Epoxide-activated substrates and hydrophobic interaction chromatography membranes made therefrom - Patents.com

Abstract

Activated membranes and derivatized membranes that can be formed from the activated membranes are disclosed. HIC systems incorporating derivatized membranes that can be used for purifying plasmid DNA using hydrophobic interaction separation methods are also disclosed. The derivatized membranes can exhibit high plasmid DNA binding capacity and short retention times. Methods for forming the activated membranes and for further derivatizing the membranes are also described.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 203,196, filed July 12, 2021, entitled "Epoxide-Activated Surface Preparation and Novel Hydrophobic Interaction Chromatography Membrane Adsorber for Polynucleotide Purification," and claims priority to U.S. Publication No. 17 / 742,956, filed May 12, 2022, entitled "Epoxide-Activated Substrates and Hydrophobic Interaction Chromatography Made Therefrom for Polynucleotide Purification," both of which are incorporated herein by reference for all purposes.

[0002] Federal Government Research Statement This invention was made with Federal support under Grant No. GM125429 awarded by the National Institutes of Health. The Federal Government has certain rights in this invention. [Background technology]

[0003] The gene and cell therapy industry is rapidly shifting to full-scale manufacturing processes due to its potential to treat a variety of serious diseases. Plasmid DNA (pDNA) is a key component in the production of viral vectors, proteins, and mRNA that are widely used in gene and cell therapy. There is an acute and urgent need for high-capacity, high-quality pDNA production. This production challenge is exacerbated by recent pandemic-related demands, as pDNA is a key component of many products addressing the SARS-CoV-2 pandemic. For example, producing one billion doses of an mRNA vaccine would require more than half of the world's pDNA production capacity.

[0004] pDNA production has been an obstacle for the industry, as it is not easy to scale up pDNA manufacturing. Currently, certified contract manufacturers have long waiting lists and significant backlogs as supply attempts to keep up with the surge in demand. Like many other biologics production schemes, pDNA production involves multiple steps and unit operations. Traditionally, downstream purification is expensive, time-consuming, and difficult to scale. For decades, resin-based chromatography columns have been the gold standard for biologics purification. Resin columns are known to require long retention times to perform adequately. Moreover, the large size of pDNA limits the accessible surface area of ​​the resin for pDNA binding. Overall, these factors combine to make pDNA purification extremely low productivity.

[0005] The purity of pDNA is important for subsequent bioprocessing. Besides the need for separation from RNA, genomic DNA, host cell proteins, etc., certain pDNA isoforms are also undesirable. There are generally five isoforms of pDNA: supercoiled (sc) pDNA, open circular (oc) pDNA, relaxed circular pDNA, linear pDNA, and supercoiled denatured pDNA. Resin-based hydrophobic interaction chromatography (HIC) columns are often used as a critical step to distinguish the desired sc pDNA from other isoforms.

[0006] Membrane adsorbers are known to provide rapid separation of biological agents because they perform well with short column retention times compared to resin columns. However, currently, no commercially available HIC membrane adsorbers are known that are effective for pDNA purification, and there are no reports in the literature of high-performance HIC membrane adsorbers for pDNA purification.

[0007] Sartorius AG produces a HIC membrane product with phenyl groups as HIC ligands, but it was not designed for pDNA purification. Studies have shown that this HIC membrane product has a very low pDNA binding capacity (less than 0.1 mg / mL for 3,000 base pairs (3 kbp) pDNA) at a retention time of 180 seconds. The binding capacity would be even lower (less than 0.04 mg / mL for 3 kbp pDNA) at higher flow rates (e.g., 36 second retention time). Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, there remains a need for HIC membrane columns that have high binding capacity for pDNA with short retention times, which would increase downstream pDNA and other polynucleotide purification productivity. [Means for solving the problem]

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

[0010] In one embodiment, a method of forming an epoxy-activated substrate is disclosed. For example, the method can include contacting a substrate, such as a porous membrane substrate, with an activation solution. The activation solution includes an activating agent, a base, and an organic solvent. The activating agent includes a reactive functional group that reacts with the surface of the substrate to form a linking group on the surface of the substrate. The activating agent also includes an epoxy functional group that can remain intact during activation such that the linking agent includes an epoxy functional group after activation.

[0011] Also disclosed is a method of further derivatizing an activated substrate comprising a linking group at a surface thereof comprising an epoxy functional group. The method may 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 functional group that reacts with the epoxy functional group of the linking group. The derivatization agent also includes a hydrophobic moiety that includes a hydrophobic ligand. After reaction, the hydrophobic ligand is attached to the substrate surface via the reacted linking group.

[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 described below. [Brief description of the drawings]

[0013] [Figure 1] The dynamic binding capacity 10% (DBC10%) values ​​of 2-mercaptopyridine (MCP) derivatized membranes developed from activated membranes formed with different activation times are compared. [Diagram 2] The DBC10% values ​​of MCP-derivatized membranes developed from activated membranes formed by different activation methods are compared. [Diagram 3] The effect of alkaline pretreatment on the derivatized membranes is compared. [Figure 4] The DBC10% values ​​of MCP-derivatized membranes formed using different alkaline pretreatment and rinsing steps are presented. [Diagram 5] 1 is an FPLC chromatogram illustrating the loading, washing and elution steps of a separation using a HIC membrane as disclosed herein. [Figure 6] FIG. 1 is a graph showing that the binding capacity of the membrane can be improved through pH adjustment of the loading buffer. [Figure 7] 1 presents the dynamic binding capacity of HIC media as described herein for plasmids of different sizes. [Figure 8] 1 presents the dynamic binding capacity of a HIC medium as described herein for different flow rates. [Figure 9] 1 presents the dynamic binding capacity of HIC media as described herein for different ammonium sulfate concentrations. [Figure 10] The chromatographic results of the resin-type HIC media are compared to the HIC membrane media as described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] 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 as an explanation of the subject matter, not as a limitation of the subject matter. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made in this disclosure without departing from the scope or spirit of the subject matter. For example, features illustrated or described as part of one embodiment can be used with another embodiment to yield still a further embodiment.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter disclosed herein belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the subject matter disclosed herein, representative methods, devices, and materials are described herein.

[0016] Unless specifically stated, terms and phrases used herein, and variations thereof, should be construed as open ended as opposed to limiting. Similarly, a group of items joined by the conjunction "and" should not be read as requiring every one of those items to be present in the group, but rather, "and / or," unless expressly stated otherwise. Similarly, a group of items joined by the conjunction "or" should not be read as requiring mutual exclusivity between the groups, but rather, "and / or," unless expressly stated otherwise.

[0017] Additionally, although items, elements, or components of the disclosure may be described or claimed in the singular, the plural is contemplated within its scope unless expressly stated to be limited to the singular. The presence in some instances of broader words and phrases, such as "one or more," "at least," "including but not limited to," or other similar phrases, should not be read to imply that a narrower case is intended or required in instances where such broader phrases may not be present.

[0018] The present disclosure generally relates to an activated membrane that, in one embodiment, can be further derivatized for use in purifying pDNA using hydrophobic interaction separation methods. Methods for forming the activated membrane and further derivatizing the activated membrane are also described. In embodiments, the derivatized membrane as described herein can exhibit high pDNA binding capacity with short retention times.

[0019] One object of the present disclosure is to provide a method for modifying a substrate to contain a dense epoxy-based chemistry, thereby forming an activated film.

[0020] It is a further object of the present disclosure to provide a method for subsequently derivatizing the activated membrane so as to incorporate a high density of ligands, and in one particular embodiment a high density of hydrophobic ligands, on the surface of the membrane.

[0021] It is a further object of the present disclosure to provide HIC membranes and methods of forming HIC membranes for rapid and efficient purification of pDNA and other polynucleic acids, including, but not limited to, polynucleotides, oligonucleotides, polynucleosides and oligonucleosides, such as messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), nucleic acid products resulting 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 concatemers, aptamers, and the like.

