Methods for coupling ligands to composite materials

Abstract

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

Description

Background Art

[0001] In the 1970s, membrane-based water treatment methods were first introduced. Since then, membrane-based separation technologies have been used in many other industries. In the pharmaceutical industry and biotechnology industry, the use of preparative chromatography, direct flow filtration (DFF) and tangential flow filtration (TFF) (including microfiltration, ultrafiltration, nanofiltration and diafiltration) is a long-established method for separating dissolved molecules or suspended particles. For separation and purification in biomolecule manufacturing, ultrafiltration (UF) membranes and microfiltration (MF) membranes have become indispensable. Biomolecule manufacturing (regardless of its scale) generally adopts one or more steps using filtration. The attraction of these membrane separations lies in several features, including, for example, high separation power and simplicity, only a pressure difference needs to be applied between the feed stream and the permeate. This simple and reliable one-step filtration of samples into two parts makes membrane separation a valuable method for separation and purification.

[0002] The ligands conjugated to the fluid accessible surface of composite materials (such as membranes) can be used for separation and purification methods. However, the chemical modification of membranes is more challenging than resins. Resins can be easily suspended in solutions, and therefore resins can be modified in large reactors (wherein reagents are promoted to diffuse into resins by stirring suspensions). It is more challenging to modify membranes 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 when the reaction kinetics are very fast, the membrane physically moves through the groove of the reaction solution. It takes a longer reaction time to modify the membrane with a slower reaction chemistry (such as the coupling of protein A ligands with active membranes). Longer reaction times make roll-to-roll membrane modification methods impractical, and the roll-to-roll membrane modification methods require extremely slow movement of the membrane and therefore require extremely long processing time.

[0003] Composite modification methods are needed in which the reaction solution flows through the support components of the composite over a long period of time. Increasing the coupling of ligands to the composite increases the binding capacity of the affinity medium. Conjugation methods should take advantage of fast, efficient and easily controlled reactions to couple ligands to the composite. Summary of the invention

[0004] In one aspect, the 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 spirally wound configuration, the method comprising the steps of:

[0005] a. providing a functionalized composite material, the functionalized composite material comprising:

[0006] i. a support member comprising a plurality of holes extending through the support member; and

[0007] ii. a macroporous cross-linked gel, wherein the macroporous cross-linked gel comprises a polymer formed by reacting one or more polymerizable monomers with 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

[0008] b. flowing a first solution substantially 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 reactive functional groups and the first ligands. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1A Depicted is a schematic diagram of an exemplary composite material stacked between interlayers (eg, screens) with fluid flowing substantially across the layers of the functionalized composite material (tangential flow).

[0010] Figure 1B Depicted is a schematic diagram of an exemplary composite material stacked between interlayers (eg, screens) wherein a fluid substantially flows through the layers of the functionalized composite material (direct flow).

[0011] Figure 2 An exemplary composite material having an interlayer is shown in a spirally wound configuration.

[0012] Figure 3 Shown is the IgG dynamic binding capacity at a flow rate of 10 membrane volumes / min for a Protein A affinity ligand membrane conjugated using the flow through method compared to a membrane conjugated using the batch method.

[0013] Figure 4 is a sketch illustrating the composite material layers (i.e., membranes), interlayers (i.e., screens), and flow distribution layers assembled in a chromatography column. This pattern of 10 membranes is repeated 9 more times until 100 membranes are assembled. Then, an additional flow distribution layer is added.

[0014] Figure 5A Shown is the dynamic binding capacity of IgG as a function of membrane position within the stack.

[0015] Figure 5B The membrane flux is shown as a function of membrane position within the stack.

[0016] Figure 6is a sketch illustrating the different positions where a circular segment of the composite material is removed from a rectangular membrane sheet after tangential flow coupling with the protein A ligand on a spiral wound roll sandwiched with a mesh. DETAILED DESCRIPTION

[0017] Overview

[0018] The capacity of affinity medium depends largely on the amount of affinity ligands that can be conjugated to the fluid accessible surface of medium (such as composite material). The conjugation method that increases ligand and composite material coupling will increase binding capacity. In some embodiments, these methods make full use of fast, effective and easy to control reaction to functionalize composite material. In some embodiments, composite material is adsorptive macroporous chromatographic membrane. In some embodiments, compared with batch or static conjugation method, it is related to make ligand solution flow directly through membrane (that is, dead-end flow) or flow across membrane (that is, tangential flow) for affinity ligand conjugation method to produce affinity membrane with improved dynamic binding capacity.

[0019] Chromatographic membrane utilizes fast convection mass transfer mechanism to promote rapid purification or separation operation. However, in order to maximize the productivity of these operations, the binding capacity of the membrane to the target compound must be maximized. In some embodiments, the present invention describes the flow-through or dead-end flow method for conjugating ligands to the membrane containing side hanging reactive functional groups under the appropriate conditions of flow velocity, buffer pH and concentration, affinity ligand concentration and exposure time. In some embodiments, the present invention describes the cross-flow or tangential flow method for conjugating ligands to the membrane containing side hanging reactive functional groups under the appropriate conditions of flow velocity, buffer pH and concentration, affinity ligand concentration and exposure time. In some embodiments, the method produces conjugated affinity chromatography membrane, and the protein binding capacity of the conjugated affinity chromatography membrane is greater than the protein binding capacity achieved by using similar buffer conditions and affinity ligands using batch, non-flow conjugation method.

[0020] The flow-through and cross-flow conjugation methods produce consistently higher membrane binding capacities and can be performed on equipment containing in-line measurement tools that allow real-time observation of reaction progress and thus optimization of the reaction process. The higher membrane binding capacity combined with the fast binding kinetics enables fast, high-productivity chromatography purification operations.

[0021] definition

[0022] For convenience, certain terms used in the specification, examples and appended claims are collected here before further describing the present invention. These definitions should be interpreted in light of the remainder of this disclosure and understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art.

[0023] In describing the present invention, various terms are used in the specification. Standard terminology is widely used in the fields of filtration, fluid delivery, and general fluid handling.

[0024] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. For example, "an element" refers to one element or more than one element.

[0025] The terms "comprise" and "comprising" are used in an inclusive, open-ended sense, meaning that additional elements may be included.

[0026] The term "including" is used to mean "including, but not limited to." "Including" and "including, but not limited to" are used interchangeably.

[0027] The term "affinity chromatography" refers to a separation method based on a specific binding interaction 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.

[0028] The term "affinity medium" refers to a material comprising a plurality of immobilized ligands, for example, a composite material comprising covalently bound ligands.

[0029] The term "polymer" refers to a macromolecule formed by the combination of repeating units (monomers). The term polymer also includes copolymers.

[0030] The term "copolymer" refers to a polymer of at least two or more different monomers. If the crosslinking agent is a bifunctional monomer, the copolymer may consist of the crosslinking agent and the monomers.

[0031] The term "functionalized composite material" refers to a macroporous cross-linked gel comprising a plurality of pendant reactive functional groups located in the pores of a support member.

[0032] The term "pendant reactive functional group" refers to a functional group that will form one or more covalent bonds with the ligand when the ligand solution contacts the functional group. Examples of pendant 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, carbodiimides, acyl azides, fluorobenzenes, carbonates, N-hydroxysuccinimide esters, imidoesters, and fluorophenyl esters. Examples of pendant 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, haloacetyl groups, pyridyl disulfides, thiosulfates, and reactive halogens.

[0033] 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.

[0034] As used herein, the term "protein A" or "PrA" refers to a bacterial protein, protein A derivative or recombinant protein A from Staphylococcus aureus, which has the ability to bind to 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 to proteins with CH2 / CH3 regions (e.g., Fc regions). 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, thereby allowing the production of a large amount of recombinant protein A and protein A derivatives.

[0035] The term "wash solution" in connection with the coupling process refers to a solution that will carry away the coupling reactants. That is, any excess polymerizable monomer and any excess ligand will be removed.

[0036] The term "quenching solution" in connection with the coupling method is used to refer to a solution containing a reactive compound that will covalently bond with any remaining pendant reactive functional groups to form non-reactive groups. That is, the reactive compound will convert any remaining pendant reactive functional groups into non-reactive groups.

[0037] The term "non-reactive group" refers to a group that will not form a covalent bond under the conditions of further coupling reactions and separation methods. For example, exposure of the 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.

[0038] The term "buffer" refers to a solution that resists pH changes through the action of its acid-base conjugate components. Various buffers that can be used for 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, such as phosphate buffers are generally used for pH between 6.0 and 8.0, and for higher pH, borate buffers can be used, and for lower pH, carbonate buffers can be used. Those of ordinary skill in the art will be able to easily confirm the suitable buffer to be used according to the pH to be maintained. Non-limiting examples of the buffer that can be used in the method according to the present 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 buffer and combinations of these.

[0039] The term "cross-flow" in connection with fluid flow and filtration is used to refer to a fluid flow or filtration configuration in which a flowing fluid is directed along a surface of a composite material (e.g., a filter media) 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).

[0040] The terms "tangential flow" or "tangential filtration" are used to refer to a fluid flow or filtration process in which a flowing fluid is directed substantially parallel (i.e., tangentially) to a surface of a composite material (e.g., filter media), and a portion of the fluid passes through such composite material to provide a permeate. The terms "tangential filtration" and "cross-flow filtration" are often used interchangeably in the art.

[0041] The term "dead end" in connection with fluid flow and filtration is used to refer to a fluid flow or filtration configuration in which a flowing fluid is directed through a composite material (e.g., a filter media) and a portion of the fluid passing through such composite material has a velocity component that is through (i.e., parallel to the direction of fluid flow through such composite material).

[0042] The terms "direct flow" or "direct filtration" are used to refer to a fluid flow or filtration process in which a flowing fluid is directed substantially through (i.e., directly to) the surface of a composite material (e.g., filter media), and a majority 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.

[0043] The term "permeate" is used to mean a portion of a fluid that passes through a filter medium and exits through a first outlet port in a filter device that is operably connected to such filter medium. The term "decantate" is used to mean a portion of a fluid that flows along the surface of a filter medium but does not pass through such filter medium and exits through a second outlet port in a filter device that is operably connected to such filter medium.

[0044] Cross-flow filtration and tangential filtration are well-known filtration methods. Reference may 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 may also be made to "Filter and Filtration Handbook", 4th Ed., T. Christopher Dickenson, Elsevier Advanced Technology, 1997 (the disclosures of which are incorporated herein by reference).

[0045] As used herein, "binding-elution mode" refers to a method of operation for chromatography, wherein buffer conditions are established so that both the target protein and the undesirable contaminants are bound to a chromatographic carrier or composite material. The fractionation of the target protein and other components is subsequently achieved by changing the conditions so that the target protein and the contaminants are eluted respectively. In certain embodiments, the membrane described herein can be used in "binding-elution mode", characterized in that the high dynamic binding capacity under high conductivity, high volume flux and selectivity. In certain embodiments, the amount of the target protein in the eluent is reduced by about 50% to about 99%. In certain embodiments, the eluent in the aggregate of the target protein is reduced by about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%.

[0046] As used herein, the term "flow-through mode" refers to a method of operation for chromatography in which buffer conditions are established so 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 a "flow-through mode" during post-protein A purification to remove key contaminants (such as DNA, host cell proteins (HCPs), leached protein A, undesirable aggregates, and viruses) in a single step.

[0047] The term "average pore size" of a macroporous cross-linked gel may be understood by one of ordinary skill in the art to be determined by any suitable method. For example, the average pore size may be estimated by an environmental scanning electron microscope (ESEM) image 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 top, cross-section, and bottom perspectives; porosity and pore size distribution can be estimated from photographs.

[0048] The "volume porosity" of the support member is determined by simple calculation. For example, for a support member made of polypropylene, the outer dimensions of the support member are measured and the total volume is calculated [e.g., for a flat disk: V = πr 2 h, the volume of the support member (if it is solid or not porous). Then, determine the mass of the support member. Because the density of polypropylene is known or can be determined from Polymer Handbook, edited by Brandrup et al., Chapter VII, Wiley and Sons, New York, 1999, the volume porosity is calculated as follows:

[0049] Volume porosity = {(volume of support member (if solid)) - [(mass of support member) / (density of polypropylene)]} / (volume of support member (if solid)).

[0050] In this calculation, the void volume of the support member = (the volume of the outer 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 .

