CN117915999A

CN117915999A - Epoxide-activated substrates and hydrophobic interaction chromatography membranes made therefrom

Info

Publication number: CN117915999A
Application number: CN202280048151.7A
Authority: CN
Inventors: 周锦翔; G·坦普尔斯; 郭斌
Original assignee: Donaldson Co Inc
Current assignee: Donaldson Co Inc
Priority date: 2021-07-12
Filing date: 2022-07-11
Publication date: 2024-04-19

Abstract

Activated films and derivatized films such as may be formed from the activated films are disclosed. HIC systems are also disclosed that incorporate derivatized membranes as may be used to purify plasmid DNA using hydrophobic interaction separation methods. The derivatized membranes may exhibit high plasmid DNA binding capacity and short residence time. Methods for forming the activated film and methods for further derivatization of the film are also described.

Description

Epoxide-activated substrates and hydrophobic interaction chromatography membranes made therefrom

Cross Reference to Related Applications

The present application requires the following commit priorities: U.S. patent application Ser. No. 17/742,956, titled "Epoxide-Activated Substrates and Hydrophobic Interaction Chromatography Made Therefrom for Polynucleotide Purification[ epoxide-activated substrate and hydrophobic interaction chromatography for polynucleotide purification made therefrom, at 2022, 5, 12, which claims the benefit of provisional patent application Ser. No. 63/203,196, titled "Epoxide-Activated Surface Preparation and Novel Hydrophobic Interaction Chromatography Membrane Adsorber for Polynucleotide Purification[ epoxide-activated surface preparation and novel hydrophobic interaction chromatography membrane adsorbate for polynucleotide purification, at 2021, 7, 12, both of which are incorporated herein by reference for all purposes.

Statement of federal research

The present invention was completed with government support under accession number GM125429 awarded by the national institutes of health (National Institutes of Health). The government has certain rights in this invention.

Background

The gene and cell therapy industry has rapidly shifted to commercialization because of its promising potential for the treatment of various destructive 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 a sudden and urgent need for high volume, high quality pDNA production. Since pDNA is a key component of many products that address the SARS-CoV-2 pandemic, the recent pandemic-related requirements further exacerbate the production challenges. For example, it would require more than half of the world's pDNA production capacity to produce billions of mRNA vaccines.

PDNA production has become a bottleneck in the industry because expansion of pDNA fabrication is not straightforward. Currently, qualified contract manufacturers have long waiting lists and a large number of outstanding orders to meet the rising demand. As with many other biological agent production schemes, pDNA production involves multiple steps and unit operations. Traditionally, downstream purification is expensive, slow and difficult to scale. Resin-based chromatography columns have been the gold standard for purifying biological agents for decades. It is known that a long residence time is required for the resin column to function adequately. Furthermore, due to the large size of the pDNA, the resin has a low accessible surface area for pDNA binding. Overall, the combination of these factors results in very low productivity of pDNA purification.

The purity of the pDNA is critical for subsequent biological processes. In addition to the need for isolation from RNA, genomic DNA, host cell proteins, etc., certain pDNA isoforms are also undesirable. pDNA generally has five isoforms: supercoiled (sc) pDNA, open-loop (oc) pDNA, relaxed circular pDNA, linear pDNA, and supercoiled denatured pDNA. Resin-based Hydrophobic Interaction Chromatography (HIC) columns are typically used as a key step to distinguish the desired sc pDNA from other isoforms.

Membrane adsorbates are known to perform well at short column residence times compared to resin columns, resulting in rapid separation of biological agents. However, there is currently no known efficient commercial HIC membrane adsorbate for pDNA purification nor is there a literature reporting high performance HIC membrane adsorbate for pDNA purification.

Sidoris corporation (Sartorius AG) produces HIC membrane products with phenyl groups as HIC ligands; however, it is not designed for pDNA purification. Studies have shown that this HIC membrane product has a very low pDNA binding capacity (for 3,000 base pairs (3 kbp) pDNA, <0.1 mg/mL) at 180 seconds residence time. Its binding capacity at higher flow rates (e.g., 36 second residence time) will be even lower (less than 0.04mg/mL for 3kbp pDNA).

Thus, there remains a need for HIC membrane columns with high binding capacities for pDNA at short residence times. Meeting this need will increase downstream pDNA and other polynucleotide purification productivity.

Disclosure of Invention

According to one embodiment, an HIC separation medium is disclosed that includes a cellulosic substrate (e.g., a porous regenerated cellulosic substrate) and a hydrophobic ligand bonded to a surface of the cellulosic substrate.

In one embodiment, a method for forming an epoxy-activated substrate is disclosed. For example, the method can include contacting a substrate (e.g., a porous membrane substrate) with an activation solution. The activation solution includes an activator, a base, and an organic solvent. The activator includes reactive functional groups that react with the surface of the substrate to form linking groups on the surface of the substrate. The activator also includes epoxy functionality that can remain intact during activation such that the linker includes epoxy functionality after activation.

Also disclosed is a method for further derivatizing an activated substrate comprising a linking group at a surface, the linking group comprising an epoxy functionality. The method may include contacting the activated substrate with a derivatizing solution. The derivatizing solution comprises a derivatizing agent, a base, and optionally an organic solvent. The derivatizing agent comprises a functional group that reacts with the epoxide functional group of the linking group. The derivatizing agent also includes a hydrophobic moiety comprising a hydrophobic ligand. After the reaction, the hydrophobic ligand is bonded to the substrate surface via the reactive linking group.

Drawings

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 wherein:

FIG. 1 compares the dynamic binding capacity 10% (DBC _10％) values of 2-Mercaptopyridine (MCP) derivatized membranes developed from activated membranes formed with different activation times.

FIG. 2 compares DBC _10％ values for MCP-derivatized films developed from activated films formed via different activation recipes.

FIG. 3 compares the effect of alkaline pretreatment on derivatized membranes.

Fig. 4 presents DBC _10％ values for MCP-derived membranes formed using different alkaline pretreatment and rinsing steps.

Fig. 5 is a FPLC chromatogram showing the loading, washing and elution phases of separation using HIC membranes as disclosed herein.

Fig. 6 is a graph showing that the binding capacity of a membrane can be improved by pH adjustment of a loading buffer.

FIG. 7 presents the dynamic binding capacity of HIC media as described herein for plasmids of different sizes.

Figure 8 presents the dynamic binding capacity of HIC media as described herein for different flow rates.

Figure 9 presents the dynamic binding capacities of HIC media as described herein for different ammonium sulfate concentrations.

FIG. 10 compares the chromatographic results of a resin type HIC medium with a HIC membrane medium as described herein.

Detailed Description

Reference will now be made in detail to the various embodiments of the disclosed subject matter, one or more examples of which are set forth below. Each of the embodiments is provided by way of explanation of the subject matter and not limitation of the subject matter. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to 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, can be used on another embodiment to yield a still further embodiment.

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

Unless specifically stated otherwise, the terms and phrases used in this document and variations thereof should be construed to be open-ended, and not limiting. Likewise, a group of items linked with the conjunction "and" should not be construed as requiring that each of these items be present in the group, but rather should be construed as "and/or" unless expressly stated otherwise. Similarly, a group of items linked with the conjunction "or" should not be construed as requiring mutual exclusivity among that group, but rather should also be construed as "and/or" unless expressly stated otherwise.

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. In some cases, the presence of extended words and phrases such as "one or more," "at least," "but not limited to," or other similar phrases should not be construed to mean that a narrower case is intended or required where such extended phrases may not be present.

The present disclosure relates generally to an activated membrane that can be further derivatized for purification of pDNA using a hydrophobic interaction separation method in one embodiment. Methods for forming the activated film and further derivatization of the activated film are also described. In embodiments, a derivatized film as described herein may exhibit high pDNA binding capacity at short residence times.

