a 7.7-cm diameter (actual available diameter of 7.3 cm) circular membrane specimen under a 100 kPa applied pressure is measured. The flux is expressed as the amount of liquid per unit time per unit surface area (kg / m 2 h).

[0338] Imaging of porous structures

[0339] To probe the gel structure and porosity, an environmental scanning electron microscope (ESEM) is used to image the film in the wet state. Small specimens (~7 × 5 mm) are wetted by soaking in distilled water for 10 - 15 minutes and then examined using an ESEM instrument (FEI Quanta FEG 250 ESEM). The sample is placed on a cooling stage to adjust the temperature to 5 °C, and the images are examined at a low pressure level (4.5 - 5.5 Torr) and a relative humidity of 50 - 55%.

[0340] To probe the film structure in the dry state, a Tescan Vega II LSU scanning electron microscope (SEM) (Tescan, PA, USA) is used to image the gold-coated film at a voltage setting of 10 - 20 kV.

[0341] Pore size measurement

[0342] The membrane pore size (diameter) is measured using a CFP-1500-AE capillary flow porosimeter (Porous Materials Inc., Ithaca, NY) operated by CapWin software (V.6).

[0343] Immerse the small discs (2.5-cm diameter) of the membrane in the wetting liquid (Porous Materials Inc., surface tension = 15.9 dynes / cm) for 10 min, then gently squeeze it between two pre-wetted filter paper discs (Whatman 5 - 70 mm) to remove the excess solution, and measure the thickness of the wetted membrane using a micrometer. Then, place the membrane disc on a 2.5-cm stainless steel mesh support disc. Place the support disc loaded with the test membrane in the designated holder with the membrane side up. Then, gently place the metal cover on the holder, and the test is carried out in the pressure range of 0 - 200 psi.

[0344] Protein A ligand density on the composite membrane

[0345] To measure the Protein A ligand density on the conjugated membrane, determine the amount of uncoupled protein remaining after the conjugation reaction and subtract it from the total ligand amount to obtain the amount of conjugated ligand, then divide it by the membrane volume (mL) to express the density as mg ligand per mL of membrane.

[0346] To determine the amount of Protein A in the solution, prepare a series of protein solutions in 0.1 M phosphate buffer (pH 7.2), measure the absorbance of each solution at 280 nm, and construct a calibration curve from which the slope is determined.

[0347] For the selected membrane formulation, cut 4 cm × 7 cm specimens and measure their thickness, from which the volume is calculated. Carry out the conjugation reaction as described above and load 20 mg onto each membrane conjugation separately. When the UV reaction is complete, collect the reaction solution in a tube, then add 3 - 5 mL of 0.1 M phosphate buffer to the reaction bag and use it to wash the membrane by shaking for 20 - 25 min, then add the resulting solution to the collection tube.

[0348] Repeat the washing cycle two more times, then measure the absorbance of the final solution and calculate the amount of uncoupled protein using the slope of the calibration curve. The amount of conjugated ligand is determined by taking the difference between the total reaction amount and the uncoupled amount.

[0349] Binding capacity measurement

[0350] Bioaffinity IgG binding capacity

[0351] A 25-mm diameter membrane disc was placed in a 25-mm Natrix stainless steel (SS) holder. Twenty milliliters of binding buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4) was passed through to achieve equilibration (∼160 - 200 bed volumes / min). In the binding step, 0.5 mg / mL polyclonal IgG in the binding buffer was passed through at a flow rate of 1 mL / min until the UV absorbance of the effluent exceeded 10% of the feed solution, and 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, the bound IgG was eluted by passing 10 - 14 mL of elution buffer (0.1 M glycine-HCl or 0.1 M sodium citrate, both at pH 3) through at a flow rate of 2 mL / min.

[0352] Cation exchange IgG binding capacity

[0353] A 25-mm membrane disc was placed in a 25-mm Natrix-SS holder, and 20 mL of binding buffer (132 mM sodium acetate, pH 5.0) was passed through to achieve equilibration. Then the protein solution (0.5 mg / mL human polyclonal IgG (Equitech-Bio Inc.) in the binding buffer) was passed through until the UV absorbance of the effluent exceeded 10% of the feed solution, and then 10 - 15 mL of buffer was passed through the cell to wash unbound protein. In the elution step, the 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.