[0051] The volume porosity ε of the composite material is an experimentally determined value for each composite material. It is calculated by mass. The macroporous cross-linked gel is incorporated into the void volume of the support member. After drying to constant weight, the mass of the combined 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:

[0052] ε = {(void volume of support member) - [(mass of gel) x (differential specific volume of gel polymer)]} / (void volume of support member)

[0053] 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). PrA ligands selectively bind to the Fc and / or Fab binding domains on mAbs while allowing most impurities (host cell proteins, DNA, residual cell culture media) to flow through the resin. PrA media are usually washed after the loading step to remove additional impurities, and then the captured mAbs are eluted at low pH. The mAb purity in the eluate is significantly greater and has also been concentrated relative to the clarified cell culture. However, affinity media (e.g., with PrA) are very expensive and must be used for many batches in a few years to reduce the cost of each batch. Ideally, affinity media can be used for the maximum number of capture chromatography cycles (~200) for purifying a single batch of target substances (e.g., mAbs), which significantly reduces the volume of PrA media required for processing a single batch. Then, PrA media can be disposed of after purifying a single batch of mAbs, thereby eliminating the costs associated with storage resins. At present, most of the PrA media used in the downstream purification of biotherapeutic mAbs are in the form of resins. The slow mass transfer of mAbs into the porous resin structure requires a long loading time, wherein the residence time ranges 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 the mAb loaded onto the chromatographic medium divided by the volume of the chromatographic medium. Longer loading times prevent the resin from being cycled more than several times (2-4 times) per batch, without extending the PrA capture chromatography step to several days.

[0054] The PrA membrane can be loaded at a much shorter residence time (e.g., 0.1-1 min), and therefore provides the opportunity to rapidly cycle the capture chromatography step. In some embodiments, relative to the same volume of PrA resin, the rapid cycle of the PrA membrane allows the purification of much more mAb in the same amount of time. Therefore, in a given time period, the rapid cycle of a small volume of PrA membrane can be used to capture the same amount of mAb as the volume of a resin with a smaller number of cycles. In some embodiments, the PrA membrane provides the potential for processing a single batch of mAb using the complete life of the PrA membrane, thereby significantly reducing the upfront cost for setting up a mAb downstream purification process and eliminating the cost associated with the storage resin.

[0055] In some embodiments, protein A is an affinity ligand. In some embodiments, protein A is a protein, peptide or recombinant protein comprising a ligand that binds to a monoclonal antibody (e.g., an IgG antibody) and a portion that can form a covalent bond with a side-hanging reactive functional group (e.g., a thiol of Cys or an amine of Lys). In some embodiments, protein A is a protein, peptide or recombinant protein comprising a ligand that binds to the Fc domain of an antibody and a portion that can form a covalent bond with a side-hanging reactive functional group. In some embodiments, protein A is a protein, peptide or recombinant protein comprising a ligand that binds to the Fab domain of an antibody and a portion that can form a covalent bond with a side-hanging reactive functional group. In some embodiments, protein A can form multiple covalent bonds with multiple side-hanging reactive functional groups. In some embodiments, protein A forms multiple covalent bonds with a functionalized composite material.

[0056] 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 resistant to degradation.

[0057] 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 comprising a chromatography solid support matrix to which Protein A is covalently attached.

[0058] Chemical modification of composites is more challenging than that of resins. For example, a roll-to-roll modification approach for membranes with slow reaction chemistries would require extremely slow movement of the membrane through the reaction solution and extremely long processing times, which is incompatible with large-scale modification.

[0059] In some embodiments, the ligand coupling methods disclosed herein can be used to modify composite materials such as membranes.

[0060] 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:

[0061] a. providing a functionalized composite material, the functionalized composite material comprising:

[0062] i. a support member comprising a plurality of holes extending through the support member; and

[0063] 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

[0064] 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;

[0065] wherein the functionalized composite material is arranged in a coplanar stack of coextensive sheets, a tubular configuration, or a spirally wound configuration.

[0066] Exemplary functionalized composite materials

[0067] Composition of gel

[0068] In some embodiments, the crosslinked gel may be formed by an in situ reaction of one or more polymerizable monomers with one or more crosslinking agents. In certain embodiments, the gel may be formed by the reaction of one or more crosslinkable polymers with one or more crosslinking agents.

[0069] In some embodiments, cross-linked polymer is macroporous.Porosity in polymer can be improved by solvent exclusion of polymer chains during polymerization by degree of crosslinking, polymer network formation or some combination of the two.In some embodiments, the cross-linking agent of increasing concentration produces macroporous cross-linked gel.In some embodiments, porosity is affected by the degree of change polymer-(diluent+monomer) interaction, the amount of cross-linking agent, the amount of diluent, initiator concentration and polymerization temperature.

[0070] The degree of crosslinking in the polymer can be adjusted by adjusting the monomer ratio. The chain length of the polymers in the polymer network and therefore the degree of crosslinking can also be controlled by using specific monomers that impart specific physicochemical properties to the final polymer and membrane. These "tuning" monomers can affect the interaction of the polymer chains with the solvent system. In addition, the hydrophilicity / hydrophobicity of these monomers will affect the final water swelling properties of the resulting gel and the hydrophilic / hydrophobic surface properties of the polymer network.

[0071] In order to minimize the formation of composite materials without holes, solvent systems and monomers are selected to ensure that there are enough driving forces to exclude the growing polymer chains from the solution at a certain point, thereby forming macropores. Specifically, the mixture of solvent and non-solvent is adjusted to provide a suitable reaction system, which can dissolve all reactants at first, but acts as a poor solvent for the cross-linked polymer chains when the cross-linked polymer chains grow to greater than a certain molecular weight. The solvent system with a high proportion of poor solvents (for polymer chains) can cause the rapid precipitation of the growing polymer chains, which reduces porosity. The size of the macropores generally depends on the properties and concentration of the cross-linking agent, the properties of one or more solvents that form the gel, the amount of any polymerization initiator or catalyst, and the properties and concentration of pore-forming agent (porogen) (if present). In certain embodiments, composite materials can have a narrow pore size distribution.

[0072] In some embodiments, the macroporous cross-linked gel is formed due to phase separation during the free radical cross-linking 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 a low molecular weight compound (monomer and diluent) to form a macroporous cross-linked polymer. In some embodiments, the macroporous cross-linked gel is only slightly swollen in a solvent.

[0073] 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). Therefore, in order to prepare mechanically suitable membranes, in certain embodiments, a composite material comprising both a porous substrate (such as a fabric substrate made of chemically inert polypropylene) and a porous cross-linked polymer is produced by synthesizing the polymer directly within the pores of the substrate.

[0074] In certain embodiments, the composite material exhibits a well-connected gel network incorporated within the fibers of the matrix when examined using environmental scanning electron microscopy (ESEM).

[0075] In some embodiments, the formation of high polymer density regions (also referred to as bundling or lateral aggregation of polymer chains) leaves macropores between the high polymer density regions. In some embodiments, the macroporous cross-linked gel has a heterogeneous appearance.

[0076] In certain embodiments, 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).

[0077] In certain embodiments, the invention relates to any one of the methods disclosed herein, wherein the functionalized composite material comprises:

[0078] i. a support member comprising a plurality of holes extending through the support member; and

[0079] 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.

[0080] In certain embodiments, the present invention relates to any one of the methods disclosed herein, wherein the macroporous cross-linked gel of the composite material has macropores with an average diameter of about 5nm to about 10000nm. In certain embodiments, the macroporous cross-linked gel has macropores with an average diameter between about 10nm and about 3000nm. In certain embodiments, the macroporous cross-linked gel has macropores with an average diameter between about 25nm and about 1500nm. In certain embodiments, the macroporous cross-linked gel has macropores with an average diameter between about 50nm and about 1000nm. In certain embodiments, the macroporous cross-linked gel has macropores with an average diameter of about 50nm, about 100nm, about 150nm, about 200nm, about 250nm, about 300nm, about 350nm, about 400nm, about 450nm, about 500nm, about 550nm, about 600nm, about 650nm or about 700nm.

[0081] 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. Because only the maximum porosity is a characteristic property of a given material, it is appropriate to define the macroporosity relative to the maximum porosity.

[0082] In certain embodiments, the present invention relates to any one of the methods disclosed herein, wherein the macroporous cross-linked gel of the composite material is a neutral hydrogel, a charged hydrogel, a polyelectrolyte gel, a hydrophobic gel, a neutral gel, or a gel comprising functional groups. In certain embodiments, the present invention relates to any one of the methods disclosed herein, wherein the macroporous cross-linked gel of the composite material is a neutral hydrogel or a charged hydrogel; and the neutral hydrogel or the charged hydrogel is selected from cross-linked poly(vinyl alcohol), poly(acrylamide), poly(isopropylacrylamide), poly(vinyl pyrrolidone), poly(hydroxymethyl acrylate), poly(ethylene oxide), copolymers of acrylic acid or methacrylic acid with acrylamide, isopropylacrylamide or vinyl pyrrolidone, copolymers of acrylamide-2-methyl-1-propanesulfonic acid with acrylamide, isopropylacrylamide or vinyl pyrrolidone, copolymers of (3-acrylamido-propyl)trimethylammonium chloride with acrylamide, isopropylacrylamide or N-vinyl-pyrrolidone, and copolymers of diallyldimethylammonium chloride with acrylamide, isopropylacrylamide or vinyl pyrrolidone. In certain embodiments, the present invention relates to any one of the methods disclosed herein, wherein the macroporous cross-linked gel of the composite material is a polyelectrolyte gel; and the polyelectrolyte gel is selected from cross-linked poly(acrylamido-2-methyl-1-propanesulfonic acid) and salts thereof, poly(acrylic acid) and salts thereof, poly(methacrylic acid) and salts thereof, poly(styrenesulfonic acid) and salts thereof, poly(vinylsulfonic acid) and salts thereof, poly(alginic acid) and salts thereof, poly[(3-acrylamidopropyl)trimethylammonium] salts, poly(diallyldimethylammonium) salts, poly(4-vinyl-N-methylpyridinium) salts, poly(vinylbenzyl-N-trimethylammonium) salts, and poly(ethyleneimine) and salts thereof. In certain embodiments, the present invention relates to any one of the methods disclosed herein, wherein the macroporous cross-linked gel of the composite material is a hydrophobic gel; and the hydrophobic gel is selected from cross-linked 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 one of the methods disclosed herein, wherein the macroporous cross-linked gel of the composite material is a neutral gel; and the neutral gel is selected from a cross-linked polymer or copolymer of acrylamide, N,N-dimethylacrylamide, N-methacryloyl acrylamide, N-methyl-N-vinylacetamide and N-vinylpyrrolidone.

[0083] In certain embodiments, the cross-linked composite material (e.g., membrane) is further grafted with chemical functional groups or molecular species to provide a functionalized composite material. In certain embodiments, the cross-linked polymer can be functionalized to form a functionalized composite material by post-polymerization modification. In certain embodiments, the functionalized composite material comprising a functionalized cross-linked polymer can be coupled to a ligand by post-polymerization modification. In this two-step method, excess pendant reactive functional groups are modified during a separate grafting step, for example, excess thiol or olefin groups produced during thiol-olefin polymerization. By controlling the feed ratio of monomer to cross-linking agent, the final polymer can have excess pendant reactive functional groups. The pendant reactive functional groups of the functionalized composite material can then be used in a coupling reaction (e.g., a click reaction) to further modify the final polymer chemical composition and chemical properties (chemistry) or functional groups. In certain embodiments, the cross-linked polymer in the composite material contains residual reactive groups (referred to as pendant reactive functional groups, such as thiol or unsaturated carbon-carbon bonds), which can be used to connect various ligands via coupling reactions. In certain embodiments, the method can be used to prepare a polymer composite material (e.g., membrane) containing various ligands that can be used for chromatography. For example, chromatographic separation of biomolecules (e.g., proteins). For example, the method can be used to introduce ion exchange functional groups (e.g., carboxylates, sulfonates, quaternary ammonium salts, amines), hydrophobic interaction moieties (e.g., by using the octyl group of 1-octyl mercaptan or 1-octene) and biomolecules for bioaffinity chromatography (e.g., cysteine-protein A for monoclonal antibody purification) into a composite material (e.g., membrane).

[0084] In some embodiments, the composite materials exhibit high selectivity and high flow rates, low back pressure, are inexpensive, and allow for long column lifetimes, short processing times, and overall operational flexibility.

[0085] In certain embodiments, the present invention relates to any one of the aforementioned composite materials, wherein the composite material is a membrane.

[0086] In certain embodiments, the present invention relates to any one of the aforementioned composite materials, wherein the water contact angle of the composite material is from about 50° to about 120°.

[0087] Porous support member

[0088] In some embodiments, the support member has a void volume; and the void volume of the support member is substantially filled with the macroporous cross-linked gel. In some embodiments, the volume porosity of the porous support member is about 40% to about 90%. In some embodiments, the volume porosity of the porous support member is 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%.

[0089] In certain embodiments, the porous support is flat.

[0090] In certain embodiments, the porous support is disc-shaped.