It is an object of the present disclosure to provide a method of modifying a substrate to include a high density of epoxy-based chemistry and thereby form an activated film.

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

It is a further object of the present disclosure to provide a HIC membrane and a method for forming the HIC membrane for rapid and efficient purification of pDNA and other polynucleic acids, including polynucleotides, oligonucleotides, polynucleic acids and oligonucleotides, such as, but not limited to, messenger RNAs (mRNA), transfer RNAs (tRNA), ribosomal RNAs (rRNA), short interfering RNAs (siRNA), nucleic acid products resulting from rolling circle amplification, micrornas (miRNA), micrornas (snRNA), piwi-interacting RNAs (piRNA), double-stranded RNAs (dsRNA), genomic DNA, single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), a-DNA, B-DNA, C-DNA, Z-DNA, DNA concatamers, aptamers, and the like.

The terms "polynucleotide" and "oligonucleotide" as used herein refer to polymers containing at least two nucleotides in single-or double-stranded form (e.g., deoxyribonucleotides or ribonucleotides) and include DNA and RNA. "nucleotide" includes sugar, base and linking groups. In some embodiments, the sugar may be deoxyribose or natural ribose (e.g., DNA and RNA, respectively). In some embodiments, the linking group may be a phosphate group. Nucleotides are linked together by a linking group to form polynucleotides and oligonucleotides. Polymers of covalently bonded linking groups may be referred to as backbones. "nucleosides" are otherwise similar to nucleotides, except that the nucleoside does not include a phosphate group. "base" or "nucleobase" includes purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogs, as well as synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications to place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates and alkyl halides. Nucleobases include modified or similar nucleobases, modified or similar sugars, and/or modified or similar linking groups. The modified nucleobase, modified sugar, and/or modified linking group can be a non-canonical/chemically modified nucleobase, sugar, and/or linking group, which can be synthetic, naturally occurring, and/or non-naturally occurring, and which has similar binding properties as the reference nucleic acid. Examples of such analogs and/or modified bases, sugars, and/or linking groups include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2' -O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

Deoxyribose oligonucleotides consist of 5-carbon deoxyribose sugars covalently bonded to a phosphate at the 5 'and 3' carbons of the sugar to form alternating unbranched polymers. The DNA may be in the form of, for example, antisense molecules, pDNA, pre-condensed DNA, PCR products, vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. Ribose oligonucleotides consist of similar repetitive structures in which the 5-carbon sugar is ribose. Thus, the terms "polynucleotide" and "oligonucleotide" may refer to polymers or oligomers of nucleotides or nucleoside monomers consisting of naturally occurring bases, sugars, and inter-sugar (backbone) linkages.

The terms "polynucleotide" and "oligonucleotide" may also include functionally similar polymers or oligomers comprising non-naturally occurring monomers or portions thereof. Such modified or substituted oligonucleotides may be superior to the natural form due to properties such as, for example, enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases. It is to be understood that the terms "polynucleotide" and "oligonucleotide" may also include polymers or oligomers comprising a combination of both deoxynucleotides and ribonucleotides, or variants thereof, in combination with backbone modifications, such as those described herein.

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 (xantine), 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyl uracil, dihydropyrimidine, β -D-galactosyl-plagioside (beta-D-galactosylqueosine), inosine, N6-isopentenyl adenine, 1-methylguanine, 1-methyl inosine, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosyl-pigtail, 5' -methoxycarboxymethyl uracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine, uracil-5-oxyacetic acid (v), huai Dingyang glycoside (wybutoxosine), pseudouracil, pigtail (queosin), 2-mercaptocytosine, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp 3) w, 2, 6-diaminopurine, and the like. In some cases, a nucleotide may include modifications in its phosphate moiety, including modifications to the triphosphate moiety. Non-limiting examples of such modifications include longer phosphate chains (e.g., phosphate chains having 4,5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications having thiol moieties (e.g., α -and β -thiotriphosphates).

Polynucleotides or oligonucleotides may be modified at the base moiety (e.g., at one or more atoms typically available to form hydrogen bonds with a complementary nucleotide and/or at one or more atoms typically unable to form hydrogen bonds with a complementary nucleotide), the sugar moiety, or the linking group (e.g., backbone). Backbone modifications may include, but are not limited to, phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilino phosphorothioates (phosphonatooates), phosphoroanilino phosphates (phosphonatoadates), phosphoramidates, and phosphorodiamidates. Phosphorothioate linkages replace non-bridging oxygen in the phosphate backbone with sulfur atoms and delay nuclease degradation of the oligonucleotide. The phosphorodiamidite linkage (N3 '. Fwdarw.P5') prevents nuclease recognition and degradation. Backbone modifications may also include peptide bonds instead of phosphorus, or linking groups including carbamates, amides, and linear and cyclic hydrocarbon groups in the backbone structure (e.g., N- (2-aminoethyl) -glycine units linked by peptide bonds in peptide nucleic acids). Oligonucleotides with modified backbones are reviewed in MICKLEFIELD, curr. Med. Chem. [ current medicinal chemistry ],8 (10): 1157-79,2001 and Lyer et al, curr. Opin. Mol. Ther. [ current point of molecular therapeutics ],1 (3): 344-358, 1999. The nucleic acid molecules described herein may contain a sugar moiety comprising ribose or deoxyribose as found in naturally occurring nucleotides, or a modified sugar moiety or sugar analogue. Modified sugar moieties include, but are not limited to, 2' -O-methyl, 2' -O-methoxyethyl, 2' -O-aminoethyl, 2' -fluoro, N3 '. Fwdarw.P 5' phosphoramidate, 2' dimethylaminooxyethoxy, 2' dimethylaminoethoxyethoxy, 2' -guanidinium (2 ' -guanidinidium), 2' -O-guanidinium ethyl, carbamate modified sugar, and bicyclic modified sugar. The 2 '-O-methyl or 2' -O-methoxyethyl modification promotes a type a or RNA-like conformation in the oligonucleotide, increases binding affinity to RNA, and has enhanced nuclease resistance. The modified sugar moiety may also include a methylene bridge having additional bridging bonds (e.g., connecting the 2'-O and 4' -C atoms of ribose in the locked nucleic acid) or sugar analogs, such as morpholino rings (e.g., as in morpholino phosphorodiamidates).

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. [ Nucleic acids research ],19:5081 (1991); ohtsuka et al, J.biol. Chem. [ J.biochemistry ],260:2605-2608 (1985); rossolini et al, mol. Cell. Probes [ molecules and cell probes ],8:91-98 (1994)).

The methods of the present disclosure encompass the isolation 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 molecule or RNA molecule is one that exists outside of its natural environment. The isolated DNA molecule or RNA molecule may be present in purified form or may be present in a non-natural 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.

In one embodiment, the membranes as disclosed herein may be used to purify polynucleic acids ranging in size from tens of base pairs (e.g., mirnas ranging in length from about 20 to about 25 bases) to hundreds of thousands of bases (e.g., about 200,000 bases) or in some embodiments even longer. For example, the membrane can be used to purify 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 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 200 bases to about 150,000 bases, such as from about 10,000 bases to about 150,000 bases, such as from about 150,000 bases to about 150,000 bases, such as from about 000 bases to about 150,000 bases.

