EP4370227A1

EP4370227A1 - Epoxide-activated substrates and hydrophobic interaction chromatography membrane made therefrom

Info

Publication number: EP4370227A1
Application number: EP22765265.8A
Authority: EP
Inventors: Jinxiang Zhou; Graham TEMPLES; Bin Guo
Original assignee: Donaldson Co Inc
Current assignee: Donaldson Co Inc
Priority date: 2021-07-12
Filing date: 2022-07-11
Publication date: 2024-05-22
Also published as: WO2023287718A1; JP2024533931A

Abstract

Disclosed are activated membranes and derivatized membranes as may be formed from the activated membranes. HIC systems incorporating the derivatized membranes as may be used for use purifying plasmid DNA using hydrophobic interaction separation methods are also disclosed. Derivatized membranes can exhibit a high plasmid DNA binding capacity and short residence times. Methods for forming the activated membranes and methods for further derivatization of the membranes are also described.

Description

EPOXIDE-ACTIVATED SUBSTRATES AND HYDROPHOBIC INTERACTION CHROMATOGRAPHY MEMBRANE MADE THEREFROM

Cross Reference to Related Application

[0001] This application claims filing priority to United States Patent Application Serial No. 17/742,956, entitled “Epoxide-Activated Substrates and Hydrophobic Interaction Chromatography Made Therefrom for Polynucleotide Purification,” having a filing date of May 12, 2022, which claims filing benefit of Provisional Patent Application Serial No. 63/203,196, entitled “Epoxide-Activated Surface Preparation and Novel Hydrophobic Interaction Chromatography Membrane Adsorber for Polynucleotide Purification,” having a filing date of July 12, 2021 , both which are incorporated herein by reference for all purposes.

Federal Research Statement

[0002] This invention was made with Government support under Grant No.

GM 125429, awarded by the National Institutes of Health. The Government has certain rights in the invention.

Background

[0003] The gene and cell therapy industry has shifted rapidly toward commercial processes, due to their promising potential to treat various devastating diseases. Plasmid DNAs (pDNA) are key components in the production of the viral vectors, proteins, and mRNAs that are used widely in gene and cell therapy. There has been a sudden and urgent requirement for high-capacity, high-quality pDNA production.

As pDNA is a key component of many products that address the SARS-CoV-2 pandemic, the recent pandemic-related demands have further exacerbated the production challenge. For example, it will require over half of the world’s pDNA production capacity to produce one billion mRNA vaccines.

[0004] pDNA production has become a bottleneck of the industry as the scale-up of pDNA manufacturing is not straightforward. Currently, qualified contract manufacturers have long waiting lists and substantial backlogs to service the skyrocketing demand. Like many other biologies production schemes, multiple steps and unit operations are involved in pDNA production. Traditionally, downstream purification has been expensive, slow, and difficult to scale. Resin based chromatography columns have been the gold standard employed to purify biologies for decades. Resin columns are known to require long residence times to perform adequately. Moreover, because of the large size of pDNA, resins have low accessible surface area for pDNA binding. Overall, the combination of these factors results in very low productivity for pDNA purification.

[0005] Purity of the pDNA is key to follow-on bioprocesses. Beyond the need to separate from RNA, genomic DNA, host cell protein, etc., certain pDNA isoforms are also undesirable. pDNA usually has five isoforms: supercoiled (sc) pDNA, open- circular (oc) pDNA, relaxed circular pDNA, linear pDNA, and supercoiled denatured pDNA. Resin based hydrophobic interaction chromatography (HIC) columns often are used as a critical step to differentiate the desired sc pDNA from other isoforms. [0006] Membrane adsorbers are known to perform well at short column residence times compared to resin columns, resulting in rapid separations for biologies. However, there is currently no known effective commercial HIC membrane adsorber for pDNA purification, nor literature reporting high performance HIC membrane adsorbers for pDNA purification.

[0007] Sartorius AG produces an HIC membrane product with phenyl groups as HIC ligands; however, it was not designed for pDNA purification. Studies have indicated that this HIC membrane product has a very low pDNA binding capacity (<0.1 mg/mL for 3,000 base pair (3 kbp) pDNA) at 180 seconds residence time. Its binding capacity at higher flowrates (e.g., 36 seconds residence time) will be even lower (less than 0.04 mg/mL for 3 kbp pDNA).

[0008] Thus, there remains a need for HIC membrane columns with high binding capacity for pDNA at short residence times. Fulfilling this need would increase downstream pDNA and other polynucleotide purification productivities.

Summary

[0009] According to one embodiment, disclosed is an HIC separation medium that includes a cellulose substrate, e.g., a porous regenerated cellulose substrate, and a hydrophobic ligand bonded to a surface of the cellulose substrate.

[0010] In one embodiment, disclosed is a method for forming an epoxy-activated substrate. For instance, a method can include contacting a substrate, e.g., a porous membrane substrate, with an activation solution. The activation solution includes an activation agent, a base, and an organic solvent. The activation agent includes a reactive functionality that reacts with a surface of the substrate to form linking groups on the surface of the substrate. The activation agent also includes an epoxy functionality that can remain intact during the activation such that the linking agent includes the epoxy functionality following the activation.

[0011 ] Also disclosed is a method for further derivatizing an activated substrate that includes linking groups at a surface, the linking groups including an epoxy functionality. The method can include contacting the activated substrate with a derivatization solution. The derivatization solution includes a derivatization agent, a base, and optionally, an organic solvent. The derivatization agent includes a functionality that reacts with the epoxy functionality of the linking group. The derivatization agent also includes a hydrophobic portion that includes a hydrophobic ligand. Following the reaction, the hydrophobic ligand is bonded to the substrate surface via the reacted linking group.

Brief Description of the Figures

[0012] A full and enabling disclosure of the present subject matter, including the best mode thereof to one of ordinary skill in the art, is set forth more particularly in the remainder of the specification, including reference to the accompanying figures in which:

[0013] FIG. 1 compares the dynamic binding capacity 10% (DBCio%) values of 2- mercaptopyridine (MCP) derivatized membranes that were developed from activated membranes formed with different activation times.

[0014] FIG. 2 compares the DBCio% values of MCP derivatized membranes that were developed from activated membranes formed via different activation formulas. [0015] FIG. 3 compares the effect of alkaline pretreatments on derivatized membranes.

[0016] FIG. 4 presents the DBCio% values of MCP derivatized membranes formed using different alkaline pre-treatments and rinsing steps.

[0017] FIG. 5 is a FPLC chromatogram illustrating the loading, wash, and elution phases of a separation using an H IC membrane as disclosed herein.

[0018] FIG. 6 is a graph showing that binding capacity of a membrane can be improved through pH adjustment of loading buffer.

[0019] FIG. 7 presents the dynamic binding capacity of H IC media as described herein for plasmids of different sizes.

[0020] FIG. 8 presents the dynamic binding capacity of H IC media as described herein for different flow rates. [0021] FIG. 9 presents the dynamic binding capacity of HIC media as described herein for different ammonium sulfate concentrations.

[0022] FIG. 10 compares the chromatography results of a resin-type HIC media with an H IC membrane media as described herein.

Detailed Description

[0023] Reference will now be made in detail to various embodiments of the disclosed subject matter, one or more examples of which are set forth below. Each embodiment is provided by way of explanation of the subject matter, not limitation thereof. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made in the present disclosure without departing from the scope or spirit of the subject matter. For instance, features illustrated or described as part of one embodiment, may be used in another embodiment to yield a still further embodiment.

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to 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 herein described.

[0025] Unless specifically stated, terms and phrases used in this document, and variations thereof, should be construed as open ended as opposed to limiting. Likewise, a group of items linked with the conjunction “and” should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as “and/or” unless expressly stated otherwise. Similarly, a group of items linked with the conjunction “or” should not be read as requiring mutual exclusivity among that group, but rather should also be read as “and/or” unless expressly stated otherwise.

[0026] Furthermore, although items, elements or components of the disclosure may be described or claimed in the singular, the plural is contemplated to be within the scope thereof unless limitation to the singular is explicitly stated. The presence of broadening words and phrases such as “one or more,” “at least,” “but not limited to” or other like phrases in some instances shall not be read to mean that the narrower case is intended or required in instances where such broadening phrases may be absent. [0027] The present disclosure generally relates to an activated membrane that can be further derivatized for use in one embodiment for purifying pDNA using hydrophobic interaction separation methods. Methods for forming the activated membrane and further derivatization of the activated membrane are also described.

In embodiments, a derivatized membrane as described herein can exhibit a high pDNA binding capacity at short residence times.

[0028] It is one object of the present disclosure to provide a method of modifying a substrate to include a high density of epoxy-based chemistries, and thereby form an activated membrane.

[0029] It is a further object of the present disclosure to provide methods of subsequently derivatizing an activated membrane to incorporate a high density of ligands at a surface of the membrane, and in one particular embodiment, a high density of hydrophobic ligands.

[0030] It is a further object of the present disclosure to provide a HIC membrane and a process for forming the HIC membrane for rapid and efficient purification of pDNA and other polynucleic acids including polynucleotides, oligonucleotides, polynucleosides, and oligonucleosides, such as, and without limitation to, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), nucleic acids products yielded from rolling circle amplification, microRNA (miRNA), small nuclear RNA (snRNA), Piwi-interacting RNA (piRNA), double stranded RNA (dsRNA), genomic DNA, single stranded DNA (ssDNA), double stranded DNA (dsDNA), A-DNA, B-DNA, C-DNA, Z-DNA, DNA concatamers, aptamers, etc.

[0031] The terms “polynucleotide” and “oligonucleotide” as used herein refer to a polymer containing at least two nucleotides (e.g., deoxyribonucleotides or ribonucleotides) in either single- or double-stranded form and includes DNA and RNA. “Nucleotides” include a sugar, a base, and a linking group. In some embodiments, the sugar may be deoxyribose or a natural ribose (e.g., DNA and RNA, respectively). In some embodiments, the linking group may be a phosphate group. Nucleotides are linked together through the linking group to form polynucleotides and oligonucleotides. A polymer of covalently bonded linking groups may be termed a backbone. “Nucleoside” is otherwise similar to a nucleotide except that a nucleoside does not include a phosphate group. “Bases” or “nucleobases” include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides. Nucleobases include modified or analog nucleobases, modified or analog sugars, and/or modified or analog linking groups. The modified nucleobases, modified sugars, and/or modified linking groups may be non-canonical/chemically modified nucleobases, sugars, and/or linking groups which may be synthetic, naturally occurring, and/or non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs and/or modified bases, sugars, and/or linking groups include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2’-0-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0032] A deoxy-ribooligonucleotide consists of a 5-carbon deoxyribose sugar joined covalently to phosphate at the 5’ and 3’ carbons of this sugar to form an alternating, unbranched polymer. DNA may be in the form of, e.g., antisense molecules, pDNA, pre-condensed DNA, a PCR product, vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. A ribooligonucleotide consists of a similar repeating structure where the 5-carbon sugar is ribose. Accordingly, the terms “polynucleotide” and “oligonucleotide” can refer to a polymer or oligomer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and inter-sugar (backbone) linkages.