[0091] Many porous substrates or films can be used as support members. In some embodiments, porous support members are made of polymer materials. In certain embodiments, support members can be polyolefins that can be obtained at low cost. In certain embodiments, polyolefins can be poly (ethylene), poly (propylene) or poly (vinylidene fluoride). Mention is made of a polyolefin film prepared by thermally induced phase separation (TIPS) or non-solvent induced phase separation. In certain embodiments, support members can be made of natural polymers (such as cellulose or its derivatives). In certain embodiments, suitable support members include polyethersulfone membranes, poly (tetrafluoroethylene) membranes, nylon membranes, cellulose ester membranes, glass fiber or filter paper. In some embodiments, support members include polymer materials selected from polysulfones, polyethersulfones, polyphenylene oxides, polycarbonates, polyesters, cellulose and cellulose derivatives.

[0092] In certain embodiments, the porous support member is composed of a woven fiber material or a nonwoven fiber material (e.g., a polyolefin, such as polypropylene). Such fiber woven or nonwoven support members can have a larger pore size than the TIPS support member, in some cases up to about 75 μm. Larger pores in the support member allow for the formation of composite materials with larger macropores in the macroporous gel. Non-polymer support members (e.g., ceramic-based supports) can also be used. The porous support member can be of various shapes and sizes.

[0093] In some embodiments, the support member is in the form of a membrane.

[0094] In some embodiments, the thickness of the support member is about 10 μm to about 2000 μm, about 10 μm to about 1000 μm, or about 10 μm to about 500 μm. In some embodiments, the thickness of the support member is 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.

[0095] In some embodiments, the average pore size of the pores of the support member is about 0.1 μm to about 50 μm. In some embodiments, the average pore size of the pores of the support member is about 0.1 μm to about 25 μm. In some embodiments, the average pore size of the pores of the support member is about 0.5 μm to about 15 μm. In some embodiments, the average pore size 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.

[0096] 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 before forming a gel in the interstices of the porous support. In another embodiment, a single support member unit is used to form a composite membrane, which is then stacked before use.

[0097] Relationship between gel and support structure

[0098] The gel may be anchored within the support member. The term "anchored" is intended to mean that the gel is held within the pores of the support member, but the term is not necessarily limited to mean that the gel is chemically bound to the pores of the support member. Although in some embodiments, the gel may be grafted to the surface of the pores of the support member, the gel may be held by physical constraints imposed thereon by entanglement and entanglement with structural elements of the support member without actually being chemically grafted to the support member.

[0099] In certain embodiments, the cross-linked gel is macroporous. In these cases, because macropores are present in the gel occupying the holes of the support member, the macropores of the gel must be smaller than the holes of the support member. Therefore, the flow characteristics and separation characteristics of the composite material depend on the characteristics of the gel, but are largely independent of the characteristics of the porous support member, provided that the size of the holes present in the support member is greater than the size of the macropores of the gel. The porosity of the composite material can be regulated by filling the support member with a gel, and the porosity of the gel depends partially or completely on the properties and amount of the monomer or polymer, crosslinking agent, reaction solvent and pore-forming agent (if used). The properties of the composite material are partially (if not completely) determined by the properties of the gel. The final result is that the present invention provides control of the macropore size, permeability and surface area of ​​the composite material.

[0100] 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 mentioned above, the influence of the pore size of the support material on the pore size of the macroporous gel is usually negligible. There are great differences in the pore size and pore size distribution of the support member and in those cases of seeking macroporous gels with very small pore size and narrow pore size distribution range, exceptions have been found. In these cases, the large change of the pore size distribution of the support member is reflected in the pore size distribution of the macroporous gel slightly. In certain embodiments, the support member with a slightly narrow pore size range can be used in these cases.

[0101] In certain embodiments, the present invention relates to any one of the aforementioned composite materials, wherein the composite material is relatively non-toxic.

[0102] Preparation of composite materials

[0103] In certain embodiments, composite materials of the present invention can be prepared by a single-step method. In certain embodiments, these methods can use water or other environmentally friendly solvents as reaction solvents. In certain embodiments, the method can be fast, and therefore can lead to a simple and / or fast manufacturing process. In certain embodiments, the preparation of composite materials can be cheap.

[0104] In certain embodiments, composite material can be prepared by mixing one or more monomers, one or more cross-linking agents, one or more initiators and one or more pore-forming agents optionally 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, and a gel-forming reaction can occur in the porous support.

[0105] In certain embodiments, a pore former may be added to the reactant mixture, where the pore former may be broadly described as a pore forming additive. In certain embodiments, the pore former may be selected from thermodynamically poor solvents and extractable polymers (eg, poly(ethylene glycol)), surfactants, and salts.

[0106] 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, hydroxyalkyl benzophenone 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.

[0107] 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 the support member containing the mixture of monomer, crosslinking agent and photoinitiator can be exposed to a wavelength of about 250nm to about 400nm UV radiation for a period of several seconds to several hours. In certain embodiments, the support member containing the mixture of monomer, crosslinking agent and photoinitiator can be exposed to a period of several seconds to several hours of UV radiation under about 350nm. In certain embodiments, the support member containing the mixture of monomer, crosslinking agent and photoinitiator can be exposed to UV radiation under about 350nm 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 so that energy can be transmitted by the support member.

[0108] In certain embodiments, the rate at which polymerization is performed may have an effect on the size of the macropores obtained in the macroporous gel. In certain embodiments, when the concentration of the crosslinker in the gel is increased 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 "macroporous". This mechanism is affected by the rate of polymerization.

[0109] 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.

[0110] Exemplary methods for coupling ligands to functionalized composite materials

[0111] 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.

[0112] 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:

[0113] a. providing a functionalized composite material, the functionalized composite material comprising:

[0114] i. a support member comprising a plurality of holes extending through the support member; and

[0115] 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

[0116] 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;

[0117] wherein the functionalized composite material is arranged in a coplanar stack of coextensive sheets, a tubular configuration, or a spirally wound configuration.

[0118] In certain embodiments, the present invention is directed to providing any of the aforementioned functionalized composite materials.

[0119] 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.

[0120] In certain embodiments, the present invention relates to any of the aforementioned methods, wherein the first solution is substantially flowed through the functionalized composite material. In some embodiments, the fluid flow path is directly through the functionalized composite material ( Figure 1B ). In some embodiments, direct current provides an increased pressure drop as the number of composite layers increases. 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. In some embodiments, direct current increases the mass transfer rate of ligands into the porous composite structure.

[0121] In certain embodiments, the cross-linked macroporous gel is further grafted with chemical functional groups or molecular species to provide a functionalized composite material. In certain embodiments, the cross-linked polymer can be functionalized by post-polymerization modification to form a functionalized composite material. In this two-step process, excess pendant reactive functional groups are modified during a separate grafting step, for example, excess thiol or olefin groups generated during thiol-olefin polymerization. By controlling the feed ratio of monomer to cross-linker, the final polymer can have an excess of pendant reactive functional groups.

[0122] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the pendant reactive functional group is selected from the group consisting of aldehydes, amines, carbon-carbon double bonds, carbon-carbon triple bonds, epoxides, hydroxyls, thiols, anhydrides, azides, reactive halogens, acid chlorides, and mixtures thereof.

[0123] In some embodiments of the method, the pendant reactive functional group is selected from a carbon-carbon double bond, a carbon-carbon triple bond and a thiol. In some embodiments, the pendant reactive functional group is derived from a molecule comprising a thiol functional group or a molecule comprising an unsaturated carbon-carbon bond. In some embodiments, the pendant reactive functional group is derived from a molecule comprising a thiol functional group; and the molecule comprising a thiol functional group is selected from 3-mercaptopropionic acid, 1-mercaptosuccinic acid, a polypeptide comprising a cysteine ​​residue, a protein comprising a cysteine ​​residue, a recombinant protein comprising a cysteine ​​residue, a bacterial immunoglobulin-binding protein comprising a cysteine ​​residue, a recombinant fusion protein comprising a cysteine ​​residue, cysteamine, 1-thiohexitol, poly(ethylene glycol) 2-mercaptoethyl ether acetic acid, poly(ethylene glycol) methyl ether thiol, 1-thio glycerol, 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.

[0124] In some embodiments, the molecule comprising a thiol functional group is selected from the group consisting of a polypeptide comprising a cysteine ​​residue, a protein comprising a cysteine ​​residue, a recombinant protein comprising a cysteine ​​residue, a bacterial immunoglobulin-binding protein comprising a cysteine ​​residue, and a recombinant fusion protein comprising a cysteine ​​residue. In some embodiments, the molecule comprising a thiol functional group is a protein comprising a cysteine ​​residue.

[0125] In some embodiments, the pendant reactive functional group is 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, amide or acrylate.

[0126] In some embodiments, the pendant reactive functional groups are selected from acid chlorides, acyl azides, aldehydes, amines, anhydrides, azides, carbonates, carbon-carbon double bonds, carbon-carbon triple bonds, carbodiimides, carboxylic acids, disulfides, epoxides, fluorobenzenes, fluorophenyl esters, haloacetyl groups, hydroxyls, imidoesters, isocyanates, isothiocyanates, maleimides, N-hydroxysuccinimide esters, pyridyl disulfides, reactive esters, reactive halogens, sulfonyl halides, thiols, and thiosulfates. In some embodiments, the pendant reactive functional groups are selected from aldehydes, amines, epoxides, hydroxyls, anhydrides, azides, reactive halogens, and acid chlorides. In some embodiments, the pendant reactive functional groups are selected from aldehydes, amines, epoxides, and hydroxyls. In some embodiments, the pendant reactive functional groups are selected from epoxides, aldehydes, carboxylic acids, reactive halogens, reactive esters, isocyanates, isothiocyanates, sulfonyl halides, carbodiimides, acyl azides, fluorobenzenes, carbonates, N-hydroxysuccinimide esters, imidoesters, and fluorophenyl esters. In some embodiments, the pendant reactive functional groups are selected from epoxides, aldehydes, carboxylic acids, reactive halogens, reactive esters, isocyanates, isothiocyanates, sulfonyl halides, carbodiimides, acyl azides, fluorobenzenes, carbonates, N-hydroxysuccinimide esters, imidoesters, and fluorophenyl esters, and react with amine groups. In some embodiments, the pendant reactive functional groups are selected from epoxides, thiols, disulfides, carbon-carbon double bonds, carbon-carbon triple bonds, maleimides, haloacetyl groups, pyridyl disulfides, thiosulfates, and reactive halogens. In some embodiments, the pendant reactive functional group is selected from the group consisting of epoxide, thiol, disulfide, carbon-carbon double bond, carbon-carbon triple bond, maleimide, haloacetyl, pyridyl disulfide, thiosulfate, and reactive halogen.

[0127] In some embodiments, the 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.

[0128] In some embodiments, the pendant reactive functional group is an aldehyde. In some embodiments, the one or more monomers comprising a pendant reactive functional group is vinyl methyl ketone.

[0129] In some embodiments, the pendant reactive functional group is an amine.

[0130] In some embodiments, the pendant reactive functional group is an epoxide. In some embodiments, the one or more monomers comprising a pendant reactive functional group is glycidyl methacrylate.

[0131] In some embodiments, the pendant reactive functional group is a hydroxyl group.

[0132] In some embodiments, the first ligand comprises a first functional group. In some embodiments, the first ligand further comprises at least one grafted 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. In some embodiments, 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.

[0133] In some embodiments, individual functional groups are included by incorporating functional monomers. In some embodiments, the relative amount of each functional group can be easily and readily adjusted to obtain optimal performance characteristics.

[0134] 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-acryloyloxysuccinimide, 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, methyl acrylate Acrylic acid, 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.

[0135] 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 the group consisting of 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 a salt of iminodiacetic acid.

[0136] In some embodiments, the metal chelating functional group is complexed with multiple metal ions. In some embodiments, the metal chelating functional group is selected from iminodicarboxylic acids, iminodiacetic acids and salts of iminodiacetic acids complexed 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 acids, iminodiacetic acids and salts of iminodiacetic acids complexed 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 complexed with multiple metal ions, wherein the metal ion is nickel or zirconium.

[0137] 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 following: albumin, lysozyme, virus, cell, γ-globulin of human and animal origin, immunoglobulin of human and animal origin, recombinant or natural source protein including, synthetic or natural source polypeptide, 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 derivative, 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 following: γ-globulin of human and animal origin, immunoglobulin of human and animal origin, recombinant or natural source protein including, synthetic or natural source polypeptide, 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 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.