[0354] Hydrophobic interaction mode IgG binding capacity

[0355] A 25-mm membrane disc was placed in a 25-mm Natrix-SS holder, and 20 mL of binding buffer (50 mM sodium phosphate, 1 M ammonium sulfate, pH 6.5) was passed through to achieve equilibration. Then, the protein solution (0.5 mg / mL human polyclonal IgG (Equitech-Bio Inc.) in the binding buffer) was passed through until the UV absorbance of the effluent exceeded 10% of the feed solution. Subsequently, 15 - 20 mL of buffer was passed through the cell to wash unbound protein. In the elution step, the bound IgG was eluted by passing 10 mL of elution buffer (50 mM sodium phosphate, pH 7.0) through.

[0356] Example 2 - Exemplary bulk coupling protocol

[0357] Protein A ligand was conjugated to the click olefin membrane

[0358] To examine the feasibility of chemically conjugating biomolecules (with thiol functional groups) to olefin membranes via hydrothiolation click reactions, an engineered protein A ligand containing cysteine residues was conjugated to one or more olefin membranes (with different chemical formulas), and the biological activity of the immobilized ligand was detected.

[0359] The freeze-dried powder of the protein A ligand (recombinant protein A-cys) was dissolved in PBS (20 mM sodium phosphate, 0.15 M NaCl, pH 7.4) to prepare a stock solution of 50 mg / mL. To prepare the conjugation solution for each membrane, 0.4 mL of the ligand stock solution was transferred to a small self-sealing plastic bag (5×8 cm), 1.6 mL of 2 M phosphate buffer (pH 7.2) was added to the self-sealing plastic bag, and then 50 μL of the initiator (4,4’-azobis(4-cyanovaleric acid), ACVA) (150 mg / mL) in DMAc was added. The reaction solution was mixed well. The final volume of the reaction solution was ∼2.0 mL and contained approximately 20 mg of ligand and approximately 7.5 mg of initiator.

[0360] Alternatively, ACVA was dissolved in the reaction buffer (2 M phosphate, pH 7.2) at a concentration of 5 mg / mL to avoid using DMAc. For low-salt experiments, the initiator was dissolved in 0.5 M phosphate at a concentration of 7.5 mg / mL.

[0361] A 4×7-cm membrane specimen (pre-wetted in water) was added to the bag loaded with the conjugation reactants. The bag was shaken for one minute and then irradiated with UV light (∼365 nm) for 10 minutes. After the irradiation was completed, the conjugation solution was decanted, then 15 - 20 mL of washing buffer solution (0.1 M phosphate, pH 7.2) was added, and the membrane was placed on an oscillator for 10 - 15 minutes. The washing cycle was repeated three times, after which the membrane was: (i) transferred to 8 mL of trehalose solution (10 wt%) and shaken for 10 - 15 minutes and dried in an oven (50 °C) for 20 - 30 min; or (ii) stored in 0.1 M phosphate buffer.

[0362] For conjugation 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. The protein A ligand was dissolved in 20 mM sodium phosphate buffer (pH 7.2) to prepare a 50 mg / mL stock solution. In each of three small bags (5×8 cm), 0.25 mL of the ligand stock solution was mixed with 0.25 mL of the initiator solution, and 50 μL of the additive was added (cysteamine-HCl was added to reaction bag B, and 1-mercaptoethanol was added to reaction bag C).

[0363] After the reaction solution was thoroughly mixed, a 25-mm diameter membrane disk was placed in each bag, and the reaction bags were shaken vigorously and then irradiated with UV light for 10 minutes. The reaction solution was decanted, and the membrane specimens were then washed three times with 0.1 M sodium phosphate buffer (pH 7.2) and shaken for 10 - 15 minutes. As described above, the composite membrane specimens were stored in the buffer (0.1 M sodium phosphate, pH 7.2), and the bioaffinity for IgG protein was tested.

[0364] Example 3 - Flow-through ligand conjugation effect

[0365] A sample disk containing a wetted membrane with pendant reactive functional groups was placed in a stainless steel holder and attached to an AKTA chromatography system. An affinity ligand solution was pumped through the membrane holder at a defined flow rate for a specified duration. Subsequently, a wash solution was pumped through the holder, followed by a quenching solution containing a reactive compound that converted the remaining membrane pendant groups to non-reactive groups. Finally, the wash solution was pumped through the holder. The pump was stopped, the holder was removed from the AKTA, and the holder was disassembled to remove 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 per minute and compared to the membrane conjugated in a batch non-flow-through method (see Figure 3 ). When using flow-through conjugation, a consistently higher membrane dynamic binding capacity was observed.

