Purification of target molecules using functionalized nonwoven membranes and chromatography devices

Abstract

Described herein are membranes, devices, methods, and kits for the purification of target molecules. In some embodiments, the membranes, devices, methods, and kits comprise a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications, and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule. In some embodiments, the devices comprise a cartridge, a cassette, or a column.

Description

[0001] PURIFICATION OF TARGET MOLECULES USING FUNCTIONALIZED NONWOVEN MEMBRANES AND CHROMATOGRAPHY DEVICES CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No.63 / 555,575, filed on February 20, 2024, which is incorporated by reference herein in its entirety. BACKGROUND The production and purification of biologics require reliable and efficient separation processes to ensure the safety, potency, and efficacy of the therapeutic. For example, therapeutic monoclonal antibodies (mAbs) remain the dominant biopharmaceutical class in the numbers of biologic approvals and sales. These mAbs have been broadly applied in the treatment of cancers, rheumatoid arthritis, multiple sclerosis, autoimmune disorders, and COVID-19, while the costs of such treatment place a huge burden on patients and healthcare systems. To enhance accessibility and affordability, significant efforts in biopharmaceutical industries have been devoted to transforming the mAb manufacturing processes with improved productivity, lower production cost and faster turn-around time. The downstream processing that accounts for over 50% of mAb production costs usually relies on two or three-step resin chromatography, i.e., Protein A chromatography for capture and one or two following ion exchange chromatography steps for purification and polishing. Protein A resin, with its high cost of around $10,000 per liter or more, accounts for more than 50% of the cost of the entire downstream processing. Moreover, it faces the challenges of ligand leaching, mAb aggregation at low pH elution and stability in alkaline cleaning solutions. Therefore, much attention has been paid to alternative non-Protein A processes such as precipitation, aqueous two-phase extraction, mixed-mode chromatography, and peptide-based affinity chromatography. Multimodal or mixed-mode chromatography that combines electrostatic, hydrophobic interactions and other secondary interactions including hydrogen binding, thiophilic and π-π interactions have been demonstrated to provide high selectivity for mAb capture via screening salt additives in wash steps and optimizing elution pH. Multimodal ligands usually exhibit salt- tolerance, meaning that the binding capacity is relatively independent of solution conductivity in low-to-moderate salt concentrations. This reduces the need for prior buffer exchange or dilution required in conventional ion exchange chromatography, resulting in a significant reduction of operating costs and processing time. Multimodal cation exchange (MMC) chromatography typically requires operating pH (usually 4.5–6.0) lower than the pI of mAb and leads to an additional filtration for the pH adjusted culture fluid to remove any possible precipitation prior to chromatography operation. Further, multimodal anion exchange (MMA) chromatography can operate in physiological-like conditions where the ligand is still positively charged, holding the potential to enable truly direct capture of mAb. The performance of four MMA resins: Capto adhere (Cytiva), PPA HyperCel (Sartorius), HEA HyperCel (Sartorius), and MEP HyperCel (Sartorius) were previously found to vary depending on the charges and hydrophobicity of ligands as well as their interactions with impurity proteins that have distinct surface charge distributions and hydrophobic zones. Although good impurity removal can be obtained, the reported binding capacity of MMA resins for mAb capture from supernatant (titer: 0.4–1.69 mg / mL) were generally low (<20 mg / mL with residence time of 2.5–6.6 min) leading to a rather low productivity. In addition to high costs, the use of resin-based chromatography methods suffers from several processing limitations which cannot be rectified through improvements to the binding chemistry including diffusional mass transport resistance, high pressure drops at high flow rates, and operational inefficiencies due to a lack of disposability. These issues can be largely mitigated through the adoption of membrane-based chromatography devices. In membrane chromatography, convective flow through the pores eliminates the diffusion limitations, and the pressure drop is significantly lower than that of resin-packed columns, resulting in a more cost- effective process. In addition, membrane chromatography is particularly suitable for large biomolecules (i.e., molecules with a molecular weight above 150 kDa), such as large proteins, viruses, viral vectors, viral vaccines, plasmids, DNA, RNA, and other oligonucleotides, cells, and exosomes, giving it an advantage over conventional resins. The pores of chromatographic resins are not large enough to allow access to such biomolecules, so the inner surface is not fully exploited, and adsorption is limited to the outer surface of the particles. However, many existing membranes cast from polymer solutions have small pores, small surface areas, very low binding capacities, and cannot be used to purify large biologics such as exosomes, viral vectors, and vaccines. Various chromatographic membranes based on microporous and fibrous membranes have been exploited in purification of biologics with availability as single use modules, high throughput, and high binding capacity at relatively short residence times. They have been regarded as effective alternatives to resin chromatography with higher productivity and lower capital expenditure for mAb capture, charge variants separation as well as host cell protein (HCP), DNA, and aggregates clearance. However, only one MMA membrane was reported in the literature, and it was aimed to adsorb impurities for mAb polishing with a low IgG binding capacity of <20 mg / mL at 1.2 and 6 sec residence time (RT). Thus, what is needed are membranes and methods for the purification of various target molecules with increased production efficiency and reduced manufacturing costs. Nonwoven membranes are composed of highly engineered fibers that can be manufactured on a large scale using a variety of polymers, and the surface chemical modification can be realized with different chemistries and ligands for different purposes. Novel processes for fiber manufacturing and multiple parameters that can be used to control the membrane properties including fiber diameter, pore size, porosity, surface area, and thickness enable the development of improved separation media for efficient purification of biomolecules. SUMMARY One embodiment described herein is a functionalized nonwoven membrane for purifying a target molecule or species, the membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N- methylethanolamine (BMEA), 2-mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof. In one aspect, the polymeric fibers comprise polyolefins, polyethylenes, polypropylenes, cellulose, polyesters, polyamides, polycarbonates, polyethersulfones, polybutylene terephthalate (PBT), polytrimethylene terephthalate (PTT), polyethylene terephthalate (PET), polyamide 6 (PA6), polyamide 6-6 (PA6-6), polyamide 11 (PA11), polyamide 12 (PA12), thermoplastic polymers, or combinations thereof. In another aspect, the surface modification comprises a grafted second polymer. In another aspect, the grafted second polymer comprises an acrylate or methacrylate polymer from one or more monomers comprising glycidyl methacrylate (GMA), methacrylic acid, 2-(diethylamino)ethyl methacrylate, [2-(methacryloyloxy)ethyl] trimethyl-ammonium chloride, 2- hydroxyethyl methacrylate, 2-acrylamido-2-methylpropane sulfonic acid, 2-(dimethylamino)ethyl methacrylate, butyl methacrylate, 3-chloro-2-hydroxypropyl methacrylate, 2-ethylhexyl methacrylate, or combinations thereof. In another aspect, the functional ligand comprises N- benzyl-N-methylethanolamine (BMEA). Another embodiment described herein is a functionalized nonwoven membrane for purifying a target molecule, the membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having a grafted second polymer; and a functional ligand coupled to the grafted second polymer and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA). In one aspect, the polymeric fibers comprise polyolefins, polyethylenes, polypropylenes, cellulose, polyesters, polyamides, polycarbonates, polyethersulfones, polybutylene terephthalate (PBT), polytrimethylene terephthalate (PTT), polyethylene terephthalate (PET), polyamide 6 (PA6), polyamide 6-6 (PA6-6), polyamide 11 (PA11), polyamide 12 (PA12), thermoplastic polymers, or combinations thereof. In another aspect, the grafted second polymer comprises an acrylate or methacrylate polymer from one or more monomers comprising glycidyl methacrylate (GMA), methacrylic acid, 2-(diethylamino)ethyl methacrylate, [2-(methacryloyloxy)ethyl] trimethyl- ammonium chloride, 2-hydroxyethyl methacrylate, 2-acrylamido-2-methylpropane sulfonic acid, 2-(dimethylamino)ethyl methacrylate, butyl methacrylate, 3-chloro-2-hydroxypropyl methacrylate, 2-ethylhexyl methacrylate, or combinations thereof. In another aspect, the grafted second polymer comprises a methacrylate polymer of polyGMA. In another aspect, the functionalized nonwoven membrane further comprises one or more additional functional ligands comprising 2- mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof. Another embodiment described herein is a functionalized nonwoven membrane for purifying a target molecule, the membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) coupled to the polyGMA. In one aspect, the membrane has a dynamic binding capacity (DBC) for target molecule capture of ˃50 mg / mL with a residence time of from about 0.5 min to about 5 min. In another aspect, the membrane has a flow permeability of about 1.0 × 10−9cm2to about 4.6 × 10−9cm2in a buffer comprising about 50 mM to about 1 M of one or more salts at about pH 7.0. Another embodiment described herein is a method for purifying a target molecule, the method comprising: contacting a sample comprising the target molecule with a functionalized nonwoven membrane to bind the target molecule to the membrane, the membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2-mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof; and (b) eluting the target molecule from the membrane and collecting the target molecule in a first eluate fraction. In one aspect, the target molecule is a biomolecule comprising a protein, a peptide, an antibody, a nucleic acid, an enzyme, a lipoprotein, a lipid, an extracellular vesicle, an exosome, a viral vector, a plasmid, a carbohydrate, a vaccine, a virus, a bacterium, a cell, an organelle, or combinations thereof. In another aspect, the sample is a biological fluid sample. In another aspect, the biological fluid sample comprises a cell culture supernatant or a sample obtained from an animal subject. In another aspect, the sample obtained from an animal subject comprises one or more of blood, plasma, serum, tears, urine, saliva, sputum, pleural effusion, or ascites. In another aspect, the method further comprises: (a)(i) performing a first wash of the membrane using a low salt buffer at about pH 5–9; and (a)(ii) performing a second wash of the membrane using a low salt buffer having a charged species at about pH 5–9. In another aspect, the steps comprise: (a)(i) performing a first wash of the membrane using 50 mM Tris-HCl with about 1 mM to about 150 mM NaCl at pH 7.9; and (a)(ii) performing a second wash of the membrane using 50 mM Tris-HCl with about 50 mM to about 125 mM arginine at pH 7.4. In another aspect, the target molecule is eluted from the membrane using one or more elution buffers or solutions at about pH 2–6. In another aspect, the target molecule is eluted from the membrane using a first elution buffer at about pH 4.5. In another aspect, the target molecule is further eluted from the membrane using a second elution buffer at about pH 4.0. In another aspect, the one or more elution buffers or solutions comprises 50 mM acetate at about pH 2–6. In another aspect, method further comprises virus inactivation comprising: (b)(i) adjusting the pH of the first eluate fraction to about pH 3–5; (b)(ii) incubating the first eluate fraction at room temperature for about 1 hour; (b)(iii) adjusting the pH of the first eluate fraction to about pH 7–8; and (b)(iv) filtering the first eluate fraction using microfiltration. In another aspect, method further comprises: (c) contacting the first eluate fraction comprising the target molecule with a second functionalized nonwoven membrane and collecting a flow-through fraction comprising the target molecule. In another aspect, method further comprises: (c)(i) performing a wash of the second functionalized nonwoven membrane using a buffer with about pH 6–9. In another aspect, method further comprises: (c)(i) performing a wash of the second functionalized nonwoven membrane using 50 mM Tris-HCl at pH 7.2. In another aspect, the second functionalized nonwoven membrane comprises a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2-mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof. In another aspect, the method further comprises: (d) contacting the flow-through fraction comprising the target molecule with a third functionalized nonwoven membrane to bind the target molecule to the third functionalized nonwoven membrane; and (e) eluting the target molecule from the third functionalized nonwoven membrane and collecting the target molecule in a second eluate fraction. In another aspect, the method further comprises: (d)(i) performing a wash of the third functionalized nonwoven membrane using a low salt buffer at about pH 4–7. In another aspect, the step comprises: (d)(i) performing a wash of the third functionalized nonwoven membrane using 50^mM acetate with 150 mM NaCl at about pH 5.5. In another aspect, the target molecule is eluted from the third functionalized nonwoven membrane using a low salt buffer at about pH 7– 10. In another aspect, the target molecule is eluted from the third functionalized nonwoven membrane using 50 mM sodium phosphate with 150 mM NaCl at about pH 7.7. In another aspect, the third functionalized nonwoven membrane comprises a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N- methylethanolamine (BMEA), 2-mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof. In another aspect, each of steps (a)–(e) has a residence time of about 0.1 min to about 5 min. Another embodiment described herein is a method for purifying a target molecule, the method comprising: contacting a sample comprising the target molecule with a functionalized nonwoven membrane to bind the target molecule to the membrane, the membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) coupled to the polyGMA; and eluting the target molecule from the membrane and collecting the target molecule in a first eluate fraction. In one aspect, the target molecule is a biomolecule comprising a protein, a peptide, an antibody, a nucleic acid, an enzyme, a lipoprotein, a lipid, an extracellular vesicle, an exosome, a viral vector, a plasmid, a carbohydrate, a vaccine, a virus, a bacterium, a cell, an organelle, or combinations thereof. Another embodiment described herein is a method for purifying a target molecule, the method comprising: contacting a sample comprising the target molecule with a first functionalized nonwoven membrane to bind the target molecule to the first membrane, the first membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) coupled to the polyGMA; eluting the target molecule from the first membrane and collecting the target molecule in a first eluate fraction; contacting the first eluate fraction comprising the target molecule with a second functionalized nonwoven membrane, the second membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising triethylamine (TEA) coupled to the polyGMA; collecting a flow-through fraction comprising the target molecule; contacting the flow- through fraction comprising the target molecule with a third functionalized nonwoven membrane to bind the target molecule to the third membrane, the third membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising 2- mercaptopyridine-3-carboxylic acid (MPCA) coupled to the polyGMA; and eluting the target molecule from the third membrane and collecting the target molecule in a second eluate fraction. In one aspect, the target molecule is a biomolecule comprising a protein, a peptide, an antibody, a nucleic acid, an enzyme, a lipoprotein, a lipid, an extracellular vesicle, an exosome, a viral vector, a plasmid, a carbohydrate, a vaccine, a virus, a bacterium, a cell, an organelle, or combinations thereof. In another aspect, the target molecule is an antibody. Another embodiment described herein is a method of making a functionalized nonwoven membrane for purifying a target molecule, the method comprising: (a) grafting polyglycidyl methacrylate (polyGMA) onto a polybutylene terephthalate (PBT) nonwoven web to generate a PBT-polyGMA nonwoven web; and (b) coupling a functional ligand comprising N-benzyl-N- methylethanolamine (BMEA) to the grafted polyGMA of the PBT-polyGMA nonwoven web to generate the functionalized nonwoven membrane. In one aspect, grafting the polyGMA onto the PBT nonwoven web is performed using ultraviolet radiation. In another aspect, a concentration of the BMEA functional ligand that is coupled to the PBT-polyGMA nonwoven web is from about 2% (v / v) to about 15% (v / v). In another aspect, the BMEA functional ligand is coupled to the PBT- polyGMA nonwoven web at about 40 °C for about 6 hours, followed by treatment with 0.1 M sulfuric acid at about 50 °C for about 16 hours. In another aspect, the method further comprises washing the functionalized nonwoven membrane with pure water and storing the functionalized nonwoven membrane in a solution of 20% ethanol / water (v / v). Another embodiment described herein is a chromatography device for purifying a target molecule, the chromatography device comprising: a housing unit comprising one or more functionalized nonwoven membranes, each membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N- methylethanolamine (BMEA), 2-mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof; and a flow device connected to the housing unit and configured to induce fluid flow of a sample comprising the target molecule through the one or more functionalized nonwoven membranes. In one aspect, the housing unit is a cartridge, a cassette, or a column. Another embodiment described herein is a method for purifying a target molecule, the method comprising: contacting a sample comprising the target molecule with a chromatography device comprising: a housing unit comprising one or more functionalized nonwoven membranes, each membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2- mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof; and a flow device connected to the housing unit and configured to induce fluid flow of the sample comprising the target molecule through the one or more functionalized nonwoven membranes; inducing fluid flow of the sample comprising the target molecule through the one or more functionalized nonwoven membranes using the flow device to bind the target molecule to the one or more functionalized nonwoven membranes; and eluting the target molecule from the one or more functionalized nonwoven membranes and collecting the target molecule in a first eluate fraction. Another embodiment described herein is a kit for purifying a target molecule, the kit comprising: one or more functionalized nonwoven membranes, each membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2-mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof; (b) optionally, columns, buffers, cartridges, cassettes, and receptacles; and (c) optionally, one or more of packaging or instructions for use. Another embodiment described herein is a method for removing ions or ionic contaminants from a liquid, the method comprising: (a) contacting a liquid comprising ions or ionic contaminants with a functionalized nonwoven membrane to bind the ions or ionic contaminants to the membrane, the membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2- mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof; and (b) collecting the liquid having the ions or ionic contaminants removed. In one aspect, the liquid is water. Another embodiment described herein is a method for removing ions or ionic contaminants from a liquid, the method comprising: (a) contacting a liquid comprising ions or ionic contaminants to a first functionalized nonwoven membrane to bind anions or anionic contaminants to the first functionalized nonwoven membrane, the first functionalized nonwoven membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) or triethylamine (TEA) coupled to the polyGMA; (b) collecting a first liquid flow-through having the anions or anionic contaminants removed; (c) contacting the first liquid flow-through to a second functionalized nonwoven membrane to bind cations or cationic contaminants to the second functionalized nonwoven membrane, the second functionalized nonwoven membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising 2-mercaptopyridine-3-carboxylic acid (MPCA) coupled to the polyGMA; and (d) collecting a second liquid flow-through having the cations or cationic contaminants removed. In one aspect, the liquid is water. Another embodiment described herein is a method for purifying water, the method comprising: (a) contacting a sample of water with a first functionalized nonwoven membrane to bind anions or anionic contaminants to the first functionalized nonwoven membrane, the first functionalized nonwoven membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) or triethylamine (TEA) coupled to the polyGMA; (b) collecting a first water flow-through having the anions or anionic contaminants removed; (c) contacting the first water flow-through with a second functionalized nonwoven membrane to bind cations or cationic contaminants to the second functionalized nonwoven membrane, the second functionalized nonwoven membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising 2-mercaptopyridine-3-carboxylic acid (MPCA) coupled to the polyGMA; and (d) collecting a second water flow-through having the cations or cationic contaminants removed. DESCRIPTION OF THE DRAWINGS FIG. 1 shows a schematic diagram of non-Protein A three-step membrane chromatography and standard three-step resin chromatography containing Protein A for mAb capture. FIG.2 shows the chemical reaction for coupling BMEA to polyGMA (pGMA) grafted PBT membranes. FIG. 3 shows the influence of BMEA content in the reaction solution on BSA binding of the prepared MMA-BMEA membranes. Initial BSA concentration in the starting solution was 10 mg / mL. FIG.4 shows the percentage of BSA desorption from MMA-BEMA membrane at elution pH from 2.2–5.0. Before elution, the BSA was adsorbed by MMA-BMEA membrane with an initial BSA concentration of 10 mg / mL in a 50 mM Tris-HCl, pH 7.0, with 0.15 M NaCl. The elution buffers used were 0.1 M citric acid, pH 2.2, 50 mM glycine-HCl pH 3.0–3.5 and 50 mM acetate, pH 4.0–5.0. FIG. 5 shows the equilibrium adsorption isotherm of BSA binding to MMA-BMEA membrane. Initial BSA solution: 0.5 to 10.5 mg / mL BSA dissolved in 50 mM Tris-HCl, pH 7.0, with 150 mM NaCl (≈ 17.5 mS / cm). The Langmuir isotherm was used to fit the experimental data. FIG. 6 shows the effect of NaCl concentration on BSA adsorption to MMA-BMEA membrane with an initial BSA concentration in the starting solution of 3 mg / mL. BMEA content in the reaction solution for ligand coupling: 15% v / v. FIG.7 shows the flow permeability of MMA-BMEA membrane in 50 mM Tris pH 7.0 with and without added 0.15 M NaCl (60 layers of MMA-BMEA membranes in a 10 mm-diameter column). BMEA content in the reaction solution for ligand coupling: 15% v / v. FIG.8A–B show SEM images of an MMA-BMEA membrane (FIG.8A) and an AEX-TEA membrane (FIG.8B). FIG.9 shows a chromatogram of DBC10%(BSA) measurement for MMA-BMEA membrane and Capto adhere resin at 1.0 min residence time (RT). The 0.24 mL MMA-BMEA membrane was packed in a 10 mm-diameter column with a membrane bed height of 0.3 cm. The 0.25 mL Capto adhere resin was packed in a 5 mm-diameter column with a bed height of 1.3 cm. FIG. 10 shows the effect of different NaCl concentrations on DBC10%of MMA-BMEA membrane at 1.0 min and 5.0 min RT. The BMEA content in the reaction solution for ligand coupling was 15% v / v. FIG. 11 shows equilibrium adsorption isotherm of IgG binding to the MMA-BMEA membrane. Initial IgG solution: 1.0 to 13.0 mg IgG / mL dissolved in pH 7.950 mM Tris-HCl with added 120 mM NaCl (≈14.5 mS / cm). The Langmuir isotherm was used to fit the experimental data. FIG.12 shows a chromatogram of DBC10%(IgG) measurement for MMA-BMEA membrane at 1.0 min RT. The 0.24 mL MMA-BMEA membrane was packed in a 10 mm-diameter column with a membrane bed height of 0.3 cm. FIG. 13 shows chromatograms for five consecutive human IgG bind-and-elute cycles at 2.0 min RT. FIG. 14 shows the determination of DBC10%for capturing mAb in CHO supernatant by MMA-BMEA membrane at 2.0 min RT. FIG.15A–C show chromatograms of mAb capture by MMA-BMEA membrane at 0.5 RT (FIG.15A), 2.0 RT (FIG.15B), and 5.0 RT (FIG.15C). FIG.16A–C show chromatograms of mAb capture by MMA-BMEA membrane with 50 mM (FIG.16A), 87.5 mM (FIG.16B), and 125 mM (FIG.16C) arginine in the wash step after sample loading. FIG.17A–C show chromatograms of capturing mAb in CHO supernatant by MMA-BMEA membrane (FIG.17A), subsequent mAb polishing by AEX-TEA membrane (FIG.17B), and final mAb polishing by MMC-MPCA membrane (FIG.17C). All steps were conducted at 0.5 min RT, except the sample loading at 2.0 min RT. FIG.18A–C show chromatograms of capturing mAb in CHO supernatant by Eshmuno® Protein A resin (FIG.18A), subsequent mAb polishing