Affinity membrane and manufacturing method

Abstract

A medium is provided that includes a macroporous support. A medium comprising a macroporous support, the macroporous support comprising covalently bound Protein A to a surface of the macroporous support according to the formula: JPEG2026041810000005.jpg2545 The medium is provided, wherein ProA is Protein A, * is the macroporous support, the medium exhibits a dynamic binding capacity at 10% breakthrough of at least 20 mg of human immunoglobulin G per mL of the macroporous support with a retention time of 6 seconds or less, and the macroporous support comprises a cellulose ester membrane, a cellulose acetate membrane, a regenerated cellulose membrane, a cellulose-based nanofiber membrane, a cellulose-based monolith, a filter paper membrane, cellulose or a derivative thereof, or a combination thereof.

Description

[Technical Field]

[0001] 1) Field of the invention The present invention relates to membranes for purifying biological products such as proteins, polypeptides, peptides, polynucleotides, nucleotides, viral vectors, and vaccines using affinity separation methods, and more particularly to membranes that provide high binding capacity for biological products with short retention times, and methods for producing the membranes. [Background technology]

[0002] 2) Description of related technologies Biologics, including monoclonal antibodies (mAbs), are key components of many treatment plans for chronic conditions such as cancer, autoimmune disorders, cardiovascular disease, and many incurable diseases. However, biologics are among the most expensive drugs. For example, recent reports indicate that the costs of research, development, and manufacturing of mAbs account for approximately 35% of the drug price. Drug research is becoming increasingly expensive, with R&D costs to develop FDA-approved drugs doubling every nine years. In addition, the industry is moving toward small-batch production as a strategy to reduce market uncertainty caused by increasing competition. Demand for small-batch production operations is growing, particularly due to competition and emerging markets such as personalized medicine and orphan drugs. However, the cost per dose of small-batch production of biologics can be 10 times higher than that of large-scale production. Technologies that can rapidly and efficiently purify biologics would contribute to improving human health by enabling affordable drug production.

[0003] The major drawback of resin-based columns is that as flow rates increase (retention times decrease), binding capacity decreases. The slow mass transfer of proteins through the small pore structure of the resin necessitates the use of long retention times to achieve high capacity. Typical resin chromatography products require retention times of 6 minutes or more to achieve optimal binding capacity. Such long retention times result in very low productivity and, in some cases, product degradation.

[0004] For example, Protein A ligands have been routinely used in industry as a platform technology for mAb capture due to their high affinity for the Fc region of antibodies. Despite strong preference for Protein A-based products for mAb purification, the capacity of most Protein A resin chromatography products is 60–80 mg mAb / mL at a 6-minute retention time. This capacity drops to 18–30 mg / mL at 1–2 minutes of retention time. Therefore, there are currently no Protein A chromatography products on the market (or known to be in development) with a binding capacity >40 mg / mL at retention times of 6 seconds or less. Similarly, there are no high-throughput affinity chromatography products available for other biologics, such as plasmid DNA, messenger RNA, viral vectors, virus particles, virus-like particles, natural proteins, recombinant proteins, and endotoxins.

[0005] Membrane chromatography addresses this issue and offers an alternative to resin-based chromatography. Adsorption membranes with large through-pores can operate at short retention times but have low binding capacities. Existing porous hydrogel membranes exhibit improved static binding capacities due to their large surface area. However, their small mesh size results in poor macromolecular accessibility, thereby resulting in low capacity at short retention times (<60 seconds). Another challenge associated with porous hydrogel membranes is the high backpressure (>3 bar) resulting from the increased flow rate associated with short retention times. Therefore, a technological gap remains for affinity columns with high binding capacities at short retention times. Such invention would economically increase downstream biological product purification productivity.

[0006] In addition to the need for long retention times, the small pore structure of conventional resin-based columns further limits their use in the purification of large biologicals. In particular, the demand for preparative purification of large biologicals is rapidly increasing with the development of the gene and cell therapy industry. Examples of such biologicals include plasmid DNA, messenger RNA, viral vectors, virus particles, virus-like particles, and some natural and recombinant proteins. These biologicals are close to or larger than the pores of the resin beads. For these large biologicals, resin-based columns typically have low binding capacities, even with long retention times. Resin-based columns are also highly susceptible to clogging or fouling. Membrane chromatography products with macroporous structures can address this issue. However, no affinity membrane chromatography products for such applications are available.

[0007] It is therefore an object of the present invention to provide membranes for rapid and efficient purification of biological products, such as antibodies, plasmid DNA, messenger RNA, viral vectors, virus particles, virus-like particles, natural proteins, recombinant proteins, endotoxins, and other biological products, particularly monoclonal antibodies, as well as methods for producing affinity membranes.

[0008] It is a further object of the present invention to provide a membrane for use in pre-packed chromatography columns that has short retention times and high binding capacities for antibody capture-step purification under low backpressure.

[0009] It is a further object of the present invention to provide a Protein A membrane that has high binding capacity with short retention time and low back pressure. Summary of the Invention

[0010] The above objects are achieved in accordance with the present invention by providing a method for producing a membrane for binding biological drug molecules, the method comprising the steps of: immersing the membrane in a first solution of a coupling reagent in a first swelling solvent solution to swell the membrane and increase the exposure of reactive sites on the membrane for binding of the coupling reagent to form coupling groups; immersing the membrane in a second solution containing adsorption groups in a second swelling solvent solution to react at least a portion of the coupling groups with the adsorption groups, which provide a concentration effect for coupling at least one selected from the group consisting of ligands, nucleotides, oligonucleotides, peptides, polypeptides, proteins, and enzymes to the coupling groups; and immersing the membrane in an incubation solution selected from the group consisting of solutions of ligands, nucleotides, oligonucleotides, peptides, polypeptides, proteins, and enzymes having affinity for biological drug target molecules, to couple one of the ligands, nucleotides, oligonucleotides, peptides, polypeptides, proteins, and enzymes to at least a portion of the coupling groups of the membrane for binding to the biological drug target molecules when exposed to the membrane.

[0011] In a further advantageous embodiment, the membrane is a regenerated cellulose membrane having a specific surface area of ​​about 0.1-20 m^2 / mL; the ligand is Protein A; the membrane has a dynamic protein binding capacity of about 20-90 mg human immunoglobulin G / mL membrane at a retention time of about 6 seconds and a back pressure of less than 3 bar; and the membrane has a static protein binding capacity of greater than 60 mg human immunoglobulin G / mL membrane.

[0012] In a further advantageous embodiment, the first and second swelling solvent solutions comprise at least one swelling solvent selected from the group consisting of dimethyl sulfoxide (DMSO), mixtures of DMSO with other solvents with a DMSO content of more than 70% by volume, organic solvents, hexamethylphosphoramide, ionic liquids, sulfolane, and combinations thereof.

[0013] In a further advantageous embodiment, the coupling reagent is selected from the group consisting of N,N'-disuccinimidyl carbonate (DSC), 1,1'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), cyanogen halides, diisocyanates, diglycidyl ethers, epichlorohydrin, tosyl chloride, glutaraldehyde, divinyl sulfone, acyl halides, triazines, anhydrides, and combinations thereof.

[0014] In a further advantageous embodiment, the adsorption groups of the second solution are selected from the group consisting of tertiary amine-containing groups, negatively charged moieties, positively charged moieties, hydrophobic, hydrophilic, and functional groups containing moieties that promote π-π stacking interactions, and combinations thereof.

[0015] In a further advantageous embodiment, the first and second swelling solvent solutions consist of dimethyl sulfoxide (DMSO), the coupling reagent consists of N,N'-disuccinimidyl carbonate (DSC), the adsorption group consists of N,N'-dimethylethylenediamine (DMEDA), and the incubation solution comprises a Protein A solution, the Protein A solution having a Protein A concentration of 10 mg / mL or less.

[0016] The above object is further achieved in accordance with the present invention by providing a method for producing an adsorption medium for binding biological drug molecules, the method comprising the steps of: providing a macroporous support; immersing the macroporous support in a first solution of a coupling reagent in a solvent solution to bind the coupling reagent and form coupling groups; immersing the macroporous support in an incubation solution comprising an organic solvent and a target binding solution selected from the group consisting of a solution of a ligand, a nucleotide, an oligonucleotide, a peptide, a polypeptide, a protein, and an enzyme having affinity for a biological drug target molecule, thereby coupling one of the ligand, nucleotide, oligonucleotide, peptide, polypeptide, protein, and enzyme to at least a portion of the coupling groups of the macroporous support for binding to the biological drug target molecule when exposed to the macroporous support.

[0017] In a further advantageous embodiment, the macroporous support is selected from the group consisting of polyolefin membranes, polyethersulfone membranes, poly(tetrafluoroethylene) membranes, nylon membranes, glass fiber membranes, hydrogel membranes, hydrogel monoliths, polyvinyl alcohol membranes; natural polymer membranes, cellulose ester membranes, cellulose acetate membranes, regenerated cellulose membranes, cellulose-based nanofiber membranes, cellulose-based monoliths, filter paper membranes, and macroporous support membranes substantially containing cellulose or its derivatives, and combinations thereof.

[0018] In a further advantageous embodiment, before or after any step in the preparation of the affinity adsorption media, the macroporous support is immersed in a swelling solvent solution to swell the macroporous support and increase the exposure of at least one of the reactive sites, coupling groups, and ligand sites.

[0019] In a further advantageous embodiment, the swelling solvent solution comprises at least one swelling solvent selected from the group consisting of dimethyl sulfoxide (DMSO), mixtures of DMSO with other solvents with a DMSO content of more than 70% by volume, organic solvents, hexamethylphosphoramide, ionic liquids, sulfolane, and combinations thereof.