Advantageously, the disclosed HIC medium may exhibit similar binding capacities independent of polynucleotide size. For example, a purification column as described herein may exhibit a difference in binding capacity (i.e., polynucleotide retained upon penetration (mg)/volume membrane bed (mL)) of about 20% or less, about 10% or less, or about 5% or less for polynucleotides that differ in size by a factor of 10 or more in some embodiments or 100 or more in some embodiments under the same conditions. For example, in some embodiments, polynucleotides ranging in size from the order of 10 ³ base pairs to the order of 10 ⁶ base pairs may exhibit a difference in binding capacity of about 20% or less, about 10% or less, or about 5% or less.

To form an activated substrate, the substrate may be contacted with an activation solution. The activation solution includes at least one epoxy-containing activator, one or more bases, and one or more organic solvents.

The substrate may be in the form of a non-porous film, a porous membrane, a nanofiber mat, a monolith (monolith) (single porous three-dimensional structure), a resin (solid polymer phase in the form of single 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, but not limited to, gel spinning, lyophilization, casting, molding, electrospinning, machining, wet spinning, dry spinning, milling, spraying, phase separation, template-assisted assembly, rolling, compaction, or any combination thereof.

In one embodiment, a thin substrate, such as a film or nanofiber mat, may have a thickness of from about 30 microns to about 2500 microns. For example, the thin substrate may have a thickness of greater than about 500 microns, greater than about 250 microns, greater than about 100 microns, greater than about 80 microns, greater than about 50 microns, or greater than about 30 microns, such as from about 30 microns to about 500 microns, from about 50 microns to about 500 microns, from about 80 microns to about 500 microns, from about 100 microns to about 500 microns, from about 250 microns to about 500 microns, from about 30 microns to about 250 microns, from about 50 microns to about 250 microns, from about 80 microns to about 250 microns, from about 100 microns to about 2500 microns, from about 30 microns to about 100 microns, from about 50 microns to about 100 microns, or from about 80 microns to about 100 microns.

As used herein, unless otherwise indicated, the thickness of a sample is measured with an automatic thickness tester. Exemplary thickness test measures the gap between two test device plates when held at a fixed pressure. An exemplary pressure for measuring thickness is 1.5PSI. One useful instrument for film sample thickness is a 49-56 micrometer (Messmer Buchel, fisher, netherlands).

In some embodiments, the substrate may be a porous film substrate, which may be a support film, e.g., a laminate comprising a porous film adjacent (e.g., adhered or attached to) a support frame or backing material (e.g., a woven or nonwoven backing material, which may be formed of the same or different material as the substrate) that exhibits greater porosity than the porous film substrate.

The nonwoven backing material (e.g., backing web) may be produced by melt blowing, wet-laying, melt spinning, solution spinning, air-laying, or electrospinning. The nonwoven web may additionally be treated by post-treatment steps such as calendaring, embossing, needling, or hydroentangling. The nonwoven backing material may also contain a structural resin having low binding affinity for biomolecules. Such resins are typically used to increase the strength of the backing material. The backing material may comprise a mixture of fibrous slurry and fibrous material. The fibers used to make the backing material may include glass, polypropylene, polyamide, polyester, cellulosic materials, and the like, as well as combinations thereof. The fibers may have an average fiber size of 0.1 μm or greater, 1 μm or greater, 2 μm or greater, or 3 μm or greater. The fibers may have an average fiber size 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 size may range from 0.1 μm to 50 μm, or from 1 μm to 25 μm. The average pore size measured by the capillary flow porosimeter can be 1 μm or more, 2 μm or more, or 3 μm or more. The average pore size may be 100 μm or less, 50 μm or less, 25 μm or less, 10 μm or less, or 8 μm or less. Suitable nonwoven substrates may have an average pore size ranging from 0.1 μm to 50 μm, from 1 μm to 10 μm, or from 3 μm to 8 μm. The average pore size of the woven substrate may be slightly larger than the nonwoven substrate and may range from 1 μm to 100 μm. The basis weight of the fibrous substrate may be 1gsm (grams per square meter) or greater, 10gsm or greater, or 20gsm or greater. The basis weight of the fibrous substrate may be 200gsm or less or 80gsm or less. The basis weight of the fibrous substrate may be in the range of 1gsm to 200gsm, or from 20gsm to 80 gsm.

In some embodiments, the porous membrane substrate may be a self-supporting membrane, i.e., no backing material is required. Of course, the self-supporting film in the other case may be held by a support, if desired. Porous film substrates as described herein encompass films prepared by casting, coating, or shaping, including but not limited to porous hydrogel films as well as fibrous films, e.g., porous films formed from nanofibers such as electrospun nanofibers.

The substrate may have a relatively high surface area. The surface area can be determined from BET (Brunauer), emmett (Emmett) and Teller) measurement evaluations using nitrogen as the adsorbate. By way of example, a substrate, for example, a porous substrate, may have a surface area from about 0.1 m/mL to about 30 m/mL, such as from about 0.1 m/mL to about 25 m/mL, from about 0.1 m/mL to about 20 m/mL, from about 0.1 m/mL to about 15 m/mL, from about 0.1 m/mL to about 10 m/mL, from about 0.5 m/mL to about 30 m/mL, from about 0.5 m/mL to about 25 m/mL, from about 0.5 m/mL to about 20 m/mL, from about 0.5 m/mL to about 15 m/mL, from about 0.5 m/mL to about 10 m/mL, from about 0.5 m/mL to about 5 m/mL, from about 1 m/mL to about 30 m/mL, from about 1 m/mL to about 25 m/mL, from about 1 m/mL to about 20 m/mL, from about 1 m/mL to about 15 m/mL, from about 1 m/mL to about 10 m/mL, from about 5m to about 5 m/mL, from about 5 m/mL to about 5 m/mL, from about 5 m/mL.

In some embodiments, the porous substrate may be a macroporous substrate, e.g., a macroporous membrane substrate. Generally, the macroporous substrate may include a specific surface area of about 1m ²/mL or greater, such as from about 1m ²/mL to about 30m ²/mL, from about 1m ²/mL to about 25m ²/mL, from about 1m ²/mL to about 20m ²/mL, from about 1m ²/mL to about 15m ²/mL, from about 1m ²/mL to about 10m ²/mL, from about 1m ²/mL to about 5m ²/mL, from about 5m ²/mL to about 30m ²/mL, from about 5m ²/mL to about 25m ²/mL, from about 5m ²/mL to about 20m ²/mL, from about 5m ²/mL to about 15m ²/mL, or from about 5m ²/mL to about 10m ²/mL. In one embodiment, the macroporous membrane substrate may exhibit a high volumetric flow rate without generating high pressure due to the micro-scale pores, may exhibit low non-specific adsorption of proteins, and may exhibit a high density of surface functional groups that may be used as reactive sites.

In those embodiments in which the substrate is porous, the porous substrate may generally have a pore size of from about 0.1 microns to about 10 microns. For example, a porous substrate, e.g., a porous membrane substrate, may exhibit a pore size of from about 0.1 microns to about 10 microns, from about 0.1 microns to about 0.2 microns, from about 0.1 microns to about 0.45 microns, from about 0.1 microns to about 1 micron, from about 0.1 microns to about 2 microns, from about 0.2 microns to about 0.45 microns, from about 0.2 microns to about 1 micron, from about 0.2 microns to about 2 microns, from about 0.2 microns to about 10 microns, from about 0.45 microns to about 1 micron, from about 0.45 microns to about 2 microns, from about 0.45 microns to about 10 microns, from about 1 micron to about 2 microns, or from about 1 micron to about 5 microns.