by Capto S cation exchange resin (FIG. 18B), and final mAb polishing by Capto Q anion exchange resin (FIG. 18C). All steps were conducted at 5.0 min RT. FIG.19 shows photomicrographs of nonwoven structures and fibrillation of fibers by SEM at low (500×) and high (5000×) magnification. Samples from 75 to 125 gsm with fibrillation by hydroentanglement in one (1P), two (2P), or three (3P) passes. FIG.20 shows the chemical structure of the DEM monomer. FIG. 21 shows an SEM photomicrograph of a polyDEM grafted PBT nonwoven (20% weight gain). FIG.22 shows a chromatogram of 100% DBC measurement for BSA binding onto DEA- polyamide 11 (PA11) membrane (20% weight gain). BSA solution: 150 mL of 2 mg / mL BSA dissolved in 20 mM Tris-HCl, pH 7.0. Equilibration buffer: 20 mM Tris-HCl, pH 7.0; Elution buffer: 20 mM Tris-HCl, pH 7.0, with 1.0 M NaCl. Residence time: 0.1–5 min. Column volume was 1.5 mL with 12 packed membrane layers. FIG.23 shows 100% DBC obtained at residence times of 0.1–5 min. FIG.24 shows the chemical structure of BMEP monomer. FIG. 25 shows an SEM photomicrograph of a polyBMEP grafted PBT nonwoven (20% weight gain). FIG.26 shows a chromatogram of 100% DBC measurement for BSA binding onto DEA- PA11 membrane (20% weight gain). BSA solution: 150 mL of 2 mg / mL BSA dissolved in 20 mM Tris-HCl, pH 7.0. Equilibration buffer: 20 mM Tris-HCl, pH 7.0; Elution buffer: 20 mM Tris-HCl, pH 7.0, with 1.0 M NaCl. Residence time: 0.1–5 min. Column volume was 1.4 mL with 12 packed membrane layers. FIG.27 shows 100% DBC obtained at residence times of 0.1–5 min. FIG.28 shows an SEM photomicrograph of a PA11 meltblown nonwoven. FIG. 29 shows an equilibrium adsorption isotherm of BSA onto DEA-PA11 membrane (15.8% weight gain). Initial BSA solution: 0.5 to 12.5 mg / mL BSA dissolved in 20 mM Tris-HCl, pH 7.0. Experimental data were compared with the fitted data based on the Langmuir model. FIG. 30 shows a chromatogram of 10% DBC measurement for BSA binding onto DEA- PA11 membrane (15.8% weight gain). BSA solution: 5.0 mg BSA / mL dissolved in 20 mM Tris- HCl, pH 7.0. Equilibration buffer: 20 mM Tris-HCl, pH 7.0; Elution buffer: pH 7.020 mM Tris-HCl with 1.0 M NaCl. Residence time: 0.2–5 min. Column volume was 0.263 mL with 10 packed membrane layers. FIG.31 shows 10% DBC obtained at residence times of 0.2–5 min. DETAILED DESCRIPTION Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein. As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein. As used herein, the terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting essentially of,” and “consisting of” the embodiments or elements presented herein, whether explicitly set forth or not. As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified. As used herein, the term “or” can be conjunctive or disjunctive. As used herein, the term “and / or” refers to both the conjunctive and disjunctive. As used herein, the term “substantially” means to a great or significant extent, but not completely. As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol “~” means “about” or “approximately.” All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. example, a range of 0.1–2.0 includes 0.1, 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ± 10% of any value within the range or within 3 or more standard deviations, including the end points. As used herein, the terms “active ingredient” or “active pharmaceutical ingredient” refer to a pharmaceutical agent, active ingredient, compound, or substance, compositions, or mixtures thereof, that provide a pharmacological, often beneficial, effect. As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells. As used herein, “sample” may include, but is not limited to, liquids, solutions, emulsions, or suspensions containing a target molecule. A sample may include a medical sample or a laboratory sample. Samples may include any biological fluid sample, cell sample, or tissue sample, including, but not limited to, a cell culture extract or supernatant, blood, whole blood, fractions of blood such as plasma and serum, muscle, interstitial fluid, sweat, saliva, urine, tears, synovial fluid, bone marrow, cerebrospinal fluid, nasal secretions, sputum, amniotic fluid, bronchoalveolar lavage fluid, gastric lavage, emesis, fecal matter, lung tissue, peripheral blood mononuclear cells, total white blood cells, lymph node cells, spleen cells, tonsil cells, cancer cells, tumor cells, bile, digestive fluid, skin, or combinations thereof. Samples can be obtained by any means known in the art. The sample can be used directly as obtained or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as is known in the art. In some embodiments, a biological fluid sample may comprise a cell culture supernatant or a sample obtained from an animal subject. In one aspect, a sample obtained from an animal subject may comprise one or more of blood, plasma, serum, tears, urine, saliva, sputum, pleural effusion, or ascites. As used herein, “target molecule” or “target analyte” refer to a particular substance to be purified or captured using the membranes and methods as disclosed herein. The target molecule may be a biological substance associated with a biological state or a biological process, such as a disease state or a diagnostic or prognostic indicator of a disease or disorder (e.g., an indicator identifying the likelihood of the existence or later development of a disease or disorder). As described herein, target molecules may include one or more biomolecules comprising proteins, peptides, enzymes, lipoproteins, nucleic acids, extracellular vesicles, exosomes, nanocarriers, viruses, viral particles, bacteria, cells (e.g., a viral cell, a bacterial cell, a fungal cell, a mammalian cell, a cancer cell, a blood cell, etc.), antigens, antibodies, DNAs, RNAs, sugars, carbohydrates, polysaccharides, ligands, small molecules, lipids, receptors, anionic or cationic chemical substances, combinations thereof, and the like. In some embodiments, a target molecule may comprise a biomolecule comprising a protein, a peptide, an antibody, a nucleic acid, an enzyme, a lipoprotein, a lipid, an extracellular vesicle, an exosome, a viral vector, a plasmid, a carbohydrate, a vaccine, a virus, a bacterium, a cell (e.g., blood cell or component), an organelle, or combinations thereof. In another aspect, the target molecule may be a contaminant in a liquid medium that is removed by adsorbing to the membrane while the liquid passes through the membrane (“the flow-through”). As used herein, “water purification” refers to the process of removing ions or contaminants from water by contacting the water to one or more functionalized nonwoven membranes as described herein. Two or more distinct membrane types can be utilized in series or parallel, or simultaneously or sequentially. In one non-limiting exemplary aspect, the water containing contaminants is contacted with the functionalized nonwoven membrane and the contaminants adsorb to the membrane and the water flows through the membrane. Multiple functionalized nonwoven membranes as described herein may be used to specifically remove anionic species, cationic species, organic or hydrophobic species, or combinations thereof. As used herein, “contaminants” comprise any compound that is desirable to remove from a liquid or water to make the liquid or water pure, purer than it currently exists, deionized, or potable for human or animal use. Exemplary contaminants include ions (e.g., sodium, potassium, magnesium, calcium, chloride, bromide, iodide, carbonate, phosphate, sulfate, nitrate, etc., and ions, oxides, or salts thereof), organic compounds, heavy metals (e.g., mercury, arsenic, iron, cobalt, nickel, zinc, cadmium, copper, lead, manganese, chromium, cesium, uranium, plutonium, gold, palladium, platinum, iridium, osmium, ruthenium, etc., and ions, oxides, or salts thereof), dyes or colorants, terrestrial contaminants (e.g., dissolved solids, calcium, magnesium, silicates, clays, minerals, etc.), drugs or drug metabolites (e.g., from human urine or waste), industrial or agricultural chemicals (e.g., per- and polyfluoroalkyl substances (PFAS) compounds, polychlorinated biphenyls (PCBs), bisphenol A (BPA), pesticides, insecticides, herbicides, fertilizers, paints, solvents, petroleum compounds such as gasoline, diesel, or kerosene, oils, tar, or asphalt), sewage (urine, feces, wash water), detergents, surfactants, biological contaminants including, but not limited to, bacteria, fungi, algae, viruses, protozoa, and parasites and toxins or metabolites thereof, or radioactive compounds. Any of the above contaminants can be removed from liquids or water by contacting the liquid or water with one or more functionalized nonwoven membranes as described herein, where the contaminants will adsorb to the membrane and the liquid or water will pass through the membrane. The membrane can be discarded or cleaned and regenerated by treating the membraned with high concentrations of ions or solvents to remove the contaminants. As used herein, “antibody” or “Ab” refers to immunoglobulins or immunoglobulin-like molecules, including, but not limited to, IgA, IgD, IgE, IgG, and IgM, and combinations thereof. In some embodiments, “antibody” may refer to, for example, intact immunoglobulins and / or antibody fragments. In some embodiments, “antibody” may refer to, for example, a monoclonal antibody (mAb) or a polyclonal antibody (pAb). The term “antibody” may also include genetically engineered forms, including, but not limited to, chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). As used herein, “nonwoven web” refers to a membrane or fabric that has a structure of individual fibers or filaments which are randomly and / or unidirectionally interlaid in a mat-like fashion. The nonwoven web can be constructed of monocomponent or multicomponent fibers and can have an average diameter of varying size, typically in the range of about 0.1 to about 100 µm (more often about 1 to about 10 µm). The nonwoven web can have an exemplary specific BET surface area of about 0.5 to about 30 m2 / g, such as about 1.0 m2 / g to about 2.0 m2 / g. As used herein, the term “fiber” is defined as a basic element of textiles which has a high aspect ratio of, for example, a ratio of length to diameter of at least about 100. In addition, “filaments / continuous filaments” are continuous fibers of extremely long lengths that possess a very high aspect ratio. The term “multicomponent fibers” refers to fibers that comprise two or more polymers that are different by physical or chemical nature including bicomponent fibers. The term “nonwoven” as used herein in reference to fibrous materials, webs, mats, batts, or sheets refers to fibrous structures in which fibers are aligned in an undefined or random orientation. The disclosed fibers can vary and include fibers having any type of cross-section, including, but not limited to, circular, rectangular, square, oval, triangular, and multi-lobal. In certain embodiments, the fibers can have one or more void spaces, wherein the void spaces can have, for example, circular, rectangular, square, oval, triangular, or multi-lobal cross-sections. Exemplary processes for the production of surface-modified fibers and nonwoven membranes are described, for example, in U.S. Pat. No.9,091,006 and U.S. Pat. No.11,027,243, which are both incorporated herein by reference for such teachings. The membranes and methods described herein provide unexpected improvements over previous techniques disclosed in the art by not requiring fiber surface UV pretreatment commonly used in the production of nonwoven fabrics, and by providing simplified and milder conditions for the purification of various target molecules, resulting in superior end results compared to industry standard practices. The methods or means for producing the disclosed nonwoven webs can vary. In general, nonwoven webs are typically produced in three stages: web formation, bonding, and finishing treatments. Web formation can be accomplished by any means known in the art. For example, webs may be formed by a drylaid process, a spunlaid process, or a wetlaid process. In various embodiments, the nonwoven web is made by a spunbond process. The spunbond can employ various types of fiber spinning processes (e.g., wet, dry, melt, or emulsion). Melt spinning is most commonly used, wherein a polymer is melted to a liquid state and forced through small orifices into cool air, such that the polymer strands solidify according to the shape of the orifices. The fiber bundles thus produced are then drawn, i.e., mechanically stretched (e.g., by a factor of 3–5) to orient the fibers. A nonwoven web is then formed by depositing the drawn fibers onto a moving belt. General spunbonding processes are described, for example, in U.S. Pat. No.4,340,563 to Appel et al., U.S. Pat. No.3,692,618 to Dorschner et al., U.S. Pat. No.3,802,817 to Matsuki et al., U.S. Pat. Nos.3,338,992 and 3,341,394 to Kinney, U.S. Pat. No.3,502,763 to Hartmann, and U.S. Pat. No. 3,542,615 to Dobo et al., which are all incorporated herein by reference for such teachings. The spunbond process typically produces a larger diameter filament than meltblowing. For example, in some embodiments, spunbonding produces fibers having an average diameter of about 5 µm or more. Various methods are available for processing multicomponent fibers to obtain fibers having smaller diameters (e.g., less than about 1.5 µm, less than about 1.0 µm, or less than about 0.5 µm). Although these methods are commonly applied to spunbonded materials, which typically have larger diameters, it is noted that they can also be applied to meltblown materials as well as fibrous materials prepared by other means. For example, in some embodiments, splittable multicomponent fibers are produced (e.g., including but not limited to, segmented pie, ribbon, islands in the sea, or multilobal) and subsequently split or fibrillated to provide two or more fibers having smaller diameters. The means by which such fibers can be split can vary and can include various processes that impart mechanical energy to the fibers, such as hydroentangling. Exemplary methods for this process are described, for example, in U.S. Pat. No. 7,981,226 to Pourdeyhimi et al., which is incorporated herein by reference for such teachings. In certain embodiments, multicomponent fibers are produced and subsequently treated (e.g., by contacting the fibers with a solvent) to remove one or more of the components. For example, in certain embodiments, an islands-in-the-sea fiber can be produced and treated to dissolve the sea component, leaving the islands as fibers with smaller diameters. Exemplary methods for this type of process are described, for example, in U.S. Pat. No.4,612,228 to Kato et al., which is incorporated herein by reference for such teachings. The nonwoven web can be formed by the meltblowing process using either a single row of capillaries (the so-called Exxon Die) or by multi-row meltblowing also referred to as “spunblowing.” In both processes the molten fibers are attenuated by high velocity jets. The fibers undergo significant stretching dur to aerodynamic forces and drag forces resulting fibers with an average diameter of varying size, typically in the range of about 0.1 to about 20 µm (more often about 0.5 to about 5 µm). The nonwoven web can have an exemplary specific BET surface area of about 0.5 to about 30 m2 / g, such as about 1.0 m2 / g to about 2.0 m2 / g. Alternatively, the meltblown-like structures can be formed by melt film fibrillation where a molten film is subjected to high velocity air that fibrillates the film resulting in a meltblown-like structure. These webs can also be homo-component or bicomponent as in the spunbond process. The fibrous webs thus produced can have varying basis weight. In some embodiments, the basis weight of the nonwoven web is about 400 g / m2or less, about 150 g / m2or less, about 100 g / m2or less, or about 50 g / m2or less. The foregoing ranges can be further defined with a minimum of about 10 g / m2. In certain embodiments, the nonwoven fabric has a basis weight of about 25 g / m2to about 125 g / m2. The basis weight of the fabric can be measured, for example, using test methods outlined in ASTM D 3776 / D 3776M-09ae2 entitled “Standard Test Method for Mass Per Unit Area (Weight) of Fabric.” This test reports a measure of mass per unit area and is measured and expressed as grams per square meter (g / m2). In some embodiments, heat induced grafting can be used with fiber-based substrates (e.g., nonwoven webs) having basis weights up to about 1,000 g / m2. As described herein, nonwoven webs suitable for surface modification (e.g., grafting) can have a width (i.e., thickness) in the range of about 1 μm up to about 1000 μm (1 mm). In certain non-limiting embodiments, nonwoven webs may have a thickness ranging from about 50 μm to about 800 μm, such as from about 100 μm to about 600 μm. In some embodiments, multiple nonwoven webs can be stacked together to create membranes having a variety of thicknesses, such as from about 600 μm up to about 2 cm, up to about 10 cm, or even greater. In some embodiments, for example, a membrane comprised of a plurality of nonwoven webs can have a thickness ranging from about 500 μm to about 2 cm. The polymeric fibers of the nonwoven web can vary, but typically comprise a thermoplastic polymer that is well-suited for surface modification (e.g., grafting). Exemplary polymers include polyolefins (e.g., polyethylene or polypropylene), polyesters, and polyamides. Polyesters are particularly useful, including polybutylene terephthalate (PBT), polytrimethylene terephthalate (PTT), polyethylene terephthalate (PET), co-polyesters, and combinations thereof. Thermoplastics, such as polyamides, likewise can be particularly useful and can include polyamide 6 (PA6), polyamide 6-6 (PA6-6), polyamide 11 (PA11), and polyamide 12 (PA12). Useful thermoplastic polymers in addition to polyamides include, for example, polycarbonates and polyethersulfones. In certain non-limiting embodiments, the polymeric fibers may comprise polyolefins, polyethylenes, polypropylenes, various types of virgin and modified celluloses, polyesters, polyamides, polycarbonates, polyethersulfones, polybutylene terephthalate (PBT), polytrimethylene terephthalate (PTT), polyethylene terephthalate (PET), polyamide 6 (PA6), polyamide 6-6 (PA6-6), polyamide 11 (PA11), polyamide 12 (PA12), thermoplastic polymers, or combinations thereof. PBT is particularly useful because the polymer structure of PBT has natural affinity for certain initiators used in grafting reactions, such as benzophenone (BP). For this reason, a pretreatment step to introduce functional ligand groups to the polymer in order to enhance affinity between the polymer and the initiator is not necessary when using PBT. In some embodiments, the surface of the polymeric fibers of the nonwoven may be modified through a grafting process through which polymeric brushes or segments are chemically attached to the fibers uniformly throughout the width and depth of the sample, and forming a conformal or uniform coating around each fiber. Thermal and UV grafting in particular may be utilized. As used herein, thermal grafting is understood to relate to a process wherein thermal free-radical initiators are adsorbed or absorbed on a substrate (e.g., fibers in a nonwoven web) prior to the addition of polymeric monomers, and heating is then applied to cause polymerization of the monomers as initiated by the thermal free-radical initiators. Initial thermal grafting conditions have an impact on the overall binding capacity of the material. Increasing initial monomer concentration for grafting typically results in a grafted layer (such as a poly(glycidyl methacrylate (“polyGMA”) layer) that binds more target molecule, and increasing polymerization temperature typically results in a grafted layer that binds less target molecule. As described herein, UV grafting processes typically include contacting a nonwoven web with a solution comprising a monomer dissolved in a suitable solvent, along with a free radical polymerization initiator, such as a photoinitiator (e.g., benzophenone (BP)). The grafting process typically also entails subjecting the nonwoven web to ultraviolet (UV) light with wavelengths between about 200 nm and about 500 nm (e.g., about 365 nm) and with intensities between about 1 mW / cm2and about 30 mW / cm2(e.g., about 5 mW / cm2) to initiate the polymerization reaction. The concentration of monomer in the grafting solution can vary but is typically about 5% v / v to about 50% v / v (e.g., about 15–25% (v / v)), and the initiator, such as BP, is typically present in a molar ratio of initiator to monomer of about 1:100 to about 1:5 (e.g., 1:20). In certain embodiments, the polymerization reaction is allowed to proceed until the weight of the grafted polymer segments is about 2% to about 50% of the weight of the nonwoven web (e.g., about 5–25% weight gain). The polymer used for grafting can vary but will typically be an acrylate or methacrylate polymer. The grafting polymer provides brush-like extensions to the fibers of the nonwoven web that can be functionalized with ligands as described herein to enhance affinity for certain target molecules. The selection of monomer for the grafted polymer can vary, and will depend in part, on the desired binding properties needed for the final membrane structure. Certain monomers will inherently carry functional groups that can be used for affinity or ion exchange binding while other monomers will require further functionalization to add the necessary binding groups. Exemplary monomers and possible uses thereof include: glycidyl methacrylate (suitable for further functionalization), methacrylic acid (weak cation exchange membranes), 2- (diethylamino)ethylmethacrylate (weak anion exchange membranes), [2- (methacryloyloxy)ethyl]trimethyl-ammonium chloride (strong anion exchange membranes), 2- hydroxyethyl methacrylate (HEMA, hydrophilic membranes), 2-acrylamido-2-methylpropane sulfonic acid (strong cation exchange membranes), 2-(dimethylamino)ethylmethacrylate (weak anion exchange membranes), butyl methacrylate (hydrophobic interaction membranes), 3-chloro- 2-hydroxypropyl methacrylate (suitable for further functionalization), 2-ethylhexyl methacrylate (hydrophobic interaction membranes), or combinations thereof. In some embodiments, the grafted polymer comprises an acrylate or methacrylate polymer from one or more monomers comprising glycidyl methacrylate (GMA), methacrylic acid, 2-(diethylamino)ethyl methacrylate, [2- (methacryloyloxy)ethyl] trimethyl-ammonium chloride, 2-hydroxyethyl methacrylate, 2- acrylamido-2-methylpropane sulfonic acid, 2-(dimethylamino)ethyl methacrylate, butyl methacrylate, 3-chloro-2-hydroxypropyl methacrylate, 2-ethylhexyl methacrylate, or combinations thereof. The UV and thermal grafting processes described herein can result in a nonwoven web with a grafted layer around each individual fiber in the nonwoven. The grafted segment thus formed can have a thickness in the range of about 0.05 μm up to about 100 μm. In certain embodiments, the grafted polymer can have a thickness of about 0.1 μm to about 10 μm, about 0.1 μm to about 5 μm, or about 0.2 μm to about 5 μm. In some embodiments, the grafted layer can be formed on a plurality of the individual fibers forming the nonwoven web. In particular, the grafted layer can be formed on substantially all of the fibers. More particularly, the grafted layer can be formed on each of the fibers forming the nonwoven web. Nonwoven fabrics (webs) can be manufactured in equipment with widths that range from about 3–4 cm for an electrospinning machine to about 6 m for melt-blowing and spun-bonding operations. Nonwovens can be produced in web sizes from 0.5 mm (~1 mm or less) to 10 m (1000 cm, 32 ft). Typical properties of the nonwoven fabrics include fiber diameter: Spun-bond (15–40 µm); Melt-blown (1–10 µm); and Electro-spun (0.04–2 µm). The overall range is from 0.04 µm to 40 µm. In one aspect, nonwoven fabrics comprise fiber diameter of 1 nm (0.001 µm) to 100 µm or greater. The basis weights (mass / surface area) comprise: Spun-bond: 8–350 gm / m2; Melt blown: 5–200 gm / m2; and Electrospun: 0.02–0.5 gm / m2. In one aspect, nonwoven fabrics comprise fiber basis weights of 0.001 gm / m2to 1000 gm / m2or greater. The nonwoven fabrics have web porosities of 0.70–0.85 (Fractional pore volume). The nonwoven fabrics have web thickness, which are the dividend of the basis mass divided by polymer density. The polymer density ranges for nonwovens are: 0.9 gm / cm3(for polypropylene) to 1.4 gm / cm3(for polyethylene terephthalate). The smallest thickness isc currently (electro-spun, 0.02 gm / m2basis weight) = 0.014 µm and the largest thickness currently (spun-bonded, 350 gm / m2basis weight) = 400 µm. For UV grafting the normal range used is 200–400 µm to allow light to penetrate the sample uniformly. For heat-induced grafting (HIG) the thickness can be much greater because the entire sample is incubated at a higher temperature. The maximum size for heat-induced grafting is up to 1 meter or more. Typical nonwoven fabrics thickness ranges from 0.001 µm to 10 meters. Grafted layers are characterized by the weight percent increase (W%) in the nonwoven fabric resulting from the addition of the polymer graft. Normally the range comprises 5% to 30% weight gain. In some aspect the weight gains comprise from 0.001% to 100% or more. The larger the weight gain the larger the grafted layer thickness. Normally, the grafted layer thicknesses around each fiber are on the order or 100 to 10,000 Å (1 µm). It would be unusual to have a grafted layer thickness to be much thicker than the fiber diameter. In some aspects, the grafted layer thicknesses of 10 Å (for the smallest fibers with small weight gains) to 1000 µm (for the largest fibers and largest weight gains). As described herein, the grafted polymer segments or brushes can be further functionalized such that each polymer segment is coupled to one or more functional ligands adapted for binding to a particular target molecule. Exemplary