[0138] 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 a protein A derivative and a recombinant protein A selected from the following: a polypeptide comprising a cysteine ​​residue, a protein comprising a cysteine ​​residue, a recombinant protein comprising a cysteine ​​residue, a bacterial immunoglobulin-binding protein comprising a cysteine ​​residue, and a recombinant fusion protein comprising a cysteine ​​residue. In some embodiments, the first functional group comprises protein A, and the protein A is selected from a protein, a peptide, or a recombinant protein, which comprises a ligand that binds to a monoclonal antibody (e.g., an IgG antibody) and a portion that can form a covalent bond with a side-hanging reactive functional group. In some embodiments, the first functional group comprises protein A, and the protein A is selected from a protein, a peptide, or a recombinant protein, which comprises a ligand that binds to the Fc domain of an antibody and a portion that can form a covalent bond with a side-hanging reactive functional group. In some embodiments, the first functional group comprises protein A, and the protein A is selected from a protein, a peptide, or a recombinant protein, which comprises a ligand that binds to the Fab domain of an antibody and a portion that can form a covalent bond with a side-hanging reactive functional group.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] In certain embodiments, the present invention relates to any one of the aforementioned methods, further comprising the following steps:

[0143] c. flowing the first wash solution substantially through or substantially across the composite material at a second flow rate to remove excess first ligand.

[0144] In certain embodiments, the present invention relates to any one of the aforementioned methods, further comprising the following steps:

[0145] 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

[0146] 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.

[0147] In some embodiments, the first solution of step b. is recirculated through or across the composite material.

[0148] In some embodiments, the quench solution of step d. is recirculated through or across the composite material.

[0149] In certain embodiments, the present invention relates to any one of the aforementioned methods, further comprising the following steps:

[0150] c. optionally, flowing the first wash solution substantially through or substantially across the composite material at a second flow rate to remove any excess first ligand;

[0151] d. flowing a second solution substantially through or substantially across the functionalized composite 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 ligands are cross-linking agents.

[0152] 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.

[0153] In some embodiments, the second ligand comprises a second functional group. In some embodiments, the second ligand further comprises at least one grafted end group; and the second 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. In some embodiments, the second 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.

[0154] 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.

[0155] 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.

[0156] In some embodiments, the polymerizable monomer comprising at least two pendant reactive functional groups is poly(ethylene glycol) divinyl ether.

[0157] In certain embodiments, the present invention relates to any one of the aforementioned methods, further comprising the following steps:

[0158] 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.

[0159] In certain embodiments, the present invention relates to any one of the aforementioned methods, further comprising the following steps:

[0160] 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

[0161] 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.

[0162] In some embodiments, the present invention relates to a method for coupling a ligand to a functionalized composite material, the method comprising the steps of:

[0163] a. providing a functionalized composite material, the functionalized composite material comprising:

[0164] i. a support member comprising a plurality of holes extending through the support member; and

[0165] 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;

[0166] 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;

[0167] 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 ligand;

[0168] 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

[0169] e. Optionally, flowing a second wash solution substantially through or substantially across the composite material at a fourth flow rate to remove any residual reactive compounds;

[0170] wherein the functionalized composite material is arranged in a coplanar stack of coextensive sheets, a tubular configuration, or a spirally wound configuration.

[0171] In some embodiments, the present invention relates to a method for coupling a ligand to a functionalized composite material, the method comprising the steps of:

[0172] a. providing a functionalized composite material, the functionalized composite material comprising:

[0173] i. a support member comprising a plurality of holes extending through the support member; and

[0174] 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;

[0175] 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;

[0176] 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 ligand;

[0177] d. flowing a second solution substantially through or substantially across the functionalized composite 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 ligands are cross-linking agents;

[0178] e. optionally, 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;

[0179] f. flowing a quenching solution 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 to non-reactive groups; and

[0180] g. Optionally, flowing a third washing solution substantially through or substantially across the composite material at a sixth flow rate to remove any residual reactive compounds;

[0181] wherein the functionalized composite material is arranged in a coplanar stack of coextensive sheets, a tubular configuration, or a spirally wound configuration.

[0182] In some embodiments, the functionalized composite material is a wetted membrane.

[0183] 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., a first solution, a first washing solution, a second solution, a quenching solution, a second washing solution, and a third washing solution) are pumped through the membrane holder.

[0184] In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the functionalized composite material is arranged in a coplanar stack of coextensive sheets, a tubular configuration, or a spirally wound configuration.

[0185] Membrane stack

[0186] In some embodiments, the composite material is arranged as 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 5 to 200 individual support members. In some embodiments, the composite material has from 5 to 100 individual support members.

[0187] In some embodiments, when the composite material is arranged as a substantially coplanar stack of substantially coextensive sheets, one or more separate support members are separated by one or more interlayers. In some embodiments, the present invention relates to any of the aforementioned methods, wherein the composite material layers and interlayers are alternating layers of composite material and interlayers (i.e., (composite material-interlayer) x or (sandwich-composite) x). In some embodiments, the composite material is layered with 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, 98, 99, and 100 separate interlayers. In some embodiments, the composite material has 1 to 50 separate interlayers. In some embodiments, the composite material has 1 to 25 separate interlayers.

[0188] 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.

[0189] In some embodiments, when the composite material is arranged into a substantially coplanar stack of substantially coextensive sheets, one or more individual support members and optionally one or more interlayers are separated by one or more flow distribution layers. In some embodiments, one or more flow distribution layers allow uniform coupling within the stack. In some embodiments, one or more flow distribution layers allow ligands to be more uniformly coupled to the composite material throughout the stack compared to coupling without a flow distribution layer. In some embodiments, the composite material has 1 to 250 individual flow distribution layers. In some embodiments, the composite material has 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 1 to 50 individual flow distribution layers. In some embodiments, the composite material has 1 to 25 individual flow distribution layers.

[0190] In some embodiments, one or more flow distribution layers are non-porous sheets. In some embodiments, one or more flow distribution layers are non-porous sheets containing holes. In some embodiments, flow distribution layers are periodically distributed within the membrane stack. In some embodiments, flow distribution layers are periodically distributed within the membrane stack comprising a composite layer and an interlayer (i.e., (composite-interlayer) x (Traffic Distribution) y or (sandwich-composite) x (Traffic Distribution) y ).

[0191] In some embodiments, the present invention relates to any one of the aforementioned methods, wherein the support member comprises a polymer material selected from the group consisting of polysulfone, polyethersulfone, polyphenylene ether, polycarbonate, polyester, cellulose and cellulose derivatives.

[0192] In some embodiments, the composite material is arranged in a tubular configuration.

[0193] Spiral Wound Configuration

[0194] In some embodiments, the composite material is arranged in a substantially spirally wound configuration. In some embodiments, the substantially spirally wound configuration comprises the composite material wound around the inner core to form a layer. In some embodiments, the substantially spirally wound configuration comprises the composite material wound around the inner core and an interlayer.

[0195] Sandwich in spiral winding configuration

[0196] In some embodiments, the present invention relates to any of the aforementioned methods, wherein the composite material layers and the interlayers are alternating layers of composite materials and interlayers (ie, (composite materials-interlayers) x or (sandwich-composite) x In certain embodiments, the present invention relates to any one of the above methods, wherein the composite material layer and the interlayer are arranged as (interlayer-first composite material-second composite material) x or (first composite material-second composite material-sandwich layer) x In certain embodiments, the invention relates to any of the above methods, wherein the composite material layers and the interlayer are arranged in a combination of the aforementioned arrangements. In certain embodiments, the first composite material and the second composite material are the same.

[0197] In some embodiments, the composite material comprises from about 3 to about 50 composite material layers surrounding an inner core.

[0198] In some embodiments, the invention relates to any of the aforementioned methods (eg, membrane stacking or spiral-wound configurations), wherein the composite material is in contact with one or more interlayers. In some embodiments, the interlayers provide some mechanical support for the composite material.

[0199] In some embodiments, the interlayer helps reduce back pressure.

[0200] In some embodiments, the present invention relates to any one of the aforementioned methods (e.g., membrane stacking or spiral winding 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.

[0201] 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 an approximately 0.45-mm mesh. In certain embodiments, the mesh interlayer is a biplanar thermoplastic mesh. In certain embodiments, the mesh interlayer is substantially similar to Naltex (a specific biplanar thermoplastic mesh) from DelStar Technologies, Inc.

[0202] In certain embodiments, the interlayer is spunbond polypropylene. In certain embodiments, the interlayer is a spunbond polypropylene having a basis weight of about 0.70 oz / yd. 2 About 0.95 oz / yd 2 In certain embodiments, the interlayer is a spunbond polypropylene having a basis weight of about 0.70 oz / yd 2 , about 0.75oz / yd 2 , about 0.80oz / yd 2 , about 0.85oz / yd 2 , about 0.90oz / yd 2 or about 0.95oz / yd 2 In certain embodiments, the interlayer is a spunbond polypropylene having a basis weight of about 0.86 oz / yd 2 of spunbond polypropylene.

[0203] In certain embodiments, the thickness of the interlayer is 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.

[0204] In certain embodiments, the volume porosity of the interlayer is about 50% to about 99%. In certain embodiments, the volume porosity of the interlayer is 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 about 80% to about 90%. In certain embodiments, the interlayer is substantially compressible.

[0205] In some embodiments, one or more interlayers are in contact with one or more flux distribution layers.

[0206] Spiral wound core

[0207] In some embodiments, the inner core is plastic. In some embodiments, the inner core is polypropylene or polysulfone.

[0208] In some embodiments, the inner core is a cylinder.In certain embodiments, the inner core is a cylinder that is capped or sealed at both ends.

[0209] 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 that is capped or sealed at one of its ends.

[0210] In some embodiments, the inner core is a screen wrapped around the cylinder.In certain embodiments, the screen provides a path through which a fluid can flow.

[0211] In some embodiments, the invention relates to any of the aforementioned methods, the composite material is a membrane.

[0212] In some embodiments, the present invention relates to any one of the aforementioned methods, wherein the cross-linked gel is a neutral hydrogel, a charged hydrogel, a polyelectrolyte gel, a hydrophobic gel, a neutral gel, or a gel comprising functional groups.

[0213] In some embodiments, the present invention relates to any one of the aforementioned methods, wherein the macroporous cross-linked gel comprises macropores having an average size of 10 nm to 3000 nm.

[0214] In some embodiments, the present invention relates to any one of the aforementioned methods, wherein the pores of the support member have an average pore size of about 0.1 μm to about 50 μm.

[0215] In some embodiments, the invention relates to any of the aforementioned methods, wherein the fluid flow path is substantially through the composite material.

[0216] In some embodiments, the invention relates to any of the aforementioned methods, wherein the fluid flow path substantially spans the composite material.

[0217] Methods of Making Exemplary Composite Materials

[0218] In certain embodiments, the invention relates to any of the aforementioned methods, wherein the ratio of pendant reactive functional groups to grafted end groups in the monomer mixture is from about 1:10 to about 2:1, for example, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, or about 2: 1. In some embodiments, the alkynyl group is equivalent to two alkenyl groups.

[0219] In certain embodiments, the present invention relates to any of the aforementioned methods, wherein the first monomer is present in the monomer mixture in an amount of about 5% to about 25% by weight of the monomer mixture. In certain embodiments, the present invention relates to any of the aforementioned methods, wherein the first monomer is present in the monomer mixture in an amount of about 5% to about 20% by weight of the monomer mixture.

[0220] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the second monomer is present in the monomer mixture in an amount of about 0.1 wt % to about 20 wt % of the monomer mixture.

[0221] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first cross-linking agent is present in the monomer mixture in an amount of about 1 wt % to about 20 wt % of the monomer mixture.

[0222] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the photoinitiator is present in the monomer mixture in an amount of about 0.1 wt % to about 2 wt % of the monomer mixture.

[0223] In certain embodiments, the present invention relates to any one of the aforementioned 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-propyl)ketone, )-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-methylphenylpropiophenone, 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.

[0224] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first solvent comprises N,N'-dimethylacetamide (DMAc), (±)-1,3-butanediol (Budiol), di(propylene glycol) methyl ether acetate (DPMA), water, di(propylene glycol) dimethyl ether (DPM), di(propylene glycol) propyl ether (DPGPE), di(propylene glycol) methyl ether (DPGME), tri(propylene glycol) butyl ether (TPGBE), 3-methyl-1,3-butanediol, 3,3-dimethyl-1,2-butanediol, 3-methoxy-1-butanol, dimethyl sulfoxide (DMSO), ethylene glycol, di(ethylene glycol), tri(ethylene glycol), tetra(ethylene glycol), hexylene glycol, sodium dodecyl sulfate, or N,N-dimethylformamide (DMF), or a mixture thereof.

[0225] In certain embodiments, the present invention relates to any of the aforementioned methods, wherein N,N'-dimethylacetamide (DMAc) is present in the monomer mixture in an amount of about 0% to about 70% by weight of the monomer mixture. In certain embodiments, the present invention relates to any of the aforementioned methods, wherein N,N'-dimethylacetamide (DMAc) is present in the monomer mixture in an amount of about 0% to about 50% by weight of the monomer mixture. In certain embodiments, the present invention relates to any of the aforementioned methods, wherein N,N'-dimethylacetamide (DMAc) is present in the monomer mixture in an amount of about 0% to about 70% by weight of the total solvent. In certain embodiments, the present invention relates to any of the aforementioned methods, wherein N,N'-dimethylacetamide (DMAc) is present in the monomer mixture in an amount of about 0% to about 50% by weight of the total solvent.