As used herein, unless otherwise indicated, pore size is determined using capillary flow porosimetry. Capillary flow porosimetry may be performed using a continuous pressure scanning mode. An exemplary applied pressure range that may be used is 0.115 bar to 3.5 bar (15 kPa to 350 kPa). It may be useful to use wetting solutions with a surface tension of 16.4 dynes/cm, including, for example, porofil ^TM wetting solutions (Kang Da instruments (Quantachrome Instruments), an Dongpa (anton paar), boynton beach (boynton beacon), florida (FL)). The sample may be initially tested in the dry state (changing the low pressure to high pressure) and then in the wet state (changing the low pressure to high pressure again). The test is typically performed at ambient temperature conditions (e.g., 20 ℃ to 25 ℃). 200 data points can be collected over the entire pressure sweep for both the dry and wet curves. The relationship between dewetting pressure (P) and pore size (D) is described by the relationship p=4τcos θγ _lv/D, where γ _lv is the surface tension of the wetting fluid, θ is the contact angle of the wetting fluid with the porous material, and τ is an empirical correction factor, called tortuosity factor. Typically, a tortuosity factor or shape factor of 0.715 is applied. The average pore size may be calculated from the average of at least three measurements. A separate measurement of the maximum pore size can be detected at the measured bubble point, where the measured bubble is determined by increasing the pressure on the wetting sample until the point where the wetting fluid is replaced and measuring the air flow. The pressure at which the air flow is measured for the first time indicates the point at which the first largest hole is dewetted allowing air flow. A single measurement of the average flow pore size can be calculated by determining the pressure at the intersection of the wet curve and the "semi-dry" curve. The semi-dry curve is obtained by dividing the mathematical value of the air flow through the dried sample by 2 as a function of diameter. One available instrument for determining pore size distribution is Porolux 500,500 (epstein technology limited (Aptco Technologies NV), basiler (Nazareth), belgium).

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 manufactured by converting natural cellulose to soluble cellulose derivatives and subsequently regenerating to form fibers or films produced by, for example, the viscose or lyocell process or by spinning cellulose from a solution thereof in an ionic liquid.

Cellulose derivatives contemplated herein may include, but are not limited to, one or more cellulose ethers, one or more cellulose esters, or any combination thereof. As examples, and without limitation, the cellulose derivatives may include alkyl celluloses (e.g., methyl 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 butyrate), inorganic acid celluloses (e.g., cellulose nitrate, cellulose sulfate), or any combination thereof. In one embodiment, the optically active material may include an alkyl cellulose, such as hydroxypropyl cellulose (HPC), methyl cellulose, ethyl cellulose, or a combination thereof.

However, it should be understood that the use of hydrophilic materials is not required and that the substrate may be formed of other materials known to those of ordinary skill in the art, such as polysulfone, polyethersulfone, polyvinylidene fluoride, polyacrylonitrile, polyetherimide, polypropylene, polyethylene, polyetherterephthalate, and the like, or any combination of materials.

To form an activated substrate, a substrate (e.g., a porous membrane substrate) may be contacted with an activation solution. In one embodiment, the substrate may be contacted by immersing the substrate in an activation solution. The activation solution may include an activator and at least one base and at least one organic solvent. After soaking, the interaction between the reactive functional groups of the activator and the surface of the substrate may form activated linker groups on the surface, each linker group comprising at least one reactive epoxy group. Interactions may include covalent bonding, ionic bonding, hydrogen bonding, and the like, or any combination thereof.

The activator may be a multifunctional agent, e.g., difunctional, trifunctional, etc., wherein 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 group that will not react during the activation step, i.e., an epoxy group that will remain active after the activation step. Thus, after interaction between the activator and the substrate, the epoxy-containing linking group may be at the surface of the activated substrate.

The activator may include, but is not limited to, epichlorohydrin, diglycidyl ether, triglycidyl ether, tetraglycidyl ether, or any combination thereof.

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.

The organic solvent component of the activation solution may be selected from, but is not limited to, a single organic solvent, an aqueous/organic solvent mixture, or a mixture of organic solvents. In some embodiments, when considering an activation solution comprising water, the water will typically 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% or less, about 10% (V/V) or less, or about 5% (V/V) or less, by volume of the activation solution.

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 promote the SN1 reaction. The protic solvent may be selected from the group including, but not limited to, alcohols (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 comprise a combination of an alcohol (e.g., ethanol) and water.

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 promote the SN2 reaction. The aprotic solvent may be selected from the group including, but not limited to, dimethylsulfoxide, dimethylformamide, acetonitrile, N-methylpyrrolidone, and the like.

In some embodiments, the concentration of the one or more organic solvents in the activation solution may range from about 1% (V/V) to about 99% (V/V) of the activation solution, 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.

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. Weak bases of the activation solution may include, but are not limited to, alkylamine (alkanamine) (e.g., methylamine, triethylamine, trimethylamine, tripropylamine, tributylamine, etc.), sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, pyridine, imidazole, benzimidazole, histidine, guanidine, phosphazene base, N-dimethylbenzylamine, 3-dimethylaminopropylamine, N-diisopropylethylamine, N-dimethylenediamine, diethylamine, or any combination thereof. A strong base may optionally be used in the base component of the activation solution. The strong base may include, but is not limited to, sodium amide, sodium hydroxide, potassium hydroxide, lithium bis (trimethylsilyl) amide, lithium t-butoxide, or any combination thereof.

In one embodiment, the weak base may be present in the 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 weak base in the organic solvent component of the activation solution may range from about 0.01M to about 5M, in some embodiments, such as from about 0.5M to about 4M, or from about 0.1M to about 1M.

In some embodiments, the strong base may be present in the activation solution at a concentration in the range of 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, when used. 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, in some embodiments, such as from about 0.01M to about 0.2M, or from about 0.02M to about 0.1M.

To effect activation of the substrate, the substrate may be contacted with (e.g., immersed in) the activation solution for a period of time. Optionally, the activation solution may be cooled or heated prior to/during contact. For example, in some embodiments, the activation step may be performed at a temperature where the activation solution is maintained in the range of from about 0 ℃ to about 100 ℃, such as at a temperature in the range of from about 10 ℃ to about 90 ℃, from about 20 ℃ to about 80 ℃, from about 20 ℃ to about 70 ℃, from about 20 ℃ to about 60 ℃, from about 30 ℃ to about 90 ℃, from about 30 ℃ to about 80 ℃, from about 30 ℃ to about 70 ℃, from about 30 ℃ to about 60 ℃, from about 30 ℃ to about 50 ℃, or at a temperature of about 40 ℃. In some embodiments, the activation step may be performed in the dark, for example, in a darkroom with little or no visible light.

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

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 contact with an alkaline solution. When included, such pretreatment may include contacting the substrate with an alkaline solution including at least one base and at least one protic solvent. The base may include, but is not limited to, sodium hydroxide, potassium carbonate, potassium bicarbonate, sodium carbonate, sodium bicarbonate, tris base, and the like, or any combination thereof. The protic solvent may include, but is not limited to, an alcohol (e.g., ethanol, methanol, propanol (1-propanol, 2-propanol), butanol (n-butanol), etc.), nitromethane, water, or any combination thereof.

In some embodiments, when included, the alkaline pretreatment step may generally be performed at a temperature ranging from about 0 ℃ to about 80 ℃, for example, at a temperature ranging from about 10 ℃ to about 70 ℃, about 25 ℃, or at room temperature. The contact time for the alkaline pretreatment may vary. In some embodiments, the alkaline pretreatment time has a contact time ranging from 1 minute to 72 hours. In some embodiments, the alkaline pretreatment time has a contact time ranging from 0.5 hours to 48 hours. In some embodiments, the alkaline pretreatment time has a contact time ranging from 10 minutes to 12 hours. In some embodiments, the alkaline pretreatment time has a contact time ranging from 1 hour to 24 hours. In some embodiments, the alkaline pretreatment time has a contact time ranging from 0.5 hours to 16 hours. In some embodiments, the alkaline pretreatment time has a contact time ranging from 0.5 hours to 4 hours. In some embodiments, the alkaline pretreatment time has a contact time ranging from 20 minutes to 2 hours. In some embodiments, the alkaline pretreatment time has a contact time ranging from 4 hours to 16 hours. As noted, alkaline pretreatment is not required in forming the activated or derivatized substrate.