[0022] The terms "polynucleotide" and "oligonucleotide," as used herein, refer to a polymer having at least two nucleotides (e.g., deoxyribonucleotides or ribonucleotides), in either single-stranded or double-stranded form, including DNA and RNA. A "nucleotide" includes a sugar, a base, and a linking group. In some embodiments, the sugar can be deoxyribose or natural ribose (e.g., DNA and RNA, respectively). In some embodiments, the linking group can be a phosphate group. Nucleotides are linked together by linking groups to form polynucleotides and oligonucleotides. A polymer of covalently bonded linking groups can be referred to as a backbone. A "nucleoside" is otherwise similar to a nucleotide, except that a nucleoside does not include a phosphate group. "Base" or "nucleobase" includes purines and pyrimidines, which further include the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and naturally occurring analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that 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. Modified nucleobases, modified sugars, and / or modified linking groups can be synthetic, naturally occurring, and / or non-naturally occurring, and can be non-standard / chemically modified nucleobases, sugars, and / or linking groups that 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'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).

[0023] Deoxyribooligonucleotides consist of 5-carbon deoxyribose sugars covalently linked to phosphates at the 5' and 3' carbons of the sugar, forming an alternating, unbranched polymer. The DNA can be in the form of, for example, an antisense molecule, pDNA, precondensed DNA, PCR product, vector, expression cassette, chimeric sequence, chromosomal DNA, or derivatives and combinations of these groups. Ribooligonucleotides consist of a similar repeating structure where the 5-carbon sugar is a ribose. Thus, the terms "polynucleotide" and "oligonucleotide" can refer to polymers or oligomers of nucleotide or nucleoside monomers composed of naturally occurring bases, sugars and intersugar (backbone) linkage groups.

[0024] The terms "polynucleotide" and "oligonucleotide" may also include similarly functional polymers or oligomers that contain non-naturally occurring monomers or portions thereof. Such modified or substituted oligonucleotides may be preferred due to properties such as enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases, as compared to native forms. It should be understood that the terms "polynucleotide" and "oligonucleotide" may also include polymers or oligomers that contain combinations of both deoxynucleotides and ribonucleotides or variants thereof in combination with backbone modifications, such as those described herein.

[0025] Polynucleotides and oligonucleotides as may be purified by the materials described herein may include one or more nucleotide variants, including one or more non-standard nucleotides, one or more non-natural nucleotides, one or more nucleotide analogs and / or modified nucleotides. Examples of modified nucleotides include, but are not limited to, diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7 ... Included are guanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueuosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocine, pseudouracil, queuosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, etc. In some cases, a nucleotide can contain modifications in its phosphate portion, including modifications to the triphosphate portion. Non-limiting examples of such modifications include modifications with longer length phosphate chains (e.g., phosphate chains having 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and thiol moieties (e.g., α-thiotriphosphate and β-thiotriphosphate).

[0026] A polynucleotide or oligonucleotide may be modified at a base moiety (e.g., one or more atoms typically available to form hydrogen bonds with a complementary nucleotide and / or one or more atoms typically incapable of forming hydrogen bonds with a complementary nucleotide), a sugar moiety, or a linking group (e.g., backbone). Backbone modifications may include, but are not limited to, phosphorothioate, phosphorodithioate, phosphoroselenate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoramidate, and phosphorodiamidate linkages. Phosphorothioate linkages replace sulfur atoms of non-bridging oxygens in the phosphate backbone, retarding nuclease degradation of oligonucleotides. Phosphorodiamidate linkages (N3'→P5') prevent recognition and degradation by nucleases. Backbone modifications can also include peptide bonds replacing phosphorus in the backbone structure (e.g., N-(2-aminoethyl)-glycine units linked by peptide bonds in peptide nucleic acids) or linking groups including carbamates, 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. Ther., 1(3):344-358, 1999. The nucleic acid molecules described herein can contain sugar moieties including ribose or deoxyribose as present in naturally occurring nucleotides or modified sugar moieties or sugar analogs. Modified sugar moieties include, but are not limited to, 2'-O-methyl, 2'-O-methoxyethyl, 2'-O-aminoethyl, 2'-fluoro, N3'→P5' phosphoramidate, 2'dimethylaminooxyethoxy, 2'2'dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars. The 2'-O-methyl or 2'-O-methoxyethyl modifications promote an A-form or RNA-like conformation in oligonucleotides, increase binding affinity for RNA, and exhibit enhanced nuclease resistance.Modified sugar moieties can also include those having an additional bridge bond (e.g., a methylene bridge connecting the 2'-O atom and the 4'-C atom of ribose in a locked nucleic acid) or a sugar analog such as a morpholine ring (e.g., as in phosphorodiamidate morpholinos).

[0027] Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can 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)).

[0028] The disclosed methods include the separation and / or purification of isolated or substantially purified nucleotides, nucleosides, nucleic acid molecules and compositions containing such molecules. As used herein, an "isolated" or "substantially purified" DNA or RNA molecule is a DNA or RNA molecule that exists away from its natural environment. An isolated DNA or RNA molecule may exist in 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 a 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 does not include sequences that naturally flank the nucleic acid in the genomic DNA of the original organism from which the nucleic acid is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid).

[0029] In one embodiment, membranes as disclosed herein may be useful in purifying polynucleic acids ranging in size from tens of base pairs, e.g., from about 20 to about 25 bases in length up to hundreds of thousands of bases, e.g., up to about 200,000 bases, or even longer in some embodiments. For example, the membrane may have a length of about 20 bases to about 250 bases, such as about 50 bases to about 200,000 bases, for example, about 50 bases to about 150,000 bases, such as about 50 bases to about 200,000 bases, for example, about 100 bases to about 200,000 bases, such as about 100 bases to about 100,000 bases, for example, about 100 bases to about 50,000 bases, such as about 200 bases to about 200,000 bases, for example, about 200 bases to about 150,000 bases, such as about 200 bases to about 100,000 bases, such as about 200 bases to about 50,000 bases, such as about 200 bases to about 25,000 bases, for example, about 200 bases to about 20,0 It may be useful in purifying polynucleic acids of about 10,000 bases, for example, about 200 bases to about 15,000 bases, for example, about 1,000 bases to about 50,000 bases, for example, about 5,000 bases to about 150,000 bases, for example, about 10,000 bases to about 150,000 bases, for example, about 15,000 bases to about 150,000 bases, for example, about 15,000 bases to about 200,000 bases, for example, about 20,000 bases to about 100,000 bases, for example, about 20,000 bases to about 200,000 bases, for example, about 20,000 bases to about 150,000 bases, for example, about 30,000 bases to about 150,000 bases.

[0030] Advantageously, the disclosed HIC media can exhibit similar binding capacities regardless of polynucleotide size. For example, under identical conditions, a purification column as described herein can exhibit a difference in binding capacity (i.e., mg of polynucleotide retained per mL of membrane bed volume at breakthrough) of about 20% or less, about 10% or less, or in some embodiments, about 5% or less for polynucleotides that differ in size by 10-fold or more, or in some embodiments, by 100-fold or more. 3 From about base pairs to 10 6Polynucleotides ranging in size down to base pairs may exhibit differences in binding capacity of about 20% or less, about 10% or less, or in some embodiments, about 5% or less.

[0031] To form an activated substrate, the substrate can be contacted with an activating solution, the activating solution including at least one epoxy-containing activator, one or more bases, and one or more organic solvents.

[0032] The substrate may 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), and the like. As used herein, the term "membrane" generally refers to a relatively thin sheet material having a porous structure. The substrate may be formed according to forming techniques as generally known in the art, including, without limitation, gel spinning, freeze-drying, casting, molding, electrospinning, machining, wet spinning, dry spinning, fulling, spraying, phase separation, mold-assisted assembly, rolling, compression, or any combination thereof.

[0033] The thin substrate, such as a membrane or nanofiber mat, in one embodiment, has a thickness of from about 30 micrometers to about 2500 micrometers. For example, a thin substrate may 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, for example, 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.

[0034] As used herein, unless otherwise indicated, sample thickness is measured with an automated thickness tester. An exemplary thickness test measures the distance between two tester plates when held at a constant pressure. An exemplary pressure for measuring thickness is 1.5 PSI. One useful instrument for film sample thickness is the 49-56 Micrometer (Messmer Buechel, Veenendaal, The Netherlands).