[0033] The terms “polynucleotide” and “oligonucleotide” can also include polymers or oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly. Such modified or substituted oligonucleotides can be preferred over native forms because of properties such as, for example, enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases. It should be understood that the terms “polynucleotide” and “oligonucleotide” can also include polymers or oligomers comprising both deoxy and ribonucleotide combinations or variants thereof in combination with backbone modifications, such as those described herein.

[0034] The polynucleotides and oligonucleotides as may be purified by materials described herein may include one or more nucleotide variants, including nonstandard nucleotide(s), non-natural nucleotide(s), nucleotide analog(s), and/or modified nucleotides. Examples of modified nucleotides include, but are not limited to diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5’-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio- N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4— thiouracil, 5- methyluracil, uracil-5- oxyacetic acid methylester, 5-methyl-2-thiouracil, 3-(3- amino- 3- N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine and the like. In some cases, nucleotides may include modifications in their phosphate moieties, including modifications to a triphosphate moiety. Non-limiting examples of such modifications include phosphate chains of greater length (e.g., a phosphate chain having, 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (e.g., alpha-thiotriphosphate and beta-thiotriphosphates).

[0035] A polynucleotide or oligonucleotide may be modified at the base moiety (e.g., at one or more atoms that typically are available to form a hydrogen bond with a complementary nucleotide and/or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide), sugar moiety, or liking group (e.g., backbone). Backbone modifications can include, but are not limited to, a phosphorothioate, a phosphorodithioate, a phosphoroselenoate, a phosphorodiselenoate, a phosphoroanilothioate, a phosphoraniladate, a phosphoramidate, and a phosphorodiamidate linkage. A phosphorothioate linkage substitutes a sulfur atom for a non-bridging oxygen in the phosphate backbone and delay nuclease degradation of oligonucleotides. A phosphorodiamidate linkage (N3’ P5’) prevents nuclease recognition and degradation. Backbone modifications can also include peptide bonds instead of phosphorous in the backbone structure (e.g., N-(2-aminoethyl)-glycine units linked by peptide bonds in a peptide nucleic acid), or linking groups including carbamate, amides, and linear and cyclic hydrocarbon groups. Oligonucleotides with modified backbones are reviewed in Micklefield, Curr. Med. Chem., 8 (10): 1157-79, 2001 and Lyer et al., Curr. Opin. Mol. Then, 1 (3): 344-358, 1999. Nucleic acid molecules described herein may contain a sugar moiety that comprises ribose or deoxyribose, as present in naturally occurring nucleotides, or a modified sugar moiety or sugar analog. Modified sugar moieties include, but are not limited to, 2’-0-methyl, 2’-0-methoxyethyl, 2’-0-aminoethyl, 2’- Flouro, N3’ P5’ phosphoramidate, 2’dimethylaminooxyethoxy, 2’ 2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-0-guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars. 2’-0-methyl or 2’-0-methoxyethyl modifications promote the A-form or RNA-like conformation in oligonucleotides, increase binding affinity to RNA, and have enhanced nuclease resistance. Modified sugar moieties can also include having an extra bridge bond (e.g., a methylene bridge joining the 2’-0 and 4’-C atoms of the ribose in a locked nucleic acid) or sugar analog such as a morpholine ring (e.g., as in a phosphorodiamidate morpholino). [0036] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al. , Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al. , J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).

[0037] The methods of the present disclosure encompass separation and/or purification of isolated or substantially purified nucleotides, nucleosides, nucleic acid molecules, and compositions containing those molecules. As used herein, an “isolated” or “substantially purified” DNA molecule or RNA molecule is a DNA molecule or RNA molecule that exists apart from its native environment. An isolated DNA molecule or RNA molecule may exist in a purified form or may exist in a non native environment such as, for example, a transgenic host cell. For example, an “isolated” or “purified” nucleic acid molecule or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an “isolated” nucleic acid is free of sequences that naturally flank the nucleic acid (i.e. , sequences located at the 5’ and 3’ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.

[0038] In one embodiment, a membrane as disclosed herein can be useful in purification of polynucleic acids of sizes ranging from tens of base pairs, e.g., miRNA of from about 20 to about 25 bases in length, to hundreds of thousands of bases, e.g., about 200,000 bases or even longer in some embodiments. For instance, a membrane can be useful in purifying polynucleic acids of from about 20 bases to about 250 bases, such as from about 50 bases to about 200,000 bases, such as from about 50 bases to about 150,000 bases, such as from about 50 bases to about 200,000 bases, such as from about 100 bases to about 200,000 bases, such as from about 100 bases to about 100,000 bases, such as from about 100 bases to about 50,000 bases, such as from about 200 bases to about 200,000 bases, such as from about 200 bases to about 150,000 bases, such as from about 200 bases to about 100,000 bases, such as from about 200 bases to about 50,000 bases, such as from about 200 bases to about 25,000 bases, such as from about 200 bases to about 20,000 bases, such as from about 200 bases to about 15,000 bases, such as from about 1 ,000 bases to about 50,000 bases, such as from about 5,000 bases to about 150,000 bases, such as from about 10,000 bases to about 150,000 bases, such as from about 15,000 bases to about 150,000 bases, such as from about 15,000 bases to about 200,000 bases, such as from about 20,000 bases to about 100,000 bases, such as from about 20,000 bases to about 200,000 bases, such as from about 20,000 bases to about 150,000 bases, such as from about 30,000 bases to about 150,000 bases.

[0039] Beneficially, disclosed HIC media can exhibit similar binding capacity independent of polynucleotide size. For instance, at identical conditions, a purification column as described herein can exhibit a difference in binding capacity (i.e. , retained polynucleotide (mg) per volume of membrane bed (ml_) at breakthrough) of about 20% or less, about 10% or less, or about 5% or less in some embodiments, for polynucleotides differing in size by a factor of 10 or greater or a factor of 100 or greater in some embodiments. For instance, polynucleotides ranging in size from an order of 10³ base pairs to an order of 10⁶ base pairs can exhibit a difference in binding capacity of about 20% or less, about 10% or less, or about 5% or less in some embodiments. [0040] To form an activated substrate, a substrate can be contacted with an activation solution. The activation solution including at least one epoxy-containing activation agent, one or more bases, and one or more organic solvents.

[0041] The substrate can be in the form of a non-porous film, a porous membrane, a nanofiber mat, a monolith (a single, porous three-dimensional structure), a resin (a solid polymeric phase in the form of individual particles, e.g., chips or beads), etc. As utilized herein, the term “membrane” generally refers to a relatively thin sheet material, having a porous structure. A substrate can be formed according to formation technologies as are generally known in the art including, without limitation, gel spinning, lyophilization, casting, molding, electrospinning, machining, wet-spinning, dry-spinning, milling, spraying, phase separation, template- assisted assembly, rolling, compaction, or any combinations thereof.

[0042] A thin substrate, e.g., a membrane or nanofiber mat, can in one embodiment have a thickness of from about 30 micrometers to about 2500 micrometers. For instance, a thin substrate can have a thickness of greater than about 500 micrometers, greater than about 250 micrometers, greater than about 100 micrometers, greater than about 80 micrometers, greater than about 50 micrometers, or greater than about 30 micrometers, such as from about 30 micrometers to about 500 micrometers, from about 50 micrometers to about 500 micrometers, from about 80 micrometers to about 500 micrometers, from about 100 micrometers to about 500 micrometers, from about 250 micrometers to about 500 micrometers, from about 30 micrometers to about 250 micrometers, from about 50 micrometers to about 250 micrometers, from about 80 micrometers to about 250 micrometers, from about 100 micrometers to about 2500 micrometers, from about 30 micrometers to about 100 micrometers, from about 50 micrometers to about 100 micrometers, or from about 80 micrometers to about 100 micrometers.

[0043] As used herein, unless indicated otherwise, sample thickness is measured with an automated thickness tester. An exemplary thickness test measures the gap between two test apparatus plates when held at a fixed pressure. An exemplary pressure to measure thickness is 1.5 PSI. One useful instrument for membrane sample thickness is a 49-56 Micrometer (Messmer BCichel, Veenendaal, Netherlands).

[0044] In some embodiments a substrate can be a porous membrane substrate that can be a supported membrane, e.g., a laminate including a porous membrane adjacent (e.g., adhered or attached) to a supporting frame or backing material that exhibits a greater porosity than the porous membrane substrate, e.g., a woven or nonwoven backing material that can be formed of the same or a different material as the substrate.

[0045] A nonwoven backing material (e.g., backing web) can be created by melt blowing, wet laying, melt spinning, solution spinning, air laying, or electrospinning. Nonwoven webs may additionally be treated through post-processing steps, such as calendaring, embossing, needle-punching, or hydroentangling. Nonwoven backing materials may also contain a structural resin that has low binding affinity to biomolecules. Such resins are typically used to increase the strength of the backing material. A backing material can contain a mixture of fiber sizes and fiber materials. The fibers used to make the backing material may include glass, polypropylene, polyamides, polyesters, cellulosic materials, and the like, and combinations thereof. The fibers may have an average fiber size of 0.1 pm or greater, 1 pm or greater, 2 pm or greater, or 3 pm or greater. The fibers may have an average fiber size of 100 pm or less, 50 pm or less, 25 pm or less, 10 pm or less, or 8 pm or less. Average fiber sizes may range from 0.1 pm to 50 pm, or from 1 pm to 25 pm. The average pore size, measured by capillary flow porometer, may be 1 pm or greater, 2 pm or greater, or 3 pm or greater. The average pore size may be 100 pm or less, 50 pm or less, 25 pm or less, 10 pm or less, or 8 pm or less. The average pore size of suitable nonwoven substrates may range from 0.1 pm to 50 pm, from 1 pm to 10 pm, or from 3 pm to 8 pm. The average pore size of woven substrates may be slightly greater than nonwoven substrates, and may range from 1 pm to 100 pm. The basis weight of the fibrous substrate may be 1 gsm (grams per square meter) or greater, 10 gsm or greater, or 20 gsm or greater. The basis weight of the fibrous substrate may be 200 gsm or less or 80 gsm or less. The basis weight of the fibrous substrate may be in a range of 1 gsm to 200 gsm, or from 20 gsm to 80 gsm.