Once formed, the activated substrate may be further derivatized via an epoxy-containing linking group. Advantageously, the activated substrate (e.g., activated film substrate) may not need to be dried after activation. However, in other embodiments, the activated film may be dried prior to further derivatization. The drying method is not particularly limited, and may include forced air drying or simple air drying. Generally, in some embodiments, the drying temperature will be such as to ensure no damage to the activated substrate, e.g., near room temperature, such as from about 20 ℃ to about 40 ℃, or from about 25 ℃ to about 30 ℃. After drying, the dried activated substrate may retain the reactive functional groups until use. For example, the dried activated film may be stored and/or transported for a period of time (e.g., from days to a month or even longer, depending on the storage conditions), and after that time the desired reactive functionality may be maintained at the surface, e.g., for further derivatization.

To further derivatize the activated substrate, the substrate may be contacted (e.g., immersed) in a derivatizing solution. The derivatizing solution can include an organic solvent, a base, and a derivatizing agent. In one embodiment, the derivatizing agent can include a hydrophobic moiety that can provide a hydrophobic ligand on the surface of the substrate upon reaction of the derivatizing agent with a reactive functional group of a linking agent that activates the substrate.

The derivatizing agent of the solution can include an epoxy-reactive functional group configured to react with the epoxy groups of the activated substrate and at least one hydrophobic moiety. The hydrophobic moiety may remain at the substrate surface as a hydrophobic ligand that is bonded to the substrate surface via a linker. For example, the epoxy-reactive functional groups may include, but are not limited to, primary or secondary amines, thioethers, epoxides, carboxylic acids, organohalides, and the like, or any combination thereof.

The hydrophobic portion of the derivatizing agent may comprise a hydrophobic ligand. Examples of hydrophobic ligands as may be bound to the activated substrate may include, but are not limited to, aliphatic chains having two or more carbons (e.g., butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl), benzyl-containing groups, phenyl-containing groups, phenol-containing groups, pyridine-containing groups, boric 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-benzenedimethylthiol, 2-phenylethanethiol), and the like, as well as any combinations thereof.

The hydrophobic ligand, which may be a component of the derivatizing agent and attached to the substrate via reaction of the derivatizing agent with the epoxy group of the linking agent, may be selected from the following ligands: these ligands follow the hydrophobic interactions of the hydrophobic interaction ligands; pi-pi stacking between the immobilized aromatic compound and the aromatic ring in the nucleobase containing the aromatic compound ligand, or electron supply/charge transfer of the thioether-containing ligand, or a combination thereof.

In one embodiment, the hydrophobic ligand of the derivatizing agent may exhibit affinity for a polynucleotide (e.g., a plasmid). However, the hydrophobic portion of the derivatizing agent is not limited to the hydrophobic ligands exemplified above, and alternative ligands may be incorporated on the activated substrate as described herein.

Examples of derivatizing agents may include, but are not limited to, thiophenol, 2-butanethiol, furfuryl mercaptan, 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-xylylene mercaptan, 1, 3-xylylene mercaptan, 1, 2-xylylene mercaptan, 4' -bis (mercaptomethyl) biphenyl, 2, 4-dichlorobenzyl mercaptan, 4-methoxybenzyl mercaptan, triphenylmethyl mercaptan, 2, 4-dimethoxythiophenol, or any combination thereof.

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

The base of the derivatizing solution may be the same as or different from the base of the activating solution. In some embodiments, the base may be a weak base. Weak bases of the derivatizing solution may include, but are not limited to, alkylamines (e.g., methylamine, triethylamine, trimethylamine, tripropylamine, tributylamine, etc.), pyridine, imidazole, benzimidazole, histidine, guanidine, phosphazene base, N-dimethylbenzylamine, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, 3-dimethylaminopropylamine, N-diisopropylethylamine, N-dimethylenediamine, diethylamine, or any combination thereof. A strong base may optionally be used in the base component of the derivatizing solution. The strong base may include, but is not limited to, sodium amide, sodium hydroxide, potassium hydroxide, lithium bis (trimethylsilyl) amide, lithium t-butoxide, or any combination thereof.

In one embodiment, the concentration of the base in the derivatization solution may 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.

The organic solvent component of the derivatizing solution may be the same as or different from the organic solvent component of the activating solution. For example, the organic solvent of the derivatizing solution may include a protic organic solvent, an aprotic organic solvent, an aqueous/organic solvent mixture, or a combination thereof. Examples of solvents that may be used as in the derivatizing solution may include, but are not limited to, alcohols (e.g., ethanol, methanol, propanol (1-propanol, 2-propanol), butanol (N-butanol), etc.), nitromethane, dimethyl sulfoxide, dimethylformamide, N-methylpyrrolidone, or any combination thereof.

In some embodiments, the concentration of the organic solvent component of the derivatizing solution may range from about 0% (V/V) of the derivatizing solution to about 99% (V/V) of the derivatizing solution, such as from about 10% (V/V) of the derivatizing solution to about 90% (V/V) of the derivatizing solution, or from about 20% (V/V) of the derivatizing solution to about 85% (V/V) of the derivatizing solution.

To effect derivatization of the activated substrate, the substrate may be contacted with (e.g., immersed in) a derivatization solution for a period of time. Optionally, the derivatizing solution may be cooled or heated prior to/during contacting. For example, in some embodiments, the derivatizing step may be performed at a temperature of the derivatizing solution ranging from about 0 ℃ to about 80 ℃, such as at a temperature ranging from about 20 ℃ to about 60 ℃ or about 30 ℃.

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

In some embodiments, the derivatized substrate may be dried prior to use. In other embodiments, the derivatized substrate may be used immediately after derivatization. The drying method at the time of bonding is not particularly limited, and may include forced air drying or simple air drying. Generally, in some embodiments, the drying temperature will be such as to ensure no damage to the derivatized substrate, e.g., near room temperature, such as from about 20 ℃ to about 40 ℃, or from about 25 ℃ to about 30 ℃. After drying, the dried derivatized substrate may retain the hydrophobic ligand until use. For example, the dried activated membrane may be stored and/or transported for a period of time (e.g., from days to months or even years, depending on storage conditions), and after that time the desired hydrophobic ligand used in the HIC scheme may be maintained.

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

As an example, multiple derivatized film substrates may be stacked to form a multi-layer arrangement to increase capacity for a given application. In one embodiment, the stacked arrangement of derivatized membranes may have a thickness of about 70 micrometers to about 10,000 micrometers, such as 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, about 100 micrometers or more, about 70 micrometers or more, 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 5,000 micrometers to about 250 micrometers, about 250 micrometers to about 250,250 micrometers, about 250 micrometers to about 250 micrometers, about 250,000 micrometers or more, about 250,000 micrometers or about 250 micrometers to about 250,250 micrometers.

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

As used herein, the term "column volume" refers to the volume of the column inner membrane bed according to standard practice. Thus, when considering the bed volume formed by the stack of derivatized film substrates, the column volume can be determined as the total volume of the film stack, i.e., stack thickness times the footprint of the stack.

In one embodiment, the separation medium may be used to purify pDNA and other polynucleotides, including but not limited to 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.

The separation system incorporating a separation medium as described herein may comprise a separation column as known in the art. As examples, separation columns contemplated herein may include, but are not limited to, syringe filtration columns, centrifuge columns, cartridges, multi-well plates, spiral wound membrane columns, and the like.