[0226] In certain embodiments, the present invention relates to any of the aforementioned methods, wherein (±)-1,3-butanediol (Budiol) is present in the monomer mixture in an amount of about 0% to about 50% by weight of the monomer mixture. In certain embodiments, the present invention relates to any of the aforementioned methods, wherein (±)-1,3-butanediol (Budiol) is present in the monomer mixture in an amount of about 0% to about 50% by weight of the total solvent.

[0227] In certain embodiments, the present invention relates to any of the aforementioned methods, wherein di(propylene glycol) methyl ether acetate (DPMA) is present in the monomer mixture in an amount of about 0% to about 60% by weight of the monomer mixture. In certain embodiments, the present invention relates to any of the aforementioned methods, wherein di(propylene glycol) methyl ether acetate (DPMA) is present in the monomer mixture in an amount of about 0% to about 60% by weight of the total solvent.

[0228] In certain embodiments, the present invention relates to any of the aforementioned methods, wherein water is present in the monomer mixture in an amount of about 0% by weight to about 50% by weight of the monomer mixture. In certain embodiments, the present invention relates to any of the aforementioned methods, wherein water is present in the monomer mixture in an amount of about 0% by weight to about 30% by weight of the monomer mixture. In certain embodiments, the present invention relates to any of the aforementioned methods, wherein water is present in the monomer mixture in an amount of about 0% by weight to about 30% by weight of the total solvent.

[0229] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the covered support member is irradiated at about 350 nm.

[0230] In certain embodiments, the invention relates to any one of the aforementioned 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.

[0231] In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the composite material comprises macropores.

[0232] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the average pore size of the macropores is smaller than the average pore size of the pores.

[0233] Pore ​​size determination

[0234] SEM and ESEM

[0235] As mentioned above, in certain embodiments, the cross-linked gel is a macroporous cross-linked gel. The average diameter of the macropores in the macroporous cross-linked gel can be estimated by one of a variety of methods. One method that can be used is scanning electron microscopy (SEM). SEM is a well-established method for determining pore size and porosity in general, and for characterizing membranes in particular. Reference is made to Marcel Mulder's book Basic Principles of Membrane Technology. (Especially Chapter IV). Mulder provides an overview of the methods used to characterize 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 top, cross-section, and bottom views. In addition, porosity and pore size distribution can be estimated from the photographs.

[0236] Environmental SEM (ESEM) is a technique that allows non-destructive imaging of wet samples by allowing a gas environment in the sample chamber. The environmental secondary detector (ESD) requires a gas background to operate and operates at about 3 torr to about 20 torr. These pressure constraints limit the ability to change the humidity in the sample chamber. For example, at 10 torr, the relative humidity at a specific temperature is as follows:

[0237] Relative humidity at 10 Torr (%) T(℃) Approx. 80 Approx. 16 Approx. 70 Approx. 18 Approx. 60 Approx. 20 Approx. 40 Approx. 24 Approx. 20 Approx. 40 Approx. 10 Approx. 50 Approx. 2 Approx. 70 Approx. 1 Approx. 100

[0238] 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 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%.

[0239] In certain embodiments, the microscope has nanometer resolution and a magnification of up to about 100,000×.

[0240] In some embodiments, the temperature in the sample chamber during imaging is 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.

[0241] In some embodiments, the pressure in the sample chamber during imaging is 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.

[0242] In certain embodiments, the working distance from the electron beam source to the sample is 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.

[0243] In certain embodiments, the voltage is 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.

[0244] In certain embodiments, the average pore size can be measured by estimating the pore size in a representative sample of images from the top or bottom of the composite material.One of ordinary skill in the art will recognize and appreciate the various experimental variables associated with obtaining ESEM images of wetted membranes and will be able to design experiments accordingly.

[0245] Capillary Flow Porometry

[0246] Capillary flow porography is an analytical technique used to measure the size of one or more pores of a porous material. In this analytical technique, a wetting liquid is used to fill the pores of the test sample, and a 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 liquid from the pores is related to the size of the pores by the following formula:

[0247] D=4×γ×cosθ / P

[0248] D = aperture

[0249] γ = liquid surface tension

[0250] θ = liquid contact angle

[0251] P = gas pressure difference.

[0252] This formula shows that the pressure required to displace liquid from a wetted sample is inversely related to the pore size. Since this technique involves the flow of liquid from the pores of the test sample under pressure, 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.

[0253] Capillary flow porography detects the presence of a pore when the gas begins to flow through it. This only occurs when the gas pressure is high enough to displace liquid from the narrowest part of the pore. Therefore, the pore size calculated using this method is the diameter of the pore at the narrowest part, and each pore is detected as a single pore with this narrow diameter. The maximum pore size (called the bubble point) is determined by the minimum gas pressure required to initiate flow through the wet sample, and the average pore size is calculated from the average flow pressure. In addition, both the narrow pore size range and the pore size distribution can be determined using this technique.

[0254] The method can be performed on a small membrane sample (eg, about 2.5 cm in diameter) immersed in a test fluid (eg, water, buffer, alcohol).The range of applied gas pressure can be selected from about 0 psi to about 500 psi.

[0255] Other methods for determining pore size

[0256] 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.

[0257] Exemplary Uses of Composite Materials

[0258] In certain embodiments, the invention relates to a method wherein a fluid is passed through a cross-linked gel of any of the above-described composite materials.Good selectivity can be obtained by adjusting the conditions for binding or fractionation.

[0259] In certain embodiments, the invention relates to a method further comprising the following steps:

[0260] f. flowing a first fluid containing a substance substantially through or across the composite material at a fifth flow rate, thereby adsorbing or absorbing a portion of the substance onto the composite material.

[0261] In certain embodiments, the invention relates to a method comprising the following steps:

[0262] h. flowing a first fluid containing a substance substantially through or across the composite material at a seventh flow rate, thereby adsorbing or absorbing a portion of the substance onto the composite material.

[0263] 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.

[0264] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the fluid flow path of the first fluid is substantially through the macropores of the composite material.

[0265] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the fluid flow path of the first fluid is substantially perpendicular to the macropores of the composite material.

[0266] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein substantially all of the substance is adsorbed or absorbed onto the composite material after the first fluid has substantially flowed through or substantially flowed across the composite material.

[0267] In certain embodiments, the present invention relates to any one of the aforementioned methods, further comprising the following steps:

[0268] g. contacting the second fluid at a sixth flow rate with the substance adsorbed or absorbed onto the composite material, thereby releasing a portion of the substance from the composite material.

[0269] In certain embodiments, the present invention relates to any one of the aforementioned methods, further comprising the following steps:

[0270] i. contacting the second fluid at an eighth flow rate with the substance adsorbed or absorbed onto the composite material, thereby releasing a portion of the substance from the composite material.

[0271] In some embodiments, the present invention relates to a method for coupling a ligand to a functionalized composite material, the method comprising the steps of:

[0272] a. providing a functionalized composite material, the functionalized composite material comprising:

[0273] i. a support member comprising a plurality of holes extending through the support member; and

[0274] 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;

[0275] 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;

[0276] 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 ligand;

[0277] d. flowing a quench solution substantially through or substantially 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;

[0278] e. Optionally, flowing a second wash solution substantially through or substantially across the composite material at a fourth flow rate to remove any residual reactive compounds;

[0279] 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; and

[0280] g. contacting the substance adsorbed or absorbed onto the composite material with a second fluid at a sixth flow rate, thereby releasing a portion of the substance from the composite material;

[0281] wherein the functionalized composite material is arranged in a coplanar stack of coextensive sheets, a tubular configuration, or a spirally wound configuration.

[0282] In some embodiments, the present invention relates to a method for coupling a ligand to a functionalized composite material, the method comprising the steps of:

[0283] a. providing a functionalized composite material, the functionalized composite material comprising:

[0284] i. a support member comprising a plurality of holes extending through the support member; and

[0285] 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;

[0286] 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;

[0287] 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 ligand;

[0288] d. flowing a second solution substantially through or substantially across the functionalized composite 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 ligands are cross-linking agents;

[0289] e. optionally, 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;

[0290] f. flowing a quenching solution 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 to non-reactive groups; and

[0291] g. Optionally, flowing a third washing solution substantially through or substantially across the composite material at a sixth flow rate to remove any residual reactive compounds;

[0292] 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; and

[0293] i. contacting the substance adsorbed or absorbed onto the composite material with a second fluid at an eighth flow rate, thereby releasing a portion of the substance from the composite material;

[0294] wherein the functionalized composite material is arranged in a coplanar stack of coextensive sheets, a tubular configuration, or a spirally wound configuration.

[0295] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the fluid flow path of the second fluid is substantially through the macropores of the composite material.

[0296] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the fluid flow path of the second fluid is substantially perpendicular to the macropores of the composite material.

[0297] In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the substance is a biomolecule, a bioion, a virus or a viral particle.

[0298] In certain embodiments, the present invention relates to methods of separating biomolecules or bioions (such as proteins or immunoglobulins) from solutions. In certain embodiments, the present invention relates to methods of purifying biomolecules or bioions (such as proteins or immunoglobulins). In certain embodiments, the present invention relates to methods of purifying proteins or monoclonal antibodies with high selectivity. In certain embodiments, the present invention relates to a method wherein the biomolecule or bioion retains its tertiary or quaternary structure, which may be important for maintaining biological activity.

[0299] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the substance is a biomolecule or bioion selected from the group consisting of albumin, lysozyme, viruses, cells, gamma-globulins of human and animal origin, immunoglobulins of human and animal origin, hIgG, proteins of recombinant and natural origin, polypeptides of synthetic and natural origin, interleukin-2 and its receptors, enzymes, monoclonal antibodies, trypsin and its inhibitors, cytochrome C, myoglobin, myosin, alpha-chymotrypsinogen, recombinant human interleukins, recombinant fusion proteins, nucleic acid-derived products, DNA of synthetic and natural origin, and RNA of synthetic and natural origin.

[0300] In some embodiments, the present invention relates to any of the aforementioned methods, wherein the substance is a biomolecule or bioion selected from the group consisting of albumin, lysozyme, virus, cell, gamma-globulin of human and animal origin, immunoglobulin of human and animal origin, hIgG, immunoglobulin M, proteins of recombinant and natural origin (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 galactase, recombinant dornase alfa, recombinant tissue-type plasminogen activator, recombinant human interferon, recombinant insulin-like growth factor 1 and recombinant asparaginase), polypeptides of synthetic and natural origin, interleukin-2 and its receptors, enzymes, monoclonal antibodies, trypsin and its inhibitors, cytochrome C, myoglobin, myosin, α-chymotrypsinogen, recombinant human interleukin, recombinant fusion proteins, factor VIII, factor IX, antithrombin III (antithrobin III), alpha-I-antitrypsin, nucleic acid-derived products, DNA of synthetic and natural origin, and RNA of synthetic and natural origin.

[0301] In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the biomolecule or bioion is lysozyme, hIgG, myoglobin, human serum albumin, soybean trypsin inhibitor, transferase, enolase, ovalbumin, ribonuclease, egg trypsin inhibitor, cytochrome c, annexin V or α-chymotrypsinogen.

[0302] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the substance is a biomolecule or bioion selected from the following: γ-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.

[0303] In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the substance is a biomolecule or bioion selected from the group consisting of: polypeptides, proteins, recombinant proteins, bacterial immunoglobulin-binding proteins, recombinant fusion proteins, protein A, protein G, protein L and peptide H.

[0304] In certain embodiments, the invention relates to methods for recovering antibody fragments from variants, impurities or contaminants associated therewith. In certain embodiments, biomolecule or bioion separation or purification can occur substantially in the cross-linked gel. In certain embodiments, when the cross-linked gel has macropores, biomolecule or bioion separation or purification can occur substantially in the macropores of the cross-linked gel.

[0305] In certain embodiments, the present invention relates to methods for the reversible adsorption of substances. In certain embodiments, the adsorbed substances can be released by changing the liquid flowing through the gel. In certain embodiments, the absorption and release of substances can be controlled by changes in the composition of the cross-linked gel.

[0306] In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first fluid is a clarified cell culture supernatant.

[0307] In certain embodiments, the present invention relates to a method, wherein material can be applied to composite material from buffer solution. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first fluid is a buffer. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the concentration of buffer in the first fluid is about 5mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 50mM, about 60mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 0.1M, about 0.11M, about 0.12M, about 0.13M, about 0.14M, about 0.15M, about 0.16M, about 0.17M, about 0.18M, ​​about 0.19M or about 0.2M.