[0035] In some embodiments, the substrate may be a porous membrane substrate, which may be a supported membrane, e.g., a laminate comprising a porous membrane adjacent to (e.g., adhered or attached to) a supporting frame or backing material that exhibits a higher porosity than the porous membrane substrate, e.g., a woven or nonwoven backing material, which may be formed of the same or a different material as the substrate.

[0036] Nonwoven backing materials (e.g., backing webs) can be made by meltblowing, wet lamination, melt spinning, solution spinning, air lamination, or electrospinning. Nonwoven webs can additionally be treated with post-processing steps such as calendaring, embossing, needle punching, or hydroentangling. Nonwoven backing materials can also contain structural resins that exhibit low binding affinity for biomolecules. Such resins are typically used to increase the strength of the backing material. Backing materials can contain a mixture of fiber diameters and fiber materials. Fibers used to make the backing material can include glass, polypropylene, polyamides, polyesters, cellulosic materials, and the like, and combinations thereof. The fibers can have an average fiber diameter of 0.1 μm or more, 1 μm or more, 2 μm or more, or 3 μm or more. The fibers can have an average fiber diameter of 100 μm or less, 50 μm or less, 25 μm or less, 10 μm or less, or 8 μm or less. The average fiber diameter may be in the range of 0.1 μm to 50 μm or 1 μm to 25 μm. The average pore diameter measured by a capillary flow porometer may be 1 μm or more, 2 μm or more, or 3 μm or more. The average pore diameter may be 100 μm or less, 50 μm or less, 25 μm or less, 10 μm or less, or 8 μm or less. The average pore diameter of a suitable nonwoven substrate may be in the range of 0.1 μm to 50 μm, 1 μm to 10 μm, or 3 μm to 8 μm. The average pore diameter of a woven substrate may be slightly larger than that of a nonwoven substrate and may be in the range of 1 μm to 100 μm. The basis weight of the fibrous substrate may be 1 gsm (grams per square meter) or more, 10 gsm or more, or 20 gsm or more. 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 can range from 1 gsm to 200 gsm, or from 20 gsm to 80 gsm.

[0037] In some embodiments, the porous membrane substrate can be a self-supporting membrane, i.e., one that does not require a backing material. Of course, a membrane that is inherently self-supporting can be held by a support if necessary. Porous membrane substrates as described herein include, but are not limited to, porous hydrogel membranes as well as fibrous membranes, such as porous membranes formed of nanofibers, e.g., electrospun nanofibers, and membranes prepared by casting, coating, or molding.

[0038] The substrate may have a relatively large surface area. The surface area may be determined by BET (Brunauer, Emmett and Teller) measurement using nitrogen as the adsorbate. By way of example, a substrate, such as a porous substrate, may have a surface area of ​​about 0.1 m. 2 / mL ~ approx. 30m 2 / mL, e.g., about 0.1 m 2 / mL ~ approx. 25m 2 / mL, approx. 0.1m 2 / mL ~ approx. 20m 2 / mL, approx. 0.1m 2 / mL ~ approx. 15m 2 / mL, approx. 0.1m 2 / mL~about 10m 2 / mL, approx. 0.5m 2 / mL ~ approx. 30m 2 / mL, approx. 0.5m 2 / mL ~ approx. 25m 2 / mL, approx. 0.5m 2 / mL ~ approx. 20m 2 / mL, approx. 0.5m 2 / mL ~ approx. 15m 2 / mL, approx. 0.5m 2 / mL~about 10m 2 / mL, approx. 0.5m 2 / mL~approx.5m 2 / mL, approx. 1m 2 / mL ~ approx. 30m 2 / mL, approx. 1m 2 / mL ~ approx. 25m 2 / mL, approx. 1m 2 / mL ~ approx. 20m 2 / mL, approx. 1m 2 / mL ~ approx. 15m 2 / mL, approx. 1m2 / mL~about 10m 2 / mL, approx. 1m 2 / mL~approx.5m 2 / mL, approx. 5m 2 / mL ~ approx. 30m 2 / mL, approx. 5m 2 / mL ~ approx. 25m 2 / mL, approx. 5m 2 / mL ~ approx. 20m 2 / mL, approx. 5m 2 / mL ~ approx. 15m 2 / mL or about 5m 2 / mL~about 10m 2 The specific surface area may be, for example, 1 / mL.

[0039] In some embodiments, the porous substrate can be a macroporous substrate, such as a macroporous membrane substrate. Generally, a macroporous substrate is about 1 m 2 / mL or more, e.g., about 1m 2 / mL ~ approx. 30m 2 / mL, approx. 1m 2 / mL ~ approx. 25m 2 / mL, approx. 1m 2 / mL ~ approx. 20m 2 / mL, approx. 1m 2 / mL ~ approx. 15m 2 / mL, approx. 1m 2 / mL~about 10m 2 / mL, approx. 1m 2 / mL~approx.5m 2 / mL, approx. 5m 2 / mL ~ approx. 30m 2 / mL, approx. 5m 2 / mL ~ approx. 25m 2 / mL, approx. 5m 2 / mL ~ approx. 20m 2 / mL, approx. 5m 2 / mL ~ approx. 15m 2 / mL or about 5m 2 / mL~about 10m 2In one embodiment, the macroporous membrane substrate can exhibit high volumetric flow rates without high pressure due to the micron-sized pores, can exhibit low non-specific protein adsorption, and can exhibit a high density of surface functional groups that can be used as reaction sites.

[0040] In embodiments in which the substrate is porous, the porous substrate may generally have a pore size of about 0.1 micrometer to about 10 micrometers. For example, the porous substrate, e.g., the porous membrane substrate, may exhibit a pore size of about 0.1 micrometer to about 10 micrometers, about 0.1 micrometer to about 0.2 micrometers, about 0.1 micrometer to about 0.45 micrometers, about 0.1 micrometer to about 1 micrometer, about 0.1 micrometer to about 2 micrometers, about 0.2 micrometer to about 0.45 micrometers, about 0.2 micrometer to about 1 micrometer, about 0.2 micrometer to about 2 micrometers, about 0.2 micrometer to about 10 micrometers, about 0.45 micrometer to about 1 micrometer, about 0.45 micrometer to about 2 micrometers, about 0.45 micrometer to about 10 micrometers, about 1 micrometer to about 2 micrometers, or about 1 micrometer to about 5 micrometers.

[0041] As used herein, unless otherwise indicated, 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 pressure 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 fluid 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 be tested first dry at low to high pressures, and then wet again at low to high pressures. This test is typically performed at ambient temperature conditions (e.g., 20°C to 25°C). For both the dry and wet curves, 200 data points may be collected over a scan range of pressures. The relationship P=4τ cosθ γ lv / D(where γ lv The relationship between dewetting pressure (P) and pore size (D) described by (P = θ θ ) (where θ is the surface tension of the wetting liquid, θ is the contact angle of the wetting liquid on the porous material, and τ is an empirical correction factor known as the tortuosity factor). Typically, a correction factor or shape factor of 0.715 is applied. The average pore size can be calculated from the average of at least three measurements. An individual measurement of the largest pore size during the bubble point measurement can be found, and the bubble measurement is determined by increasing the pressure on the wetted sample to the point where the wetting liquid is expelled and air flow is measured. The pressure at which the air flow is first measured indicates the point at which the first largest pores dewet and allow air to flow. An individual measurement of the average flow pore size can be calculated by determining the pressure at which the wetting curve intersects with the "semi-dry" curve. The semi-dry curve is obtained by mathematically dividing the air flow rate through the dry sample as a function of diameter by two. One useful instrument for determining pore size distribution is the Porolux 500 (Aptco Technologies NV, Nazareth, Belgium).

[0042] In one embodiment, the substrate may be formed of a hydrophilic material, such as cellulose, cellulose derivatives, regenerated cellulose, nylon, or other hydrophilic materials. As used herein, the term regenerated cellulose refers to a class of materials produced by converting natural cellulose to a soluble cellulose derivative and subsequently regenerating it to form fibers or films made, for example, by the viscose or lyocell process, or by spinning the cellulose from a solution thereof in an ionic liquid.