binding that can occur between such functional ligands and a target molecule, such as a protein, can include ionic bonds, hydrogen bonds, and van der Waals forces. Exemplary functional ligands may include amine groups (including primary, secondary, tertiary, or quaternary amines), sulfonic acid groups, carboxylic acid groups, phosphate groups, and the like. The derivatizing reactions to attach such functional ligands typically involve reacting an epoxy group or other reactive group on the polymer brush with a molecule containing the desired functional ligand. In some embodiments, the grafted polymeric fibers are functionalized to attach a functional ligand configured or adapted for ion exchange (cation or anion exchange) with a target molecule. The ion exchange group of the functional ligand can be a strong ion exchanger (anion or cation), or a weak ion exchanger (anion or cation), a charged multimodal ligand (anion or cation), or an anionic or cationic charged polymer. In some embodiments, functional ligands are adapted for ion exchange with a target molecule and comprise N-benzyl-N-methylethanolamine (BMEA), 2- mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof. As used herein, the term “multimodal ligand” or “MM ligand” refers to a type of charged multimodal ligand (anion or cation) for mixed mode chromatography and purification of target molecules. Disclosed multimodal ligands may comprise immobilized or coupled ligands that interact with target molecules through multiple types of interactions including, but not limited to, size exclusion, ionic, and hydrophobic interactions. The disclosed multimodal ligands are salt- tolerant meaning that they bind at high ionic strength and that there is no need to change buffer before a capture step, as might be necessary with other ion exchange resins or membranes. As used herein, the term “residence time” or “RT” refers to the amount of time for a fluid solute to pass through a membrane-based chromatography device. The residence time is determined by dividing the liquid volume inside the device by the liquid volumetric flow rate. The disclosed functionalized nonwoven membranes and chromatography devices typically operate at lower residence times (i.e., fast flow rates) as compared to conventional resin techniques because there are no diffusional limitations in the adsorption and desorption steps. As used herein, the term “dynamic binding capacity” or “DBC” refers to the maximum amount of target molecule that can bind to an amount of chromatography media (membrane) under flow conditions before there is a significant breakthrough (5% to 10% of the inlet concentration) of the target molecule. Dynamic binding capacity is determined under actual operating conditions. In some embodiments, the dynamic binding capacity of a membrane as described herein is measured at 5–10% breakthrough (DBC5%– DBC10%). The binding capacity of the disclosed functionalized nonwoven membranes can vary and can be configured as desired based upon the particular target molecule to be bound and / or the specific end use of the material. In some embodiments, a polymer-grafted membrane according to the present disclosure can exhibit an equilibrium binding capacity for a target molecule of up to about 1,000 mmoles / g of the target molecule (with a minimum equilibrium binding capacity of at least about 1 mmol / g of the target molecule). More particularly, equilibrium binding capacity for a target molecule can be about 1 mmol / g to about 1,000 mmols / g, about 5 mmols / g to about 800 mmols / g, or about 10 mmols / g to about 600 mmols / g of the target molecule. In some embodiments, the equilibrium binding capacity can be based upon the molecular weight of the target molecule. For example, the equilibrium binding capacity for a target molecule having a molecular weight of about 100 g / mol to about 1,000 g / mol can be about 50 mmols / g to about 1,000 mmols / g, or about 100 mmols / g to about 800 mmols / g of the target molecule. As a further example, the equilibrium binding capacity for a target molecule having a molecular weight of about 2,000 g / mol to about 50,000 g / mol can be about 10 mmols / g to about 300 mmols / g, or about 20 mmols / g to about 200 mmols / g of the target molecule. As yet another example, the equilibrium binding capacity for a target molecule having a molecular weight of about 60,000 g / mol to about 500,000 g / mol can be about 2 mmols / g to about 100 mmols / g, or about 5 mmols / g to about 80 mmols / g of the target molecule. In some embodiments, the disclosed functionalized nonwoven membranes can be configured for binding to a variety of target molecules. The target molecules may be defined in relation to the presence of charged groups, molecular weight, and / or affinity for certain functional ligands. In some embodiments, the disclosed functionalized nonwoven membranes can be characterized as binding significant amounts of a target molecule in short contact periods, such as reaching equilibrium binding in about 1 hour or less. In various embodiments, a functionalized nonwoven membrane according to the present disclosure can be configured for reaching a binding equilibrium for the target molecule in a time of about 1 hour or less (i.e., with a lower end understood to be 1 second, 2 seconds, or 5 seconds). In further embodiments, the time to reaching a binding equilibrium for the target molecule can be about 1 second to about 120 minutes, about 2 seconds to about 90 minutes, about 5 seconds to about 60 minutes, or about 10 seconds to about 30 minutes. In some embodiments, the disclosed functionalized nonwoven membranes can be defined in relation to a shortened time for achieving target molecule binding equilibrium. In certain embodiments comprising grafted polymers, the time for achieving binding equilibrium can depend upon the degree of grafting, which can be based upon the percent weight gain as defined herein. For example, a polymer grafted nonwoven web substrate according to the present disclosure having up to a 5% weight gain of grafted polymer can exhibit a time for achieving target molecule binding equilibrium of about 20 minutes or less, about 10 minutes or less, or about 5 minutes or less (with an understood minimum time of about 1 second, about 5 seconds, or about 15 seconds). More particularly, the time for achieving target molecule binding equilibrium under the noted conditions can be about 5 seconds to about 20 minutes, about 10 seconds to about 10 minutes, or about 15 seconds to about 8 minutes. In further examples, a polymer grafted nonwoven web substrate according to the present disclosure having about a 6% to about a 15% weight gain of grafted polymer can exhibit a time for achieving target molecule binding equilibrium of about 30 minutes or less, about 20 minutes or less, or about 10 minutes or less (with an understood minimum time of about 1 second, about 5 seconds, or about 15 seconds). More particularly, the time for achieving target molecule binding equilibrium under the noted conditions can be about 10 seconds to about 30 minutes, about 15 seconds to about 15 minutes, or about 20 seconds to about 10 minutes. In still further examples, a polymer grafted nonwoven web substrate according to the present disclosure having about a 16% to about a 25% weight gain of grafted polymer can exhibit a time for achieving target molecule binding equilibrium of about 120 minutes or less, about 90 minutes or less, or about 60 minutes or less (with an understood minimum time of about 5 seconds, about 10 seconds, or about 15 seconds). More particularly, the time for achieving target molecule binding equilibrium under the noted conditions can be about 15 seconds to about 120 minutes, about 30 seconds to about 60 minutes, or about 45 seconds to about 45 minutes. The particular buffers, salts, and pH ranges used in the disclosed methods will vary and be determined based on the specific membrane and target molecule properties. For instance, in the disclosed methods, the particular pH range, ionic strength, and salt type of the various solvents (e.g., binding buffers, washing buffers, elution buffers) will depend on the specific nonwoven membrane being used and its functional ligands, as well as the specific chemical properties of the target molecule being purified. It is understood that there are various combinations of monovalent ions, divalent ions, salt concentrations, and buffer types that are suitable and effective in binding, wash, elution, and flowthrough purification steps. Ionic strength and conductivity are important parameters for solvents used in ion exchange chromatography. In particular, increasing the ionic strength introduces more counterions to compete with charged groups on a target biomolecule for binding onto a stationary phase membrane as described herein, and also reduces the extension of the electrical double layer on both the target biomolecule and the membrane, thus allowing the desorption of the adsorbed target species. The concentration, quantity, and specific type of the ions present in a particular solvent solution as described herein (e.g., binding buffers, washing buffers, elution buffers) determine the ionic strength and conductivity of the solvent solution that affect whether binding or elution of target molecules will occur. For example, ions with higher valency (e.g., divalent ions) have a higher ionic strength and conductivity than ions with lower valency (e.g., monovalent ions). Generally, the higher the ionic strength, the higher the electrical conductivity of the solvent solution, while solvent solutions having lower ionic strengths will be less conductive. Lower ionic strength buffers are used during loading steps to favor adsorption of target molecules, while in the elution step the ionic strength is altered to desorb the adsorbed species, including the target molecule. The adsorbed species with the lowest net charge are eluted first and the ones with the highest net charge are eluted last. Conductivity is typically measured in microohms (µΩ) or microsiemens (µS). These values can be converted to parts per million or molarity using conversion factors. The molar conductivity is represented in siemens metres squared per mole (S m2mol−1). Most monovalent ions in water have molar ionic conductivities in the range of 40–80 S cm2mol−1. Protein binding occurs when the charge of the molecule is different from the charge on the surface, so the pH must be adjusted to be greater than or lower than the isoelectric point of the protein to result in a negative or positive charge on the molecule. The electrostatic forces between the surface and the molecule decrease significantly when the conductivity of the solution is high. So, adsorption usually occurs in buffers that have lower conductivity (low salt concentration), and desorption or elution occurs when the buffers have a high conductivity (high salt concentration). For example, with NaCl, a low concentration which would give a low ionic strength would be 150 mM (0.15 M NaCl). A high concentration that would result in a high conductivity would be 0.5 to 2.0 M NaCl. The figure below shows how conductivity changes with NaCl concentration. As the figure indicates, a low conductivity would be in the range of 30 mS / cm or less, and a high conductivity would be in the range of 500 mS / cm or more. See Widodo et al., AIP Conference Proceedings 2021: 050003 (2018), which is incorporated by reference herein for such teachings. As used herein, the term “low salt buffer” refers to a buffer having less than about 2.5 M concentration of one or more salts including, but not limited to, NaCl, KCl, NH4Cl, and the like. In some embodiments, the low salt buffer may comprise one or more of Tris, Tris-HCl, acetate, phosphate, MES, HEPES, or other common buffers known in the art. In certain non-limiting exemplary embodiments, disclosed membranes may be washed with a low salt buffer comprising one or more of 0.02–0.2 M acetate buffer, 0.02–0.2 M Tris-HCl buffer, 0.02–0.2 M phosphate buffer, or 0.02–0.2 M MES buffer at pH 5–9. Membranes may then be further washed with a low salt buffer comprising one or more charged species including, but not limited to, 0.5–2.0 M urea, 10%–40% (v / v) ethylene glycol, 0.05–2.0 M arginine, or 0.05–1.5 M NaCl at pH 5–9. In other certain non-limiting exemplary embodiments, a first wash of the membrane is performed using 50 mM Tris-HCl with about 1 mM to about 150 mM NaCl, pH 7.9. A second wash of the membrane may then be performed using 50 mM Tris-HCl with about 50 mM to about 125 mM arginine, pH 7.4. In some embodiments, a target molecule may be eluted from a membrane using one or more elution buffers or solutions at about pH 2–6. For example, in some embodiments, the target molecule is eluted from the membrane using a first elution buffer at about pH 4.5, and further eluted using a second elution buffer at about pH 4.0. In certain non-limiting exemplary embodiments, the one or more elution buffers or solutions comprises 50 mM acetate at about pH 2–6. Described herein is a functionalized nonwoven membrane for purifying a target molecule. In certain non-limiting embodiments, a high-performance multimodal anion exchange (MMA) nonwoven membrane is disclosed comprising an N-benzyl-N-methylethanolamine (BMEA) functional ligand coupled to a polyglycidyl methacrylate (pGMA or polyGMA) UV-grafted polybutylene terephthalate (PBT) nonwoven web (MMA-BMEA). The high-performance MMA membrane described herein can be used as a non-Protein A antibody purification approach and exhibits a high capacity for target molecule (e.g., mAb) capture. In some embodiments, described methods may involve additional chromatographic nonwoven membranes for polishing, including anion and cation exchange nonwoven membranes. Inexpensive nonwoven membranes with low pressure drops at high flowrate, sufficient surface area and easy production are promising material to develop high-throughput high-capacity disposable chromatographic membranes. The disclosed functionalized nonwoven membranes were found to have an excellent salt tolerance where about 70% of protein binding capacity remained when salt concentration increased up to 150 mM NaCl. Due to the large inter-fiber distance of the membrane, the membrane exhibited a high flow permeability of 1.0–4.6 × 10−9cm2, significantly higher than reported results of commercial strong anion exchange resins. Meanwhile, the DBC10%of the exemplary MMA-BMEA membrane (30.8–38.5 mg / mL bovine serum albumin, BSA) was 85% higher than those of MMA Capto adhere resin (16.6–20.5 mg / mL BSA) both at 0.15 M NaCl and 0.5–5.0 min residence time (RT). The prepared membrane also showed a high DBC10%of 59.2 mg / mL for directly capturing a mAb from a CHO supernatant without pH and conductivity adjustment, and the binding capacity is essentially independent of RT from 0.5 to 5 min. Under the optimized RT, arginine concentration for membrane washing and elution pH, a satisfactory recovery of 94.3%, a high HCP removal of 1.0 LRV and DNA removal of 1.8 LRV as well as a great reduction of aggregate % from 5.4% to 0.9% were achieved. Based on the described capture step using the MMA-BMEA membrane, and the following polishing steps with an anion exchange nonwoven (AEX-TEA) and a multimodal cation exchange nonwoven (MMC-MPCA), the three-step membrane chromatography reduced HCP from 327000 ppm in the supernatant to 93.5 ppm (3.5 LRV), DNA from 994.7 ppm to 6.8 ppb (5.3 LRV), percentage aggregates from 5.4% to 0.4% with no detected fragments, within common requirements for mAb products (HCP <100 ppm, DNA <10 ppb, % aggregate <1%). A side-by- side comparison with a standard mAb purification by using protein A resin and two ion exchange resins, showed that the three-step membrane processes outperformed the standard process with reduced process time (3.8 h vs.13.1 h), 14% higher overall recovery (88.3% vs.77.5%), fewer steps (no diafiltration for buffer exchange), and fewer aggregates in the final product despite the similar HCP and DNA removal. In some embodiments, the MMA membrane may be prepared through conjugating N- benzyl-N-methylethanolamine (BMEA) to pGMA that could have satisfactory protein binding capacity and that can be used in direct mAb capture at a relatively short residence time. As described herein, the influence of ligand density of MMA-BMEA membranes prepared using different ligand concentrations in the reaction solution was investigated in terms of static binding capacity (SBC) for bovine serum albumin (BSA, model protein for basic characterization of anion exchange materials). The impact of salt concentration on SBC, flow permeability and dynamic binding capacity (DBC) of the membrane were studied and the DBC10%for BSA measured at varied residence times (RTs) was compared with that of a commercial MMA resin (Capto adhere; Cytiva) under the same conditions. The DBC10%and isotherm for IgG of MMA-BMEA membrane and its reusability within five consecutive IgG bind-and-elute cycles were studied to preliminarily evaluate its performance for antibodies. Subsequently, the MMA-BMEA membrane was used to capture a mAb from CHO culture fluid. The membrane performance at different RTs, arginine concentrations in the wash step, as well as elution pH were investigated in terms of product recovery and impurity removal. With additional virus inactivation and subsequent mAb polishing by AEX-TEA membrane (anion exchange nonwoven membrane with triethylamine as ligand) and MMC-MPCA membrane (multimodal cation exchange membrane with 2-mercaptopyridine-3- carboxylic acid as ligand), a non-Protein A, all-membrane purification strategy was established (FIG. 1). The results were compared with that of a standard three-step resin chromatography which was comprised of Protein A capture, virus inactivation, and subsequent cation exchange and anion exchange chromatography for polishing (FIG.1). Also described herein are membrane-based chromatography devices for purification of target molecules. For ideal operation of the disclosed membrane-based chromatography devices, a suitable membrane holder must be utilized. A cartridge, cassette, column, or any such similarly described device may be appropriate so long as the following requirements are met. The design should be such that dead volumes, which are any internal volumes of the device not occupied by the membrane, should be minimized and not detract from the flow performance of the device. Likewise, the flow pattern should be engineered to distribute flow from the inlet evenly across the membrane area without channeling, clogging, over pressurizing or other indications of nonuniform flow. This has the additional effect of allowing for the elution or collection of the product from the device in the most efficient and economical manner. The elution of product from the device when flow is uniform therefore results in a highly concentrated pool of the smallest volume possible (< 5 equivalent volumes of the device for removal of 95% of the total product captured), resulting from complete and rapid desorption of product. The optimal embodiment of the flow device also enables scaling of the device to meet the needs of separations for processes of different operational magnitudes. Whether through the operation of multiple cartridges, cassettes, columns, etc. in parallel or in series, or from the implementation of larger internal membrane volumes which maintain the above properties, separation of product from feedstocks at the lab, pilot, or manufacturing scale should be able to be performed without loss of productivity, yield, or overall quality. One embodiment described herein is a functionalized nonwoven membrane for purifying a target molecule or species, the membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N- methylethanolamine (BMEA), 2-mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof. In one aspect, the polymeric fibers comprise polyolefins, polyethylenes, polypropylenes, cellulose, polyesters, polyamides, polycarbonates, polyethersulfones, polybutylene terephthalate (PBT), polytrimethylene terephthalate (PTT), polyethylene terephthalate (PET), polyamide 6 (PA6), polyamide 6-6 (PA6-6), polyamide 11 (PA11), polyamide 12 (PA12), thermoplastic polymers, or combinations thereof. In another aspect, the surface modification comprises a grafted second polymer. In another aspect, the grafted second polymer comprises an acrylate or methacrylate polymer from one or more monomers comprising glycidyl methacrylate (GMA), methacrylic acid, 2-(diethylamino)ethyl methacrylate, [2-(methacryloyloxy)ethyl] trimethyl-ammonium chloride, 2- hydroxyethyl methacrylate, 2-acrylamido-2-methylpropane sulfonic acid, 2-(dimethylamino)ethyl methacrylate, butyl methacrylate, 3-chloro-2-hydroxypropyl methacrylate, 2-ethylhexyl methacrylate, or combinations thereof. In another aspect, the functional ligand comprises N- benzyl-N-methylethanolamine (BMEA). Another embodiment described herein is a functionalized nonwoven membrane for purifying a target molecule, the membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having a grafted second polymer; and a functional ligand coupled to the grafted second polymer and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA). In one aspect, the polymeric fibers comprise polyolefins, polyethylenes, polypropylenes, cellulose, polyesters, polyamides, polycarbonates, polyethersulfones, polybutylene terephthalate (PBT), polytrimethylene terephthalate (PTT), polyethylene terephthalate (PET), polyamide 6 (PA6), polyamide 6-6 (PA6-6), polyamide 11 (PA11), polyamide 12 (PA12), thermoplastic polymers, or combinations thereof. In another aspect, the grafted second polymer comprises an acrylate or methacrylate polymer from one or more monomers comprising glycidyl methacrylate (GMA), methacrylic acid, 2-(diethylamino)ethyl methacrylate, [2-(methacryloyloxy)ethyl] trimethyl- ammonium chloride, 2-hydroxyethyl methacrylate, 2-acrylamido-2-methylpropane sulfonic acid, 2-(dimethylamino)ethyl methacrylate, butyl methacrylate, 3-chloro-2-hydroxypropyl methacrylate, 2-ethylhexyl methacrylate, or combinations thereof. In another aspect, the grafted second polymer comprises a methacrylate polymer of polyGMA. In another aspect, the functionalized nonwoven membrane further comprises one or more additional functional ligands comprising 2- mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof. Another embodiment described herein is a functionalized nonwoven membrane for purifying a target molecule, the membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) coupled to the polyGMA. In one aspect, the membrane has a dynamic binding capacity (DBC) for target molecule capture of ˃50 mg / mL with a residence time of from about 0.5 min to about 5 min. In another aspect, the membrane has a flow permeability of about 1.0 × 10−9cm2to about 4.6 × 10−9cm2in a buffer comprising about 50 mM to about 1 M of one or more salts at about pH 7.0. Another embodiment described herein is a method for purifying a target molecule, the method comprising: contacting a sample comprising the target molecule with a functionalized nonwoven membrane to bind the target molecule to the membrane, the membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2-mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof; and (b) eluting the target molecule from the membrane and collecting the target molecule in a first eluate fraction. In one aspect, the target molecule is a biomolecule comprising a protein, a peptide, an antibody, a nucleic acid, an enzyme, a lipoprotein, a lipid, an extracellular vesicle, an exosome, a viral vector, a plasmid, a carbohydrate, a vaccine, a virus, a bacterium, a cell, an organelle, or combinations thereof. In another aspect, the sample is a biological fluid sample. In another aspect, the biological fluid sample comprises a cell culture supernatant or a sample obtained from an animal subject. In another aspect, the sample obtained from an animal subject comprises one or more of blood, plasma, serum, tears, urine, saliva, sputum, pleural effusion, or ascites. In another aspect, the method further comprises: (a)(i) performing a first wash of the membrane using a low salt buffer at about pH 5–9; and (a)(ii) performing a second wash of the membrane using a low salt buffer having a charged species at about pH 5–9. In another aspect, the steps comprise: (a)(i) performing a first wash of the membrane using 50 mM Tris-HCl with about 1 mM to about 150 mM NaCl at pH 7.9; and (a)(ii) performing a second wash of the membrane using 50 mM Tris-HCl with about 50 mM to about 125 mM arginine at pH 7.4. In another aspect, the target molecule is eluted from the membrane using one or more elution buffers or solutions at about pH 2–6. In another aspect, the target molecule is eluted from the membrane using a first elution buffer at about pH 4.5. In another aspect, the target molecule is further eluted from the membrane using a second elution buffer at about pH 4.0. In another aspect, the one or more elution buffers or solutions comprises 50 mM acetate at about pH 2–6. In another aspect, method further comprises virus inactivation comprising: (b)(i) adjusting the pH of the first eluate fraction to about pH 3–5; (b)(ii) incubating the first eluate fraction at room temperature for about 1 hour; (b)(iii) adjusting the pH of the first eluate fraction to about pH 7–8; and (b)(iv) filtering the first eluate fraction using microfiltration. In another aspect, method further comprises: (c) contacting the first eluate fraction comprising the target molecule with a second functionalized nonwoven membrane and collecting a flow-through fraction comprising the target molecule. In another aspect, method further comprises: (c)(i) performing a wash of the second functionalized nonwoven membrane using a buffer with about pH 6–9. In another aspect, method further comprises: (c)(i) performing a wash of the second functionalized nonwoven membrane using 50 mM Tris-HCl at pH 7.2. In another aspect, the second functionalized nonwoven membrane comprises a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2-mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof. In