[0366] Example 4 - Protein A conjugation via flow-through a membrane stack without periodic splitter plates

[0367] A header containing a porous frit was attached to the bottom of a glass chromatography column with an inner diameter of 44 mm ( L laboratory column VL 44×250, catalog number: 96440250). Then, a circular membrane with a diameter of 30 mm and a thickness of approximately 350 microns (volume 0.25 mL) was placed on the frit inside the column such that it was equidistant from the column wall. The membrane had a surface containing epoxy groups. Then, a circular section of a 44-mm diameter polypropylene screen (1:2 twill weave, 500-micron mesh) was placed in the column and lowered so that it lay flat above the membrane and contacted the inner wall of the column. Then, another 30-mm diameter membrane was placed in the column and lowered so that it lay flat above the screen and was equidistant from the column wall. The process of placing a screen above the membrane and then a membrane above the screen was repeated until the column contained 100 membranes (total volume 25 mL) layered between 99 screens. Then, a header was added to the top of the column and lowered until the membrane layer and screen layer were compressed to a height of 9.5 cm.

[0368] Next, tubes are added to the inlet at the bottom of the column and also to the outlet at the top of the column. The inlet tube is connected to a peristaltic pump so that the solution can flow through the column in a controlled manner.

[0369] The air in the column and tubes is displaced with PBS buffer consisting of 20 mM sodium phosphate and 150 mM sodium chloride at pH 7.4. The inlet tube is placed into a glass bottle containing 400 mL of PBS buffer, and the outlet tube is directed to waste. Then the pump is started, and 200 mL of PBS buffer is made to flow through the column at a rate of 50 mL / min for 4 min. The pump is stopped, and the column is inverted. Then, the connections of the inlet and outlet tubes to the column are exchanged so that the inlet is again at the bottom of the column and the outlet is at the top of the column. The pump is started, and an additional 200 mL of PBS buffer is made to flow through the column at a rate of 50 mL / min for 4 min.

[0370] The pump is stopped, and the inlet tube is moved to a glass bottle containing 500 mL of coupling buffer consisting of 1.35 M potassium phosphate at pH 9.0. The outlet tube remains directed to waste. The pump is started, and 200 mL of coupling buffer is made to flow through the column at a rate of 50 mL / min for 4 min. The pump is stopped, and the outlet tube is placed in the same glass bottle as the inlet tube, which contains 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 recycled through the column multiple times. Recycling the solution through the column allows for an extended reaction time without increasing the volume of solution required.

[0371] 82.4 mL of a PrA ligand stock solution at a concentration of 25.8 g / L in water is added to the glass bottle containing the remaining 300 mL of coupling buffer. The pump is started, and then the PrA solution is recycled through the column at a flow rate of 50 mL / min for 4 hours. The total volume of PrA solution recycled through the column is calculated to be 422.4 mL, which consists of the remaining 300 mL of coupling buffer in the glass bottle, 40 mL of coupling buffer remaining in the column / tubes, and the added 82.4 mL of PrA stock solution. Considering the dilution of the 82.4 mL of PrA ligand stock solution containing 2.12 g of PrA ligand to a volume of 422.4 mL, the PrA solution recycled in the system is calculated to have a concentration of 5 g / L. The ligand loading on the membrane is calculated by dividing the total mass of 2.12 g of PrA ligand by the total membrane volume of 25 mL to obtain a ligand loading of 85 g / L.

[0372] After 4 hours, the pump is stopped, the outlet tube is directed to waste, and the outlet tube is directed to waste. The inlet tube is placed into a glass bottle containing 200 mL of PBS buffer. The pump is started, and PBS buffer is made to flow through the column at a rate of 50 mL / min for 4 min.

[0373] Then stop the pump and place the inlet tube into a glass bottle containing 800 mL of 1 M ethanolamine. Keep the outlet tube directed to waste. Start the pump and allow 200 mL of 1 M ethanolamine to flow through the column to waste at a flow rate of 50 mL / min for 4 min. Stop the pump and direct the outlet tube to a glass bottle containing the remaining 600 mL of 1 M ethanolamine solution. Then start the pump and recycle the remaining 600 mL of 1 M ethanolamine solution through the column at a flow rate of 50 mL / min for 3 h.