[0043] The 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, the cellulose derivatives can include alkyl celluloses (e.g., methyl cellulose, ethyl cellulose, ethyl methyl cellulose), hydroxyalkyl celluloses (e.g., hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, ethyl hydroxyethyl cellulose), carboxyalkyl celluloses (e.g., carboxymethyl cellulose), organic ester celluloses (e.g., cellulose acetate, cellulose triacetate, cellulose propionate, cellulose acetate propionate, cellulose acetate butyrate), inorganic acid celluloses (e.g., nitrocellulose, cellulose sulfate), or any combination thereof. In one embodiment, the optically active material can include alkyl celluloses such as hydroxypropyl cellulose (HPC), methyl cellulose, ethyl cellulose, or any combination thereof.

[0044] However, it should be understood that the use of a hydrophilic material is not required and the substrate may be formed of other materials or any combination of materials known to those skilled in the art, such as polysulfone, polyethersulfone, polyvinylidene fluoride, polyacrylonitrile, polyetherimide, polypropylene, polyethylene, polyether terephthalate, and the like.

[0045] To form an activated substrate, a substrate, such as a porous membrane substrate, can be contacted with an activation solution. In one embodiment, the substrate can be contacted by immersing the substrate in the activation solution. The activation solution can include an activating agent in addition to at least one base and at least one organic solvent. Upon immersion, when an interaction occurs between the reactive functional groups of the activating agent and the surface of the substrate, activated linker groups can be formed 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.

[0046] The activating agent can be a multifunctional agent, e.g., bifunctional, trifunctional, etc., where at least one functional group of the agent is configured to react with the 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 after the activation step, such that an interaction between the activating agent and the substrate can result in an epoxy-containing linking group on the surface of the activated substrate.

[0047] Activators can include, without limitation, epichlorohydrin, diglycidyl ethers, triglycidyl ethers, tetraglycidyl ethers, or any combination thereof.

[0048] In embodiments, the concentration of the activator in the solvent may 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.

[0049] The organic solvent component of the 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, e.g., about 40% (V / V) or less, about 30% (V / V) or less, about 20% (V / V) or less, about 10% (V / V) or less, or in some embodiments, about 5% (V / V) or less, based on the volume of the activation solution.

[0050] In one embodiment, the organic solvent component may include a protic solvent. Without wishing to be bound by any particular theory, it is understood that the inclusion of one or more protic solvents in the activation solution may facilitate the SN1 reaction. The protic solvent may be selected from the group including, but not limited to, alcohol (e.g., ethanol, methanol, propanol (1-propanol, 2-propanol), butanol (n-butanol), etc.), nitromethane, or any combination thereof. In one embodiment, the protic solvent may include a combination of alcohol (e.g., ethanol) and water.

[0051] In one embodiment, the organic solvent may include an aprotic solvent. Without wishing to be bound by any particular theory, it is understood that the inclusion of one or more aprotic solvents in the activation solution may facilitate the SN2 reaction. The aprotic solvent may be selected from the group including, but not limited to, dimethylsulfoxide, dimethylformamide, acetonitrile, and N-methylpyrrolidinone.

[0052] The concentration of the one or more organic solvents in the 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 in some embodiments, from about 5% (v / v) to about 30% (v / v) of the activation solution.

[0053] The activation solution may also include a base. In some embodiments, the base may be a weak base. As used herein, the term "weak base" generally refers to a base that does not completely dissociate in water. The weak base of the activation solution may include, without limitation, alkanamines (e.g., methylamine, triethylamine, trimethylamine, tripropylamine, tributylamine, etc.), sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, pyridine, imidazole, benzimidazole, histidine, guanidine, phosphazene base, N,N-dimethylbenzylamine, 3-dimethylaminopropylamine, N,N-diisopropylethylamine, N,N-dimethylenediamine, diethylamine, or any combination thereof. The base component in the activation solution may optionally utilize a strong base. The strong base may include, without limitation, sodium amide, sodium hydroxide, potassium hydroxide, lithium bis(trimethylsilyl)amide, lithium tert-butoxide, or any combination thereof.

[0054] In one embodiment, the weak base may be present in the activation solution at a concentration ranging 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 in some embodiments, from about 3% (v / v) to about 20% (v / v) of the activation solution. In some embodiments, the concentration of the weak base in the organic solvent component of the activation solution may range from about 0.01 M to about 5 M, such as from about 0.5 M to about 4 M or from about 0.1 M to about 1 M in some embodiments.

[0055] When utilized, the strong base may be present in the activation solution at a concentration ranging 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 in some embodiments, from about 0.01% (v / v) to about 3% (v / v) of the activation solution. In some embodiments, the concentration of the strong base in the organic solvent component of the activation solution may range from about 0.01M to about 0.5M, such as from about 0.01M to about 0.2M or from about 0.02M to about 0.1M in some embodiments.

[0056] To activate the substrate, the substrate can be contacted (e.g., immersed) with the activation solution for a period of time. Optionally, the activation solution can be cooled or heated before / during contact. For example, the activation step can be performed while the activation solution is maintained at a temperature in the range of about 0° C. to about 100° C., e.g., about 10° C. to about 90° C., about 20° C. to about 90° C., about 20° C. to about 80° C., about 20° C. to about 70° C., about 20 to about 60° C., about 30° C. to about 90° C., about 30° C. to about 80° C., about 30° C. to about 70° C., about 30° C. to about 60° C., about 30° C. to about 50° C., or in some embodiments, at about 40° C. In some embodiments, the activation step can be performed in the dark, e.g., in a dark room with little or no visible light.

[0057] The duration of the activation step may 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 hours 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 hours to 16 hours. In some embodiments, the contact time for the activation step ranges from 0.5 hours 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.

[0058] In one embodiment, the substrate may be subjected to a pretreatment prior to contact with the activation solution. For example, the substrate may be pretreated by contacting it with an alkaline solution. When included, such pretreatment may include contacting the substrate with an alkaline solution comprising at least one base and at least one protic solvent. The base may include, without limitation, sodium hydroxide, potassium hydroxide, potassium carbonate, potassium bicarbonate, sodium carbonate, sodium bicarbonate, Tris base, and the like, or any combination thereof. The protic solvent may include, without limitation, alcohol (e.g., ethanol, methanol, propanol (1-propanol, 2-propanol), butanol (n-butanol), and the like), nitromethane, water, or any combination thereof.

[0059] When included, the alkaline pretreatment step can generally be carried out at a temperature ranging from about 0° C. to about 80° C., such as a temperature ranging from about 10° C. to about 70° C., about 25° C., or in some embodiments, at room temperature. The contact time for the alkaline pretreatment can vary. In some embodiments, the contact time for the alkaline pretreatment can range from 1 minute to 72 hours. In some embodiments, the contact time for the alkaline pretreatment can range from 0.5 hours to 48 hours. In some embodiments, the contact time for the alkaline pretreatment can range from 10 minutes to 12 hours. In some embodiments, the contact time for the alkaline pretreatment can range from 1 hour to 24 hours. In some embodiments, the contact time for the alkaline pretreatment can range from 0.5 hours to 16 hours. In some embodiments, the contact time for the alkaline pretreatment can range from 0.5 hours to 4 hours. In some embodiments, the contact time for the alkaline pretreatment can range from 20 minutes to 2 hours. In some embodiments, the contact time for the alkaline pretreatment can range from 4 hours to 16 hours. As previously mentioned, alkaline pretreatment is not required in the formation of an activated substrate or a derivatized substrate.

[0060] After formation of the activated substrate, it can be further derivatized with an epoxy-containing linking group. Advantageously, the activated substrate (e.g., activated membrane substrate) may not require drying after activation. However, in other embodiments, the activated membrane can be dried before further derivatization. The drying process is not particularly limited and can include forced air drying or simple air drying. In general, the drying temperature can be a temperature that ensures that the activated substrate is not damaged, such as around room temperature, such as about 20°C to about 40°C, or in some embodiments, about 25°C to about 30°C. After drying, the dried activated substrate can maintain reactive functional groups until the time of use. For example, the dried activated membrane can be stored and / or transported for a period of time (e.g., several days to a month or even longer depending on storage conditions), and after that time, the surface can still maintain the desired reactive functional groups, such as for further derivatization.