another aspect, the method further comprises: (d) contacting the flow-through fraction comprising the target molecule with a third functionalized nonwoven membrane to bind the target molecule to the third functionalized nonwoven membrane; and (e) eluting the target molecule from the third functionalized nonwoven membrane and collecting the target molecule in a second eluate fraction. In another aspect, the method further comprises: (d)(i) performing a wash of the third functionalized nonwoven membrane using a low salt buffer at about pH 4–7. In another aspect, the step comprises: (d)(i) performing a wash of the third functionalized nonwoven membrane using 50^mM acetate with 150 mM NaCl at about pH 5.5. In another aspect, the target molecule is eluted from the third functionalized nonwoven membrane using a low salt buffer at about pH 7– 10. In another aspect, the target molecule is eluted from the third functionalized nonwoven membrane using 50 mM sodium phosphate with 150 mM NaCl at about pH 7.7. In another aspect, the third functionalized nonwoven membrane comprises a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N- methylethanolamine (BMEA), 2-mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof. In another aspect, each of steps (a)–(e) has a residence time of about 0.1 min to about 5 min. Another embodiment described herein is a method for purifying a target molecule, the method comprising: contacting a sample comprising the target molecule with a functionalized nonwoven membrane to bind the target molecule to the membrane, the membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) coupled to the polyGMA; and eluting the target molecule from the membrane and collecting the target molecule in a first eluate fraction. In one aspect, the target molecule is a biomolecule comprising a protein, a peptide, an antibody, a nucleic acid, an enzyme, a lipoprotein, a lipid, an extracellular vesicle, an exosome, a viral vector, a plasmid, a carbohydrate, a vaccine, a virus, a bacterium, a cell, an organelle, or combinations thereof. Another embodiment described herein is a method for purifying a target molecule, the method comprising: contacting a sample comprising the target molecule with a first functionalized nonwoven membrane to bind the target molecule to the first membrane, the first membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) coupled to the polyGMA; eluting the target molecule from the first membrane and collecting the target molecule in a first eluate fraction; contacting the first eluate fraction comprising the target molecule with a second functionalized nonwoven membrane, the second membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising triethylamine (TEA) coupled to the polyGMA; collecting a flow-through fraction comprising the target molecule; contacting the flow- through fraction comprising the target molecule with a third functionalized nonwoven membrane to bind the target molecule to the third membrane, the third membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising 2- mercaptopyridine-3-carboxylic acid (MPCA) coupled to the polyGMA; and eluting the target molecule from the third membrane and collecting the target molecule in a second eluate fraction. In one aspect, the target molecule is a biomolecule comprising a protein, a peptide, an antibody, a nucleic acid, an enzyme, a lipoprotein, a lipid, an extracellular vesicle, an exosome, a viral vector, a plasmid, a carbohydrate, a vaccine, a virus, a bacterium, a cell, an organelle, or combinations thereof. In another aspect, the target molecule is an antibody. Another embodiment described herein is a method of making a functionalized nonwoven membrane for purifying a target molecule, the method comprising: (a) grafting polyglycidyl methacrylate (polyGMA) onto a polybutylene terephthalate (PBT) nonwoven web to generate a PBT-polyGMA nonwoven web; and (b) coupling a functional ligand comprising N-benzyl-N- methylethanolamine (BMEA) to the grafted polyGMA of the PBT-polyGMA nonwoven web to generate the functionalized nonwoven membrane. In one aspect, grafting the polyGMA onto the PBT nonwoven web is performed using ultraviolet radiation. In another aspect, a concentration of the BMEA functional ligand that is coupled to the PBT-polyGMA nonwoven web is from about 2% (v / v) to about 15% (v / v). In another aspect, the BMEA functional ligand is coupled to the PBT- polyGMA nonwoven web at about 40 °C for about 6 hours, followed by treatment with 0.1 M sulfuric acid at about 50 °C for about 16 hours. In another aspect, the method further comprises washing the functionalized nonwoven membrane with pure water and storing the functionalized nonwoven membrane in a solution of 20% ethanol / water (v / v). Another embodiment described herein is a chromatography device for purifying a target molecule, the chromatography device comprising: a housing unit comprising one or more functionalized nonwoven membranes, each membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N- methylethanolamine (BMEA), 2-mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof; and a flow device connected to the housing unit and configured to induce fluid flow of a sample comprising the target molecule through the one or more functionalized nonwoven membranes. In one aspect, the housing unit is a cartridge, a cassette, or a column. Another embodiment described herein is a method for purifying a target molecule, the method comprising: contacting a sample comprising the target molecule with a chromatography device comprising: a housing unit comprising one or more functionalized nonwoven membranes, each membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2- mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof; and a flow device connected to the housing unit and configured to induce fluid flow of the sample comprising the target molecule through the one or more functionalized nonwoven membranes; inducing fluid flow of the sample comprising the target molecule through the one or more functionalized nonwoven membranes using the flow device to bind the target molecule to the one or more functionalized nonwoven membranes; and eluting the target molecule from the one or more functionalized nonwoven membranes and collecting the target molecule in a first eluate fraction. Another embodiment described herein is a kit for purifying a target molecule, the kit comprising: one or more functionalized nonwoven membranes, each membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2-mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof; (b) optionally, columns, buffers, cartridges, cassettes, and receptacles; and (c) optionally, one or more of packaging or instructions for use. Another embodiment described herein is a method for removing ions or ionic contaminants from a liquid, the method comprising: (a) contacting a liquid comprising ions or ionic contaminants with a functionalized nonwoven membrane to bind the ions or ionic contaminants to the membrane, the membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2- mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof; and (b) collecting the liquid having the ions or ionic contaminants removed. In one aspect, the liquid is water. Another embodiment described herein is a method for removing ions or ionic contaminants from a liquid, the method comprising: (a) contacting a liquid comprising ions or ionic contaminants to a first functionalized nonwoven membrane to bind anions or anionic contaminants to the first functionalized nonwoven membrane, the first functionalized nonwoven membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) or triethylamine (TEA) coupled to the polyGMA; (b) collecting a first liquid flow-through having the anions or anionic contaminants removed; (c) contacting the first liquid flow-through to a second functionalized nonwoven membrane to bind cations or cationic contaminants to the second functionalized nonwoven membrane, the second functionalized nonwoven membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising 2-mercaptopyridine-3-carboxylic acid (MPCA) coupled to the polyGMA; and (d) collecting a second liquid flow-through having the cations or cationic contaminants removed. In one aspect, the liquid is water. Another embodiment described herein is a method for purifying water, the method comprising: (a) contacting a sample of water with a first functionalized nonwoven membrane to bind anions or anionic contaminants to the first functionalized nonwoven membrane, the first functionalized nonwoven membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) or triethylamine (TEA) coupled to the polyGMA; (b) collecting a first water flow-through having the anions or anionic contaminants removed; (c) contacting the first water flow-through with a second functionalized nonwoven membrane to bind cations or cationic contaminants to the second functionalized nonwoven membrane, the second functionalized nonwoven membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising 2-mercaptopyridine-3-carboxylic acid (MPCA) coupled to the polyGMA; and (d) collecting a second water flow-through having the cations or cationic contaminants removed. The compositions and methods provided herein are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof. Various embodiments and aspects of the inventions described herein are summarized by the following clauses: Clause 1. A functionalized nonwoven membrane for purifying a target molecule or species, the membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2-mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof. Clause 2. The membrane of clause 1, wherein the polymeric fibers comprise polyolefins, polyethylenes, polypropylenes, cellulose, polyesters, polyamides, polycarbonates, polyethersulfones, polybutylene terephthalate (PBT), polytrimethylene terephthalate (PTT), polyethylene terephthalate (PET), polyamide 6 (PA6), polyamide 6-6 (PA6-6), polyamide 11 (PA11), polyamide 12 (PA12), thermoplastic polymers, or combinations thereof. Clause 3. The membrane of clause 1 or 2, wherein the surface modification comprises a grafted second polymer. Clause 4. The membrane of any one of clauses 1–3, wherein the grafted second polymer comprises an acrylate or methacrylate polymer from one or more monomers comprising glycidyl methacrylate (GMA), methacrylic acid, 2-(diethylamino)ethyl methacrylate, [2- (methacryloyloxy)ethyl] trimethyl-ammonium chloride, 2-hydroxyethyl methacrylate, 2- acrylamido-2-methylpropane sulfonic acid, 2-(dimethylamino)ethyl methacrylate, butyl methacrylate, 3-chloro-2-hydroxypropyl methacrylate, 2-ethylhexyl methacrylate, or combinations thereof. Clause 5. The membrane of any one of clauses 1–4, wherein the functional ligand comprises N-benzyl-N-methylethanolamine (BMEA). Clause 6. A functionalized nonwoven membrane for purifying a target molecule, the membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having a grafted second polymer; and a functional ligand coupled to the grafted second polymer and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N- methylethanolamine (BMEA). Clause 7. The membrane of clause 6, wherein the polymeric fibers comprise polyolefins, polyethylenes, polypropylenes, cellulose, polyesters, polyamides, polycarbonates, polyethersulfones, polybutylene terephthalate (PBT), polytrimethylene terephthalate (PTT), polyethylene terephthalate (PET), polyamide 6 (PA6), polyamide 6-6 (PA6-6), polyamide 11 (PA11), polyamide 12 (PA12), thermoplastic polymers, or combinations thereof. Clause 8. The membrane of clause 6 or 7, wherein the grafted second polymer comprises an acrylate or methacrylate polymer from one or more monomers comprising glycidyl methacrylate (GMA), methacrylic acid, 2-(diethylamino)ethyl methacrylate, [2- (methacryloyloxy)ethyl] trimethyl-ammonium chloride, 2-hydroxyethyl methacrylate, 2- acrylamido-2-methylpropane sulfonic acid, 2-(dimethylamino)ethyl methacrylate, butyl methacrylate, 3-chloro-2-hydroxypropyl methacrylate, 2-ethylhexyl methacrylate, or combinations thereof. Clause 9. The membrane of any one of clauses 6–8, wherein the grafted second polymer comprises a methacrylate polymer of polyGMA. Clause 10. The membrane of any one of clauses 6–9, further comprising one or more additional functional ligands comprising 2-mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof. Clause 11. A functionalized nonwoven membrane for purifying a target molecule, the membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) coupled to the polyGMA. Clause 12. The membrane of clause 11, wherein the membrane has a dynamic binding capacity (DBC) for target molecule capture of ˃50 mg / mL with a residence time of from about 0.5 min to about 5 min. Clause 13. The membrane of clause 11 or 12, wherein the membrane has a flow permeability of about 1.0 × 10−9cm2to about 4.6 × 10−9cm2in a buffer comprising about 50 mM to about 1 M of one or more salts at about pH 7.0. Clause 14. A method for purifying a target molecule, the method comprising: (a) contacting a sample comprising the target molecule with a functionalized nonwoven membrane to bind the target molecule to the membrane, the membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2- mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof; and (b) eluting the target molecule from the membrane and collecting the target molecule in a first eluate fraction. Clause 15. The method of clause 14, wherein the target molecule is a biomolecule comprising a protein, a peptide, an antibody, a nucleic acid, an enzyme, a lipoprotein, a lipid, an extracellular vesicle, an exosome, a viral vector, a plasmid, a carbohydrate, a vaccine, a virus, a bacterium, a cell, an organelle, or combinations thereof. Clause 16. The method of clause 14 or 15, wherein the sample is a biological fluid sample. Clause 17. The method of any one of clauses 14–16, wherein the biological fluid sample comprises a cell culture supernatant or a sample obtained from an animal subject. Clause 18. The method of any one of clauses 14–17, wherein the sample obtained from an animal subject comprises one or more of blood, plasma, serum, tears, urine, saliva, sputum, pleural effusion, or ascites. Clause 19. The method of any one of clauses 14–18, further comprising: (a)(i) performing a first wash of the membrane using a low salt buffer at about pH 5–9; and (a)(ii) performing a second wash of the membrane using a low salt buffer having a charged species at about pH 5–9. Clause 20. The method of any one of clauses 14–19, wherein the steps comprise: (a)(i) performing a first wash of the membrane using 50 mM Tris-HCl with about 1 mM to about 150 mM NaCl at pH 7.9; and (a)(ii) performing a second wash of the membrane using 50 mM Tris-HCl with about 50 mM to about 125 mM arginine at pH 7.4. Clause 21. The method of any one of clauses 14–20, wherein the target molecule is eluted from the membrane using one or more elution buffers or solutions at about pH 2–6. Clause 22. The method of any one of clauses 14–21, wherein the target molecule is eluted from the membrane using a first elution buffer at about pH 4.5. Clause 23. The method of clause 22, wherein the target molecule is further eluted from the membrane using a second elution buffer at about pH 4.0. Clause 24. The method of any one of clauses 14–23, wherein the one or more elution buffers or solutions comprises 50 mM acetate at about pH 2–6. Clause 25. The method any one of clauses 14–24, further comprising virus inactivation comprising: (b)(i) adjusting the pH of the first eluate fraction to about pH 3–5; (b)(ii) incubating the first eluate fraction at room temperature for about 1 hour; (b)(iii) adjusting the pH of the first eluate fraction to about pH 7–8; and (b)(iv) filtering the first eluate fraction using microfiltration. Clause 26. The method of any one of clauses 14–25, further comprising: (c) contacting the first eluate fraction comprising the target molecule with a second functionalized nonwoven membrane and collecting a flow-through fraction comprising the target molecule. Clause 27. The method of any one of clauses 14–26, further comprising: (c)(i) performing a wash of the second functionalized nonwoven membrane using a buffer with about pH 6–9. Clause 28. The method of any one of clauses 14–27, wherein the step comprises: (c)(i) performing a wash of the second functionalized nonwoven membrane using 50 mM Tris-HCl at pH 7.2. Clause 29. The method of any one of clauses 14–28, wherein the second functionalized nonwoven membrane comprises a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N- methylethanolamine (BMEA), 2-mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof. Clause 30. The method of any one of clauses 14–29, further comprising: (d) contacting the flow-through fraction comprising the target molecule with a third functionalized nonwoven membrane to bind the target molecule to the third functionalized nonwoven membrane; and (e) eluting the target molecule from the third functionalized nonwoven membrane and collecting the target molecule in a second eluate fraction. Clause 31. The method of any one of clauses 14–30, further comprising: (d)(i) performing a wash of the third functionalized nonwoven membrane using a low salt buffer at about pH 4–7. Clause 32. The method of any one of clauses 14–31, wherein the step comprises: (d)(i) performing a wash of the third functionalized nonwoven membrane using 50^mM acetate with 150 mM NaCl at about pH 5.5. Clause 33. The method of any one of clauses 14–32, wherein the target molecule is eluted from the third functionalized nonwoven membrane using a low salt buffer at about pH 7– 10. Clause 34. The method of any one of clauses 14–33, wherein the target molecule is eluted from the third functionalized nonwoven membrane using 50 mM sodium phosphate with 150 mM NaCl at about pH 7.7. Clause 35. The method of clause 30, wherein the third functionalized nonwoven membrane comprises a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2- mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof. Clause 36. The method of any one of clauses 14–35, wherein each of steps (a)–(e) has a residence time of about 0.1 min to about 5 min. Clause 37. A method for purifying a target molecule, the method comprising: (a) contacting a sample comprising the target molecule with a functionalized nonwoven membrane to bind the target molecule to the membrane, the membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) coupled to the polyGMA; and (b) eluting the target molecule from the membrane and collecting the target molecule in a first eluate fraction. Clause 38. The method of clause 37, wherein the target molecule is a biomolecule comprising a protein, a peptide, an antibody, a nucleic acid, an enzyme, a lipoprotein, a lipid, an extracellular vesicle, an exosome, a viral vector, a plasmid, a carbohydrate, a vaccine, a virus, a bacterium, a cell, an organelle, or combinations thereof. Clause 39. A method for purifying a target molecule, the method comprising: (a) contacting a sample comprising the target molecule with a first functionalized nonwoven membrane to bind the target molecule to the first membrane, the first membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) coupled to the polyGMA; (b) eluting the target molecule from the first membrane and collecting the target molecule in a first eluate fraction; (c) contacting the first eluate fraction comprising the target molecule with a second functionalized nonwoven membrane, the second membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising triethylamine (TEA) coupled to the polyGMA; (d) collecting a flow-through fraction comprising the target molecule; (e) contacting the flow-through fraction comprising the target molecule with a third functionalized nonwoven membrane to bind the target molecule to the third membrane, the third membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising 2-mercaptopyridine-3-carboxylic acid (MPCA) coupled to the polyGMA; and (f) eluting the target molecule from the third membrane and collecting the target molecule in a second eluate fraction. Clause 40. The method of clause 39, wherein the target molecule is a biomolecule comprising a protein, a peptide, an antibody, a nucleic acid, an enzyme, a lipoprotein, a lipid, an extracellular vesicle, an exosome, a viral vector, a plasmid, a carbohydrate, a vaccine, a virus, a bacterium, a cell, an organelle, or combinations thereof. Clause 41. The method of clause 39 or 40, wherein the target molecule is an antibody. Clause 42. A method of making a functionalized nonwoven membrane for purifying a target molecule, the method comprising: (a) grafting polyglycidyl methacrylate (polyGMA) onto a polybutylene terephthalate (PBT) nonwoven web to generate a PBT-polyGMA nonwoven web; and (b) coupling a functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) to the grafted polyGMA of the PBT-polyGMA nonwoven web to generate the functionalized nonwoven membrane. Clause 43. The method of clause 42, wherein grafting the polyGMA onto the PBT nonwoven web is performed using ultraviolet radiation. Clause 44. The method of clause 42 or 43, wherein a concentration of the BMEA functional ligand that is coupled to the PBT-polyGMA nonwoven web is from about 2% (v / v) to about 15% (v / v). Clause 45. The method of any one of clauses 42–44, wherein the BMEA functional ligand is coupled to the PBT-polyGMA nonwoven web at about 40 °C for about 6 hours, followed by treatment with 0.1 M sulfuric acid at about 50 °C for about 16 hours. Clause 46. The method of any one of clauses 42–45, further comprising washing the functionalized nonwoven membrane with pure water and storing the functionalized nonwoven membrane in a solution of 20% ethanol / water (v / v). Clause 47. A chromatography device for purifying a target molecule, the chromatography device comprising: (a) a housing unit comprising one or more functionalized nonwoven membranes, each membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2- mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof; and (b) a flow device connected to the housing unit and configured to induce fluid flow of a sample comprising the target molecule through the one or more functionalized nonwoven membranes. Clause 48. The chromatography device of clause 47, wherein the housing unit is a cartridge, a cassette, or a column. Clause 49. A method for purifying a target molecule, the method comprising: (a) contacting a sample comprising the target molecule with a chromatography device comprising: a housing unit comprising one or more functionalized nonwoven membranes, each membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2- mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof; and a flow device connected to the housing unit and configured to induce fluid flow of the sample comprising the target molecule through the one or more functionalized nonwoven membranes; (b) inducing fluid flow of the sample comprising the target molecule through the one or more functionalized nonwoven membranes using the flow device to bind the target molecule to the one or more functionalized nonwoven membranes; and (c) eluting the target molecule from the one or more functionalized nonwoven membranes and collecting the target molecule in a first eluate fraction. Clause 50. A kit for purifying a target molecule, the kit comprising: (a) one or more functionalized nonwoven membranes, each membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2- mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof; (b) optionally, columns, buffers, cartridges, cassettes, and receptacles; and (c) optionally, one or more of packaging or instructions for use. Clause 51. A method for removing ions or ionic contaminants from a liquid, the method comprising: (a) contacting a liquid comprising ions or ionic contaminants with a functionalized nonwoven membrane to bind the ions or ionic contaminants to the membrane, the membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2- mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof; and (b) collecting the liquid having the ions or ionic contaminants removed. Clause 52. The method of clause 51, wherein the liquid is water. Clause 53. A method for removing ions or ionic contaminants from a liquid, the method comprising: (a) contacting a liquid comprising ions or ionic contaminants to a first functionalized nonwoven membrane to bind anions or anionic contaminants to the first functionalized nonwoven membrane, the first functionalized nonwoven membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) or triethylamine (TEA) coupled to the polyGMA; (b) collecting a first liquid flow-through having the anions or anionic contaminants removed; (c) contacting the first liquid flow-through to a second functionalized nonwoven membrane to bind cations or cationic contaminants to the second functionalized nonwoven membrane, the second functionalized nonwoven membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising 2-mercaptopyridine-3-carboxylic acid (MPCA) coupled to the polyGMA; and (d) collecting a second liquid flow-through having the cations or cationic contaminants removed. Clause 54. The method of clause 53, wherein the liquid is water. Clause 55. A method for purifying water, the method comprising: (a) contacting a sample of water with a first functionalized nonwoven membrane to bind anions or anionic contaminants to the first functionalized nonwoven membrane, the first functionalized nonwoven membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) or triethylamine (TEA) coupled to the polyGMA; (b) collecting a first water flow-through having the anions or anionic contaminants removed; (c) contacting the first water flow-through with a second functionalized nonwoven membrane to bind cations or cationic contaminants to the second functionalized nonwoven membrane, the second functionalized nonwoven membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising 2-mercaptopyridine-3-carboxylic acid (MPCA) coupled to the polyGMA; and (d) collecting a second water flow-through having the cations or cationic contaminants removed.