[0374] Stop the pump and direct the outlet tube to waste. Place the inlet tube into a glass bottle containing 200 mL of PBS buffer. Start the pump and allow the PBS buffer to flow through the column at a flow rate of 50 mL / min for 4 min.

[0375] Stop the pump and remove the top column head. Remove the membrane and store it in PBS buffer. Measure the flux and IgG dynamic binding capacity of several membranes at different positions within the stack. Position #1 is closest to the column inlet, while position #100 is closest to the column outlet. No significant deviation in membrane flux was found as a function of position (Table 1). The membranes at positions 60 and 80 were found to have a much lower IgG dynamic binding capacity, indicating non-uniform flow distribution within the column.

[0376] Table 1. Membrane flux and IgG dynamic binding capacity based on the position of the membrane in the stack

[0377]

[0378] Example 5 - Protein A conjugation via flow-through a membrane stack with periodic splitter plates

[0379] Attach the head containing the porous frit to the bottom of a glass chromatography column with an inner diameter of 44 mm ( L laboratory column VL 44×250, catalog number: 96440250). Then, lower the flow distribution layer consisting of a circular impermeable plastic sheet approximately 0.025 inches thick and 44 mm in diameter (which has a 6 mm diameter hole in the center) so that it lies flat on top of the porous frit. Then, place two circular sections of a 44 mm diameter polypropylene screen (1:2 twill weave, 500 micron mesh) on top of the flow distribution layer. Then, place a circular membrane (volume 0.25 mL) with a diameter of 30 mm and a thickness of approximately 350 microns on the screen so that it is equidistant from the column wall. The membrane has a surface containing epoxy groups. Lower another circular section of the polypropylene screen so that it lies flat on top of the membrane. Repeat the process of adding a membrane layer followed by a polypropylene screen layer 9 more times until the column has the Figure 4 following composition as shown in

[0380] Repeat the process of assembling the layers in the column as described above nine more times until the column contains 100 membrane layers (total volume 25 mL), 120 screen layers, and 10 flow distribution layers. Then, add an additional flow distribution layer to the stack and add a head with a porous frit to the top of the column. Lower the head until the membrane, screen, and flow distribution layers are compressed to a height of 10.5 cm.

[0381] Next, add a tube to the inlet at the bottom of the column and also add a tube to the outlet at the top of the column. The inlet tube is connected to a peristaltic pump so that the solution can flow through the column in a controlled manner.

[0382] Replace the air in the column and the tubes with PBS buffer having a pH of 7.4 and consisting of 20 mM sodium phosphate and 150 mM sodium chloride. Place the inlet tube into a glass bottle containing 400 mL of PBS buffer and direct the outlet tube to waste. Then start the pump and allow 200 mL of PBS buffer to flow through the column at a rate of 50 mL / min for 4 min. Stop the pump and invert the column. Then, exchange the connections of the inlet and outlet tubes to the column so that the inlet is again at the bottom of the column and the outlet is at the top of the column. Start the pump and allow an additional 200 mL of PBS buffer to flow through the column at a rate of 50 mL / min for 4 min.

[0383] Stop the pump and move the inlet tube to a glass bottle containing 500 mL of coupling buffer having a pH of 9.0 and consisting of 1.35 M potassium phosphate. The outlet tube remains directed to waste. Start the pump and allow 200 mL of coupling buffer to flow through the column at a rate of 50 mL / min for 4 min. Stop the pump and place the outlet tube in the same glass bottle as the inlet tube, which contains 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 recycled through the column multiple times. Recycling the solution through the column allows for an extended reaction time without increasing the volume of solution required.

[0384] 82.4 mL of a PrA ligand stock solution with a concentration of 25.8 g / L in water was added to a glass bottle containing the remaining 300 mL of coupling buffer. The pump was started, and then 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 remaining 300 mL of coupling buffer in the glass bottle, the remaining 40 mL of coupling buffer in the column / tubing, and the added 82.4 mL of PrA stock solution. Considering the dilution of the 82.4 mL PrA ligand stock solution containing 2.12 g of PrA ligand 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 on the membrane was calculated by dividing the total mass of 2.12 g of PrA ligand by the total membrane volume of 25 mL to obtain a ligand loading of 85 g / L.

[0385] After 4 hours, the pump was stopped, and the outlet tubing was directed to waste. The inlet tubing 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 min.