[0061] To further derivatize the activated substrate, the substrate can be contacted (e.g., immersed) in a derivatization solution. The derivatization solution can include an organic solvent, a base, and a derivatization agent. In one embodiment, the derivatization agent can include a hydrophobic moiety that can provide a hydrophobic ligand to the surface of the substrate when the derivatization agent reacts with a reactive functional group of a linking agent of the activated substrate.

[0062] The derivatizing agent of the solution may include an epoxy-reactive functional group configured for reaction with the epoxy and at least one hydrophobic moiety of the activated substrate. The hydrophobic moiety may remain on the substrate surface as a hydrophobic ligand attached to the substrate surface via a linking agent. For example, the epoxy-reactive functional group may include, without limitation, a primary or secondary amine, a thioether, an epoxide, a carboxylic acid, an organohalogen compound, or the like, or any combination thereof.

[0063] The hydrophobic part of the derivatizing agent can include hydrophobic ligand.The examples of hydrophobic ligands that can be incorporated on the activated substrate can include, without limitation, aliphatic chains having 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 sulfophilic 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), and the like, and any combination thereof.

[0064] Hydrophobic ligands, which may be components of the derivatization agent and which may be attached to the substrate by reacting the derivatization agent with an epoxy of the linking agent, may be selected from ligands that rely on the hydrophobic effect for hydrophobic interaction ligands; π-π stacking between the immobilized aromatic compound and the aromatic ring of the nucleobase for aromatic-containing ligands, or electron donation / charge transfer for thioether-containing ligands, or a combination thereof.

[0065] In one embodiment, the hydrophobic ligand of the derivatization agent may exhibit affinity for polynucleotides, such as plasmids. However, the hydrophobic moiety of the derivatization agent is not limited to the hydrophobic ligands exemplified above, and may incorporate additional ligands onto the activated substrate as described herein.

[0066] Examples of derivatizing agents 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-dichlorobenzyl mercaptan, 4-methoxybenzyl mercaptan, triphenylmethanethiol, 2,4-dimethoxythiophenol, or any combination thereof.

[0067] In one embodiment, the concentration of the derivatization agent in the derivatization solution may 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 in some embodiments, from about 0.05% (W / V) to about 1% (W / V) of the derivatization solution.

[0068] The base of the derivatization solution may be the same or different from the base of the activation solution. In some embodiments, the base may be a weak base. The weak base of the derivatization solution may include, without limitation, alkanamines (e.g., methylamine, triethylamine, trimethylamine, tripropylamine, tributylamine, etc.), pyridine, imidazole, benzimidazole, histidine, guanidine, phosphazene bases, N,N-dimethylbenzylamine, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, 3-dimethylaminopropylamine, N,N-diisopropylethylamine, N,N-dimethylenediamine, diethylamine, or any combination thereof. The base component in the derivatization solution may optionally utilize a strong base. The strong base may include, without limitation, sodium amide, sodium hydroxide, potassium hydroxide, lithium bis(trimethylsilyl)amide, lithium tert-butoxide, or any combination thereof.

[0069] In one embodiment, the concentration of the base in the 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 in some embodiments, from about 0.5% (V / V) of the derivatization solution to about 30% (V / V) of the derivatization solution.

[0070] The organic solvent component of the derivatization solution can be the same or different from the organic solvent component of the activation solution. For example, the organic solvent of the derivatization solution can include a protic organic solvent, an aprotic organic solvent, an aqueous / organic solvent mixture, or a combination thereof. Examples of solvents that may be utilized in the derivatization solution can include, without limitation, alcohol (e.g., ethanol, methanol, propanol (1-propanol, 2-propanol), butanol (n-butanol), etc.), nitromethane, dimethylsulfoxide, dimethylformamide, N-methylpyrrolidinone, or any combination thereof.

[0071] The concentration of the organic solvent component of the derivatization solution can range from about 0% (V / V) of the derivatization solution 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 in some embodiments, from about 20% (V / V) of the derivatization solution to about 85% (V / V) of the derivatization solution.

[0072] To effect derivatization of the activated substrate, the substrate can be contacted (e.g., immersed) with a derivatization solution for a period of time. Optionally, the derivatization solution can be cooled or heated prior to / during contact. For example, the derivatization step can be carried out with the derivatization solution at a temperature ranging from about 0° C. to about 80° C., such as from about 20° C. to about 60° C., or in some embodiments, about 30° C.

[0073] The contact time for the derivatization step may vary. In some embodiments, the contact time for the derivatization step ranges from 1 minute to 72 hours. In some embodiments, the contact time for the derivatization step ranges from 0.5 hours to 48 hours. In some embodiments, the contact time for the derivatization step ranges from 10 minutes to 12 hours. In some embodiments, the contact time for the derivatization step ranges from 1 hour to 24 hours. In some embodiments, the contact time for the derivatization step ranges from 0.5 hours to 16 hours. In some embodiments, the contact time for the derivatization step ranges from 0.5 hours to 4 hours. In some embodiments, the contact time for the derivatization step ranges from 20 minutes to 2 hours. In some embodiments, the contact time for the derivatization step ranges from 4 hours to 16 hours.

[0074] In some embodiments, the derivatized substrate can be dried before use. In other embodiments, the derivatized substrate can be used immediately after derivatization. When a drying process is employed, it is not particularly limited and can include forced air drying or simple air drying. In general, the drying temperature can be a temperature that ensures that the derivatized substrate is not damaged, such as around room temperature, such as about 20°C to about 40°C, or in some embodiments, about 25°C to about 30°C, etc. After drying, the dried derivatized substrate can maintain the hydrophobic ligand until the time of use. For example, the dried activated membrane can be stored and / or transported for a period of time (e.g., from several days to several months or even years depending on the storage conditions), and after that time, the desired hydrophobic ligand can be maintained for use in the HIC protocol.

[0075] In one embodiment, the derivatized substrate can be further processed for use. For example, the derivatized substrate can be processed (e.g., molded, stacked, combined, held, etc.) to form a derivatized separation medium for use in a HIC protocol. In one embodiment, the derivatized separation medium can be formed with one or more derivatized membranes, e.g., multiple derivatized membranes, which can be stacked and / or molded as needed to form a separation medium for use in a HIC protocol.

[0076] As an example, multiple derivatized membrane substrates can be stacked to form multi-layer constructs that provide increased capacity for a given application.In one embodiment, the laminated structure of the derivatized membrane has a length of about 70 micrometers to about 10,000 micrometers, for example, about 10,000 micrometers or more, about 7,500 micrometers or more, about 5,000 micrometers or more, about 2,500 micrometers or more, about 1,000 micrometers or more, about 900 micrometers or more, about 800 micrometers or more, about 700 micrometers or more, about 600 micrometers or more, about 500 micrometers or more, about 400 micrometers or more, about 300 micrometers or more. or more, about 200 micrometers or more, about 100 micrometers or more, about 70 micrometers or more, for example, 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, 00 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 2,00 The thickness may be about 0 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.

[0077] The flow rate of the separation medium disclosed herein may be, for example, about 0.5 column volumes (CV) / min to about 1000 CV / min, about 1 CV / min to about 1000 CV / min, about 2 CV / min to about 1000 CV / min, about 3 CV / min to about 1000 CV / min, about 4 CV / min to about 1000 CV / min, about 5 CV / min to about 1000 CV / min, about 6 CV / min to about 1000 CV / min, about 0.5 CV / min to about 500 CV / min, about 1 CV / min to 500 CV / min, about 2 CV / min to about 500 CV / min, about 3 CV / min to about 500 CV / min, about 4 CV / min to about 500 CV / min, about 5 CV / min to about 500 CV / min, / min, about 6 CV / min to about 500 CV / min, about 0.5 CV / min to about 100 CV / min, about 1 CV / min to about 100 CV / min, about 2 CV / min to about 100 CV / min, about 3 CV / min to about 100 CV / min, about 4 CV / min to about 100 CV / min, about 5 CV / min to about 100 CV / min, about 6 CV / min to about 100 CV / min, about 0.5 CV / min to about 50 CV / min, about 1 CV / min to about 50 CV / min, about 2 CV / min to about 50 CV / min, about 3 CV / min to about 50 CV / min, about 4 CV / min to about 50 CV / min, about 5 CV / min to about 50 CV / min, or about 6 CV / min to about 50 CV / min.