[0002] EXAMPLES Example 1 Materials and Methods Polybutylene terephthalate (PBT) nonwoven membranes (basis weight: 52 g / m2, mean fiber diameter: 3.0 μm, porosity: 85%, and mean pore size: 8.0 ± 0.5 μm) were provided by Macopharma (Tourcoing, France). Glycidyl methacrylate (GMA) was purchased from Reagent World (Ontario, CA, USA). Benzophenone (BP), BMEA and BSA were purchased from Sigma- Aldrich (St. Louis, MO, USA). The organic solvents and chemicals used for making membranes and buffers were purchased from Fisher Scientific (Fairlawn, NJ, USA). Human polyclonal IgG (IgG) was purchased from Athens Research & Technology, Inc. (Athens, GA, USA). Omnifit columns with 10 mm diameter were used as membrane holders and were purchased from Diba Industries, Inc., Cambridge, UK. The FPLC system ÄKTA™pure, 5-mm diameter empty columns, Capto Adhere, Capto Q and Capto S resins were purchased from Cytiva (Marlborough, MA, USA). Eshmuno® A resin and Amicon Ultra-15 Centrifugal Filter 10 kDa MWCO were purchased from MilliporeSigma (Burlington, MA, USA). MMC-MPCA membrane and AEX-TEA membrane were prepared according to Fan et al., Sep. Purif. Technol. 317: 123920 (2023) and Fan et al., Membranes 12(10): 944 (2022). The Yarra SEC-2000 size exclusion chromatography column (300^mm × 7.8^mm) was purchased from Phenomenex Inc (Torrance, CA, USA). The Waters Alliance 2690 separation module system and Waters 2487 dual absorbance detector for HPLC analysis were purchased from Waters Corporation, Milford, MA, USA. The CHO HCP ELISA Kit (F550) was purchased from Cygnus (Southport, NC, USA). Quant-iT™ PicoGreen™ dsDNA Assay Kit was bought from Fisher Scientific (Fair-lawn, NJ, USA). The CHO supernatant was generously provided by Fujifilm Diosynth Biotechnologies (Durham, NC, USA). Membrane Preparation Glycidyl Methacrylate (GMA) Grafting on Polybutylene Terephthalate (PBT) Glycidyl methacrylate (GMA) was used to prepare a 2.0 M (26.5%, v / v) grafting solution using 1-butanol as solvent. Benzophenone (BP) was added to the grafting solution as a photoinitiator with a molar ratio of BP:GMA of 2:100 (7.3 mg / mL). A pre-weighed polybutylene terephthalate (PBT) nonwoven sample (75 × 50 mm) was sprayed evenly with GMA grafting solution until completely soaked, sandwiched between two glass slides, and then exposed to a UV lamp (model EN-180, Spectronics Corporation, Westbury, NY) with wavelength centered at 365 nm and intensity of 5 mW / cm2. The distance between the nonwoven samples and the lamp was 3 mm. After 20 min, the residual, unreacted solution, and GMA homopolymer were removed by soaking and washing the membrane in tetrahydrofuran (THF) for 30 min under sonication (Bransonic 3510, Danbury, CT) at room temperature. The sample was then sonicated in fresh methanol for 10 min twice, followed by drying in a vacuum oven. The nonwoven membrane was weighed to determine the degree of GMA grafting (percentage weight gain, %WG). Ligand Coupling N-benzyl-N-methylethanolamine (BMEA) PolyGMA grafted PBT samples (20% WG) were then soaked in N-benzyl-N- methylethanolamine (BMEA) solutions (2–15% v / v in 17% isopropyl alcohol / water (v / v)) for ligand coupling at 40 °C in a water bath for 6 h, followed by 0.1 M sulfuric acid treatment at 50 °C for 16 h to hydrolyze unreacted epoxy groups in polyGMA. The prepared multimodal anion exchange membrane (MMA-BMEA membrane) was washed three times with pure water and stored in 20% ethanol / water (v / v). Sulfite (SO3) To create cation exchange membranes, PBT-GMA samples (20% WG) were immersed into 50 g of a sodium sulfite solution containing isopropyl alcohol (IPA), and water (Na2SO3 / IPA / Water, 10 / 15 / 75%, w / w) at 80 °C without shaking, followed by 0.1 M sulfuric acid treatment at 50 °C for 16 h. Diethylamine (DEA) Polymerized GMA-grafted membranes (20% WG) were loaded into a 50% (v / v) DEA aqueous solution at 30 °C under agitation at 100 RPM. The ring-opening reaction between epoxy and DEA was used to covalently attach amino groups on the surface of the fibers. Following amination, the membranes were washed with 100 mL deionized (DI) water to remove unreacted DEA, followed by 0.1 M sulfuric acid treatment at 50 °C for 16 h. Iminodiacetic Acid (IDA) PolyGMA grafted PBT samples (20% weight gain) were then soaked in an IDA solution (66.2 mg / mL in membrane) 20% IPA / water (v / v) at pH 10 for ligand coupling (80 °C for 7 h), followed by 0.1 M sulfuric acid treatment at 50 °C for 16 h. Triethylamine (TEA) The AEX-TEA membranes were prepared by incubating the polyGMA-grafted PBT nonwovens (20% weight gain) in 50 mL of 10% v / v TEA in water (40 °C for 8h), followed by 0.1 M sulfuric acid treatment at 50 °C for 16 h. 2-mercaptopyridine-3-carboxylic acid (MPCA) PolyGMA grafted PBT samples (17–18% WG, average 17.5% WG) were soaked in MPCA solutions for ligand coupling at 60 °C in a water bath for 16 h. The solutions contained 2 mg MPCA / mL, corresponding to molar ratio of epoxy / MPCA of 1:2.2, followed by 0.1 M sulfuric acid treatment at 50 °C for 16 h. Membrane Characterization The ligand density was determined by analyzing the amine content of the prepared membranes with a PE 2400 CHN elemental analyzer (PerkinElmer Inc., Waltham, MA, USA). A scanning electron microscope (SEM, Hitachi S-3200 N, Hitachi High-Tech, Schaumburg, Illinois, USA) was used to observe the morphology of the membrane surface (accelerating voltage: 5.0 kV). For flow permeability measurements, 48 layers of MMA-BMEA membrane were packed into a 10 mm-diameter Omnifit column with a membrane bed height of 1.2 cm (0.94 mL column volume). The column pressure was measured under various flow rates by an ÄKTA™pure system using 50 mM Tris-HCl, pH 7.0 with and without 0.15 M NaCl as the mobile phase. The superficial velocities from 152.8–764.0 cm / h were used, which was corresponding to 0.09–0.47 min RT. The pressure drop ΔP across the membrane layers (kPa) was calculated by subtracting the pressure drop of the empty column from that of column with the membranes to get a pressure drop per unit bed length (ΔP / L) where L is the height of membrane packed bed (cm). The flow permeability of the membrane was calculated using Darcy’s law: ^^ ൌ ^^∆^ ఓ^ (1), where v is the superficial velocity viscosity of the aqueous solution. Membrane Performance for Protein Binding and Elution Static Binding Capacity (SBC) and Isotherm The SBCs of anion and cation exchange membranes were measured by immersing 15 mg membrane samples (~0.05 mL) in 10 mg / mL protein in 50 mM Tris-HCl, pH 7.0, with 150 mM NaCl for 16 h at room temperature. For anion exchange membranes, the membrane was immersed in BSA protein to measure the SBC of the membrane. For cation exchange membranes, the membrane was immersed in IgG protein to measure the SBC of the membrane. The binding capacity was calculated as the mass of eluted protein (when the bound protein was completely eluted) divided by the membrane volume. 0.1 M citric acid, pH 2.2, 50 mM glycine- HCl, pH 3.0–3.5, and 50 mM acetate, pH 4.0–5.0, were used to elute protein that was adsorbed on the membrane (prepared using 15% v / v BMEA) and the elution efficiency was calculated as the mass ratio of the eluted protein to the bound protein. The same procedure was used for measuring the BSA or IgG protein binding on the membrane in 50 mM Tris-HCl, pH 7.0, with different NaCl concentrations from 0 to 450 mM, except the initial BSA protein concentration of 3.0 mg / mL for protein binding. A range of BSA protein initial concentrations from 0.5 to 10.5 mg / mL (or 1.0 to 13.0 mg IgG protein / mL dissolved in 50 mM Tris-HCl, pH 7.9, with 120 mM NaCl) for SBC measurement was employed for studying the equilibrium adsorption isotherm. The Langmuir isotherm was used to fit the experimental data: ^^^^^^^^^ ൌ^^^^^(2), where qeqis the measured on membrane at equilibrium while ceq is the protein concentration in the solution at equilibrium. The maximum binding capacity (qm) and the dissociation constant (Kd) were determined by fitting the experimental data using Eq. (2). Dynamic Binding Capacity The dynamic binding capacities of the anion and cation exchange membranes at 10% breakthrough (DBC10%) were measured in flow experiments with 12 membrane layers (~0.24 mL) packed in the 10 mm-diameter Omnifit column holder using the ÄKTA™pure system. The membranes were equilibrated with 7.2 mL of binding buffer (50 mM Tris-HCl, pH 7.0, with 0, 150 mM, and 300 mM NaCl) and then 3 mg BSA / mL (BSA powders were pre-dissolved in the corresponding binding buffer) sample solution was loaded at 0.5, 1.0, and 5.0 min RT. The sample loading was stopped once the UV absorbance reached the value corresponding to 10% of the initial feed concentration. The same procedure was used for 0.25 mL Capto adhere resins packed in a 5 mm-diameter column and 50 mM Tris-HCl, pH 7.0, with 150 mM NaCl was used as binding buffer. For measuring DBC10%of MMA-BMEA membrane for IgG, and IgG solution of 3.2 mg / mL in 50 mM Tris-HCl, pH 7.9, with 120 mM NaCl (~14.5 mS / cm) was loaded at 0.5 min, 1.0 min, 2.0 min, and 5.0 min RT. The DBC10%was calculated as follows:^^^^^^^బ^^భబ%ି^బ^^^% ൌ^ (3), where c0is the initial protein ; V10%is the effluent volume when the protein concentration in the effluent reached 10% of the initial protein concentration in the feed (mL); V0is the void volume (mL) determined using a pulse of 2% acetone and V is the membrane or resin volume. Membrane Reusability Evaluation Five repeated cycles of IgG bind-and elute were carried out for reusability evaluation. 0.24 mL anion or cation exchange membrane packed in the 10 mm diameter column was equilibrated with 7.2 mL 50 mM Tris-HCl, pH 7.9, with 120 mM NaCl (used as binding buffer), then 3.75 mL of IgG solution (3.2 mg / mL in the binding buffer) was loaded to the membrane bed at 2 min RT. The membranes were then washed with 7.2 mL binding buffer till the UV absorbance returned to baseline. The bound mAb was eluted by 7.2 mL 50 mM acetate, pH 4.0, followed by membrane cleaning with 4.8 mL 0.5 M NaOH and re-equilibrated with 12 mL binding buffer. The equilibration, washing, elution, cleaning, and re-equilibration steps were all conducted at 0.5 min RT. mAb Capture from Cell Culture Supernatant by MMA-BMEA Membrane in Bind-And-Elute Mode A membrane bed of 0.24 mL packed in a 10 mm-diameter column was equilibrated with 7.2 mL 50 mM Tris-HCl, pH 7.9, with 120 mM NaCl at 0.5 min RT, then 7.5 mL of clarified CHO supernatant was loaded to the membranes at 2 min RT. The effluent was collected at every 0.5 mL during sample loading and the mAb concentration was analyzed using a Protein G column. The loading volume wen the mAb concentration in the effluent reached 10% of the initial mAb concentration in the supernatant was used to calculate DBC10%with the same equation as above. A similar procedure was used to study the effect of 0.5-, 2-, and 5-min RT for loading 4 mL of clarified CHO supernatant, which was followed by membrane washing with 7.2 mL 50 mM Tris-HCl pH 7.9 with added 120 mM NaCl) and elution with 7.2 mL 50 mM acetate pH 4. To improve impurity removal, 7.2 mL of 50 mM, 87.5 mM, and 125 mM Arginine Tris-HCl pH 7.4 as the second wash step was executed following 50 mM Tris-HCl, pH 7.9, with 120 mM NaCl (after loading 4 mL of clarified CHO supernatant at 2 min RT). A step pH elution gradient of pH 4.5 and 4.0 (7.2 mL 50 mM acetate, pH 4.5 and 4) was executed for further removal of impurities (loading 4 mL of clarified CHO supernatant at 2 min RT and 7.2 mL of 100 mM arginine Tris-HCl, pH 7.4, as the second washing step. Other steps were kept the same). The pH 4.5 eluate was collected for further purification. The equilibration, washing and elution steps in above runs were all conducted at 0.5 min RT. mAb Polishing by AEX-TEA Membrane (Flow-Through Mode) and MMC-MPCA Membrane (Bind- And-Elute Mode) The pH 4.5 eluate from MMA-BMEA membrane process was adjusted to pH 3.6 by adding 0.5 M HCl and incubated for 1.0 h at room temperature for virus inactivation. The obtained solution was then adjusted to pH 7.2 by 1.0 M Tris-HCl, pH 8.0, and filtered using a 0.22 µm filter. The details of membrane equilibration, sample loading, washing and elution for mAb polishing by AEX-TEA membrane (flow-through mode) and MMC-MPCA membrane (bind-and-elute mode) are provided in Table 1. The membranes were packed in a 10 mm diameter column. The number of membrane volumes (MVs) was used to record buffer volume. Solutions of 0.5 M HCl and 0.5 M NaOH were used to adjust pH between each step. Table 1. Details of Chromatographic Steps with Nonwoven Membranes A: MMA-BMEA Membrane (Bind / Elute) Membrane Volume: 0.24 mL (ID^=^10^mm, Bed Height^=^3 mm) Step Buffer / Samples MVs Residence Time (min) Equilibration 50 mM Tris-HCl + 120 mM NaCl, 20 0.5 pH 7.9 Load CHO supernatant 16.7 (4.0 mL) 2.0 Wash 1 50 mM Tris-HCl + 120 mM NaCl, pH 7.9 30 0.5 Wash 2 50 mM Tris-HCl + 100 mM 30 0.5 Arginine, pH 7.4 Elution 50^mM acetate, pH 4.5 30 0.5 B: AEX-TEA Membrane (Flow-through) Membrane volume: 0.24 mL (ID^=^10^mm, Bed Height^=^3 mm) Equilibration 50 mM Tris-HCl, pH 7.2 20 0.25 The eluate from MMA-BMEA after Load VI 10.4 (2.5 mL) 2.0 Wash 50 mM Tris-HCl, pH 7.2 30 0.25 Membrane Cleaning 50 mM Tris-HCl + 1.0 M NaCl, pH 30 0.25 7.2 C: MMC-MPCA Membrane (Bind / Elute) Membrane volume: 0.24 mL (ID^=^10^mm, Bed Height^=^3 mm) 50^mM acetate + 150 mM NaCl, pH Equilibration 5.5 20 0.25 Load The flow-through fraction from 14.6 (3.5 mL) 2.0 AEX-TEA Wash 1 50^mM acetate + 150 mM NaCl, pH 5.5 20 0.25 Elution 50 mM sodium phosphate + 150 0.25 mM NaCl, pH 7.7 mAb Purification by a Standard Three-Step Resin The resins were packed in a 5 mm diameter column. 0.28 mL Eshmuno® Protein A was used to capture mAb from supernatant, followed by virus inactivation with the same procedure described above. The first polishing process by 0.25 mL Capto S resin was operated in bind-and- elute mode. A 10 kDa centrifugal tubes was used to buffer exchange to decrease the conductivity of the eluate before next polishing process: 3.5 mL eluate was centrifuged at 3500 rpm for 30 min to decrease the volume to 0.25 mL, then 25 mM sodium phosphate buffer, pH 6.5, was added to a final volume of 3.5 mL. The second polishing process by 0.25 mL Capto Q resin was conducted in flow-through mode. The three-step resin chromatography was executed at 5 min RT (4–6 min RT are commonly recommended). The details of equilibration, sample loading, washing and elution steps are provided in Table 2. The number of column volumes (CVs) was used to record buffer volume. 0.5 M HCl and 0.5 M NaOH were used to adjust pH between each step. Table 2. Details of Chromatographic Steps with Resins A: Protein A Resin (Eshmuno® Protein A, Bind / Elute) Resin Volume: 0.28 mL (ID^=^5^mm, Bed Height^=^1.43 mm) Step Buffer / Samples CVs Residence Time (min) Equilibration PBS, pH 7.4 5 5.0 Load CHO supernatant 14.3 (4.0 mL) 5.0 Wash 1 PBS, pH 7.4 10 5.0 Wash 2 25 mM sodium phosphate + 1.0 M NaCl, pH 10 5.0 7.2 Wash 3 25 mM sodium phosphate, pH 7.2 10 5.0 Elution 0.1 M glycine-HCl, pH 3.6 10 5.0 B: Cation Exchange Resin (Capto S, Bind / Elute) Resin volume: 0.25 mL (ID^=^5^mm, Bed Height^=^1.27 mm) Equilibration 50^mM acetate + 50 mM NaCl, pH 5.5 5 5.0 Load The Protein A eluate after VI 20 (2.5 mL) 5.0 Wash 50^mM acetate + 50 mM NaCl, pH 5.5 10 5.0 Elution 50^mM acetate + 400 mM NaCl, pH 5.5 20 5.0 C: Anion Exchange Resin (Capto Q, Flow-through) Resin volume: 0.25 mL (ID^=^5^mm, Bed Height^=^1.27 mm) Equilibration 25 mM sodium phosphate, pH 6.5 5 5.0 Load The eluate from Capto S 20 (2.5 mL) 5.0 Wash 25 mM sodium phosphate, pH 6.5 10 5.0 Elution 25 mM sodium phosphate + 1.0 M NaCl, pH 6.5 10 5.0 Sample Assay BSA and IgG concentrations were determined by UV-vis spectrophotometer at 280 nm with a calibration curve based on known concentrations of BSA or IgG. mAb concentration was measured with a 1 mL Protein G column. The HCP and DNA content were determined using CHO HCP ELISA Kit (F550) and Quant-iT™ PicoGreen™ dsDNA Assay Kit, respectively. The log reduction values (LRV) of HCP and DNA clearance were calculated by log10 ratio of HCP (ppm) or DNA (ppm) in the feed solution to the elution fractions. The ppm (parts per million) is the mass ratio of HCP or DNA to mAb. The content of aggregates and fragments was measured using a Yarra SEC-2000 size exclusion chromatography column (300^mm × 7.8^mm). Example 2 MMA-BMEA Membrane Preparation and Static Binding Capacity for BSA To construct a high-capacity multimodal anion exchange membrane for protein capture, the polyGMA grafted PBT nonwoven prepared by UV radiation, was used to link BMEA as multimodal anion exchange ligand. FIG.2 depicts the coupling of BMEA to the epoxy in polyGMA through reaction with tertiary amine under mild conditions. The unreacted epoxy groups were converted to diols by 0.1 M sulfuric acid. The BMEA ligand was expected to bind proteins at near physiological conditions with charge, hydrophobic, and hydrogen bonding interactions with charged amine, hydrophobic phenyl, and hydroxyl moieties. To verify this, four MMA-BMEA membranes were prepared with ligand concentration from 2% to 15% (v / v) and the membrane with the best protein binding ability was used to examine the impact of NaCl concentration on protein binding. Protein binding on MMA-BMEA membrane was investigated using BSA as model protein because it has been used to study the performance of anion-exchange media. The static binding capacity (SBC) was measured in 50 mM Tris-HCl, pH 7.0, with 150 mM NaCl, where the negatively charged BSA (pI: 4.7) should be absorbed by positively charged amine in addition to other possible interactions with phenyl and hydroxyl moieties on the ligand. In FIG.3, the eluted BSA by 1.0 M NaCl was increased from 39.8 to 69.4 mg per mL of membrane volume with the increased ligand reaction concentration from 2% to 15% (v / v) in membrane preparation (resulting in increased ligand density from 0.26 to 0.49 μmol / mg); however, the mass of eluted BSA by 1.0 M NaCl was found to only account for ~60% of the bound BSA. This indicated that 1.0 M NaCl commonly used for charge screening in the elution of the traditional anion exchange chromatography was unable to completely release the bound BSA. This confirms that charge interaction is not the sole force for protein binding. Meanwhile nearly all bound BSA can be eluted by 0.1 M citric acid (pH 2.2) as the acid solution can interrupt interactions including charge (making the amine in the ligand and BSA both positively charged), hydrophobicity