[0308] In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the pH of the first fluid is about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, or about 9.

[0309] In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first fluid comprises sodium phosphate.

[0310] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the first fluid comprises salt. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the concentration of salt in the first fluid is about 50mM, about 60mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 0.1M, about 0.11M, about 0.12M, about 0.13M, about 0.14M, about 0.15M, about 0.16M, about 0.17M, about 0.18M, ​​about 0.19M, about 0.2M, about 0.25M or about 0.3M. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the salt is sodium chloride.

[0311] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the substance is a binding partner. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the composite material comprises a coupled ligand and the substance is a binding partner of the ligand.

[0312] In certain embodiments, the present invention relates to any of the aforementioned methods, wherein the concentration of the substance in the first fluid is about 0.01 mg / mL to about 1000 mg / mL. In certain embodiments, the present invention relates to any of the aforementioned methods, wherein the concentration of the substance in the first fluid is about 0.2 mg / mL to about 10 mg / mL. In certain embodiments, the present invention relates to any of the aforementioned methods, wherein the concentration of the substance in the first fluid is about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / L, about 1 mg / mL, about 1.2 mg / mL, about 1.4 mg / mL, about 1.6 mg / mL, about 1.8 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about mg / mL or about 10 mg / mL.

[0313] In certain embodiments, the present invention relates to any of the aforementioned 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 75MV / min. In certain embodiments, the present invention relates to any of the aforementioned 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 volumes (MV) / min to about 70MV / min. In certain embodiments, the present invention relates to any of the aforementioned 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 5MV / min to about 50MV / min. In certain embodiments, the present invention relates to any one of the aforementioned 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 21 MV / min, about 22 MV / min, about 23 MV / min, about 24 MV / min, about about 25MV / min, about 26MV / min, about 27MV / min, about 28MV / min, about 29MV / min, about 30MV / min, about 30MV / min, about 31MV / min, about 32MV / min, about 33MV / min, about 34MV / min, about 35MV / min, about 36MV / min, about 37MV / min, about 38MV / min, about 39MV / min, about 40MV / min, about 40MV / min, about 41MV / min, about 42MV / min, about 43MV / min, about 44MV / min, about 45MV / min, about 46MV / min, about 47MV / min, about 48MV / min, about 49MV / min and about 50MV / min. In certain embodiments, the present invention relates to any one of the aforementioned 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.

[0314] In certain embodiments, the present invention relates to any one of the aforementioned 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 0.5mL / min to about 50L / min. In certain embodiments, the present invention relates to any one of the aforementioned 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 0.5mL / min to about 25L / min. In certain embodiments, the present invention relates to any one of the aforementioned 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 0.5mL / min to about 10L / min. In certain embodiments, the present invention relates to any one of the aforementioned 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 0.5mL / min to about 1L / min. In certain embodiments, the present invention relates to any one of the aforementioned 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 0.5mL / min to about 0.5L / min. In certain embodiments, the present invention relates to any one of the aforementioned 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 0.5mL / min to about 100mL / min. In certain embodiments, the present invention relates to any one of the aforementioned 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 0.5mL / min to about 10mL / min. In certain embodiments, the present invention relates to any one of the aforementioned 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 0.5mL / min to about 2mL / min. In certain embodiments, the present invention relates to any one of the aforementioned 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.

[0315] In certain embodiments, the present invention relates to a method, wherein material can use aqueous saline solution of different concentrations and pH to elute.In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the second fluid is a buffer.In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the second fluid comprises glycine-HCl or sodium citrate.In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the second fluid comprises a concentration of about 5mM to about 2M glycine-HCl or sodium citrate.In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the second fluid comprises about 5mM, about 10mM, about 20mM, about 30mM, about 40mM, about 50mM, about 60mM, about 70mM, about 80mM, about 90mM, about 100mM, about 125mM, about 150mM, about 200mM, about 300mM or about 400mM glycine-HCl or sodium citrate.

[0316] In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the pH of the second fluid is from about 2 to about 8. In certain embodiments, the present invention relates to any one of the aforementioned methods, wherein the pH of the second fluid is about 2, about 2.2, about 2.4, about 2.6, about 2.8, about 3, about 3.2, about 3.4, about 3.6, about 3.8, about 4, about 4.2, about 4.4, about 4.6, about 4.8, about 5, about 5.2, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, and about 8.0.

[0317] In certain embodiments, the invention relates to methods that exhibit high binding capacity. In some embodiments, the binding capacity of the composite material is higher when a flow-through conjugation method is used compared to using a batch or dead-end conjugation method. In certain embodiments, the invention relates to a method that exhibits about 1 mg / mL at 10% breakthrough. 膜 , about 2mg / mL 膜 , about 3mg / mL 膜 , about 4mg / mL 膜 , about 5mg / mL 膜 , about 6mg / mL 膜 , about 7mg / mL 膜 , about 8mg / mL 膜 , about 9mg / mL 膜 , about 10mg / mL 膜 , about 12mg / mL膜 , about 14mg / mL 膜 , about 16mg / mL 膜 , about 18mg / mL 膜 , about 20mg / mL 膜 , about 30mg / mL 膜 , about 40mg / mL 膜 , about 50mg / mL 膜 , about 60mg / mL 膜 , about 70mg / mL 膜 , about 80mg / mL 膜 , about 90mg / mL 膜 , about 100mg / mL 膜 , about 110mg / mL 膜 , about 120mg / mL 膜 , about 130mg / mL 膜 , about 140mg / mL 膜 , about 150mg / mL 膜 , about 160mg / mL 膜 , about 170mg / mL 膜 , about 180mg / mL 膜 , about 190mg / mL 膜 , about 200mg / mL 膜 , about 210mg / mL 膜 , about 220mg / mL 膜 , about 230mg / mL 膜 , about 240mg / mL 膜 , about 250mg / mL 膜 , about 260mg / mL 膜 , about 270mg / mL 膜 , about 280mg / mL 膜 , about 290mg / mL 膜 , about 300mg / mL 膜 , about 320mg / mL 膜 , about 340mg / mL 膜 mg / mL 膜 , about 360mg / mL 膜 , about 380mg / mL 膜 or about 400 mg / mL 膜 The combined capacity method.

[0318] Example

[0319] The following examples are provided by way of example. However, it will be understood that the specific details given in each example have been selected for illustrative purposes and should not be construed as limiting the scope of the present disclosure. Generally, unless otherwise noted, experiments are carried out under similar conditions.

[0320] Example 1 - General Materials and Methods

[0321] protein

[0322] Recombinant protein A-cys (rProtein A-cys) was obtained from Biomedal SL (Seville, Spain) and polyclonal immunogammaglobulin IgG was obtained from Equitech-Bio Inc. (Kerrville, TX, USA).

[0323] Membrane preparation

[0324] Plan A

[0325] One or more crosslinkers and monomers (except thiol-functionalized monomers, which are added 10 min before casting) are added to the solvent mixture along with a photoinitiator (IRGACURE 2959), and the mixture is stirred long enough to dissolve all components. A pre-weighed 7" x 8" porous support substrate sheet (non-woven polypropylene mesh) is placed on the polyethylene sheet, and then ~15 g of the polymer solution is poured into the substrate sheet. Subsequently, the impregnated substrate is covered with an additional polyethylene sheet. The sheet is gently pressed in a circular motion by hand to remove excess solution and any trapped bubbles. The polymerization process is initiated by irradiating the polymer solution / substrate sandwiched between polyethylene sheets with UV light (~350 nm) for 10 min in a closed chamber. The resulting membrane is then removed from between the polyethylene sheets and subjected to a thorough washing cycle involving a 20-30 minute soaking period (2-3 times) in purified (RO) water under agitation. The clean membrane is dried by hanging freely in air for ~16 hours at room temperature.

[0326] Plan B

[0327] The membrane is prepared by polymerizing acrylate and / or acrylamide monomers in a support mesh material with a crosslinking agent 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 membrane can also be subjected to additional heat treatment steps to adjust its performance and properties.

[0328] For example, epoxy-containing membranes can be used as reactive media platforms that can be converted into bioaffinity membranes by covalently anchoring various ligands to the surface, such as protein A. Other ligands can also be conjugated to target other biomolecules or entities, such as viruses.

[0329] Mass gain, wetting and permeability of composite membranes

[0330] The weight of the dried film was measured and used to calculate the mass increase. The wetting of the film was also determined by dispensing 50 μL of distilled water drops on the film surface and measuring the time required for the drop to be absorbed in the film. In order to estimate the membrane permeability, the flux of each membrane was determined using RO water (or acetate buffer pH 5) and a 7.7-cm diameter film sample using a 100 kPa applied pressure.

[0331] To estimate membrane permeability, the flux of RO water (or 132 mM acetate buffer pH 5) as the mobile phase through each membrane was measured. The membrane was 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 test liquid that passed through a 7.7 cm diameter (actual 7.3 cm usable diameter) circular membrane sample under an applied pressure of 100 kPa was measured. The flux is expressed as the amount of liquid per surface area per time (kg / m 2 h).

[0332] Imaging of porous structures

[0333] In order to probe the gel structure and porosity, an environmental scanning electron microscope (ESEM) was used to image the film in the wet state. Small specimens (~7×5 mm) were wetted by soaking in distilled water for 10-15 minutes and then examined using an ESEM instrument (FEI Quanta FEG 250ESEM). The sample was placed on a cooling stage to adjust the temperature to 5°C, and the image was examined at a low pressure level (4.5-5.5 Torr) and a relative humidity of 50-55%.

[0334] To probe the film structure in the dry state, a Tescan Vega II LSU scanning electron microscope (SEM) (Tescan, PA, USA) was used to image the gold-coated films with voltage settings of 10-20 kV.

[0335] Hole size measurement

[0336] Membrane pore size (diameter) was measured using a CFP-1500-AE capillary flow porosimeter (Porous Materials Inc., Ithaca, NY) operated by CapWin software (V.6).

[0337] A small disk (2.5-cm diameter) of membrane was immersed in Wetting liquid (Porous Materials Inc., surface tension = 15.9 dyne / cm) for 10min, then gently squeezed between two pre-wetted filter paper discs (Whatman 5-70mm) to remove excess solution, and the thickness of the wet film was measured using a micrometer. The film disc was then placed on a 2.5-cm stainless steel mesh support disc. The support disc loaded with the test film was placed in a specified holder with the film facing up. The metal cover was then gently placed on the holder, and the test was performed within a pressure range of 0-200psi.

[0338] Protein A ligand density on the composite membrane

[0339] To measure the Protein A ligand density on the coupled membrane, the amount of uncoupled protein remaining after the coupling reaction was determined and subtracted from the total ligand amount to give the amount of coupled ligand, which was then divided by the membrane volume (mL) to express the density as mg ligand per mL of membrane.

[0340] To determine the amount of protein A in solution, a series of protein solutions in 0.1 M phosphate buffer (pH 7.2) were prepared, the absorbance of each solution at 280 nm was measured, and a calibration curve was constructed from which the slope was determined.

[0341] For the selected membrane formulation, a 4 cm × 7 cm sample was cut and its thickness was measured, from which the volume was calculated. The coupling reaction was carried out as described above, and 20 mg was loaded to each membrane coupling reaction respectively. When the UV reaction was completed, the reaction solution was collected in a tube, and then 3-5 mL of 0.1 M phosphate buffer was added to the reaction bag and used to wash the membrane by shaking for 20-25 min, and then the resulting solution was added to the collection tube.

[0342] The wash cycle was repeated an additional two times, then the final solution absorbance was measured and the amount of uncoupled protein was calculated using the calibration curve slope.The amount of coupled ligand was determined by taking the difference between the total reaction amount and the uncoupled amount.

[0343] Binding capacity measurements

[0344] Bioaffinity IgG binding capacity

[0345] The membrane disk of 25-mm diameter is placed in 25-mm Natrix stainless steel (SS) holder.20mL binding buffer (20mM sodium phosphate, 150mM NaCl, pH 7.4) is passed through to reach equilibrium (~160-200 bed volume / min).In the binding step, 0.5mg / mL polyclonal IgG in binding buffer is passed through with a flow rate of 1mL / min, until the UV absorbance of the effluent exceeds 10% of the feed solution, and then 10-15mL buffer is passed through with a flow rate of 2mL / min to remove unbound protein.In the elution step, the combined IgG is eluted by passing through 10-14mL elution buffer (0.1M glycine-HCl or 0.1M sodium citrate, both of which are in pH 3) with a flow rate of 2mL / min.

[0346] Cation Exchange IgG Binding Capacity

[0347] The 25-mm membrane disk is placed in a 25-mm Natrix-SS holder, and 20 mL of binding buffer (132 mM sodium acetate, pH 5.0) is passed through to reach equilibrium. Then a protein solution (0.5 mg / mL human polyclonal IgG (Equitech-Bio Inc.) in binding buffer) is passed through until the UV absorbance of the effluent exceeds 10% of the feed solution, and then 10-15 mL of buffer is passed through the cell to wash unbound protein. In the elution step, the bound IgG is eluted by passing through 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).