[0020] In a further advantageous embodiment, the macroporous support is a regenerated cellulose membrane having a specific surface area of ​​about 0.1 to 20 m^2 / mL; the ligand is Protein A; the macroporous support has a dynamic protein binding capacity of about 20 to 90 mg human immunoglobulin G / mL membrane at a retention time of about 6 seconds and a back pressure of less than 3 bar; and the macroporous support has a static protein binding capacity of greater than 60 mg human immunoglobulin G / mL membrane.

[0021] In a further advantageous embodiment, the coupling reagent is selected from the group consisting of N,N'-disuccinimidyl carbonate (DSC), 1,1'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), cyanogen halides, diisocyanates, diglycidyl ethers, epichlorohydrin, tosyl chloride, glutaraldehyde, divinyl sulfone, acyl halides, triazines, anhydrides, and combinations thereof.

[0022] In a further advantageous embodiment, the organic solvent is selected from the group consisting of water-miscible alcohols, ketones, ethers, amides, and combinations thereof to facilitate coupling of one of the ligands, nucleotides, oligonucleotides, peptides, polypeptides, proteins, and enzymes to the macroporous support.

[0023] In a further advantageous embodiment, the organic solvent is selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetonitrile, acetone, tetrahydrofuran (THF), dimethylformamide (DMF), and dimethyl sulfoxide (DMSO).

[0024] In a further advantageous embodiment, the first solution consists of dimethyl sulfoxide (DMSO); the coupling reagent consists of N,N'-disuccinimidyl carbonate (DSC); the incubation solution comprises at least one organic solvent selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetonitrile, acetone, tetrahydrofuran (THF), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and the incubation solution comprises a Protein A solution; and the Protein A solution has a Protein A concentration of 10 mg / mL or less.

[0025] In a further advantageous embodiment, the amount of said organic solvent in said incubation solution is substantially close to, but does not significantly exceed, the cloud point of the solution of ligand, nucleotide, oligonucleotide, peptide, polypeptide, protein, and enzyme.

[0026] The above objects are further achieved in accordance with the present invention by providing a method for preparing an adsorption medium for binding biological drug molecules, the method comprising the step of: coupling a coupling reagent selected from the group consisting of N,N'-disuccinimidyl carbonate (DSC), 1,1'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), cyanogen halides, diisocyanates, diglycidyl ethers, epichlorohydrin, tosyl chloride, glutaraldehyde, divinyl sulfone, acyl halides, triazines, anhydrides, and combinations thereof in a swelling solvent solution. immersing the membrane in a first solution to swell the membrane and increase the exposure of reactive sites on the membrane for binding of the coupling reagent to form coupling groups; immersing the membrane in an incubation solution selected from the group consisting of solutions of ligands, nucleotides, oligonucleotides, peptides, polypeptides, proteins, and enzymes having affinity for biological target molecules, to couple one of the ligands, nucleotides, oligonucleotides, peptides, polypeptides, proteins, and enzymes to at least a portion of the coupling groups on the membrane for binding to the biological target molecules when exposed to the membrane.

[0027] In a further advantageous embodiment, the incubation solution comprises a kosmotropic salt selected from the group consisting of sodium phosphate, sodium sulfate, or ammonium sulfate, and combinations thereof, to promote coupling of one of the ligands, nucleotides, oligonucleotides, peptides, polypeptides, proteins, and enzymes to the membrane.

[0028] In a further advantageous embodiment, the incubation solution comprises an organic solvent selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetonitrile, acetone, tetrahydrofuran (THF), dimethylformamide (DMF), and dimethyl sulfoxide (DMSO).

[0029] In a further advantageous embodiment, the amount of said organic solvent in said incubation solution is substantially close to, but does not significantly exceed, the cloud point of the solution of ligand, nucleotide, oligonucleotide, peptide, polypeptide, protein, and enzyme. [Brief explanation of the drawings]

[0030] A system designed to carry out the invention, together with other features, is described below. The invention will be more readily understood by reading the following specification and by referring to the accompanying drawings, which form a part thereof and which illustrate examples of the invention.

[0031] [Figure 1] FIG. 1 shows the synthesis of a Protein A membrane via reaction with DSC, partial substitution with DMEDA, and immobilization of Protein A in accordance with the present invention. [Figure 2] FIG. 2 shows the local concentration of protein A at the membrane surface and its immobilization to the membrane in accordance with the present invention. [Figure 3] FIG. 3 shows the direct modification of a cellulose membrane by DSC and subsequent immobilization of Protein A in concentrated solution according to the present invention. [Figure 4] FIG. 4 shows the direct modification of a cellulose membrane by DSC and subsequent immobilization of Protein A in a low concentration solution containing organic solvents, according to the present invention. [Figure 5] FIG. 5 shows the direct modification of a cellulose membrane by DSC and subsequent immobilization of Protein A in a low concentration solution containing a kosmotropic salt, according to the present invention. [Figure 6]FIG. 6 shows the static binding capacity of a Protein A membrane prepared using Method 1 according to the present invention. [Figure 7] FIG. 7 shows a comparison of the static binding capacity of Protein A membranes produced using methods 1 to 4 according to the present invention. [Figure 8] FIG. 8 shows the static binding capacity of Protein A membranes prepared using different organic solvents during the surface activation process according to the present invention. [Figure 9] FIG. 9 shows the static binding capacity of Protein A membranes prepared using Method 2 according to the present invention, using a DMSO / acetonitrile mixed solvent during the surface activation step. [Figure 10] FIG. 10 shows the static binding capacity of Protein A membranes prepared using Method 3 according to the present invention, using a DMSO / acetonitrile mixed solvent during the surface activation step. [Figure 11] FIG. 11 shows the static binding capacity of concanavalin A membranes prepared using method 3 according to the present invention, using a DMSO / acetonitrile mixed solvent during the surface activation step. [Figure 12] FIG. 12 shows the change in static binding capacity with increasing protein A concentration using method 2 according to the invention. [Figure 13] FIG. 13 shows the change in static binding capacity for protein A concentrations of 1 to 16.6 mg / mL using method 4 according to the present invention. [Figure 14] FIG. 14 shows the static binding capacity of Protein A membranes prepared using method 3 according to the invention with different coupling reagents and organic solvents. [Figure 15] FIG. 15 shows the static binding capacity of Protein A membranes prepared with epichlorohydrin and further modified using methods 3 and 4 according to the invention. [Figure 16] FIG. 16 shows the static binding capacity of a Protein A membrane with an additional DMSO soak according to method 5 according to the invention. [Figure 17] FIG. 17 shows the static binding capacity of a concanavalin A membrane with different amounts of ethanol in method 3 according to the invention. [Figure 18]FIG. 18 shows the dynamic binding capacity of a Protein A membrane using Method 1 according to the invention. [Figure 19] FIG. 19 shows the dynamic binding capacity of a Protein A membrane using method 2 according to the invention. [Figure 20] FIG. 20 shows the dynamic binding capacity of a Protein A membrane using method 3 according to the invention. [Figure 21] FIG. 21 shows the dynamic binding capacity of a Protein A membrane using method 3 according to the invention. [Figure 22] FIG. 22 shows a comparison of membranes according to the present invention with other commercially available membrane products. [Figure 23] FIG. 23 shows a comparison of a membrane according to the present invention with a commercially available resin product.

[0032] Those skilled in the art will understand that one or more aspects of the present invention may fulfill certain objectives, while one or more other aspects may fulfill certain other objectives. Each objective may not apply equally in all respects to all aspects of the invention. As such, the aforementioned objectives may be viewed as alternatives for any one aspect of the invention. These and other objects and features of the present invention will become more fully apparent from the following detailed description, taken in conjunction with the accompanying figures and examples. It should be understood, however, that the foregoing summary of the invention and the following detailed description are of preferred embodiments and are not intended to limit the present invention or alternative embodiments thereof. In particular, while the present invention is described herein with reference to certain specific embodiments, it should be understood that the descriptions are illustrative of the invention and are not to be construed as limiting the invention. Various modifications and applications will occur to those skilled in the art without departing from the spirit and scope of the present invention, as set forth in the appended claims. Similarly, other objects, features, benefits, and advantages of the present invention will become apparent from this summary and certain specific embodiments described below, and will be readily apparent to those skilled in the art. Such objects, features, benefits, and advantages will become apparent from the above, taken in conjunction with the accompanying examples, data, drawings, and all reasonable inferences to be drawn therefrom, either alone or together with a consideration of the references incorporated herein. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention is described in more detail below with reference to the drawings. 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 to which the presently disclosed subject matter belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter, representative methods, devices, and materials are described herein.

[0034] Unless otherwise noted, terms and phrases used in this document, and variations thereof, unless expressly stated otherwise, should be construed as open-ended as opposed to limiting. Similarly, a group of items joined by the conjunction "and" should not be read as requiring that every single one of those items be present in the group, but should be read as "and / or" unless expressly stated otherwise. Similarly, a group of items joined by the conjunction "or" should not be read as requiring that items in the group be mutually exclusive, but should also be read as "and / or" unless expressly stated otherwise.

[0035] Furthermore, although items, elements, or components of the present disclosure may be described or claimed in the singular, the plural is also intended to be within the scope of the present disclosure unless limitation to the singular is explicitly stated. The presence in some cases of broader words and phrases such as "one or more," "at least," "including but not limited to," or other similar phrases should not be read to imply that a narrower case is intended or required where such broader phrase would not be present.

[0036] The present invention includes affinity membranes that enable the rapid capture step purification of proteins, such as monoclonal antibodies (mAbs), plasmid DNA, messenger RNA, viral vectors, virus particles, virus-like particles, native proteins, recombinant proteins, and endotoxins or other target biologicals, providing higher productivity than existing resin products, such as Protein A chromatography columns. In one embodiment of producing a Protein A affinity membrane, membranes produced according to the methods described herein can provide a static protein binding capacity of 60-100 mg human immunoglobulin G / mL and a dynamic protein binding capacity of 20-90 mg human immunoglobulin G / mL with a retention time of 6 seconds or less and a backpressure of <3 bar.