and hydrogen bonding between the ligand and protein. The membrane prepared at 15% (v / v) ligand solution achieved a high static bind capacity of 109.8 mg / mL (the mass of the eluted protein by 0.1 M citric acid was regarded as that of the bound protein), which was chosen for the following investigations. Ligand concentration higher than 15% v / v was not used for membrane preparation to avoid membrane weakness at strong alkaline conditions. Since strong acid would make protein unstable, BSA desorption under higher elution pH from 3.0–5.0 was investigated. FIG. 4 shows the elution pH from 3.0 to 4.0 also enabled a complete BSA desorption, while the elution efficiency slightly decreased to 99% at pH 4.5 and decreased further at pH 5.0 with only 92% of bound BSA being eluted. Accordingly, 50 mM acetate, pH 4.0, was used as elution buffer in the following study. FIG. 5 shows the equilibrium adsorption study using a panel of BSA solutions with concentrations ranging from 0.5 to 10.5 mg / mL. The resulting adsorption curve fits well the Langmuir model in Eq. (2), with a maximum adsorption capacity qmof ~310.0 mg / g of membrane (corresponding to 103 mg / mL (1.5 mmol / L) of dry membrane) and a Kdof 2.09 mg / mL (31.2 µM). The high equilibrium binding capacity benefited from the multilayer binding sites on the membrane which dramatically improved the protein binding capacity since a monolayer of proteins adsorbed to fiber surface was only estimated about 5–6 mg protein / g of membrane. The salt tolerance is one of the advantages of the multimodal ligand thus the effect of NaCl concentration on membrane static binding capacity was studied. FIG. 6 shows that as the NaCl concentration increased from 0 to 150 mM, the binding capacity decreased from 98.9 to 74.2 mg / mL; the modest decrease of the binding capacity (~25%) reflected the good salt tolerance of the prepared membrane, as the effect of charge screen by salt was compromised by the hydrophobic and hydrogen bonding interaction. With NaCl concentration increased to 300–450 mM, the electrostatic attraction as the main binding force between the BMEA ligand and protein was greatly disrupted, leading to a decline of binding capacity. Nevertheless, the stable protein binding performance of MMA-BMEA membrane in the buffers containing 0–150 mM NaCl can be conducive to protein separation near physiological conditions. Example 3 Flow Permeability and Dynamic Binding Capacity of MMA-BMEA Membrane for BSA Previous research reported a strong anion exchange nonwoven membrane with triethylamine as ligand (AEX-TEA membrane) that had a high flow permeability of 3.5 × 10−9cm2(calculated by Equation 2) owing to the large inter-fiber distance of 3–40 μm, significantly higher than 1.1 × 10−10to 2.2 × 10−10cm2of commercial strong anion exchange resins with particle diameters of 50–90 μm. FIG. 7 shows the prepared MMA-BMEA membrane has a lower flow permeability of 1.0 × 10−9cm2compared to AEX-TEA membrane (4.6 × 10−9cm2) measured in 50 mM Tris, pH 7.0, buffer with no added NaCl. The structure of MMA-BMEA membrane (FIG.8A) and AEX-TEA membrane (FIG. 8B) were compared under SEM. The images in FIG. 8A–B present very similar fiber size and inter-fiber distance for these two membranes. The ligand density (0.45 μmol / mg) of BMEA was slightly lower than that of TEA (0.60 μmol / mg), however the lesser charge did not favor a better flow permeability for MMA-BMEA membrane. It is very likely caused by hydrophobic phenyl which would cause more flow resistance. While the obtained flow permeability is still much higher than those of conventional AEX resins mentioned above. Since MMA membranes are capable of operating in near physiological conditions, the flow permeability was also measured using 50 mM Tris-HCl with 150 mM NaCl. The flow permeability improved to 4.6 × 10−9cm2, which was greater than the AEX-TEA membrane with no NaCl in the buffer. The reason behind this could be the contracted grafted layer by charge screening with 150 mM NaCl, which resulted in a lager distance between fibers. The high flow permeability of MMA-BMEA membrane resulted in low pressure drops (from 44.2 to 201.3 kPa / cm at 152.8 to 763.9 cm / h) and observed pressures (ΔP from 53.0 to 241.5 kPa) are lower than 0.25 MPa (2.5 bars), well below those of flow devices in downstream processing (4–8 bars). The effect of NaCl concentration on DBC10%of MMA-BMEA membrane for BSA was investigated at 1.0 min and 5.0 min RT, which provided useful insights for real applications with dynamic flow. A typical chromatogram for DBC10%measurement at 1.0 min RT is shown in FIG. 9. FIG.10 displays the results obtained with 0 to 300 mM NaCl in 50 mM Tris-HCl pH 7.0 at both 1.0 min and 5.0 min RT. To compare a commercial MMA resin, i.e., Capto adhere (Cytiva; BMEA as ligand), the resin was packed in a 0.5 mm-diameter column with the same volume (0.24 mL) and measured with 150 mM Tris-HCl, pH 7.0. The DBC10%comparison for MMA-BMEA membrane and Capto adhere resin at 0.5–5 min RTs are shown in Table 3. With NaCl concentration increased from 0 to 300 mM, the DBC10%of MMA-BMEA membrane decreased from 49.1 to 17.9 mg BSA / mL at 1.0 min RT, and decreased from 61.0 to 20.9 mg BSA / mL at 5.0 min RT. Only 25–35% of binding capacity was lost at 150 mM compared to those measured without added NaCl under both RTs, indicating a good consistency of salt tolerance with that discussed in the static binding study above. The DBC10%obtained at 1.0 min RT were somewhat lower than those obtained at 5.0 min, implying a mild diffusion limitation occurred under a higher flow rate. Under the same conditions, the DBC10%of a commercial MMA Capto adhere resin (BMEA as ligand, 0.24 mL) was measured to compare the MMA-BMEA membrane (a typical chromatogram for Capto adhere at 1.0 min RT is shown in FIG.9). In Table 3, the DBC10%of MMA-BMEA membrane (30.8–38.5 mg / mL) were generally 85% higher than those of Capto adhere resin (16.6–20.5 mg / mL) from 0.5 to 5.0 min RT at 0.15 M NaCl, which could be due in part to the higher ligand density for the MMA-BMEA membrane (150 μmol / mL vs. 90–120 mol / mL). This demonstrates that the MMA-BMEA membrane can be an effective alternative to Capto adhere resin to improve productivity of protein purification operated at physiological conditions. Table 3. DBC10%(BSA) at pH 7.0 and 150 mM NaCl MMA-BMEA Membrane Capto adhere DBC10%at 0.5 min RT 30.8 mg / mL BSA 16.6 mg / mL BSA DBC10%at 1.0 min RT 37.2 mg / mL BSA 20.1 mg / mL BSA DBC10%at 5.0 min RT 38.5 mg / ml BSA 20.5 mg / mL BSA Example 4 Direct Capture of mAb from CHO Supernatant by MMA-BMEA Membrane MMA-BMEA Membrane Performance for Binding and Eluting Polyclonal Human IgG With the knowledge of basic membrane performance in terms of salt tolerance and protein binding and elution, the MMA-BMEA membrane was then applied in antibody purification. MMA resins have previously been reported to be used in mAb capture at physiological-like conditions; however, the reported mAb binding capacities were generally <20 mg / mL with RT of 2.5–6.6 min (titer in the supernatant: 0.4–1.69 mg / mL). In addition, in previous work, a multimodal cation exchange membrane (MMA-MPCA membrane) was applied to direct capture a mAb from CHO supernatant without pre-buffer exchange or dilution to decrease conductivity. This approach still required pH adjustment to 5.5 to bind mAb and the separation result of HCP clearance of 0.67 LRV needed improvement. It was anticipated that a high-capacity mAb capture step by the MMA- BMEA membrane with improved impurity removal could be achieved for mAb purification only using chromatographic membranes. This process can not only reduce the HCP, DNA, and aggregates to the acceptable levels, but can also greatly increase productivity compared to resin processes. Therefore, pure polyclonal human IgG was first used to examine the binding performance of MMA-BMEA membrane for antibodies. FIG. 11 shows the equilibrium adsorption of IgG on the membrane and the fitted curve using Langmuir model. The multilayer binding IgG returned a qmof ~400 mg / g (133.3 mg / mL (0.89 mmol / L) of dry membrane) and a Kdof 1.37 mg / mL (9.1 µM). The IgG was absorbed by MMA-BMEA membrane at pH 7.9, which was near or below the reported pI of IgG (7.1-8.2). This is not in line with the rule in conventional anion exchange chromatography that the binding pH should be higher than the pI of target protein. It was interpreted that the hydrophobic and hydrogen bonding interaction should play an important role for IgG binding in this condition. Compared to the BSA adsorption (qm: 1.5 mmol / L; Kd: 31.2 µM) at pH 7 and 17.5 mS / cm, IgG adsorption at pH 7.9 and 14.5 mS / cm had a lower molar number of maximum binding and exhibited a stronger binding on the membrane. It may have been due to the larger IgG size and the discrepancy of the role of charge and hydrophobic interactions engaging protein adsorption to the ligand. For the dynamic binding ability, the DBC10%was kept stable around 40 mg IgG / mL from 1.0 min to 5.0 min RT as shown in Table 4 (a typical chromatogram at 1.0 min RT is shown in FIG.12) and decreased to 32.8 mg / mL at 0.1 min RT due to diffusion limitations. This trend was somewhat similar to the DBC10%for BSA in Table 3. As good reusability is important to keep consistent quality of purified bioproduct between chromatographic cycles, five consecutive IgG bind-and-elute cycles were performed to evaluate the membrane’s reusability. Between each cycle, 0.5 M NaOH was used to clean and regenerate the membrane. The chromatogram in FIG.13 shows five similar UV signal patterns for each IgG bind-and-elute cycle. When comparing the mass of eluted protein and the bound protein during sample loading, 97.4 ± 2.8% of the bound IgG were found to be eluted in each cycle, which indicated a good reusability under this condition. Table 4. DBC10%(IgG) at pH 7.9 and 120 mM NaCl RT (min) MMA-BMEA membrane 0.5 32.8 mg / mL IgG 1.0 39.3 mg / mL IgG 2.0 42.3 mg / mL IgG 5.0 42.4 mg / mL IgG Determination of DBC10%of MMA-BMEA Membrane for mAb Capture from CHO Supernatant The proper conditions for polyclonal IgG were employed for mAb capture from a clarified CHO culture fluid. To avoid overloading, the DBC10%of MMA-BMEA membrane for mAb capture was measured. After membrane equilibration, the CHO supernatant (pH 7.6, 12 mS / cm) was loaded to the membrane at 2.0 min RT and the mAb concentration in every 0.5 mL effluent was measured to determine the DBC10%. The dotted curve in FIG. 14 presents the mAb breakthrough %, which is composed of the mAb concentration ratios in the effluent to that in the feed. It shows a DBC10%of 59.2 mg / mL for mAb capture at a loading volume of 4.5 mL, verifying that mAb can be directly captured from the CHO supernatant by MMA-BMEA membrane without prior pH and conductivity adjustment. The operating pH (7.9) here is higher than the pI (7.6) of mAb, meaning the electrostatic attraction also engaged in protein binding, which explained the higher binding capacity for mAb compared to DBC10%of ~40 mg / mL for IgG (Table 4). Accordingly, the less loading volume of 4 mL for further reducing mAb breakthrough was used in the following purifications. Effect of RT and Arginine Concentration in Wash Step To examine the effect of RT on the mAb recovery and HCP removal, 0.5 min, 2.0 min, and 5.0 min residence times were employed for the capture process by MMA-BMEA membrane. The resulting chromatograms were displayed in FIG.15A–C and the corresponding purification results were listed in Table 5. The elution peak area slightly increased from 0.5 to 2.0 min RT, which resulted in higher recoveries of 98.4% at 2.0 min RT and 99.6% at 5.0 min relative to a lower recovery of 84.9% at 0.5 min RT. Since nearly all bound mAb was eluted, it translates into a ~15% lower mAb binding at 0.5 min RT. This trend agreed with pure IgG binding at 0.5–5 min RT in Table 4. The lower mAb binding at 0.5 min RT could be attributed to diffusion limitations or possible interference of the impurities in the supernatant. The HCP removal of ~0.5 LRV was quite stable at each RT, reflecting a little impact of flow rate on HCP clearance. With a good recovery and HCP removal, sample loading at 2.0 min RT was applied in the following studies. Arginine has been used as an additive in the wash step of protein A chromatography for improving HCP removal, as it can weaken the charge interaction between mAb and HCPs. Therefore, a membrane wash step with arginine concentrations from 50 to 125 mM was introduced after sample loading. The membrane separation performance was examined in terms of mAb recovery and removal of HCPs and aggregates. The chromatograms in FIG. 16A–C demonstrated an increased UV absorbance during membrane washing with arginine, which confirmed the release of proteins by arginine. The UV signal became stronger with increasing arginine concentrations as more proteins were washed out. This caused a decrease of mAb recovery from 99.5% to 95.2%. With the same reason, the HCP removal increased from 0.48 to 0.64 LRV and the aggregates in the eluate slightly decreased from 3.7% to 3.4%. Membrane washing with 87.5 mM and 125 mM arginine improved removal of HCPs and aggregates compared to that without arginine washing, and it achieved a good recovery of 95.2–97.7%. Therefore, 100 mM arginine was used for membrane washing in the following studies. Table 5. Results of mAb Recovery and HCP Removal at 0.5, 2.0, and 5.0 min RT RT (min) Recovery* (%) HCP removal (LRV) 0.5 84.9 0.50 2.0 98.4 0.52 5.0 99.6 0.52 *Calculated as the mass ratio of the eluted mAb to the mAb applied in the loading solution. Elution pH Optimization Following mAb Polishing by Two Additional Nonwoven Membranes and Comparison with Standard Three-Step mAb Purification Process by Resins To further reduce HCPs in the eluate, a step pH elution at pH 4.5 and 4.0 was executed and the chromatogram is shown in FIG.17A. A mAb recovery of 94.3% was obtained in the larger elution peak at pH 4.5 with an HCP removal of 1.0 LRV. This higher HCP removal compared to those only with pH 4.0 elution (~0.6 LRV in Table 6) implies some HCPs that have stronger binding strength with BMEA ligand were unable to be eluted at pH 4.5 (isolated from pH 4.0 eluate). The aggregates content in the pH 4.5 eluate was greatly reduced to 0.9% (5.4% in the supernatant), much better than the 3.4–3.7% by pH 4.0 elution (Table 6). By analyzing the pH 4.0 eluate with size exclusion HPLC, it was confirmed by a very high aggregates content of 81.1%. This also suggested that aggregates with more charges and higher hydrophobicity bind stronger than mAb monomer. The applied elution condition for the MMA-BMEA membrane is generally milder than those used for most Protein A columns (elution at pH 3.0–3.8), which decreased the risk of forming aggregates. Table 6. mAb Recovery, HCP Removal, and Aggregate Removal with Arginine in the Wash Step (elution at pH 4.0) Arginine Conc. HCP removal (LRV) Aggregates (%) 0.48 3.7 0.58 3.4 125 95.2 0.64 3.4 The excellent impurity removal and mAb recovery in mAb capture by MMA-BMEA membrane makes it promising to develop a fast and inexpensive mAb purification process with all chromatographic nonwovens. The AEX-TEA anion exchange nonwoven membrane and MMC-MPCA multimodal cation membrane were introduced in the following mAb polishing as shown in FIG.1. The pH 4.5 eluate from MMA-BMEA membrane was adjusted to pH 3.6 and incubated for 1 h to execute virus inactivation, followed by purification with AEX-TEA membrane in flowthrough mode and subsequent MMC-MPCA membrane in bind / elute mode (2.0 min RT for sample loading and 0.25 min RT for other steps with no buffer exchange or dilution to adjust conductivity). After virus inactivation, the low pH solution was adjusted to pH 7.2 by 1.0 M Tris- HCl, pH 8.0, and filtered with 0.22 μm microfiltration membrane. It resulted in an HCP removal of 0.4 LRV and DNA removal of 0.3 LRV. While the aggregates content increased from 0.9% to 1.6%, which is likely due to the long-time exposure at pH 3.6 that triggered the aggregation. The AEX-TEA membrane was used as the first polishing process where mAb with positive charge at pH 7.2 would flow through and acidic impurities would be retained on the membrane. The resulting chromatogram in FIG.17B presents a large and broad peak in the flow-through and a much smaller elution peak by 1.0 M NaCl elution. As shown in Table 7, this membrane process achieved a good mAb recovery of 96.9% in the flow-through peak. The HCP, DNA and aggregates content were reduced from 12483.9 ppm in the feed to 1605.3 ppm (LRV: 0.9), from 9.5 ppm to 0.052 ppm (LRV: 2.3), and from 1.6% to 0.9%, respectively, demonstrating a high mAb polishing performance. FIG. 17C shows the chromatogram of the second polishing by MMA- MPCA membrane in bind / elute mode. The pH of the flow-through fraction from AEX-TEA membrane was decreased from pH 7.2 to 5.5 by 0.5 M HCl for sample loading to MMA-MPCA membrane, where the positively charged mAb should be adsorbed by the negatively charged ligands. The bound protein was then eluted by 50 mM sodium phosphate pH 7.7 with added 150 mM NaCl which decreased the net positive charges on the mAb. There was a small and shallow peak in the flow-through and a much larger elution peak which should be mainly contributed to the eluted mAb as a large fraction of impurities had been removed in the previous steps. A high mAb recovery of 98.0% was obtained in the eluate, and the purified mAb solution contains 93.5 ppm HCP (LRV: 1.23), 6.8 ppb DNA (LRV: 0.88), and 0.40% aggregates with no detected fragments (Table 7), within the accepted limits for mAb products (HCP <100 ppm, DNA <10 ppb, % aggregate <1%). The new membrane process achieved a high overall recovery of 88.3% and high overall impurity clearances: 3.5 LRV for HCP and 5.3 LRV for DNA, demonstrating a viable alternative for efficient mAb purification. Table 7. mAb Purification using Nonwoven Membranes HCPHCP DNA DNA % % % Process time Productiv. (ppm) (LRV) (ppm) (LRV) Agg Frag Recov (min) (g / L / h)* CHO 327367.6 – 994.7 – 5.4 0.8 – – Supernatant MMA-BMEA Membrane 34062.5 1.0 17.8 1.8 0.9 ND 93.5 86.1 34.8 (0.24 mL) VI 12483.9 0.4 9.5 0.3 1.6 0.6 99.4 60.0 – AEX-TEA Membrane 1605.3 0.9 0.052 2.3 0.9 0.1 96.9 40.0 72.0 (0.24 mL) MMC-MPCA Membrane 93.5 1.2 0.0068 0.9 0.4 ND 98.0 43.6 64.8 (0.24 mL) *Calculated as the mass of the eluted mAb per membrane volume divided by overall process time. All steps were conducted in duplicate or triplicate