The separation scheme using the disclosed materials can be run via standard methods. In one embodiment, a separation column incorporating a derivatized HIC membrane as described herein may be operated in a bind-elute mode using elution methods as generally known in the art.

Isolation using the disclosed HIC separation medium allows rapid and efficient purification of polynucleotides at fast flow rates. For example, polynucleotide purification methods utilizing separation media as disclosed herein can provide greater than 80% recovery of target material having 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 1mg polynucleotide (e.g., pDNA)/milliliter of separation medium at a residence time of less than 120 seconds.

The separation system as disclosed may exhibit high process productivity. The process productivity of the column can be defined using the following equation. In the denominator, V _tot is the total volume of solution that passed through the column during the entire process (including loading, washing, eluting, and regenerating steps). BV is HIC media bed volume and τ is residence time. The loading volume is proportional to the dynamic binding capacity of the HIC medium. Thus, process productivity increases with increasing combined capacity and decreasing residence time.

Dynamic binding capacity generally refers to the concentration of bound polynucleotide (milligrams bound per unit volume of membrane bed) in the separation medium in the effluent at the time of breakthrough. In an embodiment, the disclosed separation system can provide from about 1 to about 5mg/mL of medium at a residence time of 120 seconds and utilizing a 2.5M concentration of ammonium sulfate buffer, such as from about 1 to about 10mg/mL, about 1 to about 20mg/mL, about 1 to about 25mg/mL, about 1 to about 30mg/mL, about 2 to about 5mg/mL, about 2 to about 10mg/mL, about 2 to about 15mg/mL, about 2 to about 20mg/mL, about 2 to about 25mg/mL, about 2 to about 30mg/mL, about 3 to about 5mg/mL, about 3 to about 10mg/mL, about 3 to about 15mg/mL, about 3 to about 25mg/mL, about 3 to about 30mg to about 4 to about 5mg/mL, about 4 to about 5mg/mL, about 5 to about 5mg/mL, about 4 to about 5 mg/mL. As mentioned previously, the disclosed separation media may exhibit substantially similar dynamic binding capacities independent of the size of the target polynucleotide. As such, the dynamic binding capacity of the separation system may be substantially similar to the same system when purifying different polynucleotides (larger or smaller polynucleotides). Dynamic binding capacities that are substantially similar to each other may 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 in some cases exhibiting the same dynamic binding capacities.

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

The following is a listing of exemplary aspects of products and methods according to the present disclosure.

According to aspect 1, an HIC separation medium is disclosed comprising a porous cellulose membrane and a plurality of hydrophobic ligands bound to the surface of the porous cellulose membrane.

Aspect 2 is the HIC separation membrane of aspect 1, wherein the porous cellulose membrane comprises a cast membrane, a hydrogel membrane, or a fibrous membrane, such as an electrospun nanofiber membrane.

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

Aspect 4 is the HIC separation membrane of any one of the preceding aspects, wherein the porous cellulose membrane comprises a specific surface area measured from about 0.1 m/mL to about 25 m/mL, from about 0.5 m/mL to about 20 m/mL, from about 0.1 m/mL to about 15 m/mL, from about 0.1 m/mL to about 10 m/mL, from about 0.5 m/mL to about 30 m/mL, from about 0.5 m/mL to about 25 m/mL, from about 0.5 m/mL to about 20 m/mL, from about 0.5 m/mL to about 15 m/mL, from about 0.5 m/mL to about 10 m/mL, from about 0.5 m/mL to about 5 m/mL, from about 1 m/mL to about 30 m/mL, from about 1 m/mL to about 25 m/mL, from about 1 m/mL to about 20 m/mL, from about 1 m/mL to about 15 m/mL, from about 5m to about 5 m/mL, from about 5m to about 10 m/mL, from about 5m to about 5 m/mL, from about 5 m/mL to about 10 m/mL, from about 5 m/mL to about 5 m/mL, from about 5 m/mL to about 15 m/mL, or from about 5m to about 15 m/mL.

Aspect 5 is the HIC separation membrane of any one of the preceding aspects, wherein the porous cellulose membrane comprises a pore size of from about 0.1 microns to about 10 microns, from about 0.1 microns to about 0.2 microns, from about 0.1 microns to about 0.45 microns, from about 0.1 microns to about 10 microns, from about 0.1 microns to about 2 microns, from about 0.2 microns to about 0.45 microns, from about 0.2 microns to about 1 micron, from about 0.2 microns to about 2 microns, from about 0.2 microns to about 10 microns, from about 0.45 microns to about 1 micron, from about 0.45 microns to about 2 microns, from about 0.45 microns to about 10 microns, from about 1 micron to about 2 microns, or from about 1 micron to about 5 microns.

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

Aspect 7 is the HIC separation membrane of any one of the preceding aspects, 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. Furthermore, each of the plurality of hydrophobic ligands may be bonded to the surface of up to Kong Qianwei of the plain film via a linking group comprising the reaction product of an epoxy and an epoxy-reactive functional group (e.g., an amine or thioether moiety).

Aspect 8 is the HIC separation membrane of any one of the preceding aspects, wherein the medium has a residence time of greater than about 1mg/mL at 120 seconds and with a 2.5M ammonium sulfate buffer concentration, such as from about 1mg/mL to about 10mg/mL, about 1mg/mL to about 20mg/mL, about 1mg/mL to about 25mg/mL, about 1mg/mL to about 30mg/mL, about 2mg/mL to about 5mg/mL, about 2mg/mL to about 10mg/mL, about 2mg/mL to about 15mg/mL, about 2mg/mL to about 20mg/mL, about 2mg/mL to about 25mg/mL, about 2mg/mL to about 30mg/mL, about 3mg/mL to about 5mg/mL, about 3mg/mL to about 10mg/mL, about 3mg/mL to about 15mg/mL, about 3mg/mL to about 20mg/mL, about 3mg/mL to about 25mg/mL, about 3mg/mL to about 30mg/mL, about 4mg/mL to about 5mg/mL, about 4mg/mL to about 10mg/mL, about 4mg/mL to about 15mg/mL, about 4mg to about 20mg/mL, about 4mg to about 5mg/mL, about 5mg to about 25mg/mL, about 15mg to about 5mg/mL, about 15mg to about 25mg/mL, and about 15 mg/mL.

Aspect 9 is the HIC separation membrane of any one 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.

Aspect 10 is a separation column comprising the hydrophobic interaction chromatography separation medium of any of the preceding aspects. For example, wherein the separation column comprises a syringe filtration column, a centrifuge column, a cartridge, or a spiral wound membrane column.

Aspect 11 is a method for forming an activated substrate comprising contacting a substrate with an activation solution. The activation solution includes an activator, a base, and an organic solvent. The activator includes a reactive functional group configured to react with the surface of the substrate to form a linking group on the surface, the activator further including an epoxy group, the linking group including an epoxy group.

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

Aspect 13 is the method of aspect 11 or aspect 12, wherein the activator comprises epichlorohydrin, diglycidyl ether, or any combination thereof.

Aspect 14 is a method for derivatizing an activated substrate formed according to the method of aspects 11, 12, or 13, the method comprising contacting the activated substrate with a derivatizing solution comprising a derivatizing agent comprising an epoxy-reactive functional group and a hydrophobic moiety comprising a hydrophobic ligand, a base, and optionally an organic solvent.

Aspect 15 is the method of aspect 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 thiophilic group, or any combination thereof.

Aspect 16 is the method of aspect 14 or aspect 15, 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-xylylene mercaptan, 1, 3-xylylene mercaptan, 1, 2-xylylene mercaptan, 4' -bis (mercaptomethyl) biphenyl, 2, 4-dichlorobenzyl mercaptan, 4-methoxybenzyl mercaptan, triphenylmethyl mercaptan, 2, 4-dimethoxythiophenol, or any combination thereof.