[0078] As used herein, the term "column volume" refers to the volume of the membrane bed in a column according to standard practice. Thus, when considering the bed volume formed by 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.

[0079] In one embodiment, the separation medium can be utilized for the purification of 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 resulting from rolling circle amplification, snRNA, piRNA, dsRNA, genomic DNA, ssDNA, dsDNA, A-DNA, B-DNA, C-DNA, Z-DNA, aptamers, and the like.

[0080] Separation systems incorporating a separation medium 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.

[0081] Separation protocols using the disclosed materials can be carried out using standard methods. In one embodiment, a separation column incorporating a derivatized HIC membrane as described herein can be operated in bind-elute mode using elution methods as generally known in the art.

[0082] Separation using the disclosed HIC separation media can purify polynucleotides quickly and efficiently at high flow rates. For example, polynucleotide purification methods utilizing the separation media as disclosed herein can provide greater than 80% recovery of target material with a purity of about 80% or greater. For example, a separation medium (e.g., a single derivatized membrane or multiple derivatized membranes stacked together) can have a dynamic binding capacity of greater than 1 mg of polynucleotide (e.g., pDNA) per milliliter of separation medium with a retention time of less than 120 seconds.

[0083] The separation system as disclosed can exhibit high process productivity. The process productivity of a column can be defined using the following formula: tot is the total volume of solution passed through the column during the entire process, including loading, rinsing, elution and regeneration steps. BV is the HIC media bed volume, and τ is the retention time. The loading volume is proportional to the dynamic binding capacity of the HIC media. Thus, process productivity increases with increasing binding capacity and decreasing retention time.

number

[0084] Dynamic binding capacity generally refers to the concentration of polynucleotides bound to the separation medium at breakthrough in the effluent (number of milligrams bound per unit volume of the membrane bed). In an embodiment, the disclosed separation system is capable of binding polynucleotides at a retention time of 120 seconds and a 2.5 M ammonium sulfate buffer concentration of about 1 to about 5 mg per mL of medium, for example, 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, It is possible to provide a dynamic binding capacity of polynucleotide of 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 mentioned above, the disclosed separation medium can exhibit substantially similar dynamic binding capacity regardless of the target polynucleotide size. Thus, the dynamic binding capacity of a separation system can be substantially similar when the same system is purifying different polynucleotides, whether large or small polynucleotides. Dynamic binding capacities that are substantially similar to each other can be within about 10% or less of each other, 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 optionally exhibiting the same dynamic binding capacity.

[0085] Known commercial HIC column products operate at a retention time of 180 seconds, and for 3 kbp pDNA, the dynamic binding capacity is less than 0.1 mg / mL. For two media with the same dynamic binding capacity achieving the same product yield, the ratio of loading productivity can be estimated by the inverse ratio of retention times. Thus, in one embodiment, compared to a separation medium comprising a derivatized membrane as described in this disclosure with a dynamic binding capacity of 4 mg / mL at a retention time of about 2 seconds or less, the loading productivity of the separation medium described herein can be 3600 times (=(4 / 0.1)×(180s / 2s)) higher than currently known commercial HIC column products for plasmid purification. No known column products currently available approach such productivity as realized by the present invention.

[0086] Below is a list of exemplary embodiments of the products and methods according to the present disclosure.

[0087] According to embodiment 1, an HIC separation medium is disclosed that includes a porous cellulose membrane and a plurality of hydrophobic ligands bound to the surface of the porous cellulose membrane.

[0088] Example 2 is the HIC separation membrane according to Example 1, wherein the porous cellulose membrane comprises a cast membrane, a hydrogel membrane, or a fibrous membrane such as an electrospun nanofiber membrane.

[0089] Aspect 3 is the HIC separation membrane according to Aspect 1 or 2, wherein the porous cellulose membrane comprises regenerated cellulose or a cellulose derivative.

[0090] In embodiment 4, the porous cellulose membrane has a pore size of about 0.1 m as determined by BET measurements using nitrogen as the adsorbate. 2 / mL ~ approx. 30m 2 / mL, approx. 0.1m 2 / mL ~ approx. 25m 2 / mL, approx. 0.1m 2 / mL ~ approx. 20m 2 / mL, approx. 0.1m 2 / mL ~ approx. 15m 2 / mL, approx. 0.1m 2 / mL~about 10m 2 / mL, approx. 0.5m 2 / mL ~ approx. 30m 2 / mL, approx. 0.5m 2 / mL ~ approx. 25m 2 / mL, approx. 0.5m 2 / mL ~ approx. 20m 2 / mL, approx. 0.5m 2 / mL ~ approx. 15m 2 / mL, approx. 0.5m 2 / mL~about 10m 2 / mL, approx. 0.5m 2 / mL~approx.5m 2 / mL, approx. 1m 2 / mL ~ approx. 30m 2 / mL, approx. 1m 2 / mL ~ approx. 25m 2 / mL, approx. 1m 2 / mL ~ approx. 20m 2 / mL, approx. 1m 2 / mL ~ approx. 15m 2 / mL, approx. 1m 2 / mL~about 10m 2 / mL, approx. 1m 2 / mL~approx.5m 2 / mL, approx. 5m 2 / mL ~ approx. 30m 2 / mL, approx. 5m 2 / mL ~ approx. 25m 2 / mL, approx. 5m 2 / mL ~ approx. 20m 2 / mL, approx. 5m 2 / mL ~ approx. 15m 2 / mL or about 5m 2 / mL~about 10m 2 / mL specific surface area.

[0091] Aspect 5 is the HIC separation membrane according to any one of Aspects 1 to 4, wherein the porous cellulose membrane comprises a pore size of about 0.1 micrometers to about 10 micrometers, about 0.1 micrometers to about 0.2 micrometers, about 0.1 micrometers to about 0.45 micrometers, about 0.1 micrometers to about 10 micrometers, about 0.1 micrometers to about 2 micrometers, about 0.2 micrometers to about 0.45 micrometers, about 0.2 micrometers to about 1 micrometer, about 0.2 micrometers to about 2 micrometers, about 0.2 micrometers to about 10 micrometers, about 0.45 micrometers to about 1 micrometer, about 0.45 micrometers to about 2 micrometers, about 0.45 micrometers to about 10 micrometers, about 1 micrometer to about 2 micrometers, or about 1 micrometer to about 5 micrometers.

[0092] Example 6 is the HIC separation membrane of any one of Examples 1 to 5, comprising a plurality of membranes stacked together, the stack comprising a porous cellulose membrane.For example, the laminate may be 70 micrometers to 10,000 micrometers, for example, about 10,000 micrometers or more, about 7,500 micrometers or more, about 5,000 micrometers or more, about 2,500 micrometers or more, about 1,000 micrometers or more, about 900 micrometers or more, about 800 micrometers or more, about 700 micrometers or more, about 600 micrometers or more, about 500 micrometers or more, about 400 micrometers or more, about 300 micrometers or more, about 200 micrometers or more. or more, about 100 micrometers or more, about 70 micrometers or more, for example, 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 The thickness of the nanofiber may be about 250 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.

[0093] Aspect 7 is the HIC separation membrane according to any one of Aspects 1 to 6, wherein the hydrophobic ligand comprises an aliphatic chain having 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 sulfophilic group, or any combination thereof. Moreover, each of the plurality of hydrophobic ligands can be bound to the surface of the porous cellulose membrane via a linking group, the linking group comprising a reaction product of an epoxy and an epoxy-reactive functional group, such as an amine or a thioether moiety.

[0094] Aspect 8 utilizes a retention time of 120 seconds and a 2.5 M ammonium sulfate buffer concentration to obtain a concentration of greater than about 1 mg / mL, for example, 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 ... The HIC separation membrane according to any one of Aspects 1 to 7 has a polynucleotide dynamic binding capacity of 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.

[0095] Example 9 is the HIC separation membrane according to any one of Examples 1 to 8, wherein the porous cellulose membrane is a self-supporting membrane or the medium comprises a porous cellulose membrane and a backing material.