[0046] In some embodiments, a porous membrane substrate can be a self- supporting membrane, i.e. , not requiring a backing material. Of course, an otherwise self-supporting membrane can be retained by a support if desired. Porous membrane substrates as described herein encompass membranes prepared by casting, coating, or forming, including, but not limited to, porous hydrogel membranes as well as fibrous membranes, e.g., porous membranes formed of nanofibers such as electrospun nanofibers. [0047] The substrate can have a relatively high surface area. Surface area can be determined according to the BET (Brunauer, Emmett, and Teller) measurement evaluation using nitrogen as the adsorbate. By way of example, a substrate, e.g., a porous substrate, can have a specific surface area of from about 0.1 m²/ml_ to about 30 m²/ml_, such as from about 0.1 m²/ml_ to about 25 m²/ml_, from about 0.1 m²/ml_ to about 20 m²/ml_, from about 0.1 m²/ml_ to about 15 m²/ml_, from about 0.1 m²/ml_ to about 10 m²/ml_, from about 0.5 m²/ml_ to about 30 m²/ml_, from about 0.5 m²/ml_ to about 25 m²/ml_, from about 0.5 m²/ml_ to about 20 m²/ml_, from about 0.5 m²/ml_ to about 15 m²/ml_, from about 0.5 m²/ml_ to about 10 m²/ml_, from about 0.5 m²/ml_ to about 5 m²/ml_, from about 1 m²/ml_ to about 30 m²/ml_, from about 1 m²/ml_ to about to about 25 m²/ml_, from about 1 m²/ml_ to about 20 m²/ml_, from about 1 m²/ml_ to about 15 m²/ml_ , from about 1 m²/ml_ to about 10 m²/ml_, from about 1 m²/ml_ to about 5 m²/ml_, from about 5 m²/ml_ to about 30 m²/ml_, from about 5 m²/ml_ to about to about 25 m²/ml_, from about 5 m²/ml_ to about 20 m²/ml_, from about 5 m²/ml_ to about 15 m²/ml_, or from about 5 m²/ml_ to about 10 m²/ml_.

[0048] In some embodiments, a porous substrate can be a macroporous substrate, e.g., a macroporous membrane substrate. In general, a macroporous substrate can include a specific surface area of about 1 m²/ml_ or greater, such as from about 1 m²/ml_ to about 30 m²/ml_, from about 1 m²/ml_ to about to about 25 m²/ml_, from about 1 m²/ml_ to about 20 m²/ml_, from about 1 m²/ml_ to about 15 m²/ml_ , from about 1 m²/ml_ to about 10 m²/ml_, from about 1 m²/ml_ to about 5 m²/ml_, from about 5 m²/ml_ to about 30 m²/ml_, from about 5 m²/ml_ to about to about 25 m²/ml_, from about 5 m²/ml_ to about 20 m²/ml_, from about 5 m²/ml_ to about 15 m²/ml_, or from about 5 m²/ml_ to about 10 m²/ml_. In one embodiment, macroporous membrane substrates can exhibit high volumetric flow rates without high pressure due to micron-size pores, can exhibit low non-specific adsorption of proteins, and can exhibit a high density of surface functional groups that can be used as reactive sites.

[0049] In those embodiments in which the substrate is porous, the porous substrate can generally have a pore size from about 0.1 micrometers to about 10 micrometers. For instance a porous substrate, e.g., a porous membrane substrate, can exhibit a pore size of from about 0.1 micrometer to about 10 micrometers, from about 0.1 micrometer to about 0.2 micrometers, from about 0.1 micrometer to about 0.45 micrometers, from about 0.1 micrometer to about 1 micrometer, from about 0.1 micrometer to about 2 micrometers, from about 0.2 micrometer to about 0.45 micrometers, from about 0.2 micrometers to about 1 micrometer, from about 0.2 micrometer to about 2 micrometers, from about 0.2 micrometer to about 10 micrometers, from about 0.45 micrometer to about 1 micrometer, from about 0.45 micrometers to about 2 micrometers, from about 0.45 micrometer to about 10 micrometers, from about 1 micrometer to about 2 micrometers, or from about 1 micrometer to about 5 micrometers.

[0050] As used herein, unless indicated otherwise, pore size is determined using capillary flow porometry. Capillary flow porometry may be performed using a continuous pressure scan mode. An exemplary range of applied pressures that may be used is 0.115 bar to 3.5 bar (15 kPa to 350 kPa). It may be useful to use a wetting liquid having a surface tension of 16.4 dynes/cm, including, for example Porofil™ Wetting Solution (Quantachrome Instruments, Anton Paar, Boynton Beach, FL). The sample may initially be tested dry, varying low pressure to high pressure, and then tested wet, again varying low pressure to high pressure. The test is typically performed at ambient temperature conditions (for example, 20°C to 25°C). 200 data points may be collected across the range of the scan of the pressures for both the dry curve and the wet curve. The relationship between de-wetting pressure (P) and pore size (D) described by the relationship P = 4t cos Q Yi_v/D, where y is the surface tension of the wetting liquid, Q is the contact angle of the wetting liquid with the porous material, and r is an empirical correction factor known as a tortuosity factor. Typically, a tortuosity factor or a shape factor of 0.715 is applied.

An average pore size may be calculated from the mean of at least three measurements. Individual measurements of maximum pore size may be detected at a measured bubble point, where the measured bubble is determined by increasing the pressure on a wetted sample until the point at which the wetting fluid is displaced and air flow is measured. The pressure at which air flow is first measured indicates the point at which the first and largest pore is de-wetted allowing for air flow. Individual measurements of mean flow pore size may be calculated by determining the pressure where the wet curve and the “half-dry” curve cross. The half-dry curve is obtained by the mathematical division by 2 of the air flow through a dry sample as a function of diameter. One useful instrument for determining pore size distribution is a Porolux 500 (Aptco Technologies NV, Nazareth, Belgium). [0051] In one embodiment the substrate can be formed from a hydrophilic material, such as cellulose, cellulose derivatives, regenerated cellulose, nylon, or other hydrophilic materials. As utilized herein, the term regenerated cellulose refers to a class of materials manufactured by the conversion of natural cellulose to a soluble cellulosic derivative and subsequent regeneration to form a fiber or a film produced by, e.g., the viscose or the lyocell method or by spinning cellulose from solution thereof in ionic liquids.

[0052] Cellulose derivatives encompassed herein can include, without limitation, one or more cellulose ethers, one or more cellulose esters, or any combination thereof. By way of example, and without limitation, a cellulose derivative can include an alkyl cellulose (e.g., methyl cellulose, ethyl cellulose, ethyl methyl cellulose), a hydroxy alkyl cellulose (e.g., hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, ethyl hydroxyethyl cellulose), a carboxy alkyl cellulose (e.g., carboxymethyl cellulose), an organic ester cellulose (e.g., cellulose acetate, cellulose triacetate, cellulose propionate, cellulose acetate propionate, cellulose acetate butyrate), an inorganic acid cellulose (e.g., nitrocellulose, cellulose sulfate), or any combination thereof. In one embodiment, an optically active material can include an alkyl cellulose such as hydroxypropyl cellulose (HPC), methyl cellulose, ethyl cellulose, or combinations thereof.

[0053] It is to be understood, however, that the use of a hydrophilic material is not required, and the substrate can be formed from other materials known to those of ordinary skill in the art, such as a polysulfone, a polyether sulfone, a polyvinylidene fluoride, a polyacrylonitrile, a polyetherimide, a polypropylene, a polyethylene, a polyether terephthalate, etc., or any combination of materials.

[0054] To form an activated substrate, a substrate, e.g., a porous membrane substrate, can be contacted with an activation solution. In one embodiment, the substrate can be contacted by immersing a substrate in an activation solution. The activation solution can include an activation agent in addition to at least one base and at least one organic solvent. Upon the immersion, interaction between a reactive functionality of the activation agent and a surface of the substrate can form activated linker groups on the surface, each linker group including at least one reactive epoxy group. The interaction can include covalent bonding, ionic bonding, hydrogen bonding, etc., or any combination thereof. [0055] The activation agent can be a multi-functional agent, e.g., bifunctional, trifunctional, etc., in which at least one functional group of the agent is configured to react with a surface of the substrate and at least one functional group is an epoxy that will not react during the activation step, i.e. , an epoxy group that will remain active following the activation step. As such, upon the interaction between the activation agent and the substrate, an epoxy-containing linking group can be at the surface of the activated substrate.

[0056] The activation agent can include, without limitation, an epichlorohydrin, a diglycidyl ether, triglycidyl ether, tetraglycidyl ether, or any combination thereof. [0057] In embodiments, the concentration of the activation agent in the solvent can range from about 0.1 % (V/V) to about 60% (V/V) of the solution, such as from about 2% (V/V) to about 40% (V/V) of the solution, or from about 5% (V/V) to about 30% (V/V) of the solution.

[0058] The organic solvent component of an activation solution can be selected from, but is not limited to, a single organic solvent, an aqueous/organic solvent mixture, or a mixture of organic solvents. When considering an activation solution that includes water, the water will generally be present in an amount of about 50% (V/V) or less by volume of the activation solution, e.g., about 40% (V/V) or less, about 30% (V/V) or less, about 20% or less, about 10% (V/V) or less, or about 5% (V/V) or less in some embodiments.

[0059] In one embodiment, an organic solvent component can include a protic solvent. Without wishing to be held to any particular theory, it is understood that inclusion of one or more protic solvents in an activation solution can promote SN1 reactions. Protic solvents can be selected from a group including but not limited to an alcohol (e.g., ethanol, methanol, propanol (1 -propanol, 2-propanol), butanol (n- butanol), etc.), nitromethane, or any combination thereof. In one embodiment, a protic solvent can include a combination of an alcohol (e.g., ethanol) with water. [0060] In one embodiment, an organic solvent can include an aprotic solvent. Without wishing to be held to any particular theory, it is understood that inclusion of one or more aprotic solvents in an activation solution can promote SN2 reactions. Aprotic solvents can be selected from a group including but not limited to dimethyl sulfoxide, dimethyl formamide, acetonitrile, and N-methylpyrrolidinone, etc.

[0061] The concentration of one or more organic solvents in an activation solution can range from about 1 % (V/V) to about 99% (V/V) of the activation solution in some embodiments, such as from about 10% (V/V) to about 95% (V/V) of the activation solution or from about 5% (V/V) to about 30% (V/V) of the activation solution in some embodiments.