[0348] Hydrophobic interaction mode IgG binding capacity

[0349] The 25-mm membrane disk is 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) is passed through to reach equilibrium. Then, a protein solution (0.5 mg / mL human polyclonal IgG (Equitech-Bio Inc.) in binding buffer) is passed through until the UV absorbance of the effluent exceeds 10% of the feed solution. Subsequently, 15-20 mL of buffer is passed through the pool to wash unbound protein. In the elution step, the combined IgG is eluted by passing through 10 mL of elution buffer (50 mM sodium phosphate, pH 7.0).

[0350] Example 2 - Exemplary bulk coupling protocol

[0351] Conjugation of Protein A ligand to click olefin membranes

[0352] To examine the feasibility of chemically binding biomolecules (with thiol functional groups) to olefin membranes via a hydrothiolation click reaction, engineered protein A ligands containing cysteine ​​residues were coupled to one or more olefin membranes (with different chemical formulas), and the biological activity of the immobilized ligands was detected.

[0353] The protein A ligand lyophilized powder (recombinant protein A-cys) was dissolved in PBS (20 mM sodium phosphate, 0.15 M NaCl, pH 7.4) to prepare a 50 mg / mL stock solution. To prepare the coupling 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 thoroughly. The volume of the final reaction solution was ~2.0 mL and contained approximately 20 mg of ligand and approximately 7.5 mg of initiator.

[0354] Alternatively, ACVA was dissolved in reaction buffer (2M phosphate, pH 7.2) at a concentration of 5 mg / mL to avoid the use of DMAc. For low salt experiments, the initiator was dissolved in 0.5 M phosphate at a concentration of 7.5 mg / mL.

[0355] A 4×7-cm membrane sample (pre-wetted in water) was added to the bag loaded with the coupling reactants. The bag was shaken for one minute and then irradiated with UV light (~365nm) for 10 minutes. After the irradiation was complete, the coupling solution was decanted, and then 15-20mL of washing buffer solution (0.1M 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 8mL of trehalose solution (10 wt%), shaken for 10-15 minutes, and dried in an oven (50°C) for 20-30min; or (ii) stored in 0.1M phosphate buffer.

[0356] For coupling in the presence of additives, ACVA was dissolved in 0.5 M potassium phosphate (pH 7.2) to prepare a solution at a concentration of 7.5 mg / mL. 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 ligand stock solution was mixed with 0.25 mL of initiator solution, and 50 μL of additives were added (cysteamine-HCl was added to reaction B bag, 1-mercaptoethanol was added to reaction C bag).

[0357] After fully mixing the reaction solution, a 25-mm diameter film disk is placed in each bag, and the reaction bag is fully vibrated, then irradiated with UV light for 10 minutes. The reaction solution is decanted, and then the film sample is washed three times and vibrated for 10-15 minutes using 0.1M sodium phosphate buffer (pH 7.2). As mentioned above, the composite film sample is stored in buffer (0.1M sodium phosphate, pH 7.2), and the bioaffinity to the IgG protein is tested.

[0358] Example 3 - Flow-through ligand conjugation effects

[0359] The sample tray of the wetted membrane containing the side-hanging reactive functional groups is placed in a stainless steel holder and attached to an AKTA chromatography system. The affinity ligand solution is pumped through the membrane holder for a specified duration at a limited flow rate. Subsequently, a wash solution is pumped through the holder, followed by a quenching solution containing a reactive compound, which converts the residual membrane side-hanging groups into non-reactive groups. Finally, the wash solution is pumped through the holder. The pump is stopped, the holder is removed from the AKTA, and the holder is disassembled to take out the conjugated membrane. The human IgG dynamic binding capacity of the conjugated membrane (protein A affinity ligand) is measured at a flow rate of 10 membrane volumes / minute and compared with the membrane conjugated in a batch non-circulation method (see Figure 3 ). When flow-through conjugation was used, the membrane dynamic binding capacity was observed to be consistently higher.

[0360] Example 4 - Protein A coupling via flow through a membrane stack without periodic splitters

[0361] A header containing a porous glass frit was attached to a glass chromatography column with an inner diameter of 44 mm ( L laboratory column VL 44 × 250, catalog number: 96440250) bottom. Then, a circular membrane (volume 0.25mL) with a diameter of 30mm and a thickness of about 350 microns is placed on the glass frit inside the column so that it is at an equal distance from the column wall. The membrane has a surface containing epoxy groups. Then, a circular section of a polypropylene mesh (1:2 twill weave, 500 micron mesh) with a diameter of 44mm is placed in the column and lowered so that it is flat on top of the membrane and contacts the inner wall of the column. Then, another 30mm diameter membrane is placed in the column and lowered so that it is flat on top of the mesh and is at an equal distance from the column wall. Repeat the process of placing the mesh above the membrane and then placing the membrane above the mesh until the column contains 100 membranes (total volume 25mL) layered between 99 meshes. Then, a head is added to the top of the column and lowered until the membrane layer and mesh layer are compressed to a height of 9.5cm.

[0362] Next, tubing was added to the inlet at the bottom of the column, and tubing was also added to the outlet at the top of the column. The inlet tubing was connected to a peristaltic pump, allowing controlled flow of solution through the column.

[0363] Be the PBS buffer replacement post and the air in the pipe that is composed of 20mM sodium phosphate and 150mM sodium chloride of 7.4 with pH.The inlet pipe is placed in the glass bottle containing 400mL PBS buffer, and the outlet pipe is directed to waste material.Then start the pump, and make the PBS buffer of 200mL flow through the post 4min with the speed of 50mL / min.Stop the pump, and invert the post.Then, exchange the connection of inlet pipe and outlet pipe with the post, so that the inlet is positioned at the bottom of the post again and the outlet is positioned at the top of the post.Start the pump, and make the PBS buffer of extra 200mL flow through the post 4min with the speed of 50mL / min.

[0364] The pump is stopped, and the inlet pipe is moved to a glass bottle containing 500mL of a coupling buffer consisting of 1.35M potassium phosphate at pH 9.0. The outlet pipe remains directed to waste. The pump is started, and 200mL of coupling buffer is flowed through the post at a rate of 50mL / min for 4min. The pump is stopped, and the outlet pipe is placed in a glass bottle identical to the inlet pipe, which contains remaining 300mL of coupling buffer. In this configuration of the inlet pipe and the outlet pipe in the same bottle, the solution can be recirculated through the post many times. Recirculating the solution through the post allows for extended reaction times without increasing the volume of required solution.

[0365] 82.4mL of the PrA ligand stock solution with a concentration of 25.8g / L in water was added to the glass bottle containing the remaining 300mL coupling buffer. The pump was started, and then the PrA solution was recirculated through the post for 4 hours at a flow rate of 50mL / min. The total PrA solution volume recirculated through the post was calculated to be 422.4mL, which was composed of 300mL coupling buffer remaining in the glass bottle, 40mL coupling buffer remaining in the post / tube and the 82.4mL PrA stock solution added. Considering that the 82.4mL PrA ligand stock solution containing 2.12g PrA ligand was diluted to a volume of 422.4mL, the PrA solution recirculated in the system was calculated to have a concentration of 5g / L. The ligand loading on the membrane was calculated by dividing the gross mass of the 2.12g PrA ligand by the total membrane volume 25mL to obtain a ligand loading of 85g / L.

[0366] After 4 hours, stop the pump, guide the outlet pipe to waste, and guide the outlet pipe to waste. The inlet pipe is placed in a glass bottle containing 200mL PBS buffer. Start the pump, and make the PBS buffer flow through the post for 4min with a flow rate of 50mL / min.

[0367] The pump was then stopped, and the inlet pipe was placed in the glass bottle containing 800mL 1M ethanolamine. The outlet pipe remained directed to waste. The pump was started, and 200mL 1M ethanolamine was flowed through the post to waste for 4min at a flow rate of 50mL / min. The pump was stopped, and the outlet pipe was directed to the glass bottle containing the remaining 600mL 1M ethanolamine solution. The pump was then started, and the remaining 600mL 1M ethanolamine solution was recirculated through the post for 3 hours at a flow rate of 50mL / min.

[0368] 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.

[0369] The pump is stopped, and the top column head is removed. The film is taken out, and stored in PBS buffer. The flux and the IgG dynamic binding capacity of several films at different positions in the stack are measured. Position #1 is closest to the column entrance, and position #100 is closest to the column outlet. It is found that along with position changes, the film flux does not have significant deviation (table 1). It is found that the film at position 60 and position 80 has a much lower IgG dynamic binding capacity, thereby showing that the flow distribution in the column is uneven.

[0370] Table 1. Membrane flux and IgG dynamic binding capacity based on the position of the membrane in the stack

[0371]

[0372] Example 5 - Protein A coupling via flow through a membrane stack with periodic flow dividers

[0373] The head containing the porous glass frit was attached to a glass chromatography column with an inner diameter of 44 mm ( L laboratory column VL 44×250, catalog number: 96440250). Then, a flow distribution layer consisting of a circular impermeable plastic sheet with a thickness of approximately 0.025 inches and a diameter of 44 mm (which has a hole with a diameter of 6 mm in the center) is lowered so that it lies flat on top of the porous glass. Then, two circular sections of a polypropylene screen (1:2 twill weave, 500 micron mesh) with a diameter of 44 mm are placed over the flow distribution layer. Then, a circular membrane (volume 0.25 mL) with a diameter of 30 mm and a thickness of approximately 350 microns is placed on the screen so that it is an equal distance from the column wall. The membrane has a surface containing epoxy groups. Another circular section of the polypropylene screen is lowered so that it lies flat on top of the membrane. The process of adding a membrane layer followed by a polypropylene screen layer is repeated 9 more times until the column has Figure 4 The following composition is shown in .

[0374] The process of assembling the layers in the column as described above was repeated 9 more times until the column contained 100 membrane layers (total volume 25 mL), 120 screen layers, and 10 flow distribution layers. Additional flow distribution layers were then added to the stack, and a head with a porous glass frit was added to the top of the column. The head was lowered until the membranes, screens, and flow distribution layers were compressed to a height of 10.5 cm.

[0375] Next, tubing was added to the inlet at the bottom of the column, and tubing was also added to the outlet at the top of the column. The inlet tubing was connected to a peristaltic pump, allowing controlled flow of solution through the column.

[0376] Be the PBS buffer replacement post and the air in the pipe that is composed of 20mM sodium phosphate and 150mM sodium chloride of 7.4 with pH.The inlet pipe is placed in the glass bottle containing 400mL PBS buffer, and the outlet pipe is directed to waste material.Then start the pump, and make 200mL PBS buffer flow through post 4min with the speed of 50mL / min.Stop the pump, and invert the post.Then, exchange the connection of inlet pipe and outlet pipe with post, make the inlet be positioned at the bottom of post again and outlet be positioned at the top of post.Start the pump, and make extra 200mL PBS buffer flow through post 4min with the speed of 50mL / min.

[0377] The pump is stopped, and the inlet pipe is moved to a glass bottle containing 500mL of a coupling buffer consisting of 1.35M potassium phosphate at pH 9.0. The outlet pipe remains directed to waste. The pump is started, and 200mL of coupling buffer is flowed through the post at a rate of 50mL / min for 4min. The pump is stopped, and the outlet pipe is placed in a glass bottle identical to the inlet pipe, which contains remaining 300mL of coupling buffer. In this configuration of the inlet pipe and the outlet pipe in the same bottle, the solution can be recirculated through the post many times. Recirculating the solution through the post allows for extended reaction times without increasing the volume of required solution.

[0378] 82.4mL of the PrA ligand stock solution with a concentration of 25.8g / L in water was added to the glass bottle containing the remaining 300mL coupling buffer. The pump was started, and then the PrA solution was recirculated through the post for 4 hours at a flow rate of 50mL / min. The total PrA solution volume recirculated through the post was calculated to be 422.4mL, which was composed of 300mL coupling buffer remaining in the glass bottle, 40mL coupling buffer remaining in the post / tube and the 82.4mL PrA stock solution added. Considering that the 82.4mL PrA ligand stock solution containing 2.12g PrA ligand was diluted to a volume of 422.4mL, the PrA solution recirculated in the system was calculated to have a concentration of 5g / L. The ligand loading on the membrane was calculated by dividing the gross mass of the 2.12g PrA ligand by the total membrane volume 25mL to obtain a ligand loading of 85g / L.

[0379] 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 allow the PBS buffer to flow through the column at a flow rate of 50 mL / min for 4 min.