[0096] Aspect 10 is a separation column comprising the hydrophobic interaction chromatography separation medium according to any one of aspects 1 to 9. For example, the separation column comprises a syringe filter column, a spin column, a cassette or a spiral wound membrane column.

[0097] Example 11 is a method of forming an activated substrate comprising contacting a substrate with an activating solution comprising an activating agent, a base, and an organic solvent, the activating agent comprising a reactive functional group configured to react with a surface of the substrate to form a linking group on the surface, the activating agent further comprising an epoxy group, the linking group comprising an epoxy group.

[0098] Example 12 is the method of example 11, wherein the substrate comprises a film, a porous membrane, a monolith, a nanofiber mat, or a resin, and the substrate comprises cellulose, regenerated cellulose, a cellulose derivative, nylon, polysulfone, polyethersulfone, polyvinylidene fluoride, polyacrylonitrile, polyetherimide, polypropylene, polyethylene, or polyether terephthalate.

[0099] Example 13 is the method of example 11 or 12, wherein the active agent comprises epichlorohydrin, a diglycidyl ether, or any combination thereof.

[0100] Example 14 is a method of derivatizing an activated substrate formed by the method of any one of Examples 11, 12, or 13, comprising contacting the activated substrate with a derivatization solution comprising a derivatization agent, a base, and optionally an organic solvent, wherein the derivatization agent comprises an epoxy-reactive functional group and a hydrophobic moiety, and wherein the hydrophobic moiety comprises a hydrophobic ligand.

[0101] Example 15 is the method of example 14, wherein the hydrophobic ligand comprises an aliphatic chain having 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 sulfophilic group, or any combination thereof.

[0102] Example 16 is the method of example 14 or example 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-dichlorobenzyl mercaptan, 4-methoxybenzyl mercaptan, triphenylmethanethiol, 2,4-dimethoxythiophenol, or any combination thereof.

[0103] Example 17 is the method of any one of Examples 11 to 16, wherein the organic solvent comprises a protic organic solvent or an aprotic organic solvent or a combination thereof, for example, the organic solvent comprises an alcohol, nitromethane, or any combination thereof.

[0104] Example 18 is a method according to any one of Examples 11 to 17, wherein the base comprises an alkanamine (e.g., methylamine, triethylamine, trimethylamine, tripropylamine, tributylamine), pyridine, imidazole, benzimidazole, histidine, guanidine, a phosphazene base, N,N-dimethylbenzylamine, 3-dimethylaminopropylamine, N,N-diisopropylethylamine, N,N-dimethylenediamine, diethylamine, sodium amide, sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, or potassium bicarbonate.

[0105] The present disclosure may be better understood with reference to the examples provided below. EXAMPLES

[0106] Test Method To form the chromatography column of this example, two layers of 24 mm circular HIC membrane disks were packed into a membrane housing (membrane volume = 0.055 mL) to form a DBC 10% The values ​​were measured.

[0107] Chromatography tests were performed using a fast protein liquid chromatography system (AEKTA™ pure 25, Cytiva). Four sizes of plasmids were tested, for example. Plasmids 1) pRP[Exp]-CMV>EGFP, 3657bp 2) pRP[Exp]-Alb>hCas9, 8956bp and 3) pLV[Exp]-EGFP:T2A:Bsd-Alb>dCas9 / VPR, 16129bp are purified using the Qiagen® Mega kit (catalog no. 12281). pALD-HELP, 11584bp, was purchased from a distributor. Plasmid stocks were diluted to concentrations between 6 and 13 μg / mL. Various concentrations of ammonium sulfate in 40 mM 1×TE (Tris-HCl) were used as loading buffer.

[0108] The dynamic binding capacity was determined as the plasmid concentration retained in the effluent at breakthrough (milligrams of plasmid bound per unit volume of membrane bed). 10% ) corresponds to the mass bound per unit volume of the membrane bed when the plasmid concentration in the effluent from the membrane bed reaches 10% of the feed solution.

[0109] Example 1 Activation: A 10 x 60 cm regenerated cellulose membrane sheet with a nominal pore size of 1 μm was activated by immersing 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 carried out overnight in the dark at room temperature with shaking at 120 rpm. After 20-24 hours, the membrane was rinsed with EtOH for 5 min, followed by two 5-min acetone rinses. The membrane sheet was then allowed to dry at room temperature.

[0110] MCP uptake: Activated membranes were treated with 82 mM MCP dissolved in a solution containing 80.7% (v / v) methanol, 8.6% (v / v) deionized (DI) water, 9.6% (v / v) 5 M sodium hydroxide (NaOH), and 1.1% (v / v) TEA. The reaction was carried out overnight in the dark at room temperature with shaking at 100 rpm. After 20-24 hours, the membranes were rinsed with ethanol for 5 min, followed by two 5-min acetone rinses. The membrane sheets were then dried at room temperature.

[0111] Example 2 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: 1 hour reaction time for Membrane 1, 3 hours reaction time for Membrane 2 and overnight (20-24 hours) reaction time for Membrane 3. The reactions were carried out at room temperature on a shaker at 120 rpm and in the dark. After the reaction, the membranes were rinsed and dried following the same procedure as described in Example 1.

[0112] MCP incorporation: Dried activated membranes were derivatized as in Example 1.

[0113] Figure 1 shows the DBC 10% Figure 1 shows that the HIC membrane binding capacity increased with increasing reaction time in terms of . For the same activation solution and same uptake treatment, a reaction time of 20-24 hours resulted in a 59% increase in binding capacity compared to that resulting from a 1 hour reaction.

[0114] Example 3 Activation: Two epichlorohydrin-activated membranes (Membrane 4 and Membrane 5) were prepared in the same manner as Membrane 2 in Example 1 with an activation time of 3 hours. However, two modifications were made to the formulation: 1) Membrane 4 was soaked in an activation solution with twice the EPI concentration of 16.8% v / v. 2) Membrane 5 was soaked in an activation solution where EtOH was replaced with the same amount of 2-propanol (IPA).

[0115] MCP incorporation: Dried activated membranes were derivatized as in Example 1.

[0116] As shown in Figure 2, doubling the EPI concentration resulted in a 25% increase in binding capacity for Membrane 4 when compared to Membrane 2. Also, replacement of EtOH with IPA resulted in little or no change in binding capacity between Membrane 5 and Membrane 2, indicating that in some circumstances IPA can be used to replace EtOH in making the membranes.

[0117] Example 4 Alkaline treatment: 0.3 M NaOH in 100% ethanol was prepared the day before the reaction and left on a stir plate overnight. Just before the reaction, the solution was clarified using a 47 mm 0.22 μm regenerated cellulose membrane filter.

[0118] Four regenerated cellulose membrane strips and two alkaline treatments were used: Set I) Membranes A and B were soaked in 0.3 M NaOH in 100% ethanol for 30 minutes at room temperature on a shaker at 80 rpm, then transferred to 0.1 M NaOH in DI for an additional 30 minutes at room temperature on a shaker at 80 rpm. Set II) Membranes C and D were soaked in 0.3 M NaOH in 100% ethanol for 30 minutes on a shaker at 80 rpm and at room temperature.

[0119] Membranes A and C were rinsed with 200 mM Tris pH 7 for 5 minutes, followed by DI water for 5 minutes, followed by two 5-minute acetone rinses. The membranes were then dried at room temperature under suitable forced air conditions. Meanwhile, membranes B and D were rinsed with 200 mM Tris pH 7 for 5 minutes, DI water for 5 minutes, followed by ethanol for 5 minutes. Membranes B and D were then transferred to the activation solution.

[0120] Activation: The membrane was activated in the same manner as in Example 1.

[0121] MCP incorporation: Dried activated membranes were coupled in the same manner as in Example 1.

[0122] Alkaline treatment improved the structural integrity of the membrane, as shown in Figure 3. Figure 4 compares the binding capacity of membranes prepared with different alkaline pretreatments, where the membrane treated with 0.3 M NaOH in EtOH had a similar binding capacity as the membrane similarly treated with 0.1 M NaOH in water.

[0123] Example 5 Alkaline treatment: The day before this process, 0.3 M NaOH in 100% ethanol (EtOH) was prepared and left on a stir plate overnight. Just before the reaction, the solution was clarified using a 47 mm 0.22 μm cellulose acetate (CA) membrane filter.