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

Aspect 18 is the method of any one of aspects 11-17, wherein the base comprises an alkylamine (e.g., methylamine, triethylamine, trimethylamine, tripropylamine, tributylamine), pyridine, imidazole, benzimidazole, histidine, guanidine, phosphazene base, N-dimethylbenzylamine, 3-dimethylaminopropylamine, N-diisopropylethylamine, N-dimethylenediamine, diethylamine, sodium amide, sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, or potassium bicarbonate.

The present disclosure may be better understood with reference to the examples provided below.

Test method

To form the chromatographic column of the example, layers of two 24mm circular HIC membrane discs were packed into a membrane housing (membrane volume=0.055 mL) to measure DBC _10％ values.

Chromatographic test using a rapid protein liquid chromatography systemPure 25, situo Va (Cytiva)). Four sizes of plasmids were tested as examples. Plasmid 1) pRP [ Exp ] -CMV > EGFP,3657bp, 2) pRP [ Exp ] -Alb > hCAS9, 8956bp and 3) pLV [ Exp ] -EGFP: T2A: bsd-Alb > dCAS9/VPR,16129bp were usedMega kit (class number 12281) was purified. pALD-HELP,11584bp was purchased from a supplier. The plasmid stock was diluted to a concentration of 6-13. Mu.g/mL. Ammonium sulfate was used as loading buffer at different concentrations in 40mM 1xTE (Tris-HCl).

The dynamic binding capacity was determined as the concentration of plasmid retained in the effluent at breakthrough (milligrams of bound plasmid per unit volume of membrane bed). The dynamic binding capacity at 10% breakthrough (DBC _10％) represents the binding mass per unit volume of the membrane bed when the plasmid concentration in the effluent from the membrane bed reaches 10% of the feed.

Example 1

Activating: the 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 at room temperature overnight with shaking at 120rpm in the dark. After 20-24 hours, the membrane was rinsed with EtOH for five minutes followed by two five minute acetone rinses. Subsequently, the film sheet was dried at room temperature.

MCP binding: the activated membrane was treated with 82mM MCP dissolved in a solution containing 80.7% (v/v) methanol, 8.6% (v/v) Deionized (DI) water, 9.6% (v/v) 5M sodium hydroxide (NaOH) and 1.1% (v/v) TEA. The reaction was carried out at room temperature overnight with shaking at 100rpm in the dark. After 20-24 hours, the membranes were rinsed with ethanol for five minutes, followed by two five-minute acetone rinses. Subsequently, the film sheet was dried at room temperature.

Example 2

Activating: 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: the reaction time for membrane 1 was one hour, for membrane 2 was three hours and for membrane 3 was overnight (20-24 hours). The reaction was carried out on a shaker at 120rpm and at room temperature in the dark. After the reaction, the membrane was rinsed and dried following the same procedure as described in example 1.

MCP binding: the dried activated film was derivatized in the same manner as in example 1.

Fig. 1 shows that HIC membrane binding capacity in terms of DBC _10％ increases with increasing reaction time. For the same activation solution and the same binding process, the reaction time of 20-24 hours was increased by 59% compared to the binding capacity obtained from the 1 hour reaction.

Example 3

Activating: 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 changes were made to the formulation: 1) Immersing membrane 4 in an activation solution of double EPI concentration of 16.8% v/v; 2) The membrane 5 was immersed in an activation solution in which EtOH was replaced with the same amount of 2-propanol (IPA).

As shown in fig. 2, doubling the EPI concentration resulted in a 25% increase in the binding capacity of membrane 4 as compared to membrane 2. In addition, substitution of EtOH with IPA resulted in little or no change in the binding capacity between membrane 5 and membrane 2, indicating that in some cases IPA may be used in place of EtOH to prepare the membranes.

Example 4

Alkaline treatment: 0.3M NaOH in 100% ethanol was prepared the day before the reaction and allowed to sit on a stir plate overnight. The solution was clarified just prior to the reaction using a 47mm 0.22 μm regenerated cellulose membrane filter.

Four regenerated cellulose membrane strips and two alkaline treatments were used:

Group I) membranes a and B were immersed in 0.3M NaOH dissolved in 100% ethanol at 80rpm on a shaker at room temperature for 30 minutes and then transferred to be immersed in 0.1M NaOH in DI at 80rpm and room temperature on a shaker for another 30 minutes;

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

Membranes a and C were rinsed with 200mm Tris pH 7 for five minutes, followed by DI water for five minutes, and then acetone rinsed twice for five minutes. Subsequently, these films were dried at room temperature under appropriate forced air conditions. At the same time, membranes B and D were rinsed with 200mM Tris pH 7 for five minutes, with DI water for five minutes, followed by ethanol for five minutes. Subsequently, membranes B and D were transferred into the activation solution.

Activating: the membrane was activated in the same manner as in example 1.

MCP binding: the dried activated film was coupled in the same manner as in example 1.

As shown in fig. 3, the alkaline treatment improves the structural integrity of the membrane. Figure 4 compares the binding capacities of membranes prepared using different alkaline pretreatments. In fig. 4, the membrane treated with 0.3M NaOH in EtOH has a similar binding capacity as the membrane treated in the same way with 0.1M NaOH in water.

Example 5

Alkaline treatment: 0.3M NaOH in 100% ethanol (EtOH) was prepared the day before the process and allowed to stand on a stir plate overnight. Just prior to the reaction, the solution was clarified using 47mm 0.22 μm Cellulose Acetate (CA) membrane filter.

Three membrane strips and two alkaline treatments were used:

Group I) membranes C and F were immersed in 0.3M NaOH dissolved in 100% etoh on a shaker at 80rpm and at room temperature for 30 minutes.

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

After alkaline treatment, membranes C and G were rinsed with 200mm Tris pH 7 for five minutes, followed by DI for five minutes, and then acetone twice. Film C and film G were then dried at room temperature under forced air conditions and then activated.

After alkaline treatment, membranes F and H were rinsed with 200mm Tris pH 7 for five minutes, followed by DI for five minutes and then EtOH for five minutes. Membranes F and H were not dried prior to activation.

Activating: membranes C and G were rinsed with EtOH and acetone and then transferred to the binding solution in example 1.

Membranes F and H were rinsed with EtOH for five minutes followed by two DI rinses, each for five minutes, and then transferred directly into the binding solution in example 1.

MCP binding: the film was derivatised in the same manner as in example 1.

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

Example 6

Chromatographic testing was performed using a column equipped with membrane 3 from example 2 as a medium. The plasmid size was 3657bp. As loading buffer, 3M ammonium sulfate in 40mM Tris-HCl, pH 8 was used. Figure 5 shows the resulting chromatograms (showing the loading, washing and elution phases of the separation). In all tests, the elution peaks were similar. The UV ₂₆₀ absorbance and buffer volume vary from film to film. The UV ₂₆₀ absorbance and buffer volume vary from film to film.

The chromatographic test was run again under the same conditions, but the loading buffer was reduced from pH 8 to pH 5. Fig. 6 shows the results of binding capacity, showing the 30% increase in binding capacity observed when the pH of the loading buffer was reduced.

Example 7

Chromatography testing was performed under various conditions using a column with membrane 3 of example 2 as medium to further test the material.

Initially, the test medium was used for plasmids of different sizes, including 3kbp, 8kbp and 16kbp. The loading buffer used for all runs was 2.5M ammonium sulfate and 1xTE buffer (pH 7.5 and flow rate 2mL/min (36.4 CV/min)). The results of Dynamic Binding Capacity (DBC) are provided in table 1 below and fig. 7.