[0062] The activation solution can also include a base. In some embodiments, the base can be a weak base. As utilized herein, the term “weak base” generally refers to a base that does not completely dissociate in water. A weak base of an activation solution can include, without limitation, an alkanamine (e.g., methylamine, triethylamine, trimethylamine, tripropylamine, tributylamine, etc.), sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, a pyridine, an imidazole, a benzimidazole, a histidine, a guanidine, a phosphazene base, N,N- dimethylbenzyl amine, 3-dimethylaminopropyl amine, N,N-diisopropylethylamine, N,N-dimethylene diamine, diethylamine, or any combination thereof. A strong base can optionally be utilized in the base component of the activation solution. A strong base can include, without limitation, sodium amide, sodium hydroxide, potassium hydroxide, lithium bis(trimethylsilyl)amide, lithium tert-butoxide, or any combination thereof.

[0063] In one embodiment, a weak base can be present in an activation solution at a concentration in a range from about 0.1 % (V/V) to about 50% (V/V) of the activation solution, such as from about 1% (V/V) to about 30% (V/V) of the activation solution, or from about 3% (V/V) to about 20% (V/V) of the activation solution, in some embodiments. In some embodiments, the concentration of the weak base in the organic solvent component of the activation solution can range from about 0.01 M to about 5 M in some embodiments, such as from about 0.5 M to about 4 M, or from about 0.1 M to about 1 M.

[0064] When utilized, a strong base can be in the activation solution at a concentration in a range from about 0.01% (V/V) to about 10% (V/V) of the activation solution, such as from about 0.01 % (V/V) to about 5% (V/V) of the activation solution, or from about 0.01% (V/V) to about 3% (V/V) of the activation solution, in some embodiments. In some embodiments, the concentration of a strong base in the organic solvent component of the activation solution can range from about 0.01 M to about 0.5 M in some embodiments, such as from about 0.01 M to about 0.2 M, or from about 0.02 M to about 0.1 M.

[0065] To carry out the activation of the substrate, the substrate can be contacted with (e.g., immersed in) the activation solution for a period of time. Optionally, the activation solution can be cooled or heated prior to/during the contact. For instance, the activation step can be carried out with the activation solution held at a temperature ranging from about 0°C to about 100°C, for instance at a temperature ranging from about 10°C to about 90°C, from about 20°C to about 90°C, from about 20°C to about 80°C, from about 20°C to about 70°C, from about 20°C to about 60°C, from about 30°C to about 90°C, from about 30°C to about 80°C, from about 30°C to about 70°C, from about 30°C to about 60°C, from about 30°C to about 50°C, or at a temperature of about 40°C in some embodiments. In some embodiments, the activation step can be carried out in darkness, e.g., in a darkened room with little or no visible light.

[0066] The time for the activation step can vary. In some embodiments the contact time for the activation step ranges from 1 minute to 72 hours. In some embodiments the contact time for the activation step ranges from 0.5 hour to 48 hours. In some embodiments the contact time for the activation step ranges from 10 minutes to 12 hours. In some embodiments the contact time for the activation step ranges from 1 hour to 24 hours. In some embodiments the contact time for the activation step ranges from 0.5 hour to 16 hours. In some embodiments the contact time for the activation step ranges from 0.5 hour to 4 hours. In some embodiments the contact time for the activation step ranges from 20 minutes to 2 hours. In some embodiments the contact time for the activation step ranges from 4 hours to 16 hours.

[0067] In one embodiment, a substrate can be subjected to a pre-treatment prior to contact with the activation solution. For instance, a substrate can be pretreated by contact with an alkaline solution. When included, such a pretreatment can include contacting a substrate with an alkaline solution that includes at least one base and at least one protic solvent. A base can include, without limitation, sodium hydroxide, potassium hydroxide, potassium carbonate, potassium bicarbonate, sodium carbonate, sodium bicarbonate, tris base, etc., or any combination thereof. A protic solvent can include, without limitation, an alcohol (e.g., ethanol, methanol, propanol (1-propanol, 2-propanol), butanol (n-butanol), etc.), nitromethane, water, or any combination thereof.

[0068] When included, an alkaline pretreatment step can generally be carried out a temperature ranging from about 0°C to about 80°C, for instance, at a temperature ranging from about 10°C to about 70°C, about 25°C, or at room temperature in some embodiments. The time of contact for an alkaline pretreatment can vary. In some embodiments the contact time for an alkaline pretreatment time ranges from 1 minute to 72 hours. In some embodiments the contact time for an alkaline pretreatment time ranges from 0.5 hour to 48 hours. In some embodiments the contact time for an alkaline pretreatment time ranges from 10 minutes to 12 hours.

In some embodiments the contact time for an alkaline pretreatment time ranges from 1 hour to 24 hours. In some embodiments the contact time for an alkaline pretreatment time ranges from 0.5 hour to 16 hours. In some embodiments the contact time for an alkaline pretreatment time ranges from 0.5 hour to 4 hours. In some embodiments the contact time for an alkaline pretreatment time ranges from 20 minutes to 2 hours. In some embodiments the contact time for an alkaline pretreatment time ranges from 4 hours to 16 hours. As stated, an alkaline pretreatment is not required in forming an activated substrate or a derivatized substrate.

[0069] Once formed, an activated substrate can be further derivatized via the epoxy-containing linking groups. Beneficially, an activated substrate (e.g., an activated membrane substrate) may not need to be dried following activation. However, in other embodiments, an activated membrane can be dried prior to further derivatization. A drying process is not particularly limited, and can include forced air drying or simple air drying. In general, a drying temperature will be such so as to ensure no damage to the activated substrate, e.g., around room temperature, such as from about 20°C to about 40°C, or from about 25°C to about 30°C in some embodiments. Following drying, a dried activated substrate can maintain the reactive functionality until use. For instance, a dried activated membrane can be stored and/or transported for a period of time (e.g., from a few days to a month or even longer depending upon storage conditions) and following that time can maintain the desired reactive functionality at a surface, for instance for further derivatization. [0070] To further derivatize an activated substrate, a substrate can be contacted (e.g., immersed) in a derivatization solution. A derivatization solution can include an organic solvent, a base, and a derivatization agent. In one embodiment, a derivatization agent can include hydrophobic portion that, upon reaction of the derivatization agent with the reactive functionality of the linking agent of the activated substrate can provide a hydrophobic ligand on a surface of the substrate. [0071] The derivatization agent of a solution can include an epoxy-reactive functionality configured for reaction with an epoxy of the activated substrate and at least one hydrophobic portion. The hydrophobic portion can remain at the substrate surface as a hydrophobic ligand bonded to the substrate surface via the linking agent. For instance, the epoxy-reactive functionality can include, without limitation, a primary or secondary amine, a thioether, an epoxide, a carboxylic acid, an organohalide, etc., or any combination thereof.

[0072] The hydrophobic portion of the derivatization agent can include the hydrophobic ligand. Examples of hydrophobic ligands as may be incorporated on an activated substrate can include, without limitation, aliphatic chains with two or more carbons (e.g., butyl, pentyl, hexyl, septyl, octyl, nonyl, decyl, undecyl, dodecyl), benzyl-containing groups, phenyl-containing groups, phenol-containing groups, pyridine-containing groups, boronic acid groups, branched polymers (e.g., polypropylene glycol), sulfur-containing thiophilic groups (e.g., propanethiol, 2- butanethiol, 3,6-dioxa-1,8-octanedithiol, octanethiol, benzyl mercaptan, 2- mercaptopyridine, thiophenol, 1 ,2-ethanedithiol, 1,4-benzenedimethanethiol, 2- phenylethanethiol), etc., as well as any combination thereof.

[0073] Hydrophobic ligands as may be a component of a derivatization agent and attached to a substrate via reaction of the derivatization agent with an epoxy of the linking agent can be selected from ligands that follow the hydrophobic effect for hydrophobic interaction ligands; pi-pi stacking between immobilized aromatics and the aromatic rings in the nucleobase for aromatic containing ligands, or with electron donation/charge transfer for ligands that contain thioethers, or a combination thereof. [0074] In one embodiment, a hydrophobic ligand of a derivatization agent can exhibit an affinity to polynucleotides, e.g., plasmids. However, the hydrophobic portion of a derivatization agent is not limited to the above-exemplified hydrophobic ligands, and alternative ligands can be incorporated on an activated substrate as described herein.

[0075] Examples of derivatization agents can include, but are not limited to, thiophenol, 2-butanethiol, furfurylthiol, 6-mercaptopurine, 2-mercaptopyridine, 4- mercaptopyridine, 2-mercapto-benzothiazole, propanethiol, cyclopentanethiol, o- mercaptobenzoic acid, dithiothreitol, 1 ,2-ethanedithiol, 3,6-dioxa-1 ,8-octanedithiol,

1 ,4-benzenedimethanethiol, 1 ,3-benzenedimethanethio, 1 ,2-benzenedimethanethio, 4,4’-bis(mercaptomethyl)biphenyl, 2,4-dichlorobenzylmercaptan, 4- methoxybenzylmercaptan, triphenylmethanethiol, 2,4-dimethoxythiophenol, or any combination thereof.

[0076] In one embodiment, the concentration of the derivatization agent in the derivatization solution can range from about 0.01% (W/V) to about 5% (W/V) of the derivatization solution, such as from about 0.03% (W/V) to about 3% (W/V) of the derivatization solution or from about 0.05% (W/V) to about 1 % (W/V) of the derivatization solution in some embodiments.

[0077] The base of the derivatization solution can be the same or differ from the base of an activation solution. In some embodiments, the base can be a weak base. A weak base of a derivatization solution can include, without limitation, an alkanamine (e.g., methylamine, triethylamine, trimethylamine, tripropylamine, tributylamine, etc.), a pyridine, an imidazole, a benzimidazole, a histidine, a guanidine, a phosphazene base, N,N-dimethylbenzyl amine, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, 3- dimethylaminopropyl amine, N,N-diisopropylethylamine, N,N-dimethylene diamine, diethylamine, or any combination thereof. A strong base can optionally be utilized in the base component of the derivatization solution. A strong base can include, without limitation, sodium amide, sodium hydroxide, potassium hydroxide, lithium bis(trimethylsilyl)amide, lithium tert-butoxide, or any combination thereof.

[0078] In one embodiment, the concentration of the base in a derivatization solution can range from about 0.01 % (V/V) of the derivatization solution to about 99% (V/V) of the derivatization solution, such as from about 0.1% (V/V) of the derivatization solution to about 50% (V/V) of the derivatization solution or from about 0.5% (V/V) of the derivatization solution to about 30% (V/V) of the derivatization solution in some embodiments.