[0037] The Protein A column operates in bind-elute mode. The productivity of the process can be defined using the following equation: Vtot = BV / T (Vtot) / T (T) (Vtot) where Vtot is the total volume of solution passed through the column during the entire process, including loading, rinsing, elution, and regeneration steps. BV is the volume of the Protein A media bed, and T is the retention time. The loading volume is proportional to the dynamic binding capacity of the Protein A media. Therefore, the productivity of the process increases with increasing binding capacity and decreasing retention time. JPEG2026041810000002.jpg23170

[0038] The mainstream resin column product on the market operates at a retention time of 360 seconds and has a dynamic binding capacity of 80 mg / mL. For two media with the same dynamic binding capacity and achieving the same product yield, the ratio of packing productivity can be calculated by the inverse ratio of the retention times. Thus, compared to the membrane of the present invention, which has a dynamic binding capacity of 60 mg / mL at a retention time of ≦6 seconds, the packing productivity of the membrane described herein can be 45 times (= 60 / 80 × 360 seconds / 6 seconds) higher than that of the mainstream resin column product for capturing and purifying mAbs. Currently, there are no resin or membrane products available that approach the productivity levels achieved by the present invention.

[0039] According to the present invention, the production of membranes for use in affinity separation procedures involves different manufacturing methods. In one embodiment, affinity membranes produced using these methods are differentiated from competing technologies based on their superior binding capacity for proteins, such as antibodies, including monoclonal antibodies (mAbs), with short retention times. In an exemplary embodiment described herein, the invention involves the use of ligands, nucleotides, oligonucleotides, peptides, polypeptides, proteins, or enzymes, such as oligodeoxythymidine, protein A, concanavalin A, trypsin, proteases, or endonucleases, chemically bound to the membrane, providing, in the case of protein A, a static protein binding capacity of 60-100 mg human immunoglobulin G / mL and a dynamic protein binding capacity of 20-90 mg human immunoglobulin G / mL with a retention time of 6 seconds or less and a backpressure of <3 bar. In one application, the membrane is used in the capture step of protein purification through a bind-elute procedure.

[0040] Membrane Fabrication Method 1: This method comprises fabricating a membrane for binding a biological agent, the method comprising the steps of: 1) immersing the membrane in a first solution comprising a coupling reagent in a swelling solvent to swell the membrane and increase the exposure of reactive sites on the membrane for binding of the coupling reagent; 2) immersing the membrane in a second solution comprising adsorption groups in a second swelling solvent solution to react at least a portion of the coupling groups with the adsorption groups, which provide a concentrating effect for coupling at least one of a ligand, a nucleotide, an oligonucleotide, a peptide, a polypeptide, a protein, and an enzyme to the coupling groups; and 3) immersing the membrane in an incubation solution selected from the group consisting of a solution of a ligand, a nucleotide, an oligonucleotide, a peptide, a polypeptide, a protein, and an enzyme having affinity for a biological agent target molecule to couple the one of the ligand, the nucleotide, the oligonucleotide, the peptide, and the enzyme to at least a portion of the coupling groups of the membrane for binding to the biological agent target molecule when exposed to the membrane. This embodiment of manufacturing a Protein A affinity membrane can provide a membrane capable of a high static protein binding capacity of >60 mg human immunoglobulin G / mL.

[0041] 1 and 2, in one embodiment, the present invention involves fabricating a ligand-incorporated membrane by combining a membrane swelling solvent, pre-immobilized couplings, and adsorption groups. In some embodiments, the membrane is selected from a group including, but not limited to, materials such as polyolefins, polyethersulfone membranes, poly(tetrafluoroethylene) membranes, nylon membranes, glass fiber membranes, hydrogel membranes, hydrogel monoliths, polyvinyl alcohol membranes; natural polymers such as cellulose or its derivatives, including, but not limited to, cellulose ester membranes, cellulose acetate membranes, regenerated cellulose membranes, cellulose-based nanofiber membranes, cellulose-based monoliths, or filter paper; or macroporous supports substantially containing cellulose or its derivatives. In some embodiments, the swelling solvent is selected from a group including, but not limited to, chemicals such as dimethyl sulfoxide (DMSO), acetonitrile, tetrahydrofuran (THF), and organic solvents such as dimethylformamide (DMF), hexamethylphosphoramide, ionic liquids, sulfolane, or mixtures thereof. In the detailed embodiment below, the membrane comprises regenerated cellulose (RC), and the swelling solvent is DMSO. However, the membrane may also comprise stabilized regenerated cellulose or other cellulose-based membranes having Protein A ligands incorporated therein, although the method is not limited to this membrane chemistry, as will be appreciated by those skilled in the art.

[0042] In one embodiment, the pore size is about 0.1 to 10.0 μm, 0.1 μm to 0.2 μm, 0.1 μm to 0.45 μm, 0.1 μm to 1 μm, 0.1 μm to 2 μm, 0.2 to 0.45, 0.2 to 1 μm, 0.2 to 2 μm, 0.2 to 10 μm, 0.45 μm to 1 μm, 0.45 μm to 2 μm, 0.45 μm to 10 μm, 1 μm to 2 μm, or 1 μm to 5 μm, >500 μm, >250 μm Membranes with thicknesses of >100 μm, >80 μm, >50 μm, >30 μm, 30 μm-500 μm, 50 μm-500 μm, 80 μm-500 μm, 100 μm-500 μm, 250 μm-500 μm, 30 μm-250 μm, 50 μm-250 μm, 80 μm-250 μm, 100 μm-2500 μm, 30 μm-100 μm, 50 μm-100 μm, and 80 μm-100 μm have been used. In accordance with the present invention, membranes with pore sizes of 1 μm, 0.45 μm, and 0.2 μm have been tested, achieving retention times of <6 seconds and backpressures of <3 bar. Membranes may be macroporous or fiber-based. Membranes may be stacked in multi-layer arrangements to increase capacity for any application. In one embodiment, stacked arrangements of membranes may be in the range of approximately 70 μm to 10,000 μm, >10,000 μm, >7,500 μm, >5,000 μm, >2,500 μm, >1,000 μm, >900 μm, >800 μm, >700 μm, >600 μm, >500 μm, >400 μm, >300 μm, >200 μm, >100 μm, >70 μm, 70 μm to 100 μm, 70 μm to 200 μm, 70 μm to 300 μm, 70 μm to 400 μm, 70 μm to 500 μm, 70 μm to 750 μm, 70 μm to 100 μm. 0μm, 70μm to 2000μm, 70μm to 3000μm, 70μm to 4000μm, 70μm to 5000μm, 250μm to 300μm, 250μm to 400μm, 250μm to 500μm, 250μm to 750μm, 250μm to 1000μm, 250 to 2000μm, 250 to 3000μm, 250 to 4000μm, 250 to 5000μm, 500μm to 1000μm, 500 to 2000μm, 500 to 3000μm, 500 to 4000μm, 500 to 5000μm thickness. Preferably, the membrane is a regenerated cellulose membrane with a pore size of 0.2 to 5.0 μm, a thickness of 70 to 2000 μm, and a stack array height of approximately 70 to 10000 μm.

[0043] Step 1: Membrane surface activation in a highly swelling solvent:

[0044] In the first step of an exemplary embodiment, a regenerated cellulose membrane is immersed in a mixture of N,N'-disuccinimidyl carbonate (DSC), triethylamine (TEA), and dimethyl sulfoxide (DMSO). DMSO is the preferred swelling solvent, but other swelling solvents, such as acetonitrile, tetrahydrofuran (THF), and dimethylformamide (DMF), may also be used. As shown in Figure 1, hydroxyl groups on the regenerated cellulose support membrane react with DSC to form an amino-reactive carbonate intermediate (-NHS). Membranes fabricated using DMSO as the preferred solvent during the surface activation phase have significantly higher binding capacities than membranes fabricated using other organic solvents. The swelling solvent increases the number of accessible hydroxyl groups for reaction with DSC, and therefore also increases the number of sites for subsequent protein-ligand coupling. Other solvents for cellulose result in less swelling, resulting in reduced surface area and protein-ligand coupling sites.

[0045] In this exemplary embodiment, the first step process can be carried out using 0.1-120 mg / mL DSC and 5-100 μL / mL triethylamine (TEA) in DMSO, acetonitrile, tetrahydrofuran (THF), and dimethylformamide (DMF), hexamethylphosphoramide, sulfolane, or any other solvent / solution that swells the membrane, for about 1-1800 minutes at a temperature of about 10-60° C. For example, a membrane with a diameter of 47 mm and a thickness of 70 μm is immersed in 10 mL DMSO containing 300 mg DSC and 139 μL TEA dissolved therein at 40° C. for 16 hours.

[0046] Depending on the membrane material, the solvent may cause varying amounts of swelling. Therefore, a solvent that causes a high degree of swelling should be selected. For cellulose-based membranes, DMSO is the preferred solvent, whether used alone or in combination with other solvents, including water. However, other solvents that can be used with cellulose-based membranes include, but are not limited to, acetonitrile, tetrahydrofuran (THF), and other organic solvents such as dimethylformamide (DMF), hexamethylphosphoramide, ionic liquids, sulfolane, or mixtures thereof.

[0047] Suitable coupling reagents other than DSC that can be used include, but are not limited to, 1,1'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), cyanogen halides, diisocyanates, diglycidyl ethers, epichlorohydrin, tosyl chloride, glutaraldehyde, divinyl sulfone, acyl halides, triazines, anhydrides, or mixtures thereof.

[0048] Step 2: Modification of a portion of the activated surface with adsorbing groups:

[0049] In the second step shown in Figure 1 of one exemplary embodiment, the DSC-activated membrane from step 1 is immersed in a solution of N,N-dimethylethylenediamine (DMEDA) in dimethyl sulfoxide (DMSO) solvent to replace a portion of the coupling groups with a ligand containing a tertiary amine group. DMEDA adsorbs the Protein A ligand (see Figure 2), which aids in coupling the Protein A ligand to the membrane in lower concentration solutions.