[0380] The pump was then stopped, and the inlet pipe was placed in the glass bottle containing 800mL 1M ethanolamine. The outlet pipe remained directed to waste. The pump was started, and 200mL 1M ethanolamine was flowed through the post to waste for 4min at a flow rate of 50mL / min. The pump was stopped, and the outlet pipe was directed to the glass bottle containing the remaining 600mL 1M ethanolamine solution. The pump was then started, and the remaining 600mL 1M ethanolamine solution was recirculated through the post for 3 hours at a flow rate of 50mL / min.

[0381] 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.

[0382] Stop the pump and remove the top column head. Take out the membrane and store it in PBS buffer. Determine the flux and IgG dynamic binding capacity of several membranes at different positions in the stack. Position #1 is closest to the column inlet, and position #100 is closest to the column outlet. It is found that the membrane flux has no significant deviation as the position changes (Table 2). It is also found that the IgG dynamic binding capacity has no significant deviation, thereby indicating that the flow distribution is relatively uniform throughout the column. The addition of the flow distribution layer provides uniform coupling of all epoxide membranes with the PrA ligand in the stack ( Figure 5A and Figure 5B ).

[0383] Table 2. Membrane flux and IgG dynamic binding capacity based on membrane position in the stack

[0384]

[0385] Example 6 - Protein A coupling via tangential flow through spirally wound membrane rolls

[0386] The rectangular film (total film volume is 53.6mL) with a width of 24.5cm, a length of 62.5cm and a thickness of 0.035cm and a plastic rectangular section of a polypropylene screen (1:2 twill weave, 500 micron mesh) with a width of 25.4cm are wound around a cylindrical core with a diameter of 1.6cm and a length of 25.4cm. The film is centered so that there is 0.45cm between the edge of the film and the edge of the screen. The film and the screen are wound around the core, wherein the screen contacts the core. Roll up the film and the screen until all the films are completely covered by the screen layer. The screen layer is continued to be wound around the roll until the diameter of the roll is 32mm. Then, the screen layer is cut off. Then, the roll is slid to the glass chromatographic column ( L laboratory column VL 32×250, catalog number: 96320250) so that the edge of the roll is 2.5 cm from both ends of the column. Then, a head containing a porous glass frit is attached to the bottom and top of the column.

[0387] Next, tubing was added to the inlet at the bottom of the column, and tubing was also added to the outlet at the top of the column. The inlet tubing was connected to a peristaltic pump, allowing controlled flow of solution through the column.

[0388] Be the PBS buffer replacement post and the air in the pipe that is composed of 20mM sodium phosphate and 150mM sodium chloride of 7.4 with pH.The inlet pipe is placed in the glass bottle containing 400mL PBS buffer, and the outlet pipe is directed to waste material.Then start the pump, and make 200mL PBS buffer flow through post 5min with the speed of 40mL / min.Stop the pump, and invert the post.Then, exchange the connection of inlet pipe and outlet pipe with post, make the inlet be positioned at the bottom of post again and outlet be positioned at the top of post.Start the pump, and make extra 200mL PBS buffer flow through post 5min with the speed of 40mL / min.

[0389] The pump is stopped, and the inlet pipe is moved to a glass bottle containing 1064mL of a coupling buffer consisting of 1.35M potassium phosphate at a pH of 9.0. The outlet pipe remains directed to waste. The pump is started, and 300mL of coupling buffer is flowed through the post at a rate of 40mL / min for 7.5min. The pump is stopped, and the outlet pipe is placed in a glass bottle identical to the inlet pipe, which contains the remaining 764mL coupling buffer. In this configuration of the inlet pipe and the outlet pipe in the same bottle, the solution can be recirculated through the post many times. Recirculating the solution through the post allows for extended reaction times without increasing the volume of the required solution.

[0390] 205.6mL of PrA ligand stock solution with a concentration of 25.8g / L in water was added to the glass bottle containing the remaining 764mL coupling buffer. The pump was started, and then the PrA solution was recirculated through the post for 4 hours at a flow rate of 40mL / min. The total PrA solution volume recirculated through the post was calculated to be 1062.6mL, which was composed of 764mL coupling buffer remaining in the glass bottle, 93mL coupling buffer remaining in the post / tube and the 205.6mL PrA stock solution added. Considering that the 205.6mL PrA ligand stock solution containing 5.3gPrA ligand was diluted to a volume of 1064mL, the PrA solution recirculated in the system was calculated to have a concentration of 5g / L. The ligand loading on the membrane was calculated by dividing the total mass of 5.3g PrA ligand by the total membrane volume 53.6mL to obtain a ligand loading of 99g / L.

[0391] After 4 hours, the pump was stopped and the outlet tube was directed to waste. The inlet tube was placed in 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 40 mL / min for 5 min.

[0392] The pump was then stopped, and the inlet pipe was placed in a glass bottle containing 600mL 1M ethanolamine. The outlet pipe remained directed to waste. The pump was started, and 200mL 1M ethanolamine was flowed through the post to waste for 10min at a flow rate of 20mL / min. The pump was stopped, and the outlet pipe was directed to the glass bottle containing the remaining 400mL 1M ethanolamine solution. The pump was then started, and the remaining 400mL 1M ethanolamine solution was recirculated through the post for 170 minutes at a flow rate of 20mL / min.

[0393] 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 allow the PBS buffer to flow through the column at a flow rate of 40 mL / min for 10 min.

[0394] Stop the pump and remove the top and bottom column heads. Then, remove the wound roll of membrane and screen from the column. Unroll the membrane and separate it from the screen. Then, remove the circular section of membrane with a diameter of 30 mm from the rectangular membrane sheet. Remove five circular sections of membrane from each of the following positions ( Figure 6 ):

[0395] 1. Center: Located at the overall center of the rectangular membrane sheet.

[0396] 2. Core: Located in the middle of the edge of the rectangular membrane sheet closest to the core during the coupling reaction.

[0397] 3. Shell: Located in the middle of the edge of the rectangular membrane sheet closest to the column side during the coupling reaction.

[0398] 4. Inlet: Located in the middle of the edge of the rectangular membrane sheet closest to the column inlet during the coupling reaction.

[0399] 5. Outlet: Located in the middle of the edge of the rectangular membrane sheet closest to the column inlet during the coupling reaction.

[0400] 6. The five membranes taken out 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 / minute or 10 mL / min. It was found that the 1 mL device had very similar pressure drop and IgG dynamic binding capacity at all five different positions. These results show 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 mesh.

[0401] Table 3. Pressure drop and IgG dynamic binding capacity based on membrane position in the spirally wound rectangular sheet.

[0402]

[0403] Incorporated by Reference

[0404] All U.S. patents and U.S. patent application publications cited herein are hereby incorporated by reference.

[0405] Equivalent

[0406] Those skilled in the art will recognize, or be able to ascertain using no more than 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 functionalized composite material, the method comprising the following steps: a. Providing a functionalized composite material, wherein the functionalized composite material is arranged as a coplanar stack of coextensive sheets, the coplanar coextensive sheets being periodically separated by a screen, the functionalized composite material comprising: i. a support member comprising a plurality of holes extending through the support member; and 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 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, The coplanar stack of coextensive sheets further comprises one or more flow distribution layers, which are non-porous sheets containing holes.

2. The method of claim 1, wherein the pendant reactive functional group is selected from the group consisting of aldehydes, amines, carbon-carbon double bonds, carbon-carbon triple bonds, epoxides, hydroxyls, thiols, anhydrides, azides, reactive halogens, acid chlorides, and mixtures thereof.

3. The method of claim 1, wherein the pendant reactive functional group is selected from the group consisting of a carbon-carbon double bond, a carbon-carbon triple bond, and a thiol.

4. The method of claim 1, wherein the pendant reactive functional group is derived from a molecule comprising a thiol functional group or a molecule comprising an unsaturated carbon-carbon bond.

5. The method of claim 4, wherein the pendant reactive functional group is derived from a molecule comprising a thiol functional group; and the molecule comprising a thiol functional group is selected from the group consisting of 3-mercaptopropionic acid, 1-mercaptosuccinic acid, a polypeptide comprising a cysteine ​​residue, a protein comprising a cysteine ​​residue, cysteamine, 1-thiohexitol, poly(ethylene glycol) 2-mercaptoethyl ether acetic acid, poly(ethylene glycol) methyl ether thiol, 1-thioglycerol, 2-naphthalenethiol, biphenyl-4-thiol, 3-amino 1-[2-(dimethylamino)ethyl]-1H-tetrazolyl-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.

6. The method of claim 4, wherein the molecule comprising a thiol functional group is a recombinant protein comprising a cysteine ​​residue.

7. The method of claim 4, wherein the molecule comprising a thiol functional group is a recombinant fusion protein comprising a cysteine ​​residue.

8. The method of claim 5, wherein the molecule comprising a thiol functional group is a polypeptide comprising a cysteine ​​residue or a protein comprising a cysteine ​​residue.

9. The method of claim 4, wherein the molecule comprising a thiol functional group is a bacterial immunoglobulin-binding protein comprising a cysteine ​​residue.

10. The method of claim 8, wherein the molecule comprising a thiol functional group is a protein comprising a cysteine ​​residue.

11. The method of claim 4, wherein the pendant reactive functional group is 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 or acrylate.

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

13. The method of claim 12, wherein the one or more monomers containing pendant reactive functional groups are selected from the group consisting of glycidyl methacrylate, acrylamide oxime, acrylic anhydride, azelaic anhydride, maleic anhydride, hydrazide, acryloyl chloride, 2-bromoethyl methacrylate, and vinyl methyl ketone.

14. The method of claim 12, wherein the pendant reactive functional group is an amine.

15. The method of claim 12, wherein the pendant reactive functional group is an epoxide.

16. The method of claim 12, wherein the pendant reactive functional group is a hydroxyl group.

17. The method of any one of the preceding claims, wherein the first ligand comprises a first functional group.

18. The method of claim 17, wherein the first ligand further comprises at least one grafted end group; and the first functional group is selected from the group consisting of 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.

19. The method of claim 18, wherein the first functional group is selected from the group consisting of 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.

20. The method of claim 18, wherein the molecule comprises a first functional group and is selected from the group consisting of 2-(diethylamino)ethyl methacrylate, 2-aminoethyl methacrylate, 2-carboxyethyl acrylate, 2-(methylthio)ethyl methacrylate, acrylamide, N-acryloyloxysuccinimide, 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 methyl Acrylic acid ester, methacrylamide, methacrylic anhydride, propyl acrylate or propyl methacrylate, N-isopropylacrylamide, styrene, 4-vinylpyridine, vinylsulfonic acid, N-vinyl-2-pyrrolidone, 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.

21. The method of claim 18, wherein the first functional group is a metal chelating functional group.

22. The method of claim 18, wherein the first functional group comprises a metal chelating functional group selected from the group consisting of an octadentate functional group, a hexadentate functional group, a tetradentate functional group, a tridentate functional group, a bidentate functional group, iminodicarboxylic acid, and iminodiacetic acid.

23. The method of claim 18, wherein the first functional group comprises a biomolecular or bioionic functional group selected from the group consisting of a virus, a cell, a protein of recombinant or natural origin, and a nucleic acid-derived product.

24. The method of claim 18, wherein the first functional group comprises albumin.

25. The method of claim 18, wherein the first functional group comprises lysozyme.

26. The method of claim 18, wherein the first functional group comprises gamma-globulin of human and animal origin.

27. The method of claim 18, wherein the first functional group comprises immunoglobulins of human and animal origin.

28. The method of claim 18, wherein the first functional group comprises a polypeptide of synthetic or natural origin.

29. The method of claim 18, wherein the first functional group comprises interleukin-2 or a receptor thereof.

30. The method of claim 18, wherein the first functional group comprises an enzyme.

31. The method of claim 18, wherein the first functional group comprises a monoclonal antibody.

32. The method of claim 18, wherein the first functional group comprises an antigen.

33. The method of claim 18, wherein the first functional group comprises a lectin.

34. The method of claim 18, wherein the first functional group comprises a bacterial immunoglobulin-binding protein.

35. The method of claim 18, wherein the first functional group comprises trypsin or an inhibitor thereof.

36. The method of claim 18, wherein the first functional group comprises cytochrome C.

37. The method of claim 18, wherein the first functional group comprises myosin.

38. The method of claim 18, wherein the first functional group comprises a recombinant human interleukin.

39. The method of claim 18, wherein the first functional group comprises a recombinant fusion protein.

40. The method of claim 18, wherein the first functional group comprises Protein A.

41. The method of claim 18, wherein the first functional group comprises protein G.

42. The method of claim 18, wherein the first functional group comprises protein L.

43. The method of claim 18, wherein the first functional group comprises peptide H.

44. The method of claim 18, wherein the first functional group comprises DNA of synthetic or natural origin.

45. The method of claim 18, wherein the first functional group comprises RNA of synthetic or natural origin.

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