[0079] The organic solvent component of a derivatization solution can be the same or differ from an organic solvent component of an activation solution. For instance, an organic solvent of a derivatization solution can include a protic organic solvent, an aprotic organic solvent, an aqueous/organic solvent mixture, or a combination thereof. Examples of solvents as may be utilized in a derivatization solution can include, without limitation, an alcohol (e.g., ethanol, methanol, propanol (1 -propanol, 2-propanol), butanol (n-butanol), etc.), nitromethane, dimethyl sulfoxide, dimethyl formamide, N-methylpyrrolidinone, or any combination thereof. [0080] The concentration of the organic solvent component of a derivatization solution can range from about 0% (V/V) of the derivatization to about 99% (V/V) of the derivatization solution, such as from about 10% (V/V) of the derivatization solution to about 90% (V/V) of the derivatization solution, or from about 20% (V/V) of the derivatization solution to about 85% (V/V) of the derivatization solution in some embodiments.

[0081 ] To carry out a derivatization of the activated substrate, the substrate can be contacted with (e.g., immersed in) the derivatization solution for a period of time. Optionally, the derivatization solution can be cooled or heated prior to/during the contact. For instance, the derivatization step can be carried out with the derivatization solution a temperature ranging from about 0°C to about 80°C, such as at a temperature ranging from about 20°C to about 60°C or about 30°C in some embodiments.

[0082] The time of contact for the derivatization step can vary. In some embodiments the time of contact for the derivatization step ranges from 1 minute to 72 hours. In some embodiments time of contact for the derivatization step ranges from 0.5 hour to 48 hours. In some embodiments time of contact for the derivatization step ranges from 10 minutes to 12 hours. In some embodiments time of contact for the derivatization step ranges from 1 hour to 24 hours. In some embodiments time of contact for the derivatization step ranges from 0.5 hour to 16 hours. In some embodiments the time of contact for the derivatization step ranges from 0.5 hour to 4 hours. In some embodiments time of contact for the derivatization step ranges from 20 minutes to 2 hours. In some embodiments time of contact for the derivatization step ranges from 4 hours to 16 hours.

[0083] In some embodiments, the derivatized substrate can be dried prior to use. In other embodiments, the derivatized substrate can be used immediately following derivatization. A drying process when incorporated is not particularly limited, and can include forced air drying or simple air drying. In general, a drying temperature will be such so as to ensure no damage to the derivatized substrate, e.g., around room temperature, such as from about 20°C to about 40°C, or from about 25°C to about 30°C in some embodiments. Following drying, a dried derivatized substrate can maintain the hydrophobic ligand until use. For instance, a dried activated membrane can be stored and/or transported for a period of time (e.g., from a few days to several months or even several years, depending on storage conditions) and following that time can maintain the desired hydrophobic ligand for use in an HIC protocol.

[0084] In one embodiment a derivatized substrate can be further processed for use. For instance, a derivatized substrate can be processed (e.g., shaped, stacked, combined, retained, etc.) to form a derivatized separation medium for use in an HIC protocol. In one embodiment, a derivatized separation medium can be formed from one or more derivatized membranes, for instance a plurality of derivatized membranes that can be stacked and/or shaped as desired to form a separation medium for use in an HIC protocol.

[0085] By way of example, multiple derivatized membrane substrates can be stacked to form a multi-layer arrangement to increase capacity for a given application. In one embodiment, a stacked arrangement of derivatized membranes can have a thickness of about 70 micrometers to about 10,000 micrometers, such as about 10,000 micrometers or greater, about 7,500 micrometers or greater, about 5,000 micrometers or greater, about 2,500 micrometers or greater, about 1 ,000 micrometers or greater, about 900 micrometers or greater, about 800 micrometers or greater, about 700 micrometers or greater, about 600 micrometers or greater, about 500 micrometers or greater, about 400 micrometers or greater, about 300 micrometers or greater, about 200 micrometers or greater, about 100 micrometers or greater, about 70 micrometers or greater, such as about 70 micrometers to about 100 micrometers, about 70 micrometers to about 200 micrometers, about 70 micrometers to about 300 micrometers, about 70 micrometers to about 400 micrometers, about 70 micrometers to about 500 micrometers, about 70 micrometers to about 750 micrometers, about 70 micrometers to about 1 ,000 micrometers, about 70 micrometers to about 2,000 micrometers, about 70 micrometers to about 3,000 micrometers, about 70 micrometers to about 4,000 micrometers, about 70 micrometers to about 5,000 micrometers, about 250 micrometers to about 300 micrometers, about 250 micrometers to about 400 micrometers, about 250 micrometers to about 500 micrometers, about 250 micrometers to about 750 micrometers, about 250 micrometers to about 1 ,000 micrometers, about 250 micrometers to about 2,000 micrometers, about 250 micrometers to about 3,000 micrometers, about 250 micrometers to about 4,000 micrometers, about 250 micrometers to about 5,000 micrometers, about 500 micrometers to about 1 ,000 micrometers, about 500 micrometers to about 2,000 micrometers, about 500 micrometers to about 3,000 micrometers, about 500 micrometers to about 4,000 micrometers, or about 500 micrometers to about 5,000 micrometers.

[0086] Flow rates of separation media disclosed herein can be, for example, from about 0.5 column volumes (CV)/min to about 1000 CV/min, from about 1 CV/min to about 1000 CV/min, from about 2 CV/min to about 1000 CV/min, from about 3 CV/min to about 1000 CV/min, from about 4 CV/min to about 1000 CV/min, from 5 about CV/min to about 1000 CV/min, from 6 about CV/min to about 1000 CV/min, from about 0.5 CV/min to about 500 CV/min, from about 1 CV/min to 500 CV/min, from about 2 CV/min to about 500 CV/min, from about 3 CV/min to about 500 CV/min, from about 4 CV/min to about 500 CV/min, from about 5 CV/min to about 500 CV/min, from about 6 CV/min to about 500 CV/min, from about 0.5 CV/min to about 100 CV/min, from about 1 CV/min to about 100 CV/min, from about 2 CV/min to about 100 CV/min, from about 3 CV/min to about 100 CV/min, from about 4 CV/min to about 100 CV/min, from about 5 CV/min to about 100 CV/min, from about 6 CV/min to about 100 CV/min, from about 0.5 CV/min to about 50 CV/min, from about 1 CV/min to about 50 CV/min, from about 2 CV/min to about 50 CV/min, from about 3 CV/min to about 50 CV/min, from about 4 CV/min to about 50 CV/min, from about 5 CV/min to about 50 CV/min, or from about 6 CV/min to about 50 CV/min.

[0087] As utilized herein, the term “column volume” refers to the volume of the membrane bed within the column according to standard practice. Thus, when considering a bed volume formed from a stack of derivatized membrane substrates, the column volume can be determined as the total volume of the membrane stack, i.e. , the stack thickness times the footprint area of the stack.

[0088] In one embodiment, a separation medium can be utilized for purifying pDNA and other polynucleotides including, without limitation, pDNA (including sc pDNA, oc pDNA, relaxed circular pDNA, linear pDNA, and supercoiled denatured pDNA), mRNA, tRNA, rRNA, miRNA, siRNA, nucleic acid products yielded from rolling circle amplification, snRNA, piRNA, dsRNA, genomic DNA, ssDNA, dsDNA, A-DNA, B-DNA, C-DNA, Z-DNA, aptamers, etc.

[0089] Separation systems incorporating separation media as described herein can include separation columns as known in the art. By way of example, separation columns encompassed herein can include, without limitation, syringe filter columns, spin columns, cassettes, multi-well plates, and spiral-wound membrane columns, etc.

[0090] A separation protocol using disclosed materials can be run via standard methodologies. In one embodiment, a separation column incorporating a derivatized HIC membrane as described herein can operate in a bind-and-elute mode using elution methods as are generally known in the art.

[0091] Separations using disclosed HIC separation media can purify polynucleotides rapidly and efficiently at a fast flow rate. For instance, a polynucleotide purification method utilizing separation media as disclosed herein can provide a higher than 80% recovery of targeted materials having a purity of about 80% or greater. For instance, a separation medium (e.g., a single derivatized membrane or multiple derivatized membranes stacked together) can have a dynamic binding capacity greater than 1 mg polynucleotide (e.g., pDNA) per milliliter separation media at residence time of less than 120 seconds.

[0092] Separation systems as disclosed can exhibit high process productivity. Process productivity of a column can be defined using the equation below. In the denominator, Vtot is the total volume of solution passing through the column during the whole process, including load, rinse, elution, and regeneration steps. BV is the HIC medium bed volume, and t is residence time. Loading volume is proportional to dynamic binding capacity of the HIC medium. Thus, process productivity increases with increasing binding capacity and decreasing residence time.

Polynucleotides captured

Productivity = Cost of time

Loading volume x polynucleotides concentration x yield

[0093] Dynamic binding capacity generally refers to the concentration of bound polynucleotide on the separation media (milligram bound per unit volume of membrane bed) at breakthrough in the effluent. In embodiments, disclosed separation systems can provide a dynamic binding capacity for a polynucleotide at a residence time of 120 seconds and utilizing an ammonium sulfate buffer concentration of 2.5 M of from about 1 to about 5 mg per mL media, such as from about 1 mg/mL to about 10 mg/mL, about 1 mg/mL to about 20 mg/mL, about 1 mg/mL to about 25 mg/mL, about 1 mg/mL to about 30 mg/mL, about 2 mg/mL to about 5 mg/mL, about 2 mg/mL to about 10 mg/mL, about 2 mg/mL to about 15 mg/mL, about 2 mg/mL to about 20 mg/mL, about 2 mg/mL to about 25 mg/mL, about 2 mg/mL to about 30 mg/mL, about 3 mg/mL to about 5 mg/mL, about 3 mg/mL to about 10 mg/mL, about 3 mg/mL to about 15 mg/mL, about 3 mg/mL to about 20 mg/mL, about 3 mg/mL to about 25 mg/mL, about 3 mg/mL to about 30 mg/mL, about 4 mg/mL to about 5 mg/mL, about 4 mg/mL to about 10 mg/mL, about 4 mg/mL to about 15 mg/mL, about 4 mg/mL to about 20 mg/mL, about 4 mg/mL to about 25 mg/mL, about 4 mg/mL to about 30 mg/mL, about 5 mg/mL to about 10 mg/mL, about 5 mg/mL to about 15 mg/mL, about 5 mg/mL to about 20 mg/mL, about 5 mg/mL to about 25 mg/mL, or about 5 mg/mL to about 30 mg/mL. As previously mentioned, disclosed separation media can exhibit a substantially similar dynamic binding capacity independent of target polynucleotide size. As such, dynamic binding capacity of a separation system can be substantially similar to the same system when purifying and different polynucleotide, either a larger or a smaller polynucleotide. Dynamic binding capacities that are substantially similar to one another can be within about 10% of one another or less, such as within about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, or about 3% or less, including, in some cases, exhibiting an identical dynamic binding capacity.