and the average data was reported with less than 5% errors. ND is Not Detected. To investigate the advantages of the new all membrane process for mAb purification, a comparison with a standard three-step resin chromatography process was performed (shown in FIG. 1). A RT of 5.0 min was applied for all resins as 4–6 min RT is recommended to obtain satisfactory performance in terms of protein binding capacity and separation resolution. The purification chromatograms are presented in FIG. 18A–C, and the corresponding results are displayed in Table 8. The mAb capture step by Eshmuno® protein A column obtained a high HCP removal of 2.3 LRV and DNA removal of 2.5 LRV. To avoid mAb aggregates in the elution, the elution pH used is 3.6 while the actual pH detected in the collected eluate is 4.0 (elevated by the pH 7.2 washing buffer remained in the column). The aggregates content was reduced to 0.9%, the same level obtained by MMA-BMEA membrane. While the recovery of 90.6% is lower than that of MMA-BMEA membrane (94.3%). After virus inactivation, a strong cation exchange chromatography (Capto S) was used for mAb polishing in bind / elute mode at pH 5.0, where mAb with positive charges can be absorbed by negatively charged sulfonate. The elution was performed at 0.4 M NaCl that decreased the charge interaction between mAb and ligand. This step further removed partial HCP, aggregates, and DNA, while the remaining HCP content of 150.9 ppm did not meet the common requirement of <100 ppm for mAb products. Therefore, the second polishing process by a strong anion exchange chromatography (Capto Q) was introduced, and a standard three-step mAb purification process was established. The flow-through mode at pH 6.5 was applied where only acidic impurities with negative charges can be adsorbed by the resin. Anion exchange was not used as the first polishing process under the consideration that the conductivity of Protein A eluate is ~8–9 mS / cm where acidic impurities may not be completely absorbed with flow-through mode around pH 7. Since the anion exchange resin is not salt tolerant, the eluate from the prior CEX column containing 400 mM NaCl requires buffer exchange to reduce conductivity before loading to the AEX column. A salt tolerant anion exchange resin (Capto adhere) was tried to avoid buffer exchange, but the resin can also bind nearly all mAb (data not shown) which makes it not applicable in flow-through mode. However, the buffer exchange step performed with an ultra-centrifugal filter increased the aggregates content to 10%. The dead-end filtration with high sheer speed could be the reason for producing aggregates, which might be largely mitigated if using a crossflow filter under a proper filtration speed, but it would take a longer process time. The strong anion exchange chromatography was unable to remove aggregates although it reduced the final HCP content to 20.0 ppm. This overall process that took 13.1 h to complete (Table 8), achieved an overall HCP removal of 4.0 LRV, overall DNA removal of >4.7 LRV with an overall recovery of 77.5%. In comparison, the whole membrane process (3.8 h) reduced 71% of processing time with 4x productivity in the capture step and 5–6x productivity in the polishing step (Tables 7 and 8) and obtained a 14% higher overall recovery with fewer steps, comparable HCP and DNA clearance, as well as fewer aggregates in the final product. Table 8. mAb Purification using Resins HCPHCP DNA DNA % Agg % Frag Process Prod. (ppm) (LRV) (ppm) (LRV) % Recov time (min) (g / L / h) CHO 21978 supernatant 5.1 – 647.8 – 5.4 0.8 – – – Protein A Resin (Eshmuno® Protein A, 0.28 959.2 2.3 2.2 2.5 0.9 ND 90.6 296.4 8.4 mL) VI 386.4 0.4 0.5 0.6 1.0 1.0 97.8 60 – Cation Exchange Resin (Capto S, 0.25 150.9 0.4 0.01 1.6 0.1 0.1 95.8 225.0 11.6 mL) Diafiltration (Centrifugal Filter 10 KDa 147.0 ~0 0.01 ~0 10.0 ND 96.8 30 – MWCO) Anion Exchange Resin (Capto Q, 20.4 0.9 ND >1.0 15.3 ND 94.3 175.0 13.6 0.25 mL) All steps were conducted in duplicate or triplicate and the average data was reported with less than 5% errors. ND is Not Detected. The downstream processing in mAb manufacturing is facing the challenges of increasing production efficiency and lowering production cost, while mAb capture from the harvest by Protein A is the most expensive process. In this study, a strategy of non-protein A three-step nonwoven membrane chromatography was used for monoclonal antibody purification. An MMA-BMEA nonwoven membrane was prepared based on a pGMA UV-grafted PBT nonwoven membrane. It exhibited an excellent salt tolerance in protein binding near physiological conditions, high flow permeability, good reusability, and a much better binding capacity than the counterpart MMA resin under the same conditions. These features ensured a high DBC10%of 59.2 mg mAb / mL for directly capturing a mAb from a CHO supernatant without pH and conductivity adjustment, and the performance is essentially independent of RT from 0.5 min to 5.0 min. Under the optimized conditions, a satisfactory recovery of 94.3%, a high HCP removal of 1.0 LRV and DNA removal of 1.8 LRV as well as a great reduction of aggregate % from 5.4% to 0.9% were achieved. Along with the following mAb polishing by AEX-TEA anion exchange nonwoven membrane and MMC- MPCA multimodal cation exchange nonwoven membrane, the three-step membrane chromatography reduced HCP from 327,000 ppm in the supernatant to 93.5 ppm (3.5 LRV), DNA from 994.7 ppm to 6.8 ppb (5.3 LRV), % aggregates from 5.4% to 0.4% with no detected fragments. These impurity residues are within the common requirement (HCP < 100 ppm, DNA < 10 ppb, % aggregate < 1%) for mAb products. Compared to the standard resin process which consists of protein A chromatography and two additional ion exchange chromatography, the new non-protein A three-step membrane process reduced 71% of processing time, increased 14% overall recovery, and required no diafiltration for buffer exchange. This work not only provides a high-productivity MMA-BMEA membrane for mAb capture, but also demonstrated a strategy for mAb purification relying on all single-use chromatographic nonwoven membranes which holds a great potential to greatly increase production efficiency and reduce the mAb manufacturing cost. Example 5 Dynamic Binding Capacity of pIgG via MPCA MML on Fibrillated Cellulose Nonwovens Materials and Methods TencelTMregenerated cellulose staple fibers (mean fiber diameter: 10 mm, mean fiber length: 38 mm) were provided by Lenzing Fibers, Inc. (Lenzing, Austria). Glycidyl methacrylate (GMA), benzophenone (BP), BMEA and BSA were purchased from Sigma-Aldrich (St. Louis, MO, USA). The organic solvents and chemicals used for making membranes and buffers were purchased from Fisher Scientific (Fairlawn, NJ, USA). Human polyclonal IgG (IgG) was purchased from Athens Research & Technology, Inc. (Athens, GA, USA). Omnifit columns with 10 mm diameter were used as membrane holders and were purchased from Diba Industries, Inc., Cambridge, UK. MMC-MPCA membranes were prepared according to Fan et al., Sep. Purif. Technol.317: 123920 (2023). Scanning electron microscopy (SEM) was performed on a Hitachi S-3200 N (Hitachi High-Tech, Schaumburg, IL, USA). The air permeability experiments were conducted using TEXTEST Air Permeability Tester (Schwerzenbach, Switzerland). Membrane Preparation Fibrillated Cellulose Nonwoven Preparation Cellulose staple fibers were prepared for nonwoven production from bales of raw materials by the fiber carding process. In this process bulk staple fibers are passed through a series of fine wire rollers to open or separate the fibers. As part of this process the fibers were aligned, and a rough web was formed. After fiber laydown, the web was bonded by entangling through the use of high-pressure water jets, i.e., hydroentangling. The water jets penetrate the loose structure perpendicular to the surface of the web, compressing the nonwoven and forming bonds between fibers as some were pushed through the structure and entangled. This process also exerts significant force on the fibers themselves, causing fibrillation of the regenerated cellulose fiber surface and generating nanoscale cellulose fibrils. By repeated exposure of the nonwoven to the hydroentangling process increased degradation of the fibers is achieved and more of nonwoven structure is converted to fibrils. Glycidyl Methacrylate (GMA) Grafting on Fibrillated Cellulose Glycidyl methacrylate (GMA) was used to prepare a 2.0 M (26.5%, v / v) grafting solution using 1-butanol as solvent. Benzophenone (BP) was added to the grafting solution as a photoinitiator with a molar ratio of BP:GMA of 2:100 (7.3 mg / mL). A pre-weighed cellulose nonwoven sample (75 × 50 mm) was sprayed evenly with GMA grafting solution until completely soaked, sandwiched between two glass slides, and then exposed to a UV lamp (model EN-180, Spectronics Corporation, Westbury, NY) with wavelength centered at 365 nm and intensity of 5 mW / cm2. The distance between the nonwoven samples and the lamp was 3 mm. After 20 min, the residual, unreacted solution, and GMA homopolymer were removed by soaking and washing the membrane in tetrahydrofuran (THF) for 30 min under sonication (Bransonic 3510, Danbury, CT) at room temperature. The sample was then sonicated in fresh methanol for 10 min twice, followed by drying in a vacuum oven. The nonwoven membrane was weighed to determine the degree of GMA grafting (percentage weight gain, %WG). Coupling of the 2-mercaptopyridine-3-carboxylic acid (MPCA) Ligand PolyGMA grafted fibrillated cellulose samples were soaked in MPCA solutions for ligand coupling at 60 °C in a water bath for 16 h. The solutions contained 2 mg MPCA / mL, corresponding to molar ratio of epoxy / MPCA of 1:2.2, followed by 0.1 M sulfuric acid treatment at 50 °C for 16 h. Analysis of the Fibrillated Cellulose Membrane Following production, multiple variations on the fibrillated cellulose membrane structure were characterized to understand their properties and performance as membrane-based chromatography devices. The influence of nonwoven areal density or basis weight (g / m2or “gsm”) and the number of hydroentangling passes were investigated. In this study, a “pass” was considered to be the complete processing of a roll of nonwoven membrane material through the hydroentangling device. As shown in FIG.19, fibrillation of cellulose fibers was clear at higher magnification. Lower magnification was insufficient to clearly resolve individual nanofibers, but voids between fibers became smaller and less well defined as the number of passes increased and basis weight decreased. This aligns with expectations as fibrillation should be more complete with increased hydroentangling passes, and the resultant force exerted upon the nonwoven is practically higher when the force from a pressurized water jet at static pressure is distributed over fewer fibers. In addition to the qualitative visual inspection, a more quantitative approach for fiber diameter evaluation was pursued in the form of air permeability measurements. The air permeability of a membrane can be related to the fiber diameter of the structure through Darcy’s Law as was derived by Davies, “The Separation of Airborne Dust and Particles,” Proceedings of the Institution of Mechanical Engineers, Part B: Management and Engineering Manufacture 1(1– 12): 185–213 (1953) in Equation 4: ∆^^ ൌ ^^^^^^ఓ௧^ (4) , ^ where ^^^^^^ ൌ 64^^^.ହ ^1 ^ 56^^ଷ^ (5). In these expressions, dfis the effective fiber diameter of the membrane when ΔP is the pressure drop in Pa; µ is the viscosity of the fluid in Pa^s; t is the thickness in meters; and q is the permeability of the membrane in m3 / m2 / s. The variable c is the packing density of the material, or solid volume fraction. Using this relationship and experimentally determined air permeability measurements, effective fiber diameters were calculated as shown in Table 9. As was seen in the SEM images of all samples in FIG.19, a single value for fiber diameter cannot accurately describe the wide range of actual fibers comprising a nonwoven web, and this is of particular importance in this embodiment where the use of hydroentanglement intrinsically produces a significant number of nanoscale fibers from a population of much larger fibers. This resulted in a strongly bimodal distribution of fiber sizes with an order of magnitude or more of separation between the sizes of the two groups. However, the practical representation of this reality was that the effective fiber diameter with respect to air flow through the membrane was decreased when there was a greater proportion of nanoscale fibers present in the structure. This resulted in a decreased effective fiber diameter with each hydroentangling pass experienced by the material. The increased susceptibility of lower basis weight fabrics to fibrillation also resulted in decreased effective fiber diameter, though this effect was less pronounced. Table 9. Air Permeability of Fibrillated Nonwoven Structures and Associated Fiber Diameter Estimations Sample ID Air perm. (m3 / m2 / min) Effective fiber diameter, df(µm) 125 gsm 1P 15.9 9.53 ± 0.37 125 gsm 2P 13.8 8.79 ± 0.36 100 gsm 1P 18.1 9.31 ± 0.40 100 gsm 2P 14.1 8.09 ± 0.30 75 gsm 1P 32.9 10.0 ± 0.41 75 gsm 3P 16.7 7.20 ± 0.26 The resulting effectiveness of the membrane-based chromatographic separation device based upon this fibrillated structure is shown in Table 10. In this example, the sample with greatest degree of fibrillation as observed in the effective fiber diameter testing was the 75 gsm with three hydroentangling passes. The single pass example of the same basis weight was tested as well for purposes of comparison. As previously described, the structures were grafted with pGMA to ~10% weight gain by a UV grafting method and functionalized with the multi-modal MPCA ligand. The membranes were evaluated under the 10% capacity breakthrough test method with a pure solution of polyclonal IgG at 5 mg / mL in 50 mM acetate buffer at pH 9.0 and including 150 mM NaCl. Under these conditions, the fibrillated structure produced with a three-pass treatment on 75 gsm regenerated cellulose nonwoven captured 69 mg pIgG / mL at a 1 min residence time, an increase of 28% over the non-fibrillated example. At a 0.2 min residence time, the test sample captured 63 mg / mL, a loss of only 10% capacity for 5× increased flowrate. Table 10. Dynamic Binding Capacity at 10% Breakthrough for 75 gsm Samples DBC 10% (mg pIgG / mL) RT (min) 1P 75 gsm 3P 75 gsm 1 54 69 0.75 50 69 0.5 48 66 0.2 47 62 0.1 46 51 Example 6 UV Grafting of One Charged Monomer on PBT Nonwoven The PBT nonwoven fabric was composed of fibers with a diameter of 2.8 μm. It had a surface area of 1.4 m2 / g with a thickness of 190 μm. Monomer 2-(Dimethylamino) ethyl methacrylate (DEM) (chemical structure shown in FIG. 20) with charged tertiary amine was grafted to the PBT nonwoven by UV radiation to construct weak anion exchange membrane for protein separation. The grafting solution was a mixture of DEM, benzophenone, and butanol. The UV grafting device was the same with the one for grafting of GMA in other examples. The grafting time was optimized to obtain a grafting weight gain of 20%. The SEM image in FIG.21 shows the polyDEM was coated on the fibers and some bridges caused by polymerization were observed between fibers. Membranes were cut into 2.5 cm discs. 12 layers of polyDEM grafted PBT were packed into 2.5 cm diameter column (DIBA Omnifit) fitted with a bottom end piece. The column was then connected to AKTA FPLC for determination of 100% dynamic binding capacity at residence times of 0.1, 2, and 5 min (FIG.22). The prepared anion exchange membrane showed 100% dynamic binding capacities of 50 mg BSA / mL at 0.1 min RT, 80 mg BSA / mL at 2.0 min RT, and 90 mg BSA / mL at 5.0 min RT (FIG.23). The same PBT nonwoven was used to prepare cation exchange nonwoven by UV grafting of Bis(2-(methacryloyloxy)ethyl) phosphate (BMEP) (chemical structure shown in FIG.24). The grafting solution is a mixture of BMEP, benzophenone, and butanol. The grafting time was optimized to obtain a grafting weight gain of 20%. SEM image in FIG.25 shows the polyBMEP was grafted on the nonwoven and some bridges also existed between fibers. Membranes were cut into 2.5 cm discs. 12 layers of polyBMEP grafted PBT were packed into 2.5 cm diameter column (DIBA Omnifit) fitted with a bottom end piece. The column was then connected to ÄKTA FPLC for determination of 100% dynamic binding capacity at residence times of 0.1, 2, and 5 min (FIG. 26–27). The prepared membrane showed 100% dynamic binding capacities of 40 mg IgG / mL at 0.1 min RT, 66 mg IgG / mL at 2.0 min RT, and 82 mg IgG / mL at 5.0 min RT (FIG.27). Example 7 High-Capacity Chromatographic Nonwoven Membrane Prepared Based on Meltblown PA11 The fiber diameter of polyamide 11 (PA11) observed from SEM was around 1.0 μm as shown in FIG.28. The PA11 nonwoven had a surface area of 3.9 m2 / g with a thickness of 400 μm. The PA11 nonwoven membrane was grafted with polyGMA by UV radiation to get 18.3% weight gain. Subsequently, 2-mercaptopyridine-3-carboxylic acid (MPCA) was coupled to polyGMA to develop a multimodal cation exchange chromatographic nonwoven membrane. A 1.0 cm-diameter column was used to pack the prepared membranes for testing 10% Dynamic Binding Capacity (DBC). Table 11 shows that the 10% DBC of the prepared membrane was 50.3–68.4 mg IgG / mL at residence times from 0.25 to 1.0 min with pressure drop of 0.002–0.035 MPa / cm. Table 11. 10% DBC Measurement of PA11-MPCA Membrane Delta Membrane Membrane Residence 10% DBC for P / Membrane Thickness (cm) Volume (mL) Time (min) IgG (mg / mL) Thickness (MPa / cm) 0.66 0.52 1.0 68.4 0.002 Equilibration buffer: 50 mM acetate pH 5.0 + 0.15 M NaCl; Elution buffer: 50 mM carbonate pH 9.5 + 0.5 M NaCl. Initial IgG concentration: 3.3 mg / mL. In FIG.29, the maximum binding capacity for BSA obtained by fitting with Langmuir model reached 640.38 mg / g (160 mg / mL), indicating a multi-layer protein binding due to the charged grafts. FIG. 30–31 show the 10% DBC of the prepared membrane was around 115 mg / mL measured at residence times from 0.2 to 5.0 min. The stable binding capacity at varied residence times implied that the flow rate has minor influence on protein binding to the membrane which is due to the lack of diffusion limitation. The exhibited high protein binding capacities at short residence times enable the prepared chromatographic nonwoven membrane to purify biologics with a high productivity.