[0124] Three membrane strips and two alkaline treatments were used: Set I) Membranes C and F were soaked in 0.3 M NaOH in 100% EtOH for 30 minutes on a shaker at 80 rpm and room temperature. Set II) Membranes G and H were soaked in 0.1 M NaOH in DI for 30 minutes on a shaker at 80 rpm and at room temperature.

[0125] After alkaline treatment, Membranes C and G were rinsed with 200 mM Tris pH 7 for 5 min, followed by DI rinse for 5 min, then two acetone rinses. Membranes C and G were then dried at room temperature under forced air conditions before activation.

[0126] After alkaline treatment, Membranes F and H were rinsed with 200 mM Tris pH 7 for 5 min, followed by DI for 5 min, then EtOH for 5 min. Membranes F and H were not dried prior to activation.

[0127] Activation: Membranes C and G were rinsed with EtOH and acetone and then transferred to uptake solution as in Example 1.

[0128] Membranes F and H were rinsed with EtOH for 5 min, followed by 2 rinses with DI for 5 min each rinse, then transferred directly into uptake solution as in Example 1.

[0129] MCP uptake: Membranes were derivatized as in Example 1.

[0130] Figure 4 shows that the membranes prepared without a drying step (F and H) had higher binding capacity than the membranes prepared with a drying step (C and G). For alkaline treatment, the membrane treated with 0.3 M NaOH in 100% ethanol (F) had a slightly higher binding capacity than the membrane treated with 0.1 M NaOH dissolved in DI (H).

[0131] Example 6 Chromatographic tests were performed using a column packed with Membrane 3 from Example 2 as the medium. The plasmid size was 3657 bp. 3 M ammonium sulfate in 40 mM Tris-HCl, pH 8 was used as the loading buffer. Figure 5 shows the resulting chromatogram illustrating the loading, washing and elution steps of the separation. The elution peaks were similar between all tests. UV 260 The absorbance values ​​and buffer volumes vary from membrane to membrane. 260 The absorbance values ​​and buffer volumes varied from membrane to membrane.

[0132] Chromatographic experiments were again performed under the same conditions, but with the loading buffer reduced from pH 8 to pH 5. Figure 6 shows the binding capacity results and illustrates that a 30% increase in binding capacity is observed when the pH of the loading buffer is reduced.

[0133] Example 7 These materials were further investigated by carrying out chromatographic studies under various conditions using columns packed with Membrane 3 of Example 2 as the medium.

[0134] Initially, the media was examined for use with different sized plasmids including 3 kbp, 8 kbp and 16 kbp. For all runs, the loading buffer utilized 2.5 M ammonium sulfate and 1× TE buffer at a pH of 7.5 and a flow rate of 2 mL / min (36.4 CV / min). Dynamic binding capacity (DBC) results are provided in Table 1 below and in FIG. 7.

[0135] [Table 1]

[0136] As noted, the dynamic binding capacity of the medium was essentially independent of plasmid size.

[0137] The media was examined for the effect on dynamic binding capacity for changes in flow rate. An 8 kbp plasmid was utilized. The loading buffer was 2.5 M ammonium sulfate and 1× TE buffer at pH 7.5. The results are provided in Table 2 below and in FIG. 8.

[0138] [Table 2]

[0139] The effect of different ammonium sulfate concentrations on dynamic binding capacity was also examined. The 8 kbp plasmid was utilized and the flow rate was set at 2 mL / min for all runs. The results are provided in Table 3 below and in Figure 9.

[0140] [Table 3]

[0141] The Membrane 3 chromatography column was compared to a commercially available pre-packed resin column (Cytiva HiScreen™ PlasmidSelect, 4.7 mL). The loading buffer was 3M ammonium sulfate and 1×TE at pH 7.5. The plasmid was 11 kbp. The results are shown in Table 4 below and in FIG. 10.

[0142] [Table 4]

[0143] Although particular embodiments of the disclosed subject matter have been described using specific language, it is to be understood that such description is for purposes of illustration only, and that changes and modifications can be made without departing from the spirit or scope of the subject matter.

Claims

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

2. The hydrophobic interaction chromatography separation medium according to claim 1, wherein the porous cellulose membrane comprises a fiber membrane such as a cast membrane, a hydrogel membrane or an electrospun nanofiber membrane.

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

4. The porous cellulose membrane is 0.1 mm ２ / mL to 30m ２ 3. The hydrophobic interaction chromatography separation medium according to claim 1 or 2, comprising a specific surface area of ​​0.1 μm to 10 μm / mL and / or a pore size of 0.1 μm to 10 μm.

5. A hydrophobic interaction chromatography separation medium according to claim 1 or 2, comprising a plurality of membranes laminated together, the lamination comprising the porous cellulose membrane and having a thickness of 70 micrometers to 10,000 micrometers.

6. The hydrophobic ligand comprises an aliphatic chain having 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 sulfurophilic group or any combination thereof, and each of the plurality of hydrophobic ligands is bound to the surface of the porous cellulose membrane via a linking group, the linking group comprising a reaction product of an epoxy and an epoxy-reactive functional group. The hydrophobic interaction chromatography separation medium according to claim 1 or 2.

7. The hydrophobic interaction chromatography separation medium according to claim 1 or 2, having a polynucleotide dynamic binding capacity of 1 mg / mL to 30 mg / mL at a retention time of 120 seconds and using a 2.5 M ammonium sulfate buffer concentration.

8. The hydrophobic interaction chromatography separation medium according to claim 1 or 2, wherein the porous cellulose membrane is a self-supporting membrane or the medium comprises the porous cellulose membrane and a backing material.

9. A separation column comprising the hydrophobic interaction chromatography separation medium according to claim 1 or 2, the separation column comprising a syringe filter column, a spin column, a cassette or a spiral membrane column. **Claim 10**: A method for forming an activated substrate, comprising contacting the substrate with an activation solution, wherein the activation solution comprises an activator, a base, and an organic solvent, the activator comprises a reactive functional group configured to react with the surface of the substrate to form a linking group on the surface, the activator further comprises an epoxy group, and the linking group comprises the epoxy group. **Claim 11**: The method according to claim 10, wherein the substrate comprises a film, a porous membrane, a monolith, a nanofiber mat, or a resin, and the substrate comprises cellulose, regenerated cellulose, a cellulose derivative, nylon, polysulfone, polyethersulfone, polyvinylidene fluoride, polyacrylonitrile, polyetherimide, polypropylene, polyethylene, or polyether terephthalate. **Claim 12**: The method according to claim 10 or 11, wherein the activator comprises epichlorohydrin, diglycidyl ether, or any combination thereof. **Claim 13**: A method for derivatizing the activated substrate formed by the method according to claim 10 or 11, comprising contacting the activated substrate with a derivatization solution, wherein the derivatization solution comprises a derivatizing agent and a base, the derivatizing agent comprises an epoxy-reactive functional group and a hydrophobic moiety, and the hydrophobic moiety comprises a hydrophobic ligand. **Claim 14**: The method according to claim 13, wherein the hydrophobic ligand comprises an aliphatic chain having 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. **Claim 15**: The method according to claim 13, wherein the derivatizing agent comprises thiophenol, 2-butanethiol, furfuryl mercaptan, 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-benzenedimethanethiol, 1,2-benzenedimethanethiol, 4,4'-bis(mercaptomethyl)biphenyl, 2,4-dichlorobenzyl mercaptan, 4-methoxybenzyl mercaptan, triphenylmethanethiol, 2,4-dimethoxythiophenol, or any combination thereof. **Claim 16**: The method according to claim 10 or 11, wherein the organic solvent comprises a protic organic solvent, an aprotic organic solvent, or a combination thereof. **Claim 17**: The method according to claim 10 or 11, wherein the base comprises an alkaneamine, pyridine, imidazole, benzimidazole, histidine, guanidine, a phosphazene base, N,N-dimethylbenzylamine, 3-dimethylaminopropylamine, N,N-diisopropylethylamine, N,N-dimethylene diamine, diethylamine, sodium amide, sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, or potassium bicarbonate.

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