[0050] In this exemplary embodiment, the second step process is carried out in a solvent / solution such as DMSO, other organic solvents such as acetonitrile, tetrahydrofuran (THF), and dimethylformamide (DMF), hexamethylphosphoramide, sulfolane, etc., at a concentration of about 1-100 μL / mL, <100 μL / mL, <75 μL / mL, <50 μL / mL, <20 μL / mL, <10 μL / mL, 1-10 μL / mL, This can be done by using 1-20 μL / mL, 1-50 μL / mL, 1-75 μL / mL, 1-100 μL / mL, 10-20 μL / mL, 10-50 μL / mL, 10-75 μL / mL, 10-100 μL / mL, 20-50 μL / mL, 20-75 μL / mL, 20-100 μL / mL, 50-75 μL / mL, or 50-100 μL / mL of DMEDA at approximately 10-60°C for approximately 1 minute to 24 hours. For example, place the membrane in a 15 μL / mL solution of DMEDA in DMSO at room temperature for 30 minutes.

[0051] Suitable adsorption groups other than tertiary amine-containing groups include functional groups that may include, but are not limited to, negatively charged moieties, positively charged moieties, hydrophobic, hydrophilic, or moieties that promote π-π stacking interactions, or mixtures thereof, depending on the ligand to be coupled.

[0052] Step 3: Ligand coupling, in one embodiment the ligand is Protein A:

[0053] In one exemplary embodiment, in the third step shown in Figure 1, the DMEDA / DSC-modified membrane is incubated in a Protein A solution. In this step, the DMEDA group can enhance protein coupling efficiency through physical adsorption. The incorporation of the DMEDA group allows for the use of low concentrations of Protein A (approximately 0.5-5 mg / mL) in this step due to the concentrating effect shown in Figure 2. While this exemplary embodiment is described with respect to a Protein A solution, solutions of other ligands, nucleotides, oligonucleotides, peptides, polypeptides, proteins, or enzymes may be used for any target, including, but not limited to, antibodies, plasmid DNA, messenger RNA, viral vectors, virus particles, virus-like particles, natural proteins, recombinant proteins, endotoxins, and other biologics. For example, a Protein A solution can be used to target immunoglobulin G, an oligonucleotide solution can be used to target plasmid DNA or messenger RNA, and a Concanavalin A solution can be used to target glycoproteins.

[0054] In an exemplary embodiment, the process of the third step is 0.1 to 20 mg / mL, <0.1 mg / mL, <0.5 mg / mL, <0.75 mg / mL, <1 mg / mL, <2.5 mg / mL, <5 mg / mL, <10 mg / mL, <20 mg / mL, <45 mg / mL, 0.1 to 0.5 mg / mL, 0.1 to 0.75 mg / mL, 0.1 to 1 mg / mL, 0.1 ~2.5mg / mL, 0.1~5mg / mL, 0.1~10mg / mL, 0.1~20mg / mL, 0.1~45mg / mL, 0.5~0.75mg / mL, 0.5~1mg / m L, 0.5~2.5mg / mL, 0.5~5mg / mL, 0.5~10mg / mL, 0.5~20mg / mL, 0.5~45mg / mL, 0.75~1mg / mL, 0.75~2. Protein A concentrations of 5 mg / mL, 0.75 to 5 mg / mL, 0.75 to 10 mg / mL, 0.75 to 20 mg / mL, 0.75 to 45 mg / mL, 1 to 2.5 mg / mL, 1 to 5 mg / mL, 1 to 10 mg / mL, 1 to 20 mg / mL, 1 to 45 mg / mL, 2.5 to 5 mg / mL, 2.5 to 10 mg / mL, 2.5 to 20 mg / mL, 2.5 to 45 mg / mL, 5 to 10 mg / mL, 5 to 20 mg / mL, 5 to 45 mg / mL, 10 to 20 mg / mL, 10 to 45 mg / mL, or 20 to 45 mg / mL can be used together with a buffer solution of about 0.01 to 1 M Tris base, phosphate, or carbonate buffer at pH 7.0, at a temperature of 0 to 45°C, for any time period from about 1 minute to 24 hours. For example, the membrane is placed in a protein A solution with a protein A concentration of 5 mg / mL with 20 mM Tris base at pH=7.0 for 16 hours at room temperature.

[0055] The protein A ligand coupled to the membrane in the third step of an exemplary embodiment contains a site capable of binding antibodies, including mAbs. In one embodiment, four layers of 70 μm-thick membranes prepared using Method 1 are stacked in a syringe filter-like column. This configuration yielded a target biological binding capacity of approximately 20-90 mg human immunoglobulin G / mL with a retention time of ≤6 seconds and a backpressure of <3 bar. A widely recognized advantage of membrane chromatography is that it is not subject to the same diffusional mass transfer limitations as resin or gel chromatography. As a result, macroporous adsorption membranes reduce the flow rate dependence of dynamic binding capacity over a wide range of retention times. A limitation exists at sufficiently short retention times, when the characteristic time for protein adsorption is longer than the retention time of flow through the column. However, as long as the reaction rate of the target biological agent is sufficiently fast compared to the convective rate of mass transfer, dynamic capacity is not significantly affected by retention time.

[0056] Membrane Fabrication Method 2: This method comprises fabricating a membrane for binding a biological agent, and includes the steps of: 1) immersing the membrane in a solution containing a coupling reagent in a swelling solvent to swell the membrane and increase the exposure of reactive sites on the membrane for binding of the coupling reagent; and 2) incubating the membrane in a solution having a high concentration of a ligand, nucleotide, oligonucleotide, peptide, polypeptide, protein, or enzyme selected from the group consisting of ligands, nucleotides, oligonucleotides, peptides, polypeptides, proteins, and enzymes, for coupling to the membrane. In one embodiment of fabricating a Protein A affinity membrane, the concentration of the Protein A ligand solution is at least 30 mg / mL.

[0057] While Method 1 allows for high binding capacities using low concentrations of ligand, nucleotide, oligonucleotide, peptide, polypeptide, protein, or enzyme solution during coupling to the membrane, Method 2 focuses on high concentrations of ligand, nucleotide, oligonucleotide, peptide, polypeptide, protein, or enzyme solution. Referring to Figure 3, the same high binding capacity can also be achieved with short retention times and low backpressure through direct modification of the membrane with, for example, DSC and subsequent ligand, nucleotide, oligonucleotide, peptide, polypeptide, protein, and enzyme without the DMEDA adsorbent group step (Step 2 of Method 1 described above). However, for highly concentrated ligands, oligonucleotides, peptides, polypeptides, proteins, or enzyme solutions (e.g., for Protein A, approximately >30 mg / mL, >45 mg / mL, >75 mg / mL, >100 mg / mL, 125 mg / mL, >150 mg / mL, >175 mg / mL, 30-45 mg / mL, 30-100 mg / mL, 30-125 mg / mL, 30-150 mg / mL, 30-200 mg / mL, In one embodiment, a Protein A concentration of >80 mg / mL is selected.

[0058] Step 1: Membrane surface activation in a highly swelling solvent:

[0059] In one exemplary embodiment, in the first step, a regenerated cellulose membrane is immersed in a mixture of N,N'-disuccinimidyl carbonate (DSC), triethylamine (TEA), and dimethyl sulfoxide (DMSO). DMSO is the preferred swelling solvent, but other swelling solvents, such as acetonitrile, tetrahydrofuran (THF), and dimethylformamide (DMF), may also be used. As shown in Figure 8, when prepared using Method 2, membranes prepared using DMSO, acetonitrile, DMF, or THF as swelling solvents have static binding capacities of 90, 50, 51, and 50 mg human immunoglobulin G / mL. Hydroxyl groups on the regenerated cellulose support membrane react with DSC to form an amino-reactive carbonate intermediate (-NHS). Membranes prepared using DMSO as the preferred solvent during the surface activation phase have significantly higher binding capacities than membranes prepared using other organic solvents. The swelling solvent increases the number of accessible hydroxyl groups for reaction with DSC and therefore the number of sites for subsequent protein-ligand coupling. In the case of other solvents for cellulose, swelling is reduced, reducing the surface area and protein-ligand coupling sites.

[0060] In one exemplary embodiment, the first step process can be carried out using 0.1-120 mg / mL DSC and 5-100 μL / mL triethylamine (TEA) in DMSO, other organic solvents such as acetonitrile, tetrahydrofuran (THF), and dimethylformamide (DMF), hexamethylphosphoramide, sulfolane, etc., or any other solvent / solution that will swell the membrane, for about 1-1800 minutes at a temperature of about 10-60° C. For example, a membrane with a diameter of 47 mm and a thickness of 70 μm is immersed in 10 mL of DMSO containing 300 mg DSC and 139 μL TEA dissolved therein at 40° C. for 16 hours.

[0061] Depending on the membrane material, the solvent may cause varying amounts of swelling. Therefore, a solvent that causes a high degree of swelling should be selected. For cellulose-based membranes, DMSO is the preferred solvent, whether used alone or in combination with other solvents, including water. However, other solvents that can be used with cellulose-based membranes include, but are not limited to, organic solvents such as acetonitrile, tetrahydrofuran (THF), and dimethylformamide (DMF), hexamethylphosphoramide, ionic liquids, sulfolane, or mixtures thereof.

[0062] Suitable coupling reagents other than DSC that can be used include, but are not limited to, 1,1'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), cyanogen halides, diisocyanates, diglycidyl ethers, epichlorohydrin, tosyl chloride, glutaraldehyde, divinyl sulfone, acyl halides, triazines, anhydrides, or mixtures thereof.

[0063] Step 2: Ligand coupling using a high concentration of affinity ligand, in one embodiment the ligand is Protein A:

[0064] In one exemplary embodiment, in this second step, the DSC-modified membrane is directly incubated in a Protein A solution, omitting step 2 of Method 1 described above, but this requires a high ligand concentration, e.g., a Protein A solution concentration of >45 mg / mL, as detailed herein.