[0386] Then the pump was stopped, and the inlet tubing was placed into a glass bottle containing 800 mL of 1 M ethanolamine. The outlet tubing remained directed to waste. The pump was started, and 200 mL of 1 M ethanolamine was flowed through the column to waste at a flow rate of 50 mL / min for 4 min. The pump was stopped, and the outlet tubing was directed to a glass bottle containing the remaining 600 mL of 1 M ethanolamine solution. Then 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.

[0387] The pump was stopped, and the outlet tubing was directed to waste. The inlet tubing 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 min.

[0388] The pump was stopped, and the top column head was removed. The membrane was taken out and stored in PBS buffer. The flux and IgG dynamic binding capacity of several membranes at different positions within the stack were measured. Position #1 was closest to the column inlet, while position #100 was closest to the column outlet. It was found that there was no significant deviation in membrane flux with position change (Table 2). It was also found that there was no significant deviation in IgG dynamic binding capacity, indicating that the flow distribution was relatively uniform throughout the column. The addition of the flow distribution layer provided uniform coupling of all the epoxide membranes in the stack with the PrA ligand ( Figure 5A and Figure 5B ).

[0389] Table 2. Membrane flux and IgG dynamic binding capacity based on the position of the membrane in the stack

[0390]

[0391] Example 6 - Protein A conjugation via tangential flow through a helically wound membrane roll

[0392] A rectangular membrane with a width of 24.5 cm, a length of 62.5 cm, and a thickness of 0.035 cm (total membrane volume of 53.6 mL) and a plastic rectangular section of a polypropylene sieve mesh with a width of 25.4 cm (1:2 twill weave, 500 micron mesh openings) are wound around a cylindrical core with a diameter of 1.6 cm and a length of 25.4 cm. The membrane is centered such that there is 0.45 cm between the edge of the membrane and the edge of the sieve mesh. The membrane and the sieve mesh are wound around the core, with the sieve mesh contacting the core. The membrane and the sieve mesh are rolled up until the entire membrane is completely covered by the sieve mesh layer. The sieve mesh layer is continued to be wound around the roll until the diameter of the roll is 32 mm. Then, the sieve mesh layer is cut. Then, the roll is slid into a glass chromatography column ( L laboratory column VL 32×250, catalog number: 96320250) such that the edges of the roll are 2.5 cm from the ends of the column. Then, heads containing porous frits are attached to the bottom and the top of the column.

[0393] Next, a tube is added to the inlet at the bottom of the column and a tube is also added to the outlet at the top of the column. The inlet tube is connected to a peristaltic pump so that a solution can flow through the column in a controlled manner.

[0394] The air in the column and the tubes is displaced with PBS buffer composed of 20 mM sodium phosphate and 150 mM sodium chloride with a pH of 7.4. The inlet tube is placed into a glass bottle containing 400 mL of PBS buffer and the outlet tube is directed to waste. Then the pump is started and 200 mL of PBS buffer is made to flow through the column at a rate of 40 mL / min for 5 min. The pump is stopped and the column is inverted. Then, the connections of the inlet tube and the outlet tube to the column are exchanged so that the inlet is again at the bottom of the column and the outlet is at the top of the column. The pump is started and an additional 200 mL of PBS buffer is made to flow through the column at a rate of 40 mL / min for 5 min.

[0395] The pump is stopped and the inlet tube is moved to a glass bottle containing 1064 mL of coupling buffer composed of 1.35 M potassium phosphate with a pH of 9.0. The outlet tube remains directed to waste. The pump is started and 300 mL of the coupling buffer is made to flow through the column at a rate of 40 mL / min for 7.5 min. The pump is stopped and the outlet tube is placed in the same glass bottle as the inlet tube, which contains the remaining 764 mL of coupling buffer. In this configuration where the inlet tube and the outlet tube are in the same bottle, the solution can be recycled through the column multiple times. Recycling the solution through the column allows for an extended reaction time without increasing the volume of solution required.

[0396] Add 205.6 mL of the PrA ligand stock solution at a concentration of 25.8 g / L in water to a glass bottle containing the remaining 764 mL of coupling buffer. Start the pump, and then recycle the PrA solution through the column at a flow rate of 40 mL / min for 4 hours. The total volume of the PrA solution recycled through the column is calculated to be 1062.6 mL, which consists of the 764 mL of coupling buffer remaining in the glass bottle, 93 mL of coupling buffer remaining in the column / tube, and the added 205.6 mL of PrA stock solution. Considering the dilution of the 205.6 mL of PrA ligand stock solution containing 5.3 g of PrA ligand to a volume of 1064 mL, the PrA solution recycled in the system is calculated to have a concentration of 5 g / L. The ligand loading on the membrane is calculated by dividing the total mass of 5.3 g of PrA ligand by the total membrane volume of 53.6 mL to obtain a ligand loading of 99 g / L.