[0094] Known commercial HIC column products operate at a residence time of 180 seconds, with a dynamic binding capacity of less than 0.1 mg/mL for 3k bps pDNA. For two media with the same dynamic binding capacity that achieve the same product yield, the ratio of load productivities can be estimated by the inverse ratio of residence times. Thus, in one embodiment, compared to separation media including a derivatized membrane as described in the present disclosure having a 4 mg/mL dynamic binding capacity at about 2 seconds or less residence time, the load productivity of separation media herein described can be 3600 times (= (4/0.1) x (180 s/2 s)) that of currently known commercial HIC column products for plasmid purification. There are no known column products currently available that approach such productivity as achieved by the present invention.

[0095] The following is a list of exemplary aspects of the products and methods according to the present disclosure. [0096] According to aspect 1 , an HIC separation medium comprising a porous cellulose membrane and a plurality of a hydrophobic ligand bonded to a surface of the porous cellulose membrane is disclosed.

[0097] Aspect 2 is the HIC separation membrane of aspect 1 , wherein the porous cellulose membrane includes a casted membrane, a hydrogel membrane or a fibrous membrane, such as an electrospun nanofibrous membrane.

[0098] Aspect 3 is the HIC separation membrane of aspect 1 or aspect 2, wherein the porous cellulose membrane includes regenerated cellulose or a cellulose derivative.

[0099] Aspect 4 is the HIC separation membrane of any of the preceding aspects, wherein the porous cellulose membrane comprises a specific surface area as determined according to BET measurement using nitrogen as the adsorbate of from about 0.1 m²/ml_ to about 30 m²/ml_, from about 0.1 m²/ml_ to about 25 m²/ml_, from about 0.1 m²/ml_ to about 20 m²/ml_, from about 0.1 m²/ml_ to about 15 m²/ml_, from about 0.1 m²/ml_ to about 10 m²/ml_, from about 0.5 m²/ml_ to about 30 m²/ml_, from about 0.5 m²/ml_ to about 25 m²/ml_, from about 0.5 m²/ml_ to about 20 m²/ml_, from about 0.5 m²/ml_ to about 15 m²/ml_, from about 0.5 m²/ml_ to about 10 m²/ml_, from about 0.5 m²/ml_ to about 5 m²/ml_, from about 1 m²/ml_ to about 30 m²/ml_, from about 1 m²/ml_ to about to about 25 m²/ml_, from about 1 m²/ml_ to about 20 m²/ml_, from about 1 m²/ml_ to about 15 m²/ml_ , from about 1 m²/ml_ to about 10 m²/ml_, from about 1 m²/ml_ to about 5 m²/ml_, from about 5 m²/ml_ to about 30 m²/ml_, from about 5 m²/ml_ to about to about 25 m²/ml_, from about 5 m²/ml_ to about 20 m²/ml_, from about 5 m²/ml_ to about 15 m²/ml_, or from about 5 m²/ml_ to about 10 m²/ml_. [00100] Aspect 5 is the HIC separation membrane of any of the preceding aspects, wherein the porous cellulose membrane comprises a pore size of from about 0.1 micrometer to about 10 micrometers, from about 0.1 micrometer to about 0.2 micrometers, from about 0.1 micrometer to about 0.45 micrometers, from about 0.1 micrometer to about 10 micrometers, from about 0.1 micrometer to about 2 micrometers, from about 0.2 micrometer to about 0.45 micrometers, from about 0.2 micrometers to about 1 micrometer, from about 0.2 micrometer to about 2 micrometers, from about 0.2 micrometer to about 10 micrometers, from about 0.45 micrometer to about 1 micrometer, from about 0.45 micrometers to about 2 micrometers, from about 0.45 micrometer to about 10 micrometers, from about 1 micrometer to about 2 micrometers, or from about 1 micrometer to about 5 micrometers.

[00101 ] Aspect 6 is the HIC separation membrane of any of the preceding aspects, wherein the medium comprises a plurality of membranes stacked together, the stack comprising the porous cellulose membrane. For instance, the stack can have a thickness of from 70 micrometers to 10,000 micrometers, such as about 10,000 micrometers or greater, about 7,500 micrometers or greater, about 5,000 micrometers or greater, about 2,500 micrometers or greater, about 1 ,000 micrometers or greater, about 900 micrometers or greater, about 800 micrometers or greater, about 700 micrometers or greater, about 600 micrometers or greater, about 500 micrometers or greater, about 400 micrometers or greater, about 300 micrometers or greater, about 200 micrometers or greater, about 100 micrometers or greater, about 70 micrometers or greater, such as about 70 micrometers to about 100 micrometers, about 70 micrometers to about 200 micrometers, about 70 micrometers to about 300 micrometers, about 70 micrometers to about 400 micrometers, about 70 micrometers to about 500 micrometers, about 70 micrometers to about 750 micrometers, about 70 micrometers to about 1 ,000 micrometers, about 70 micrometers to about 2,000 micrometers, about 70 micrometers to about 3,000 micrometers, about 70 micrometers to about 4,000 micrometers, about 70 micrometers to about 5,000 micrometers, about 250 micrometers to about 300 micrometers, about 250 micrometers to about 400 micrometers, about 250 micrometers to about 500 micrometers, about 250 micrometers to about 750 micrometers, about 250 micrometers to about 1 ,000 micrometers, about 250 micrometers to about 2,000 micrometers, about 250 micrometers to about 3,000 micrometers, about 250 micrometers to about 4,000 micrometers, about 250 micrometers to about 5,000 micrometers, about 500 micrometers to about 1 ,000 micrometers, about 500 micrometers to about 2,000 micrometers, about 500 micrometers to about 3,000 micrometers, about 500 micrometers to about 4,000 micrometers, or about 500 micrometers to about 5,000 micrometers..

[00102] Aspect 7 is the HIC separation membrane of any of the preceding aspects, wherein the hydrophobic ligands comprise an aliphatic chain with two or more carbons, a benzyl-containing group, a phenyl-containing group, a phenol-containing group, a pyridine-containing group, a boronic acid group, a branched polymer, a sulfur-containing thiophilic group, or any combination thereof. Moreover, each of the plurality of hydrophobic ligands can be bonded to the surface of the porous cellulose membrane via a linking group, the linking group comprising the reaction product of an epoxy and an epoxy-reactive functionality, for instance an amine or a thioether moiety.

[00103] Aspect 8 is the HIC separation membrane of any of the preceding aspects, wherein the medium has a polynucleotide dynamic binding capacity at a residence time of 120 seconds and utilizing an ammonium sulfate buffer concentration of 2.5 M that is greater than about 1 mg/mL, such as from about 1 mg/mL to about 10 mg/mL, about 1 mg/mL to about 20 mg/mL, about 1 mg/mL to about 25 mg/mL, about 1 mg/mL to about 30 mg/mL, about 2 mg/mL to about 5 mg/mL, about 2 mg/mL to about 10 mg/mL, about 2 mg/mL to about 15 mg/mL, about 2 mg/mL to about 20 mg/mL, about 2 mg/mL to about 25 mg/mL, about 2 mg/mL to about 30 mg/mL, about 3 mg/mL to about 5 mg/mL, about 3 mg/mL to about 10 mg/mL, about 3 mg/mL to about 15 mg/mL, about 3 mg/mL to about 20 mg/mL, about 3 mg/mL to about 25 mg/mL, about 3 mg/mL to about 30 mg/mL, about 4 mg/mL to about 5 mg/mL, about 4 mg/mL to about 10 mg/mL, about 4 mg/mL to about 15 mg/mL, about 4 mg/mL to about 20 mg/mL, about 4 mg/mL to about 25 mg/mL, about 4 mg/mL to about 30 mg/mL, about 5 mg/mL to about 10 mg/mL, about 5 mg/mL to about 15 mg/mL, about 5 mg/mL to about 20 mg/mL, about 5 mg/mL to about 25 mg/mL, or about 5 mg/mL to about 30 mg/mL.

[00104] Aspect 9 is the HIC separation membrane of any of the preceding aspects, wherein the porous cellulose membrane is a self-supporting membrane or wherein the medium comprises the porous cellulose membrane and a backing material. [00105] Aspect 10 is a separation column comprising the hydrophobic interaction chromatography separation medium of any of the preceding aspects. For instance, wherein the separation column comprises a syringe filter column, a spin column, a cassette, or a spiral-wound membrane column.

[00106] Aspect 11 is a method for forming an activated substrate that includes contacting a substrate with an activation solution. The activation solution includes an activation agent, a base, and an organic solvent. The activation agent includes a reactive functionality configured to react with a surface of the substrate to form a linking group on the surface, the activation agent further includes an epoxy group, the linking group comprising the epoxy group. [00107] Aspect 12 is the method of aspect 11 , wherein the substrate comprises a film, a porous membrane, a monolith, a nanofiber mat, or a resin, and wherein the substrate comprises cellulose, regenerated cellulose, a cellulose derivative, a nylon, a polysulfone, a polyether sulfone, a polyvinylidene fluoride, a polyacrylonitrile, a polyetherimide, a polypropylene, a polyethylene, ora polyether terephthalate.

[00108] Aspect 13 is the method of aspect 11 or aspect 12, wherein the active agent comprises an epichlorohydrin, a diglycidyl ether, or any combination thereof. [00109] Aspect 14 is a method for derivatizing the activated substrate formed according to the method of aspect 11 , 12, or 13, the method comprising contacting the activated substrate with a derivatization solution, the derivatization solution comprising a derivatization agent, a base, and optionally comprising an organic solvent, the derivatization agent comprising an epoxy-reactive functionality and a hydrophobic portion, the hydrophobic portion comprising a hydrophobic ligand.

[00110] Aspect 15 is the method of aspect 14, wherein the hydrophobic ligand comprises an aliphatic chain with two or more carbons, a benzyl-containing group, a phenyl-containing group, a phenol-containing group, a pyridine-containing group, a boronic acid group, a branched polymer, a sulfur-containing thiophilic group, or any combination thereof.