[0065] In one exemplary embodiment, the second step process is performed at a concentration of about >30 mg / mL, >45 mg / mL, >75 mg / mL, >100 mg / mL, 125 mg / mL, >150 mg / mL, >175 mg / mL, 30-45 mg / mL, 30-100 mg / mL, 30-125 mg / mL, 30-150 mg / mL, 30-200 mg / mL, 45-100 mg / mL, 45-125 mg / mL, 45-150 mg / mL, 45-200 mg / mL, 75-100 mg / mL, This can be done by using Protein A concentrations of 75-125 mg / mL, 75-150 mg / mL, 75-200 mg / mL, 100-125 mg / mL, 100-150 mg / mL, 100-200 mg / mL, 125-150 mg / mL, 125-200 mg / mL, or 150-200 mg / mL, together with a buffer concentration of 0.01-1 M Tris base, phosphate, or carbonate buffer at a pH level of about 6.0-10.0, at a temperature of 0-45°C for about 1 minute to 48 hours. For example, the membrane is placed in a Protein A solution with a Protein A concentration of about 45-160 mg / mL, together with about 20-200 mM Tris base at pH 8.0, at room temperature for 16 hours. Although this illustrated embodiment is described with respect to a Protein A solution, solutions of ligands, nucleotides, oligonucleotides, peptides, polypeptides, proteins, or enzymes may be used against any target, including, but not limited to, antibodies, plasmid DNA, messenger RNA, viral vectors, virus particles, virus-like particles, natural proteins, recombinant proteins, endotoxins, and other biologics. For example, a Protein A solution can be used to target immunoglobulin G, an oligonucleotide solution to target plasmid DNA or messenger RNA, and a Concanavalin A solution to target glycoproteins.

[0066] Membrane Production Method 3: This method includes producing a membrane for binding a biological agent, and includes the steps of: 1) immersing the membrane in a solution containing a coupling reagent in a swelling solvent to swell the membrane and increase the exposure of reactive sites on the membrane for binding of the coupling reagent; and 2) incubating the membrane in a solution containing an organic solvent and a target binding solution selected from the group consisting of a ligand, an oligonucleotide, a peptide, a polypeptide, a protein, and an enzyme to couple one of the ligand, oligonucleotide, peptide, polypeptide, protein, and enzyme to the membrane. In one embodiment of producing a Protein A affinity membrane, the concentration of the Protein A solution is 10 mg / mL or less.

[0067] Method 1 allows for high binding capacities despite the use of low ligand solution concentrations (<5 mg / mL) during ligand coupling. Method 2 allows for high binding capacities but requires high ligand concentrations during ligand coupling (step 2). Referring to Figure 4, Method 3 uses water-miscible organic solvents, such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and any other water-miscible organic solvents, such as other alcohols, ketones, ethers, amides, and combinations thereof, as components of the immobilization solution to enhance affinity ligand coupling efficiency, thereby enabling the use of low ligand concentrations in the coupling solution. Method 3 uses increasing percentages of organic solvent (20% to 80% by volume, depending on the organic solvent used) to bring the solution closer to the cloud point, the point at which increasing the organic solvent concentration causes the protein solution to become cloudy. In Method 3, the organic solution displaces water molecules from the protein's solvation shell, which may promote stronger interactions between the ligand and the membrane. Further addition of organic solution beyond the cloud point can worsen the assembly and aggregation kinetics of the ligands, which can relatively reduce the efficiency of the coupling reaction.

[0068] Step 1: Membrane surface activation in a highly swelling solvent:

[0069] In an exemplary embodiment, in the first step, a regenerated cellulose membrane is immersed in a mixture of N,N'-disuccinimidyl carbonate (DSC), triethylamine (TEA), and dimethyl sulfoxide (DMSO). DMSO is the preferred swelling solvent, but other swelling solvents, such as acetonitrile, tetrahydrofuran (THF), and dimethylformamide (DMF), may also be used. Hydroxyl groups on the regenerated cellulose support membrane react with DSC to form an amino-reactive carbonate intermediate (-NHS). Membranes prepared using DMSO as the preferred solvent during the surface activation phase have significantly higher binding capacities than membranes prepared using other organic solvents. The swelling solvent increases the number of accessible hydroxyl groups for reaction with DSC, and therefore also increases the number of sites for subsequent protein-ligand coupling. Other solvents for cellulose result in less swelling, resulting in reduced surface area and protein-ligand coupling sites.

[0070] In this exemplary embodiment, the first step process can be carried out using 0.1-120 mg / mL DSC and 5-100 μL / mL triethylamine (TEA) in DMSO, other organic solvents such as acetonitrile, tetrahydrofuran (THF), and dimethylformamide (DMF), hexamethylphosphoramide, sulfolane, etc., or any other solvent / solution that will swell the membrane, for about 1-1800 minutes at a temperature of about 10-60° C. For example, a membrane with a diameter of 47 mm and a thickness of 70 μm is immersed in 10 mL of DMSO containing 300 mg DSC and 139 μL TEA dissolved therein for 16 hours at 40° C.

[0071] Depending on the membrane material, the solvent may cause varying amounts of swelling. Therefore, a solvent that causes a high degree of swelling should be selected. For cellulose-based membranes, DMSO is the preferred solvent, whether used alone or in combination with other solvents, including water. However, other solvents that can be used with cellulose-based membranes include, but are not limited to, acetonitrile, tetrahydrofuran (THF), and other organic solvents such as dimethylformamide (DMF), hexamethylphosphoramide, ionic liquids, sulfolane, or mixtures thereof.

[0072] Suitable coupling reagents other than DSC that can be used include, but are not limited to, 1,1'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), cyanogen halides, diisocyanates, diglycidyl ethers, epichlorohydrin, tosyl chloride, glutaraldehyde, divinyl sulfone, acyl halides, triazines, anhydrides, or mixtures thereof.

[0073] Step 2: Ligand coupling using low concentrations of ligand, in one embodiment the ligand is Protein A:

[0074] In one exemplary embodiment, in this second step, the DSC-, tosyl chloride-, or epichlorohydrin-modified membrane is directly incubated in a low concentration Protein A solution containing an organic solvent, including methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetonitrile, or acetone.

[0075] In one exemplary embodiment, the second step process is carried out at a concentration of about 0.1 to 20 mg / mL, <0.1 mg / mL, <0.5 mg / mL, <0.75 mg / mL, <1 mg / mL, <2.5 mg / mL, <5 mg / mL, <10 mg / mL, <20 mg / mL, <45 mg / mL, 0.1 to 0.5 mg / mL, 0.1 to 0.75 mg / mL, 0.1 to 1 mg / mL, 0.1 to 2 .5mg / mL, 0.1~5mg / mL, 0.1~10mg / mL, 0.1~20mg / mL, 0.1~45mg / mL, 0.5~0.75mg / mL, 0.5~1mg / mL, 0.5 ~2.5mg / mL, 0.5~5mg / mL, 0.5~10mg / mL, 0.5~20mg / mL, 0.5~45mg / mL, 0.75~1mg / mL, 0.75~2.5mg / mL, This can be done by using affinity ligand concentrations of 0.75 to 5 mg / mL, 0.75 to 10 mg / mL, 0.75 to 20 mg / mL, 0.75 to 45 mg / mL, 1 to 2.5 mg / mL, 1 to 5 mg / mL, 1 to 10 mg / mL, 1 to 20 mg / mL, 1 to 45 mg / mL, 2.5 to 5 mg / mL, 2.5 to 10 mg / mL, 2.5 to 20 mg / mL, 2.5 to 45 mg / mL, 5 to 10 mg / mL, 5 to 20 mg / mL, 5 to 45 mg / mL, 10 to 20 mg / mL, 10 to 45 mg / mL, or 20 to 45 mg / mL, together with a buffer concentration of 0.01 to 1 M Tris base, phosphate, or carbonate buffer at a pH level of about 6.0 to 10.0 mixed with a significant portion of organic solvent, at a temperature of about 0 to 45°C, for a period of about 1 minute to 48 hours. For example, the membrane is placed in a protein A solution with a protein A concentration of about 0.1-20 mg / mL, together with about 20-200 mM Tris base at pH 8.0, for 16 hours at room temperature. However, higher concentrations of protein A may be used (20-175 mg / mL). The percentage of organic solvent ranges from 1% to 99% by volume, depending on the amount needed to bring the solution closer to the cloud point, the point at which the protein solution begins to become turbid as the organic solvent concentration increases. Organic solvents suitable for use in the present invention include, but are not limited to, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetonitrile, acetone, tetrahydrofuran (THF), and dimethylformamide (DMF).

[0076] Those skilled in the art will understand that the optimal ratio will depend on the ligand and organic solvent. Furthermore, there is an additional advantage to this method when using a water-labile linker, since the addition of an organic solvent reduces the rate of hydrolysis relative to the rate of the amine coupling reaction, thereby improving coupling efficiency. While this illustrated embodiment is described with respect to a Protein A solution, other ligand, nucleotide, oligonucleotide, peptide, polypeptide, protein, or enzyme solutions may be used for any target, including, but not limited to, antibodies, plasmid DNA, messenger RNA, viral vectors, virus particles, virus-like particles, natural proteins, recombinant proteins, endotoxins, and other biologicals. For example, a Protein A solution may be used to target immunoglobulin G, an oligonucleotide solution may be used to target plasmid DNA or messenger RNA, and a Concanavalin A solution may be used to target glycoproteins.