[0397] After 4 hours, stop the pump and direct the outlet tube to waste. Place the inlet tube into a glass bottle containing 200 mL of PBS buffer. Start the pump and flow the PBS buffer through the column at a flow rate of 40 mL / min for 5 min.

[0398] Then stop the pump and place the inlet tube into a glass bottle containing 600 mL of 1 M ethanolamine. The outlet tube remains directed to waste. Start the pump and flow 200 mL of 1 M ethanolamine through the column to waste at a flow rate of 20 mL / min for 10 min. Stop the pump and direct the outlet tube to a glass bottle containing the remaining 400 mL of 1 M ethanolamine solution. Then start the pump and recycle the remaining 400 mL of 1 M ethanolamine solution through the column for 170 minutes at a flow rate of 20 mL / min.

[0399] Stop the pump and direct the outlet tube to waste. Place the inlet tube into a glass bottle containing 400 mL of PBS buffer. Start the pump and flow the PBS buffer through the column at a flow rate of 40 mL / min for 10 min.

[0400] Stop the pump and remove the top and bottom column heads. Then, remove the wound roll of the membrane and the screen from the column. Unroll the membrane and separate it from the screen. Then, remove a circular section of the membrane with a diameter of 30 mm from the rectangular membrane sheet. Remove five circular sections of the membrane from each of the following positions ( Figure 6 ):

[0401] 1. Center: Located at the overall center of the rectangular membrane sheet.

[0402] 2. Core: Located at the middle of the edge of the rectangular membrane sheet closest to the core during the coupling reaction.

[0403] 3. Shell: Located at the middle of the edge of the rectangular membrane sheet closest to the column side during the coupling reaction.

[0404] 4. Inlet: Located at the middle of the edge of the rectangular membrane sheet closest to the column inlet during the coupling reaction.

[0405] 5. Outlet: Located at the middle of the edge of the rectangular membrane sheet closest to the column inlet during the coupling reaction.

[0406] 6. Five membranes taken 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 measured at a flow rate of 10 membrane volumes per minute or 10 mL / min. It was found that the 1 mL devices all had very similar pressure drops and IgG dynamic binding capacities at all five different positions. These results indicate that the tangential flow coupling of the epoxide membrane with the PrA ligand can be achieved uniformly across the membrane when it is sandwiched with a polypropylene screen.

[0407] Table 3. Pressure Drop and IgG Dynamic Binding Capacity Based on the Position of the Membrane in the Helically Wound Rectangular Sheet

[0408]

[0409] Incorporated by reference

[0410] All U.S. patents and U.S. patent application publications cited herein are hereby incorporated by reference.

[0411] Equivalents

[0412] Those skilled in the art will recognize or be able to ascertain using only routine experimentation many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method for coupling a ligand to a composite material, the method comprising the steps of: a. providing a composite material, wherein the composite material is arranged as a coplanar stack of coextensive sheets, or a helically wound configuration, the coextensive coplanar sheets being periodically separated by a screen, the helically wound composite material being wound with a screen, the composite material comprising: i. a support member comprising a plurality of pores extending therethrough; and ii. a macroporous crosslinked gel, wherein the macroporous crosslinked gel comprises a polymer formed by the reaction of one or more polymerizable monomers with one or more crosslinking agents; 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; and b. flowing one or more solutions sequentially through or across the composite material, wherein the one or more solutions react with the surface of the composite material to form a functionalized composite material having pendant reactive functional groups on the surface, c. flowing a first solution through or across the functionalized composite material at a first flow rate, wherein the first solution comprises a plurality of first ligands such that a plurality of covalent bonds are formed between the pendant reactive functional groups and the first ligands, wherein the pendant reactive functional groups are selected from aldehyde, amine, carbon-carbon double bond, carbon-carbon triple bond, thiol, epoxide, hydroxyl, acid anhydride, azide, reactive halogen, acyl chloride, and mixtures thereof.