Claims

CLAIMS What is claimed:

1. A functionalized nonwoven membrane for purifying a target molecule or species, the membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2-mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof.

2. The membrane of claim 1, wherein the polymeric fibers comprise polyolefins, polyethylenes, polypropylenes, cellulose, polyesters, polyamides, polycarbonates, polyethersulfones, polybutylene terephthalate (PBT), polytrimethylene terephthalate (PTT), polyethylene terephthalate (PET), polyamide 6 (PA6), polyamide 6-6 (PA6-6), polyamide 11 (PA11), polyamide 12 (PA12), thermoplastic polymers, or combinations thereof.

3. The membrane of claim 1, wherein the surface modification comprises a grafted second polymer.

4. The membrane of claim 3, wherein the grafted second polymer comprises an acrylate or methacrylate polymer from one or more monomers comprising glycidyl methacrylate (GMA), methacrylic acid, 2-(diethylamino)ethyl methacrylate, [2-(methacryloyloxy)ethyl] trimethyl-ammonium chloride, 2-hydroxyethyl methacrylate, 2-acrylamido-2- methylpropane sulfonic acid, 2-(dimethylamino)ethyl methacrylate, butyl methacrylate, 3- chloro-2-hydroxypropyl methacrylate, 2-ethylhexyl methacrylate, or combinations thereof.

5. The membrane of claim 1, wherein the functional ligand comprises N-benzyl-N- methylethanolamine (BMEA).

6. A functionalized nonwoven membrane for purifying a target molecule, the membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having a grafted second polymer; and a functional ligand coupled to the grafted second polymer and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N- methylethanolamine (BMEA).

7. The membrane of claim 6, wherein the polymeric fibers comprise polyolefins, polyethylenes, polypropylenes, cellulose, polyesters, polyamides, polycarbonates, polyethersulfones, polybutylene terephthalate (PBT), polytrimethylene terephthalate (PTT), polyethylene terephthalate (PET), polyamide 6 (PA6), polyamide 6-6 (PA6-6), polyamide 11 (PA11), polyamide 12 (PA12), thermoplastic polymers, or combinations thereof.

8. The membrane of claim 6, wherein the grafted second polymer comprises an acrylate or methacrylate polymer from one or more monomers comprising glycidyl methacrylate (GMA), methacrylic acid, 2-(diethylamino)ethyl methacrylate, [2-(methacryloyloxy)ethyl] trimethyl-ammonium chloride, 2-hydroxyethyl methacrylate, 2-acrylamido-2- methylpropane sulfonic acid, 2-(dimethylamino)ethyl methacrylate, butyl methacrylate, 3- chloro-2-hydroxypropyl methacrylate, 2-ethylhexyl methacrylate, or combinations thereof.

9. The membrane of claim 8, wherein the grafted second polymer comprises a methacrylate polymer of polyGMA.

10. The membrane of claim 6, further comprising one or more additional functional ligands comprising 2-mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof.

11. A functionalized nonwoven membrane for purifying a target molecule, the membrane comprising:a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) coupled to the polyGMA.

12. The membrane of claim 11, wherein the membrane has a dynamic binding capacity (DBC) for target molecule capture of ˃50 mg / mL with a residence time of from about 0.5 min to about 5 min.

13. The membrane of claim 11, wherein the membrane has a flow permeability of about 1.0 × 10−9cm2to about 4.6 × 10−9cm2in a buffer comprising about 50 mM to about 1 M of one or more salts at about pH 7.

0.

14. A method for purifying a target molecule, the method comprising: (b) contacting a sample comprising the target molecule with a functionalized nonwoven membrane to bind the target molecule to the membrane, the membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2- mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof; and (b) eluting the target molecule from the membrane and collecting the target molecule in a first eluate fraction.

15. The method of claim 14, wherein the target molecule is a biomolecule comprising a protein, a peptide, an antibody, a nucleic acid, an enzyme, a lipoprotein, a lipid, an extracellular vesicle, an exosome, a viral vector, a plasmid, a carbohydrate, a vaccine, a virus, a bacterium, a cell, an organelle, or combinations thereof.

16. The method of claim 14, wherein the sample is a biological fluid sample.

17. The method of claim 16, wherein the biological fluid sample comprises a cell culture supernatant or a sample obtained from an animal subject.

18. The method of claim 17, wherein the sample obtained from an animal subject comprises one or more of blood, plasma, serum, tears, urine, saliva, sputum, pleural effusion, or ascites.

19. The method of claim 14, further comprising: (a)(i) performing a first wash of the membrane using a low salt buffer at about pH 5–9; and (a)(ii) performing a second wash of the membrane using a low salt buffer having a charged species at about pH 5–9.

20. The method of claim 19, wherein the steps comprise: (a)(i) performing a first wash of the membrane using 50 mM Tris-HCl with about 1 mM to about 150 mM NaCl at pH 7.9; and (a)(ii) performing a second wash of the membrane using 50 mM Tris-HCl with about 50 mM to about 125 mM arginine at pH 7.

4.

21. The method of claim 14, wherein the target molecule is eluted from the membrane using one or more elution buffers or solutions at about pH 2–6.

22. The method of claim 21, wherein the target molecule is eluted from the membrane using a first elution buffer at about pH 4.

5.

23. The method of claim 22, wherein the target molecule is further eluted from the membrane using a second elution buffer at about pH 4.

0.

24. The method of claim 21, wherein the one or more elution buffers or solutions comprises 50 mM acetate at about pH 2–6.

25. The method of claim 14, further comprising virus inactivation comprising:(b)(i) adjusting the pH of the first eluate fraction to about pH 3–5; (b)(ii) incubating the first eluate fraction at room temperature for about 1 hour; (b)(iii) adjusting the pH of the first eluate fraction to about pH 7–8; and (b)(iv) filtering the first eluate fraction using microfiltration.

26. The method of claim 14, further comprising: (c) contacting the first eluate fraction comprising the target molecule with a second functionalized nonwoven membrane and collecting a flow-through fraction comprising the target molecule.

27. The method of claim 26, further comprising: (c)(i) performing a wash of the second functionalized nonwoven membrane using a buffer with about pH 6–9.

28. The method of claim 27, wherein the step comprises: (c)(i) performing a wash of the second functionalized nonwoven membrane using 50 mM Tris-HCl at pH 7.

2.

29. The method of claim 26, wherein the second functionalized nonwoven membrane comprises a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2- mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof.

30. The method of claim 26, further comprising: (d) contacting the flow-through fraction comprising the target molecule with a third functionalized nonwoven membrane to bind the target molecule to the third functionalized nonwoven membrane; and (e) eluting the target molecule from the third functionalized nonwoven membrane and collecting the target molecule in a second eluate fraction.

31. The method of claim 30, further comprising: (d)(i) performing a wash of the third functionalized nonwoven membrane using a low salt buffer at about pH 4–7.

32. The method of claim 31, wherein the step comprises: (d)(i) performing a wash of the third functionalized nonwoven membrane using 50^mM acetate with 150 mM NaCl at about pH 5.

5.

33. The method of claim 30, wherein the target molecule is eluted from the third functionalized nonwoven membrane using a low salt buffer at about pH 7–10.

34. The method of claim 33, wherein the target molecule is eluted from the third functionalized nonwoven membrane using 50 mM sodium phosphate with 150 mM NaCl at about pH 7.

7.

35. The method of claim 30, wherein the third functionalized nonwoven membrane comprises a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2- mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof.

36. The method of claim 30, wherein each of steps (a)–(e) has a residence time of about 0.1 min to about 5 min.

37. A method for purifying a target molecule, the method comprising: (a) contacting a sample comprising the target molecule with a functionalized nonwoven membrane to bind the target molecule to the membrane, the membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; anda functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) coupled to the polyGMA; and (b) eluting the target molecule from the membrane and collecting the target molecule in a first eluate fraction.

38. The method of claim 37, wherein the target molecule is a biomolecule comprising a protein, a peptide, an antibody, a nucleic acid, an enzyme, a lipoprotein, a lipid, an extracellular vesicle, an exosome, a viral vector, a plasmid, a carbohydrate, a vaccine, a virus, a bacterium, a cell, an organelle, or combinations thereof.

39. A method for purifying a target molecule, the method comprising: (a) contacting a sample comprising the target molecule with a first functionalized nonwoven membrane to bind the target molecule to the first membrane, the first membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) coupled to the polyGMA; (b) eluting the target molecule from the first membrane and collecting the target molecule in a first eluate fraction; (c) contacting the first eluate fraction comprising the target molecule with a second functionalized nonwoven membrane, the second membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; and a functional ligand comprising triethylamine (TEA) coupled to the polyGMA; (d) collecting a flow-through fraction comprising the target molecule; (e) contacting the flow-through fraction comprising the target molecule with a third functionalized nonwoven membrane to bind the target molecule to the third membrane, the third membrane comprising: a nonwoven web comprising a plurality of polybutylene terephthalate (PBT) fibers, each PBT fiber having polyglycidyl methacrylate (polyGMA) grafted thereto; anda functional ligand comprising 2-mercaptopyridine-3-carboxylic acid (MPCA) coupled to the polyGMA; and (f) eluting the target molecule from the third membrane and collecting the target molecule in a second eluate fraction.

40. The method of claim 39, wherein the target molecule is a biomolecule comprising a protein, a peptide, an antibody, a nucleic acid, an enzyme, a lipoprotein, a lipid, an extracellular vesicle, an exosome, a viral vector, a plasmid, a carbohydrate, a vaccine, a virus, a bacterium, a cell, an organelle, or combinations thereof.

41. The method of claim 40, wherein the target molecule is an antibody.

42. A method of making a functionalized nonwoven membrane for purifying a target molecule, the method comprising: (a) grafting polyglycidyl methacrylate (polyGMA) onto a polybutylene terephthalate (PBT) nonwoven web to generate a PBT-polyGMA nonwoven web; and (b) coupling a functional ligand comprising N-benzyl-N-methylethanolamine (BMEA) to the grafted polyGMA of the PBT-polyGMA nonwoven web to generate the functionalized nonwoven membrane.

43. The method of claim 42, wherein grafting the polyGMA onto the PBT nonwoven web is performed using ultraviolet radiation.

44. The method of claim 42, wherein a concentration of the BMEA functional ligand that is coupled to the PBT-polyGMA nonwoven web is from about 2% (v / v) to about 15% (v / v).

45. The method of claim 42, wherein the BMEA functional ligand is coupled to the PBT- polyGMA nonwoven web at about 40 °C for about 6 hours, followed by treatment with 0.1 M sulfuric acid at about 50 °C for about 16 hours.

46. The method of claim 42, further comprising washing the functionalized nonwoven membrane with pure water and storing the functionalized nonwoven membrane in a solution of 20% ethanol / water (v / v).

47. A chromatography device for purifying a target molecule, the chromatography device comprising: a housing unit comprising one or more functionalized nonwoven membranes, each membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2- mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof; and a flow device connected to the housing unit and configured to induce fluid flow of a sample comprising the target molecule through the one or more functionalized nonwoven membranes.

48. The chromatography device of claim 47, wherein the housing unit is a cartridge, a cassette, or a column.

49. A method for purifying a target molecule, the method comprising: (a) contacting a sample comprising the target molecule with a chromatography device comprising: a housing unit comprising one or more functionalized nonwoven membranes, each membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N- methylethanolamine (BMEA), 2-mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine,polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof; and a flow device connected to the housing unit and configured to induce fluid flow of the sample comprising the target molecule through the one or more functionalized nonwoven membranes; (b) inducing fluid flow of the sample comprising the target molecule through the one or more functionalized nonwoven membranes using the flow device to bind the target molecule to the one or more functionalized nonwoven membranes; and (c) eluting the target molecule from the one or more functionalized nonwoven membranes and collecting the target molecule in a first eluate fraction.

50. A kit for purifying a target molecule, the kit comprising: (a) one or more functionalized nonwoven membranes, each membrane comprising: a nonwoven web comprising a plurality of polymeric fibers, each polymeric fiber having one or more surface modifications; and one or more functional ligands coupled to the one or more surface modifications and adapted for ion exchange with the target molecule, the functional ligand comprising N-benzyl-N-methylethanolamine (BMEA), 2- mercaptopyridine-3-carboxylic acid (MPCA), triethylamine (TEA), diethylamine (DEA), iminodiacetic acid (IDA), sulfonate, polypropionic acid, polyethyleneimine, polyethyleneamidoamine, quaternary amine, carboxylate, or combinations thereof; (b) optionally, columns, buffers, cartridges, cassettes, and receptacles; and (c) optionally, one or more of packaging or instructions for use.

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