[0077] The amount of organic solvent in the incubation solution should be substantially close to, but not significantly exceed, the cloud point. It is possible to define a range of suitable amounts of organic solvent in the incubation solution, with an upper limit being [V% cp +a(100%-V% cp )] and the lower limit is [V% cp -bV% cp ], where "V% cp " is the volume percent of organic solvent in the ligand solution at the cloud point, "a" is the upper tolerance from the cloud point, and "b" is the lower tolerance from the cloud point. For example purposes, the volume percent of organic solvent in the ligand solution at the cloud point (V% cp) is 60%, and upper and lower limits are defined by a=0.3 and b=0.5, the corresponding suitable amount of organic solvent in the incubation solution would be in the range of 30% to 72% by volume of organic solvent. In one exemplary embodiment, the second step process is one in which "a" is about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.99, and "b" is This can be achieved by using amounts of organic solvent in the incubation solution that are approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 0.99. In the case of 5 mg / mL Protein A in 100 mM Tris, the volume percentages of methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetonitrile, and acetone to keep the solution slightly below the cloud point were found to be approximately 74%, 62%, 50%, 57%, 20%, 20%, 43%, and 50%, respectively. Although this illustrated embodiment is described with respect to a Protein A solution, other ligand, nucleotide, oligonucleotide, peptide, polypeptide, protein, or enzyme solutions may be used for any target, including, but not limited to, antibodies, plasmid DNA, messenger RNA, viral vectors, virus particles, virus-like particles, natural proteins, recombinant proteins, endotoxins, and other biologics. For example, a Protein A solution may be used to target immunoglobulin G, an oligonucleotide solution may be used to target plasmid DNA or messenger RNA, and a Concanavalin A solution may be used to target glycoproteins.

[0078] Membrane Fabrication Method 4: This method is similar to Method 3, but involves the use of kosmotropic salts instead of organic solvents. Method 1 allows for high binding capacities while using low ligand solution concentrations during ligand coupling. Method 2 allows for high binding capacities but requires high ligand concentrations during ligand coupling (step 2). Method 3 uses water-miscible organic solvents as components of the immobilization solution to improve ligand coupling efficiency, thereby allowing the use of low ligand, nucleotide, oligonucleotide, peptide, polypeptide, protein, and enzyme concentrations in the coupling solution. Referring to Figure 5, in Method 4, this high capacity can also be achieved by direct membrane modification by DSC and subsequent coupling of Protein A ligands using kosmotropic salts, including, but not limited to, sodium phosphate, sodium sulfate, or ammonium sulfate.

[0079] Method 4 uses an increasing proportion of kosmotropic salt to bring the ligand solution substantially close to, but not significantly above, the cloud point, the point at which increasing the concentration of kosmotropic salt causes the protein solution to become turbid. Further addition of salt beyond this point can worsen the ligand assembly and aggregation kinetics, which can relatively reduce the efficiency of the coupling reaction. In Method 4, the kosmotropic salt disrupts the solvation shell of the protein, which can promote stronger interactions between Protein A and the membrane. Near the cloud point, the electrostatic repulsive interactions between ligand molecules are alleviated by the compression of the solvation layer and the interactions between charged groups in the protein. The salt strengthens the interaction between the now-exposed hydrophobic portion of the protein and the membrane, enhancing the localization of the ligand along the membrane / solution interface, which can increase the efficiency of the coupling reaction.

[0080] Step 1: Membrane surface activation in a highly swelling solvent:

[0081] In an exemplary embodiment, in the first step, a regenerated cellulose membrane is immersed in N,N'-disuccinimidyl carbonate (DSC), triethylamine (TEA), and dimethyl sulfoxide (DMSO). DMSO swells cellulose significantly more than many other organic solvents, such as acetonitrile, tetrahydrofuran (THF), and dimethylformamide (DMF). Hydroxyl groups on the regenerated cellulose support membrane react with DSC to form an amino-reactive carbonate intermediate (-NHS). Membranes fabricated using DMSO as the preferred solvent during the surface activation phase have significantly higher binding capacities than membranes fabricated using other organic solvents. The swelling solvent increases the number of accessible hydroxyl groups for reaction with DSC, and therefore also increases the number of sites for subsequent protein-ligand coupling. Other solvents for cellulose result in less swelling, resulting in reduced surface area and protein-ligand coupling sites.

[0082] In this exemplary embodiment, the first step process can be carried out using 0.1-120 mg / mL DSC and 5-10 μL / mL triethylamine (TEA) in DMSO, other organic solvents such as acetonitrile, tetrahydrofuran (THF), and dimethylformamide (DMF), hexamethylphosphoramide, sulfolane, etc., or any other solvent / solution that will swell the membrane, for about 1-1800 minutes at a temperature of about 10-60° C. For example, a membrane with a diameter of 47 mm and a thickness of 70 μm is immersed in 10 mL DMSO containing 300 mg DSC and 139 μL TEA dissolved therein for 16 hours at 40° C.

[0083] Depending on the membrane material, the solvent may cause varying amounts of swelling. Therefore, a solvent that causes a high degree of swelling should be selected. For cellulose-based membranes, DMSO is the preferred solvent, whether used alone or in combination with other solvents, including water. However, other solvents that can be used with cellulose-based membranes include, but are not limited to, acetonitrile, tetrahydrofuran (THF), and other organic solvents such as dimethylformamide (DMF), hexamethylphosphoramide, ionic liquids, sulfolane, or mixtures thereof.

[0084] Suitable coupling reagents other than DSC that can be used include, but are not limited to, 1,1'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), cyanogen halides, diisocyanates, diglycidyl ethers, epichlorohydrin, tosyl chloride, glutaraldehyde, divinyl sulfone, acyl halides, triazines, anhydrides, or mixtures thereof.

[0085] Step 2: Ligand coupling using low concentrations of affinity ligand, in one embodiment the ligand is Protein A:

[0086] In one exemplary embodiment, in this second step, the DSC-modified membrane is directly incubated in a low concentration solution of Protein A in a concentrated kosmotropic salt solution.

[0087] In one exemplary embodiment, the second step process is carried out at a concentration of about 1-20 mg / mL, <1 mg / mL, <2.5 mg / mL, <5 mg / mL, <10 mg / mL, <20 mg / mL, <45 mg / mL, 1-2.5 mg / mL, 1-5 mg / mL, 1-10 mg / mL, 1-20 mg / mL, 1-45 mg / mL, 2.5-5 mg / mL, 2.5-10 mg / mL, 2.5-20 mg / mL, 2.5-45 mg / mL, 5-10 mg / mL, 5-20 mg / mL, 5-45 mg / mL, 5-45 mg / mL, 5-5 ... This can be done by using ligand concentrations of 5 mg / mL, 10-20 mg / mL, 10-45 mg / mL, or 20-45 mg / mL with salt concentrations of 0.5-3 M, 0.5-1 M, 0.5-2 M, 0.5-2.5 M, 0.5-3 M, 1-2 M, 1-2.5 M, 1-3 M, 1.5-2 M, 1.5-2.5 M, 1.5-3 M, 2-2.5 M, 2-3 M, or 2.5-3 M at temperatures of about 0-45°C for about 1 minute to 48 hours. For example, the membrane is placed in a Protein A solution with a Protein A concentration of about 5 mg / mL, in about 2 M NaSO at pH 6.5, for 16 hours at room temperature. However, higher concentrations of Protein A (20-175 mg / mL) may also be used. The kosmotropic salt concentration can range from 0.5 to 3 M to bring the solution close to, but preferably below, the cloud point, the point at which increasing the concentration of kosmotropic salt causes the protein solution to become turbid (as detailed in Method 3 above). One skilled in the art will appreciate that the optimal ratio will depend on the ligand and kosmotropic salt. While this illustrated embodiment is described with respect to a Protein A solution, other ligand, nucleotide, oligonucleotide, peptide, polypeptide, protein, or enzyme solutions may be used for any target, including, but not limited to, antibodies, plasmid DNA, messenger RNA, viral vectors, virus particles, virus-like particles, natural proteins, recombinant proteins, endotoxins, and other biologicals.For example, a protein A solution can be used to target immunoglobulin G, an oligonucleotide solution can be used to target plasmid DNA or messenger RNA, and a concanavalin A solution can be used to target glycoproteins.

[0088] Membrane Fabrication Method 5: This method involves preparing a membrane for binding biological agents by using a swelling solvent before or after any of the steps in the affinity membrane fabrication described in Methods 1-4. In one embodiment, the swelling solvent is DMSO, the ligand is Protein A, and the regenerated cellulose membrane is pre-soaked in DMSO, followed by an activation step and subsequent immobilization of the Protein A ligand. In another embodiment, the activation step is followed by immersion of the regenerated cellulose membrane in DMSO, followed by immobilization of the Protein A ligand. In another embodiment, a regenerated cellulose membrane functionalized with Protein A is immersed in DMSO. Results showed that swelling in these various treatments can increase binding capacity by 45% compared to unswollen membranes.

[0089] In one embodiment, a regenerated cellulose membrane was pre-soaked in DMSO, followed by an activation step and subsequent immobilization of Protein A ligand. For example, a membrane with a diameter of 47 mm and a thickness of 70 μm was soaked in 10 mL of DMSO at 40° C. for 16 hours.

[0090] In another embodiment, the activation step was followed by immersion of the regenerated cellulose membrane in DMSO followed by immobilization of Protein A ligand. For example, an NHS-activated membrane with a diameter of 47 mm and a thickness of 70 μm was immersed in 10 mL of DMSO at 40° C. for 16 hours.

[0091] In another embodiment, a regenerated cellulose membrane functionalized with Protein A was soaked in DMSO. For example, a Protein A functionalized membrane with a diameter of 47 mm and a thickness of 70 μm was soaked in 10 mL of DMSO at 40° C. for 16 hours.

[0092] In Method 5, it is not necessary to complete the activation step in a swelling solvent, because a swelling step before or after either step can increase the exposure of reactive, coupling, or ligand sites sufficiently to obtain a membrane with high binding capacity.