[00111 ] Aspect 16 is the method of aspect 14 or aspect 15, wherein the derivatization agent comprises thiophenol, 2-butanethiol, furfurylthiol, 6- mercaptopurine, 2-mercaptopyridine, 2-mercapto-benzothiazole, propanethiol, cyclopentanethiol, o-mercaptobenzoic acid, dithiothreitol, 1 ,2-ethanedithiol, 3,6- dioxa-1 ,8-octanedithiol, 1 ,4-benzenedimethanethiol, 1 ,3-benzenedimethanethio, 1,2- benzenedimethanethio, 4,4’-bis(mercaptomethyl)biphenyl, 2,4- dichlorobenzylmercaptan, 4-methoxybenzylmercaptan, triphenylmethanethiol, 2,4- dimethoxythiophenol, or any combination thereof.

[00112] Aspect 17 is the method of any of aspects 11-16, wherein the organic solvent comprises a protic organic solvent or an aprotic organic solvent or a combination thereof, for instance wherein the organic solvent comprises an alcohol, nitromethane, or any combination thereof.

[00113] Aspect 18 is the method of any of aspects 11-17, wherein the base comprises an alkanamine (e.g., methylamine, triethylamine, trimethylamine, tripropylamine, tributylamine), a pyridine, an imidazole, a benzimidazole, a histidine, a guanidine, a phosphazene base, N,N-dimethylbenzyl amine, 3- dimethylaminopropyl amine, N,N-diisopropylethylamine, N,N-dimethylene diamine, diethylamine, sodium amide, sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, or potassium bicarbonate. [00114] The present disclosure may be better understood with reference to the examples, provided below.

Testing Methods

[00115] To form the chromatographic columns of the Examples, two layers of 24 mm circular HIC membrane disks were packed into a membrane housing (membrane volume = 0.055 ml_) to measure DBCio% values.

[00116] The chromatography tests were conducted using Fast Protein Liquid chromatography system (AKTA™ pure 25, Cytiva). Four sizes of plasmid were tested for examples. Plasmid 1) pRP[Exp]-CMV>EGFP, 3657 bp 2) pRP[Exp]- Alb>hCas9, 8956 bp and 3) pLV[Exp]-EGFP:T2A:Bsd-Alb>dCas9/VPR, 16129 bp are purified using Qiagen^® Mega kit (Cat. No. 12281). pALD-HELP, 11584bp was purchased from vendor. Plasmid stock was diluted to a concentration of 6-13 pg/mL. Various concentrations of ammonium sulfate in 40 mM 1xTE (Tris-HCI), were used as loading buffer.

[00117] Dynamic binding capacity was determined as the retained plasmid concentration (milligram plasmid bound per unit volume of membrane bed) at breakthrough in the effluent. A dynamic binding capacity at 10% breakthrough (DBCio%) represents the mass bound per unit volume of membrane bed when the plasmid concentration in the effluent from the membrane bed reached 10% of the feed.

Example 1

[00118] Activation: A 10x60 cm regenerated cellulose membrane sheet with a nominal pore size of 1 pm was activated by soaking it in a solution containing 9.2% (v/v) epichlorohydrin (EPI), 5.4% (v/v) triethylamine (TEA) and 85.4% (v/v) ethanol (EtOH). The reaction was performed with overnight shaking at 120 rpm at room temperature in darkness. After 20-24 hours, the membrane was rinsed with EtOH for five minutes followed by two, five-minute acetone rinses. Subsequently, the membrane sheet was dried at room temperature.

[00119] MCP incorporation: The activated membrane was treated with 82 mM MCP dissolved in a solution containing 80.7% (v/v) methanol, 8.6% (v/v) deionized (Dl) water, 9.6% (v/v) 5M sodium hydroxide (NaOH) and 1.1% (v/v) TEA. The reaction was performed with overnight shaking at 100 rpm at room temperature in darkness. After 20-24 hours, the membrane was rinsed with ethanol for five minutes followed by two, five-minute acetone rinses. Subsequently, the membrane sheet was dried at room temperature.

Example 2

[00120] Activation: Three epichlorohydrin activated membranes (Membrane 1 , Membrane 2, and Membrane 3) were prepared in the same manner as described in Example 1 , except that three reaction times were applied: one hour reaction time for membrane 1 , three hours reaction time for membrane 2 and overnight (20-24 hours) reaction time for membrane 3. Reactions were performed on a shaker at 120 rpm and room temperature in darkness. After reaction, membranes were rinsed and dried following the same procedure as described in Example 1.

[00121] MCP incorporation: Dried activated membranes were derivatized in the same manner as in Example 1.

[00122] FIG. 1 shows that the HIC membrane binding capacity in terms of DBCio% increased with the extended reaction time. For the same activation solution and the same incorporation processes, 20-24 hours reaction time brought a 59% increase of binding capacity compared to that yielded from 1 hour reaction.

Example 3

[00123] Activation: Two epichlorohydrin activated membranes (Membrane 4 and Membrane 5) were prepared in the same manner as Membrane 2 in Example 1 with 3 hours activation time. Flowever, two changes were applied to the formula: 1 ) Membrane 4 was soaked in activation solution with double EPI concentration of 16.8% v/v; 2) Membrane 5 was soaked in activation solution with EtOFI replaced by the same amount of 2-propanol (IPA).

[00124] MCP incorporation: Dried activated membranes were derivatized in the same manner as in Example 1.

[00125] As shown in FIG. 2, double EPI concentration resulted in an 25% increase of binding capacity for Membrane 4 as compared to Membrane 2. Also, the replacement of EtOH by IPA caused little or no change in binding capacity between Membrane 5 and Membrane 2, which indicates that IPA can be used to replace EtOH to make said membranes in some situations. Example 4

[00126] Alkaline treatment: 0.3 M NaOH in 100% ethanol was prepared one day before the reaction and left on stir plate overnight. A 47 mm 0.22 pm regenerated cellulose membrane filter was used to clarify the solution right before the reaction. [00127] Four regenerated cellulose membrane strips and two alkaline treatments were used:

Set I) membrane A and B were soaked in 0.3 M NaOH dissolved in 100% ethanol for 30 minutes on a shaker at 80 rpm at room temperature, and then were transferred to soak in 0.1 M NaOH in Dl for additional 30 minutes on shaker at 80 rpm and room temperature;

Set II) membrane C and D were soaked in 0.3 M NaOH dissolved in 100% ethanol for 30 minutes on a shaker at 80 rpm and room temperature.

[00128] Membranes A and C were rinsed with 200 mM Tris pH 7 for five minutes followed by Dl water for five minutes, and then two, five-minute acetone rinses. Subsequently, these membranes were dried at room temperature under a proper forced air condition. Meanwhile, membrane B and D were rinsed with 200 mM Tris pH 7 for five minutes, Dl water for five minutes followed by ethanol for five minutes. Subsequently, membrane B and D were transferred to activation solution.

[00129] Activation: Membranes were activated in the same manner as in Example 1.

[00130] MCP incorporation: Dried activated membranes were coupled in the same manner as in Example 1.

[00131] As shown in FIG. 3, the alkaline treatment improved the structural integrity of the membrane. FIG. 4 compares the binding capacities of membranes prepared using different alkaline pre-treatments. In FIG.4, membranes treated by 0.3 M NaOH in EtOH had similar binding capacity as membranes treated by 0.1 M NaOH in water in the same manner.

Example 5

[00132] Alkaline treatment: 0.3 M NaOH in 100% ethanol (EtOH) was prepared one day before the process and left on a stir plate overnight. A 47 mm 0.22 pm cellulose acetate (CA) membrane filter was used to clarify the solution right before the reaction.

[00133] Three membrane strips and two alkaline treatments were used: Set I) Membrane C and F was soaked in 0.3 M NaOH dissolved in 100% EtOH for 30 minutes on a shaker at 80 rpm and room temperature.

Set II) Membrane G and Membrane H were soaked in 0.1 M NaOH dissolved in Dl for 30 minutes on a shaker at 80 rpm and room temperature.

[00134] Following the alkaline treatment, Membrane C and Membrane G were rinsed with 200 mM Tris pH 7 for five minutes, followed by Dl rinsing for five minutes, and then two acetone rinses. Membrane C and Membrane G were subsequently dried at room temperature under a forced air condition prior to activation.

[00135] Following the alkaline treatment, Membrane F and H was rinsed with 200 mM Tris pH 7 for five minutes, followed by Dl rinsing for five minutes, and then EtOH rinsing for five minutes. Membrane-F and-H were not dried prior to activation.

[00136] Activation: Membrane C and Membrane G were rinsed with EtOH and acetone and then transferred to an incorporation solution as in Example 1.

[00137] Membrane F and H were rinsed with EtOH for five minutes followed by two Dl rinses each rinse being for five minutes and then transferred directly to an incorporation solution as in Example 1.

[00138] MCP incorporation: Membranes were derivatized in the same manner as in Example 1.

[00139] FIG. 4 shows that membranes (F and H) prepared without drying steps had higher binding capacities than membranes (C and G) prepared with drying steps. For alkaline treatment, the membrane (F) treated by 0.3 M NaOH in 100% ethanol had slightly higher binding capacity than membrane (H) which was treated by 0.1 M NaOH dissolved in Dl.

Example 6

[00140] Chromatography tests were conducted using columns packed with Membrane 3 from Example 2 as the media. Plasmid size was 3657 bp. 3 M ammonium sulfate in 40 mM Tris-HCI, pH 8 was used as loading buffer. FIG. 5 shows a resulting chromatogram illustrated the loading, washing, and elution phases of a separation. Elution peaks were similar among all the tests. UV260 absorbance values and buffer volumes vary from membrane to membrane. UV260 absorbance values and buffer volumes varied from membrane to membrane.

[00141 ] The chromatography test was run again under the same conditions but for a decrease in the loading buffer from pH 8 to pH 5. FIG. 6 shows the results for binding capacity, illustrating a 30% increase of binding capacity observed when the pH of the loading buffer was decreased.

Example 7

[00142] Chromatography tests were conducted under various conditions using columns packed with Membrane 3 of Example 2 as the media to further examine the materials.

[00143] Initially, the media was examined for use with plasmids of different sizes, including 3 kbp, 8 kbp, and 16 kbp. The loading buffer for all runs utilized 2.5 M ammonium sulfate and 1xTE buffer at a pH of 7.5 and a flow rate of 2 mL/min (36.4 CV/min). Results for the dynamic binding capacity (DBC) are provided in Table 1 , below, and FIG. 7.

Table 1

[00144] As indicated, the dynamic binding capacity of the media was essentially independent of plasmid size.

[00145] Media was examined for variation in flow rate for effect on dynamic binding capacity. An 8 kbp plasmid was utilized. The loading buffer was 2.5 M ammonium sulfate and 1xTE buffer at a pH of 7.5. Results are provided in Table 2, below, and FIG. 8.