2. The method of claim 1, wherein the pendant reactive functional groups are selected from carbon-carbon double bond, carbon-carbon triple bond, and thiol.

3. The method of claim 1, wherein the pendant reactive functional groups are derived from a molecule comprising an unsaturated carbon-carbon bond; and the molecule comprising an unsaturated carbon-carbon bond is selected from 1-octene, 1-hexyne, 4-bromo-1-butene, allyldiphenylphosphine, allylamine, allyl alcohol, 3,4-dihydroxy-1-butene, 7-octene-1,2-diol, 3-allyloxy-1,2-propanediol, 3-butenoic acid, 3,4-dehydro-L-proline, vinyl laurate, 1-vinyl-2-pyrrolidone, vinyl cinnamate, acrylamide, and acrylate.

4. The method of claim 1, wherein the pendant reactive functional groups are selected from aldehyde, amine, epoxide, hydroxyl, acid anhydride, azide, reactive halogen, and acyl chloride.

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

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

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

8. The method of claim 4, wherein the pendant reactive functional group is a hydroxyl.

9. The method according to any one of the preceding claims, 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 grafting end group; and the first functional group is selected from a cationic functional group, an anionic functional group, a hydrophobic functional group, a hydrophilic functional group, a thiophilic functional group, a hydrogen bond donating functional group, a hydrogen bond accepting functional group, a π-π bond donating functional group, a π-π bond accepting functional group, a metal chelating functional group, a biomolecule, and a bioion.

11. The method according to claim 10, wherein the first functional group is selected from a cationic functional group, an anionic functional group, a hydrophobic functional group, a hydrophilic functional group, a thiophilic functional group, a hydrogen bond donating functional group, a hydrogen bond accepting functional group, a π-π bond donating functional group, and a π-π bond accepting functional group.

12. The method according to claim 10, wherein the first ligand comprises a first functional group, and the first ligand is selected from 2-(diethylamino)ethyl methacrylate, 2-aminoethyl methacrylate, 2-carboxyethyl acrylate, 2-(methylthio)ethyl methacrylate, acrylamide, N-acryloxysuccinimide, butyl acrylate or butyl methacrylate, N,N-diethylacrylamide, N,N-dimethylacrylamide, 2-(N,N-dimethylamino)ethyl acrylate or 2-(N,N-dimethylamino)ethyl methacrylate, N-[3-(N,N-dimethylamino)propyl]methacrylamide, N,N-dimethylacrylamide, ethyl acrylate or ethyl methacrylate, 2-ethylhexyl methacrylate, hydroxypropyl methacrylate, glycidyl acrylate or glycidyl methacrylate, ethylene glycol phenyl ether methacrylate, methacrylamide, methacrylic anhydride, propyl acrylate or propyl methacrylate, N-isopropylacrylamide, styrene, 4-vinylpyridine, vinylsulfonic acid, N-vinyl-2-pyrrolidone (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, methacryloyloxyethyltrimethylammonium halide, 3-sulfopropyl methacrylate, 2-(2-methoxy)ethyl acrylate or 2-(2-methoxy)ethyl methacrylate, hydroxyethylacrylamide, N-(3-methoxypropylacrylamide), N-[tris(hydroxymethyl)methyl]acrylamide, N-phenylacrylamide, N-tert-butylacrylamide, and diacetoneacrylamide.

13. The method according to claim 10, wherein the first functional group is a metal chelating functional group.

14. The method according to claim 10, wherein the first functional group comprises a metal chelating functional group selected from an octadentate functional group, a hexadentate functional group, a tetradentate functional group, a tridentate functional group, a bidentate functional group, iminodiacarboxylic acid, and iminodiacetic acid.

15. The method according to claim 10, wherein the first functional group comprises a biomolecule or bioionic functional group selected from the following: albumin, lysozyme, virus, cell, gamma globulin from human and animal sources, immunoglobulin from human and animal sources, recombinant or natural source proteins including, synthetic or natural source polypeptides, interleukin-2 and its receptor, enzyme, monoclonal antibody, antigen, lectin, bacterial immunoglobulin-binding protein, trypsin and its inhibitor, cytochrome C, myosin, recombinant human interleukin, recombinant fusion protein, protein A, protein G, protein L, peptide H, nucleic acid derivatives, synthetic or natural source DNA, and synthetic or natural source RNA.

16. The method according to claim 1, wherein the coplanar stack of the coextensive sheets further comprises one or more flow splitters.

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