[0093] Membrane performance:

[0094] Important performance measures for columns with membranes prepared according to the methods described above are the static binding capacity (SBC) and the dynamic binding capacity at 10% breakthrough (DBC 10% In one embodiment, the primary biological product examined during testing was purified polyclonal human immunoglobulin G (hIgG), because it is often used in the industry as a model antibody for standardizing performance testing of Protein A-based products. These proteins are antibodies with a molecular weight of approximately 150,000 Da. The isoelectric points of these molecules were not specifically identified during testing, but are in the range of 6.1 to 9.4.

[0095] In another embodiment, concanavalin A (ConA) was used as the affinity ligand. ConA has been used to purify glycosylated proteins. To evaluate the performance of the ConA affinity membrane, the industry standard hIgG or porcine thyroglobulin was used.

[0096] i: SBC Measurement: Static binding capacity (SBC) testing provides a good benchmark for initial screening experiments. SBC was measured by mass balance using the initial and equilibrium hIgG concentrations measured using a Nanodrop UV spectrophotometer. Figure 6 shows that the SBC of Protein A membranes prepared by Method 1 varies depending on the concentration of DMEDA in step 2 (Method 1). The Protein A concentration used in this experiment was 5 mg / mL in 20 mM Tris, pH 7.0. hIgG was used as a model antibody. DMSO was used as the solvent in all of the examples described. The highest SBC obtained using Method 1 is over 100 mg / mL.

[0097] Figure 7 compares the SBC of Protein A membranes with 1, 0.45, and 0.2 μm pore sizes produced using Methods 1, 2, 3, and 4. In this example, a 5 mg / mL Protein A solution was used during ligand coupling in Methods 1, 2, and 3. Method 4 uses a 16.6 mg / mL Protein A solution. Overall, Methods 1, 3, and 4 achieve significantly higher SBCs than Method 2, which uses a lower concentration Protein A solution. The difference in SBC performance is more pronounced for support membranes with smaller pore sizes. Therefore, Method 2 requires a Protein A concentration greater than 45 mg / mL to achieve similar results as Methods 1, 3, and 4. Data were not collected for 1 μm pore-size membranes using Method 4.

[0098] Figure 8 shows that membranes prepared using DMSO as the swelling solvent during surface activation have significantly higher hIgG binding capacities than membranes prepared using acetonitrile, DMF, or THF using Method 2, which were prepared using 90 mg / mL Protein A in 100 mM Tris base at pH 8.0–9.0. Figure 9 shows the IgG binding capacities of membranes activated using Method 2 with different volumetric percentages of DMSO-acetonitrile mixed solvents (0, 30%, 50%, 70%, and 100% DMSO) using 90 mg / mL Protein A in 100 mM Tris base at pH 8.0–9.0. The membrane binding capacity increases with DMSO content because the swelling solvent increases the number of accessible hydroxyl groups for reaction with DSC and therefore increases the number of sites for subsequent protein-ligand coupling. As shown in Figure 10, the same trend was observed when Method 3 was used to prepare the Protein A affinity membrane adsorbent, which was prepared using a DMSO-acetonitrile mixed solvent during step 1 (Method 3) and 5 mg / mL Protein A in 100 mM Tris base at pH 8.0-9.0 with approximately 60% by volume of ethanol during the ligand coupling step.

[0099] In Figure 11, ConA membranes were prepared by Method 3 using different volumetric ratios of DMSO-acetonitrile (0, 25%, 50%, 75%, and 100% DMSO) in the membrane activation step. Ethanol (19.6% by volume) was used as the organic solvent in the ligand coupling step, and the concentration of ConA ligand was 5 mg / mL. Porcine thyroglobulin was used as the probe protein for SBC measurements in 20 mM Tris, pH 7.4, 0.5 M NaCl, 1 mM MnCl2, and 1 mM CaCl2. Binding capacity also generally increased with the volumetric percentage of DMSO used.

[0100] Table 1 shows the SBC of Protein A membranes activated with DMSO / acetonitrile mixed solvents followed by immobilization of Protein A under different buffer conditions. Overall, the SBC decreased as the solvent used in the activation step changed from DMSO to acetonitrile because the swelling solvent increased the number of accessible hydroxyl groups for reaction with DSC and, therefore, the number of sites for subsequent protein-ligand coupling. Table 1: SBC of membranes prepared using Method 3 with 100 mM of the indicated buffer and 5 mg / mL Protein A at the indicated pH, using a DMSO-acetonitrile mixed solvent during step 1 and approximately 60% by volume of ethanol during the ligand coupling step.

[0101] Figure 12 shows that achieving an SBC of >80 mg hIgG / mL requires the use of a Protein A concentration of >100 mg / mL in the ligand coupling step of Method 2. In comparison, Methods 1, 3, and 4 require a Protein A solution with a concentration of <20 mg / mL to produce membranes with an SBC of >80 mg hIgG / mL. As a result, Methods 1, 3, and 4 require a reduced protein concentration to produce membranes with comparable binding capacities, significantly reducing membrane production costs.

[0102] In Figure 13, membranes were prepared by Method 4. This figure shows that the SBC is similarly maintained across various Protein A concentrations with 2M sodium sulfate. A 0.2 um pore size support membrane was soaked in 5 mg DSC / mL DMSO during the activation step.

[0103] Figure 14 shows that high-capacity Protein A membranes can be obtained using various coupling reagents using Method 3. In this example, regenerated cellulose membranes with 0.2 μm pore size were activated using DSC, tosyl chloride, and epichlorohydrin. These membranes were immersed in the following solutions: a solution containing 5 mg DSC / mL DMSO; a solution containing 0.12 mL of 1 M NaOH, 0.13 mL of epichlorohydrin / mL DMSO; and a solution containing 22.5 mg tosyl chloride / mL DMSO.

[0104] In Figure 15, a 0.2 μm pore size membrane was activated in two different epichlorohydrin solutions, each with a different base catalyst. Solution A was 1.45 mg sodium amide, 0.132 mL epichlorohydrin / mL DMSO, and Solution B was 0.067 mL TEA, 0.132 mL epichlorohydrin / mL DMSO. The activated membrane was then immersed in a Protein A solution using the compositions described in Methods 3 and 4.

[0105] Previous examples have shown that immersing membranes in a swelling solvent, such as DMSO, during surface activation can increase binding capacity. Figure 16 shows that swelling membranes before or after any step in affinity membrane fabrication according to Methods 1-4, including before or after surface activation and after ligand immobilization, can also increase binding capacity. In this example, regenerated cellulose membranes were activated with either DSC / DMSO or DSC / acetonitrile. The protein A coupling solution contained approximately 60% ethanol and 5 mg / mL protein A in 100 mM Tris-base buffer at pH 8.0-9.0. Fabricated membranes were immersed in DMSO at 40°C for 15 hours before surface activation, after surface activation, or after ligand coupling. Results showed that swelling can increase binding capacity by 45% compared to unswollen membranes. Swelling membranes increase binding capacity by increasing the exposure of reactive, coupling, or ligand sites.

[0106] Figure 17 shows the SBC of ConA membranes prepared using different amounts of ethanol (0%, 24.5%, 29.4%, and 40% ethanol by volume) with a ligand concentration of 5 mg / mL in the ligand coupling step of Method 3. hIgG was used as the test glycoprotein for SBC measurements in 20 mM Tris, pH 7.4, 0.5 M NaCl, 1 mM MnCl2, and 1 mM CaCl2. The SBC increases with increasing amounts of ethanol added. The maximum SBC in this example is obtained near the cloud point at 40% ethanol by volume.

[0107] ii:DBC 10% Measurement: DBC 10% represents the mass of protein bound per unit volume of the membrane layer when the protein concentration in the eluate from the membrane layer reaches 10% of the feed concentration. The membrane was packed into a plastic prototype mini-column (membrane volume = 0.08-0.1 mL) and used for DBC analysis. 10% The DBC values ​​were measured using an AKTA Pure chromatography system. Flow rates of 10 to 100 column volumes per minute (CV / min) were used, corresponding to retention times of 6 to 0.6 seconds.10% The test solutions were human IgG at different concentrations in 1x PBS buffer at pH 7.3.

[0108] Figure 18 shows the DBC of a Protein A membrane packed into a syringe filter-like membrane holder. 10% DBC 10% were collected using hIgG solutions of different concentrations. Data shown are the average of triplicates, and error bars indicate standard error. These membranes were prepared by Method 1. 0.45 μm pore size membranes were activated in a solution of 50 mg DSC / mL DMSO, then further modified in a solution of 50 μL DMEDA / mL DMSO, followed by ligand coupling using 5 mg / mL Protein A in 20 mM Tris-base buffer, pH 7.0.

[0109] Figure 19 shows the DBC of a Protein A membrane packed into a syringe filter-like membrane holder. 10% DBC 10% were collected using hIgG solutions of different concentrations. These membranes were prepared by Method 2. Membranes with a pore size of 0.45 μm were activated in a solution of 30 mg DSC / mL DMSO, followed by ligand coupling using 120 mg / mL Protein A in 100 mM Tris-base buffer at pH 8.0–9.0.

[0110] Figure 20 shows the DBC of a Protein A membrane packed into a syringe filter-like membrane holder. 10% DBC 10% The data were collected using hIgG solutions of different concentrations. Data shown are the average of triplicates, and error bars indicate standard error. These membranes were prepared by Method 3. Membranes with pore sizes of 0.2 μm and 0.45 μm were separately activated in a solution of 5 mg DSC / mL DMSO, followed by ligand coupling using 5 mg / mL Protein A in approximately 60% ethanol, 100 mM Tris base, pH 8.0–9.0.

[0111] Figure 21 shows the DBC of Protein A membranes prepared using Method 3.10% Two different IgG concentrations were used with a retention time of 2.32 seconds. Data shown are the average of triplicates, and error bars indicate standard error. A 0.2 μm pore size regenerated cellulose membrane was activated in a solution of 0.0665 mL TEA, 0.131 mL epichlorohydrin / mL DMSO. The subsequent coupling solution contained 5 mg / mL Protein A in approximately 60% ethanol, 100 mM Tris base, pH 8.0-9.0.