TABLE 1

Plasmid size (kbp)	DBC(mg/mL)
		3	4.7
8	5.0
		16	5.0

As indicated, the dynamic binding capacity of the medium is substantially independent of plasmid size.

The effect of flow rate variation of the media on dynamic binding capacity was tested. An 8kbp plasmid was used. The loading buffer was 2.5M ammonium sulfate and 1xTE buffer (pH 7.5). The results are provided in table 2 below and fig. 8.

TABLE 2

The effect of different ammonium sulfate concentrations on dynamic binding capacity was also tested. For all runs, the 8kbp plasmid was used and the flow rate was set at 2mL/min. The results are provided in table 3 below and fig. 9.

TABLE 3 Table 3

(NH₄)₂SO₄(M)	DBC(mg/mL)
		2.5	5.0
2.2	3.6
		2	2.0
1.8	2.1

The membrane 3 chromatography column was compared to a pre-packed commercial resin column (CYTIVA HISCREEN ^TM PLASMIDSELECT,4.7 mL). The loading buffer was 3M ammonium sulfate and 1xTE (pH 7.5). The plasmid was 11kbp. The results are shown in table 4 below and fig. 10.

TABLE 4 Table 4

Column type	DBC(mg/mL)
		Resin composition	0.31
Film 3	4.2

Although 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

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 of claim 1 wherein the porous cellulose membrane comprises a cast membrane, a hydrogel membrane or a fibrous membrane, such as an electrospun nanofiber 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 from about 0.1 m/mL to about 30 m/mL, from about 0.1 m/mL to about 25 m/mL, from about 0.1 m/mL to about 20 m/mL, from about 0.1 m/mL to about 15 m/mL, from about 0.1 m/mL to about 10 m/mL, from about 0.5 m/mL to about 30 m/mL, from about 0.5 m/mL to about 25 m/mL, from about 0.5 m/mL to about 20 m/mL, from about 0.5 m/mL to about 15 m/mL, from about 0.5 m/mL to about 10 m/mL, from about 0.5 m/mL to about 5 m/mL, from about 1 m/mL to about 30 m/mL, from about 1 m/mL to about 25 m/mL, from about 1 m/mL to about 20 m/mL, from about 1 m/mL to about 5 m/mL, from about 5 m/mL to about 15 m/mL, from about 5 m/mL to about 5 m/mL, from about 5 m/mL to about 15 m/mL, from about 5 m/mL to about 5 m/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 microns to about 10 microns, from about 0.1 microns to about 0.2 microns, from about 0.1 microns to about 0.45 microns, from about 0.1 microns to about 10 microns, from about 0.1 microns to about 2 microns, from about 0.2 microns to about 0.45 microns, from about 0.2 microns to about 1 micron, from about 0.2 microns to about 2 microns, from about 0.2 microns to about 10 microns, from about 0.45 microns to about 1 micron, from about 0.45 microns to about 2 microns, from about 0.45 microns to about 10 microns, from about 1 micron to about 2 microns, or from about 1 micron to about 5 microns.

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 pore size of from 70 microns to 10,000 microns, such as about 10,000 microns or greater, about 7,500 microns or greater, about 5,000 microns or greater, about 2,500 microns or greater, about 1,000 microns or greater, about 900 microns or greater, about 800 microns or greater, about 700 microns or greater, about 600 microns or greater, about 500 microns or greater, about 400 microns or greater, about 300 microns or greater, about 200 microns or greater, about 100 microns or greater, about 70 microns or greater, such as from about 70 microns to about 100 microns, from about 70 microns to about 200 microns, from about 70 microns to about 300 microns, from about 70 microns to about 400 microns, from about 70 microns to about 500 microns, from about 70 microns to about 750 microns, from about 70 microns to about 1,000 microns, from about 70 microns to about 2,000 microns, from about 70 microns to about 3,000 microns, from about 70 microns to about 4,000 microns, from about 70 microns to about 5,000 microns, from about 250 microns to about 300 microns, from about 250 microns to about 400 microns, from about 250 microns to about 500 microns, from about 250 microns to about 750 microns, from about 250 microns to about 1,000 microns, from about 250 microns to about 2,000 microns, from about 250 microns to about 3,000 microns, from about 500 microns to about 1,000 microns, from about 500 microns to about 2,000 microns, from about 500 microns to about 3,000 microns, from about 500 microns to about 4,000 microns, or from about 500 microns to about 5,000 microns.

7. The hydrophobic interaction chromatography separation medium of any of the preceding claims wherein the hydrophobic ligands comprise aliphatic chains having two or more carbons, benzyl-containing groups, phenyl-containing groups, phenol-containing groups, pyridine-containing groups, boric acid groups, branched polymers, sulfur-containing thiophilic groups, 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 comprising the reaction product of an epoxy and an epoxy-reactive functional group, such as an amine or thioether moiety.

8. The hydrophobic interaction chromatography separation medium of any of the preceding claims wherein the medium has a residence time of greater than about 1mg/mL, such as from about 1mg/mL to about 10mg/mL, about 1mg/mL to about 20mg/mL, about 1mg/mL to about 25mg/mL, about 1mg/mL to about 30mg/mL, about 2mg/mL to about 5mg/mL, about 2mg/mL to about 10mg/mL, about 2mg/mL to about 15mg/mL, about 2mg/mL to about 20mg/mL, about 2mg/mL to about 25mg/mL, about 2mg/mL to about 30mg/mL, about 3mg/mL to about 5mg/mL, about 3mg/mL to about 10mg/mL, about 3mg/mL to about 15mg/mL, about 3mg/mL to about 20mg/mL, about 3mg/mL to about 25mg/mL, about 2mg/mL to about 5mg/mL, about 4mg to about 5mg/mL, about 5mg to about 5mg/mL, about 4mg to about 5mg/mL, about 5mg to about 5 mg/mL.

9. The hydrophobic interaction chromatography separation medium of any one 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 one of the preceding claims, wherein the separation column comprises a syringe filter column, a centrifuge column, a cartridge, or a spiral wound membrane column.

11. A method for forming an activated substrate, the method comprising contacting a substrate with an activation solution comprising an activator, a base, and an organic solvent, the activator comprising a reactive functional group configured to react with a surface of the substrate to form a linking group on the surface, the activator further comprising 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, nylon, polysulfone, polyethersulfone, polyvinylidene fluoride, polyacrylonitrile, polyetherimide, polypropylene, polyethylene, or polyether terephthalate.

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

14. A method for derivatizing an activated substrate formed according to the method of any one of claims 11-13, the method comprising contacting the activated substrate with a derivatizing solution comprising a derivatizing agent comprising an epoxy-reactive functional group and a hydrophobic moiety comprising a hydrophobic ligand, a base, and optionally an organic solvent.

15. The method of claim 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 thiophilic group, or any combination thereof.

16. The method of claim 14 or claim 15, 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-xylylene mercaptan, 1, 3-xylylene mercaptan, 1, 2-xylylene mercaptan, 4' -bis (mercaptomethyl) biphenyl, 2, 4-dichlorobenzyl mercaptan, 4-methoxybenzyl mercaptan, triphenylmethyl mercaptan, 2, 4-dimethoxythiophenol, or any combination thereof.

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

18. The method of any one of claims 11-17, wherein the base comprises an alkylamine (e.g., methylamine, triethylamine, trimethylamine, tripropylamine, tributylamine), pyridine, imidazole, benzimidazole, histidine, guanidine, phosphazene base, N-dimethylbenzylamine, 3-dimethylaminopropylamine, N-diisopropylethylamine, N-dimethylenediamine, diethylamine, sodium amide, sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, or potassium bicarbonate.