Table 2

[00146] Different ammonium sulfate concentrations were also examined for effect on dynamic binding capacity. An 8 kbp plasmid was utilized with the flow rate set to 2 mL/min for all runs. Results are provided in Table 3, below, and FIG. 9.

Table 3

[00147] The Membrane 3 chromatography column was compared to a pre-packed commercial resin column (Cytiva HiScreen™ PlasmidSelect, 4.7 ml_). The loading buffer was 3 M ammonium sulfate and 1xTE at a pH of 7.5. The plasmid was 11 kbp. Results are shown in Table 4, below and FIG. 10.

Table 4

[00148] While certain embodiments of the disclosed subject matter have been described using specific terms, such description is for illustrative purposes only, and it is to be understood that changes and variations may be made without departing from the spirit or scope of the subject matter.

Claims

WHAT IS CLAIMED IS:

1. A hydrophobic interaction chromatography separation medium comprising a porous cellulose membrane and a plurality of a hydrophobic ligand bonded to a surface of the porous cellulose membrane.

2. The hydrophobic interaction chromatography separation medium of claim 1 , wherein the porous cellulose membrane comprises a casted membrane, hydrogel membrane or a fibrous membrane, such as an electrospun nanofibrous membrane.

3. The hydrophobic interaction chromatography separation medium of claim 1 or claim 2, wherein the porous cellulose membrane comprises cellulose, regenerated cellulose or a cellulose derivative.

4. The hydrophobic interaction chromatography separation medium of any of the preceding claims, wherein the porous cellulose membrane comprises a specific surface area of from about 0.1 m²/mL to about 30 m²/mL, from about 0.1 m²/mL to about 25 m²/mL, from about 0.1 m²/mL to about 20 m²/mL, from about 0.1 m²/mL to about 15 m²/mL, from about 0.1 m²/mL to about 10 m²/mL, from about 0.5 m²/mL to about 30 m²/mL, from about 0.5 m²/mL to about 25 m²/mL, from about 0.5 m²/mL to about 20 m²/mL, from about 0.5 m²/mL to about 15 m²/mL, from about 0.5 m²/mL to about 10 m²/mL, from about 0.5 m²/mL to about 5 m²/mL, from about 1 m²/mL to about 30 m²/mL, from about 1 m²/mL to about to about 25 m²/mL, from about 1 m²/mL to about 20 m²/mL, from about 1 m²/mL to about 15 m²/mL , from about 1 m²/mL to about 10 m²/mL, from about 1 m²/mL to about 5 m²/mL, from about 5 m²/mL to about 30 m²/mL, from about 5 m²/mL to about to about 25 m²/mL, from about 5 m²/mL to about 20 m²/mL, from about 5 m²/mL to about 15 m²/mL, or from about 5 m²/mL to about 10 m²/mL.

5. The hydrophobic interaction chromatography separation medium of any of the preceding claims, wherein the porous cellulose membrane comprises a pore size of from about 0.1 micrometer to about 10 micrometers, from about 0.1 micrometer to about 0.2 micrometers, from about 0.1 micrometer to about 0.45 micrometers, from about 0.1 micrometer to about 10 micrometers, from about 0.1 micrometer to about 2 micrometers, from about 0.2 micrometer to about 0.45 micrometers, from about 0.2 micrometers to about 1 micrometer, from about 0.2 micrometer to about 2 micrometers, from about 0.2 micrometer to about 10 micrometers, from about 0.45 micrometer to about 1 micrometer, from about 0.45 micrometers to about 2 micrometers, from about 0.45 micrometer to about 10 micrometers, from about 1 micrometer to about 2 micrometers, or from about 1 micrometer to about 5 micrometers.

6. The hydrophobic interaction chromatography separation medium of any of the preceding claims, wherein the medium comprises a plurality of membranes stacked together, the stack comprising the porous cellulose membrane, the stack having a thickness of from 70 micrometers to 10,000 micrometers, such as about 10,000 micrometers or greater, about 7,500 micrometers or greater, about 5,000 micrometers or greater, about 2,500 micrometers or greater, about 1 ,000 micrometers or greater, about 900 micrometers or greater, about 800 micrometers or greater, about 700 micrometers or greater, about 600 micrometers or greater, about 500 micrometers or greater, about 400 micrometers or greater, about 300 micrometers or greater, about 200 micrometers or greater, about 100 micrometers or greater, about 70 micrometers or greater, such as about 70 micrometers to about 100 micrometers, about 70 micrometers to about 200 micrometers, about 70 micrometers to about 300 micrometers, about 70 micrometers to about 400 micrometers, about 70 micrometers to about 500 micrometers, about 70 micrometers to about 750 micrometers, about 70 micrometers to about 1 ,000 micrometers, about 70 micrometers to about 2,000 micrometers, about 70 micrometers to about 3,000 micrometers, about 70 micrometers to about 4,000 micrometers, about 70 micrometers to about 5,000 micrometers, about 250 micrometers to about 300 micrometers, about 250 micrometers to about 400 micrometers, about 250 micrometers to about 500 micrometers, about 250 micrometers to about 750 micrometers, about 250 micrometers to about 1 ,000 micrometers, about 250 micrometers to about 2,000 micrometers, about 250 micrometers to about 3,000 micrometers, about 250 micrometers to about 4,000 micrometers, about 250 micrometers to about 5,000 micrometers, about 500 micrometers to about 1 ,000 micrometers, about 500 micrometers to about 2,000 micrometers, about 500 micrometers to about 3,000 micrometers, about 500 micrometers to about 4,000 micrometers, or about 500 micrometers to about 5,000 micrometers.

7. The hydrophobic interaction chromatography separation medium of any of the preceding claims, wherein the hydrophobic ligands comprise an aliphatic chain with two or more carbons, a benzyl-containing group, a phenyl-containing group, a phenol-containing group, a pyridine-containing group, a boronic acid group, a branched polymer, a sulfur-containing thiophilic group, or any combination thereof, and wherein each of the plurality of hydrophobic ligands is bonded to the surface of the porous cellulose membrane via a linking group, the linking group comprising the reaction product of an epoxy and an epoxy-reactive functionality, for instance an amine or a thioether moiety.

8. The hydrophobic interaction chromatography separation medium of any of the preceding claims, wherein the medium has a polynucleotide dynamic binding capacity at a residence time of 120 seconds and utilizing an ammonium sulfate buffer concentration of 2.5 M that is greater than about 1 mg/mL, such as from about 1 mg/mL to about 10 mg/mL, about 1 mg/mL to about 20 mg/mL, about 1 mg/mL to about 25 mg/mL, about 1 mg/mL to about 30 mg/mL, about 2 mg/mL to about 5 mg/mL, about 2 mg/mL to about 10 mg/mL, about 2 mg/mL to about 15 mg/mL, about 2 mg/mL to about 20 mg/mL, about 2 mg/mL to about 25 mg/mL, about 2 mg/mL to about 30 mg/mL, about 3 mg/mL to about 5 mg/mL, about 3 mg/mL to about 10 mg/mL, about 3 mg/mL to about 15 mg/mL, about 3 mg/mL to about 20 mg/mL, about 3 mg/mL to about 25 mg/mL, about 3 mg/mL to about 30 mg/mL, about 4 mg/mL to about 5 mg/mL, about 4 mg/mL to about 10 mg/mL, about 4 mg/mL to about 15 mg/mL, about 4 mg/mL to about 20 mg/mL, about 4 mg/mL to about 25 mg/mL, about 4 mg/mL to about 30 mg/mL, about 5 mg/mL to about 10 mg/mL, about 5 mg/mL to about 15 mg/mL, about 5 mg/mL to about 20 mg/mL, about 5 mg/mL to about 25 mg/mL, or about 5 mg/mL to about 30 mg/mL.

9. The hydrophobic interaction chromatography separation medium of any of the preceding claims, wherein the porous cellulose membrane is a self-supporting membrane or wherein the medium comprises the porous cellulose membrane and a backing material.

10. A separation column comprising the hydrophobic interaction chromatography separation medium of any of the preceding claims, wherein the separation column comprises a syringe filter column, a spin column, a cassette, or a spiral-wound membrane column.

11. A method for forming an activated substrate comprising contacting a substrate with an activation solution, the activation solution comprising an activation agent, a base, and an organic solvent, the activation agent including a reactive functionality configured to react with a surface of the substrate to form a linking group on the surface, the activation agent further including an epoxy group, the linking group comprising the epoxy group.

12. The method of claim 11 , wherein the substrate comprises a film, a porous membrane, a monolith, a nanofiber mat, or a resin, and wherein the substrate comprises cellulose, regenerated cellulose, a cellulose derivative, a nylon, a polysulfone, a polyether sulfone, a polyvinylidene fluoride, a polyacrylonitrile, a polyetherimide, a polypropylene, a polyethylene, or a polyether terephthalate.

13. The method of claim 11 or claim 12, wherein the active agent comprises an epichlorohydrin, a diglycidyl ether, or any combination thereof.

14. A method for derivatizing the activated substrate formed according to the method of any of claim 11-13, the method comprising contacting the activated substrate with a derivatization solution, the derivatization solution comprising a derivatization agent, a base, and optionally comprising an organic solvent, the derivatization agent comprising an epoxy-reactive functionality and a hydrophobic portion, the hydrophobic portion comprising a hydrophobic ligand.

15. The method of claim 14, wherein the hydrophobic ligand comprises an aliphatic chain with two or more carbons, a benzyl-containing group, a phenyl- containing group, a phenol-containing group, a pyridine-containing group, a boronic acid group, a branched polymer, a sulfur-containing thiophilic group, or any combination thereof.

16. The method of claim 14 or claim 15, wherein the derivatization agent comprises thiophenol, 2-butanethiol, furfurylthiol, 6-mercaptopurine, 2- mercaptopyridine, 2-mercapto-benzothiazole, propanethiol, cyclopentanethiol, o- mercaptobenzoic acid, dithiothreitol, 1,2-ethanedithiol, 3,6-dioxa-1 ,8-octanedithiol,

17. The method of any of claims 11-16, wherein the organic solvent comprises a protic organic solvent or an aprotic organic solvent or a combination thereof, for instance wherein the organic solvent comprises an alcohol, nitromethane, or any combination thereof.

18. The method of any of claims 11-17, wherein the base comprises an alkanamine (e.g., methylamine, triethylamine, trimethylamine, tripropylamine, tributylamine), a pyridine, an imidazole, a benzimidazole, a histidine, a guanidine, a phosphazene base, N,N-dimethylbenzyl amine, 3-dimethylaminopropyl amine, N,N- diisopropylethylamine, N,N-dimethylene diamine, diethylamine, sodium amide, sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, or potassium bicarbonate.