[0112] Figures 22 and 23 compare the performance of Protein A membrane columns with commercially available products identified as Comp1, Comp2, and Comp3. The membranes produced according to the present invention showed excellent DBC values ​​of 40, 54, and 66 mg hIgG / mL with retention times between 0.6 and 6 seconds. 10% This significantly outperforms commercially available Protein A membrane products Comp1 and Comp2 (Figure 22) and another Protein A resin product, Comp3 (Figure 23), which compares performance with an industry-leading resin column, which only achieved 25 mg hIgG / mL at a 60 second retention time.

[0113] Table 2 shows the effect of different treatments on the specific surface area (SSA) in square meters per mL of membrane volume for regenerated cellulose membranes. Data were obtained from BET analysis. JPEG2026041810000004.jpg83170Table 2: Specific surface area per membrane volume (m^2 / mL) of membranes treated with a combination of DSC, DMSO, and acetonitrile.

[0114] While the present subject matter has been described in detail with reference to specific exemplary embodiments and methods thereof, it will be understood that those skilled in the art, upon understanding the foregoing, may readily make modifications, variations, and equivalents to such embodiments. Accordingly, the scope of the present disclosure is by way of example and not limitation, and the disclosure of the present subject matter is not intended to exclude such modifications, variations, and / or additions to the present subject matter as would be readily apparent to one of ordinary skill in the art using the teachings disclosed herein.

Claims

1. 1. A method for producing a membrane for binding biologic molecules, comprising: immersing the membrane in a first solution of a coupling reagent in a first swelling solvent solution to swell the membrane and increase exposure of reactive sites on the membrane for attachment of the coupling reagent to form coupling groups; immersing the membrane in a second solution containing adsorbent groups in a second swelling solvent solution to react at least a portion of the coupling groups with the adsorbent groups that provide a concentrating effect for coupling at least one selected from the group consisting of a ligand, a nucleotide, an oligonucleotide, a peptide, a polypeptide, a protein, and an enzyme to the coupling groups; and immersing the membrane in an incubation solution selected from the group consisting of a solution of a ligand, a nucleotide, an oligonucleotide, a peptide, a polypeptide, a protein, and an enzyme having affinity for a biologic target molecule, to couple one of the ligand, the nucleotide, the oligonucleotide, the peptide, the polypeptide, the protein, and the enzyme to at least a portion of the coupling groups of the membrane for binding with the biologic target molecule when exposed to the membrane.

2. 10. A membrane produced by the method of claim 1, wherein the membrane is a regenerated cellulose membrane having a specific surface area of ​​about 0.1 to 20 m^2 / mL; the ligand is protein A; the membrane has a dynamic protein binding capacity of about 20-90 mg human immunoglobulin G / mL membrane with a retention time of about 6 seconds and a back pressure of less than 3 bar; and The membrane has a static protein binding capacity of greater than 60 mg human immunoglobulin G / mL membrane.

3. 2. The method of claim 1, wherein the first and second swelling solvent solutions comprise at least one swelling solvent selected from the group consisting of dimethyl sulfoxide (DMSO), a mixture of DMSO and another solvent, wherein the DMSO content is greater than 70% by volume, an organic solvent, hexamethylphosphoramide, an ionic liquid, sulfolane, and combinations thereof.

4. 2. The method of claim 1, wherein the coupling reagent is selected from the group consisting of N,N'-disuccinimidyl carbonate (DSC), 1,1'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), cyanogen halides, diisocyanates, diglycidyl ethers, epichlorohydrin, tosyl chloride, glutaraldehyde, divinyl sulfone, acyl halides, triazines, anhydrides, and combinations thereof.

5. 2. The method of claim 1, wherein the adsorbing groups of the second solution are selected from the group consisting of tertiary amine-containing groups, negatively charged moieties, positively charged moieties, hydrophobic, hydrophilic, and functional groups containing moieties that promote π-π stacking interactions, and combinations thereof.

6. 2. The method of claim 1, wherein the first and second swelling solvent solutions comprise dimethyl sulfoxide (DMSO), the coupling reagent comprises N,N'-disuccinimidyl carbonate (DSC), the adsorptive group comprises N,N'-dimethylethylenediamine (DMEDA), and the incubation solution comprises a Protein A solution, the Protein A solution having a Protein A concentration of 10 mg / mL or less.

7. 1. A method for producing an adsorption medium for binding biologic molecules, comprising: providing a macroporous support; immersing the macroporous support in a first solution of a coupling reagent in a solvent solution to bind the coupling reagent to form coupling groups; immersing the macroporous support in an incubation solution comprising an organic solvent and a target binding solution selected from the group consisting of a solution of a ligand, a nucleotide, an oligonucleotide, a peptide, a polypeptide, a protein, and an enzyme having affinity for a biologic target molecule, to couple one of the ligand, nucleotide, oligonucleotide, peptide, polypeptide, protein, and enzyme to at least a portion of the coupling groups on the macroporous support for binding with the biologic target molecule when exposed to the macroporous support.

8. 8. The method of claim 7, wherein the macroporous support is selected from the group consisting of polyolefin membranes, polyethersulfone membranes, poly(tetrafluoroethylene) membranes, nylon membranes, glass fiber membranes, hydrogel membranes, hydrogel monoliths, polyvinyl alcohol membranes; natural polymer membranes, cellulose ester membranes, cellulose acetate membranes, regenerated cellulose membranes, cellulose-based nanofiber membranes, cellulose-based monoliths, filter paper membranes, and macroporous support membranes substantially containing cellulose or its derivatives, and combinations thereof.

9. 8. The method of claim 7, wherein the macroporous support is immersed in a swelling solvent solution before or after any step in the preparation of the affinity adsorption media to swell the macroporous support and increase the exposure of at least one of reactive sites, coupling groups, and ligand sites.

10. 10. The method of claim 9, wherein the swelling solvent solution comprises at least one swelling solvent selected from the group consisting of dimethyl sulfoxide (DMSO), a mixture of DMSO and another solvent, wherein the DMSO content is greater than 70% by volume, an organic solvent, hexamethylphosphoramide, an ionic liquid, sulfolane, and combinations thereof.

11. 8. A macroporous support produced by the method of claim 7, wherein the macroporous support is a regenerated cellulose membrane having a specific surface area of ​​about 0.1 to 20 m^2 / mL; the ligand is protein A; the macroporous support has a dynamic protein binding capacity of about 20-90 mg human immunoglobulin G / mL membrane with a retention time of about 6 seconds and a back pressure of less than 3 bar; and A macroporous support, wherein the macroporous support has a static protein binding capacity of greater than 60 mg human immunoglobulin G / mL membrane.

12. 8. The method of claim 7, wherein the coupling reagent is selected from the group consisting of N,N'-disuccinimidyl carbonate (DSC), 1,1'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), cyanogen halides, diisocyanates, diglycidyl ethers, epichlorohydrin, tosyl chloride, glutaraldehyde, divinyl sulfone, acyl halides, triazines, anhydrides, and combinations thereof.

13. 8. The method of claim 7, wherein the organic solvent is selected from the group consisting of water-miscible alcohols, ketones, ethers, amides, and combinations thereof to facilitate coupling of the ligand, nucleotide, oligonucleotide, peptide, polypeptide, protein, and enzyme to the macroporous support.

14. 14. The method of claim 13, wherein the organic solvent is selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetonitrile, acetone, tetrahydrofuran (THF), dimethylformamide (DMF), and dimethyl sulfoxide (DMSO).

15. the first solution consists of dimethyl sulfoxide (DMSO); the coupling reagent comprises N,N'-disuccinimidyl carbonate (DSC); the incubation solution comprises at least one organic solvent selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetonitrile, acetone, tetrahydrofuran (THF), dimethylformamide (DMF), and dimethyl sulfoxide (DMSO); the incubation solution comprises a protein A solution; and 8. The method of claim 7, wherein the Protein A solution has a Protein A concentration of 10 mg / mL or less.

16. 8. The method of claim 7, wherein the amount of the organic solvent in the incubation solution is substantially close to, but does not significantly exceed, the cloud point of the ligand, nucleotide, oligonucleotide, peptide, polypeptide, protein, or enzyme solution.

17. 1. A method for producing an adsorption medium for binding biologic molecules, comprising: immersing the membrane in a first solution of a coupling reagent selected from the group consisting of N,N'-disuccinimidyl carbonate (DSC), 1,1'-carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), cyanogen halides, diisocyanates, diglycidyl ethers, epichlorohydrin, tosyl chloride, glutaraldehyde, divinyl sulfone, acyl halides, triazines, anhydrides, and combinations thereof in a swelling solvent solution to swell the membrane and increase the exposure of reactive sites on the membrane for attachment of the coupling reagent to form coupling groups; immersing the membrane in an incubation solution selected from the group consisting of a solution of a ligand, a nucleotide, an oligonucleotide, a peptide, a polypeptide, a protein, and an enzyme having affinity for a biologic target molecule, to couple one of the ligand, nucleotide, oligonucleotide, peptide, polypeptide, protein, and enzyme to at least a portion of the coupling groups on the membrane for binding with the biologic target molecule when exposed to the membrane.

18. 18. The method of claim 17, wherein the incubation solution comprises a kosmotropic salt selected from the group consisting of sodium phosphate, sodium sulfate, or ammonium sulfate, and combinations thereof, to promote coupling of the one of the ligand, nucleotide, oligonucleotide, peptide, polypeptide, protein, and enzyme to the membrane.

19. 18. The method of claim 17, wherein the incubation solution comprises an organic solvent selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetonitrile, acetone, tetrahydrofuran (THF), dimethylformamide (DMF), and dimethyl sulfoxide (DMSO).

20. 18. The method of claim 17, wherein the amount of the organic solvent in the incubation solution is substantially close to, but does not significantly exceed, the cloud point of the ligand, nucleotide, oligonucleotide, peptide, polypeptide, protein, or enzyme solution.

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