Separation media for nucleotides and nucleotide components and purification methods using same

Abstract

The separation media includes a membrane and a plurality of ligands immobilized on the membrane, including anion exchange ligands, cation exchange ligands, thiophilic ligands, hydrophilic ligands, hydrophobic interaction ligands, or a combination thereof. The separation media may be multimodal. The separation medium may be configured for separating a target molecule including a nucleic acid, a nucleotide, a nucleoside, a nucleobase, or an analog or derivative thereof from the reaction mixture. The separation media may be configured for use with an organic solvent. The separation device includes a separation medium. Materials including nucleic acids, nucleotides, nucleosides, nucleobases, or analogs and derivatives thereof can be purified at high speeds using separation devices.

Description

Technical Field

[0001] The present disclosure relates to separation media that can be used to separate biomolecules and ions such as nucleotides, nucleosides or nucleobases from solutions, suspensions or dispersions. The separation media of the present disclosure can be used for separations in membrane chromatography. The present disclosure further relates to methods of making and using separation media. Priority application

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 203,198, filed on July 12, 2021, the disclosure of which is incorporated herein by reference in its entirety. Background Art

[0003] The use of nucleotides, nucleosides and their analogs in therapeutics is a rapidly growing industrial sector. As nucleic acid therapeutics develop and their production scales up, improved separation and purification methods are needed. Summary of the invention

[0004] Disclosed are separation media that can be used to separate target molecules from solutions, suspensions, or dispersions. The target molecules can be biomolecules or ions including nucleotides or nucleosides. The separation media of the present disclosure can be used for separations in membrane chromatography. The present disclosure further relates to methods of making and using the separation media.

[0005] According to an embodiment, the separation medium includes a membrane; and a plurality of ligands fixed on the membrane. The plurality of ligands may include an anion exchange ligand, a cation exchange ligand, a thiophilic ligand, a hydrophobic interaction ligand, a hydrophilic ligand, or a combination thereof. The separation medium may be configured to separate a target molecule including nucleotides, nucleosides, nucleobases, derivatives and analogs thereof, and combinations thereof from a reaction mixture. The separation medium may be configured to be used with an organic solvent.

[0006] The plurality of ligands may include anion exchange ligands comprising aliphatic diamines or triamines containing 1 to 18 carbons between adjacent amines. The anion exchange ligands may include N,N-dimethylethylenediamine, N,N-dimethylpropylenediamine, N,N-dimethylpropylenediamine, N,N-diethylpropylenediamine, or a combination thereof.

[0007] The plurality of ligands may include cation exchange ligands including aminocarboxylic acids, aminosulfonic acids, or combinations thereof. The cation exchange ligands may include aminobenzoic acid, aminodiacetic acid, aminopropionic acid, 3-amino-1-propanesulfonic acid, 3-amino-1-ethanesulfonic acid, or combinations thereof.

[0008] Various ligands can include two or more of anion exchange ligands, cation exchange ligands, sulfophilic ligands, hydrophilic ligands and hydrophobic interaction ligands. Various ligands can include ligands with cation exchange functional groups and sulfophilic functional groups. Various ligands can include mercaptobenzoic acid, mercaptosulfonic acid, its salt or its combination. Preferably, various ligands can include 3-mercapto-1-propanesulfonic acid sodium.

[0009] A separation device may include: a housing; and a separation medium disposed in the housing. The separation medium may include a membrane and a plurality of ligands fixed on the membrane, wherein the plurality of ligands include an anion exchange ligand, a cation exchange ligand, a thiophilic ligand, a hydrophobic interaction ligand, a hydrophilic ligand, or a combination thereof. The housing may include a box or a column. The separation medium may be configured to separate a target molecule including a nucleotide, a nucleoside, a nucleobase, a derivative or analog thereof, or a combination thereof from a reaction mixture. The separation medium may be configured to be used with an organic solvent.

[0010] The method for purifying a target molecule may include passing a solution containing the target molecule through a membrane chromatography device. The target molecule may include nucleotides, nucleosides, nucleobases, derivatives or analogs thereof, or a combination thereof. The membrane chromatography device may include a housing; and a separation medium disposed within the housing. The separation medium may include a membrane and a plurality of ligands fixed on the membrane, the plurality of ligands including anion exchange ligands, cation exchange ligands, thiophilic ligands, hydrophobic interaction ligands, hydrophilic ligands, or a combination thereof. The housing may include a box or a column. The separation medium may be configured to separate a target molecule including nucleotides, nucleosides, nucleobases, derivatives or analogs thereof, or a combination thereof from a reaction mixture. The separation medium may be configured to be used with an organic solvent.

[0011] The target molecule can be purified from a solution comprising a reaction mixture after synthesizing nucleotides, nucleosides, nucleobases, derivatives or analogs thereof, or combinations thereof. The solution can comprise an organic solvent. The residence time of the solution in the membrane chromatography device can be 60 seconds or less. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1A is a schematic diagram of a separation medium according to an embodiment.

[0013] Figure 1B Yes Figure 1A Schematic perspective view of a separation device with a separation medium.

[0014] Figure 2 is a graphical representation of the dynamic binding capacity data from Example 3.

[0015] Figures 3A-3C is a graphical representation of the data from Example 4.

[0016] Figure 4 is a graphical representation of the dynamic binding capacity and pressure data from Example 4.

[0017] Figure 5A and 5B is a graphical representation of the binding-elution data from Example 5.

[0018] Fig. 6A is the chromatogram from Example 6.

[0019] Figure 6B is the DBC from instance 6 10％ and graphical representation of recovery data.

[0020] Fig. 7A is a graphical representation of the dynamic binding capacity data from Example 7.

[0021] Figure 7B is the chromatogram from Example 7.

[0022] Fig. 8A Overlaid chromatograms from Example 8 are shown.

[0023] Figure 8B is a thin layer chromatography plate of a sample from Example 8.

[0024] Figures 9A-9C is a graphical representation of the binding-elution data from Example 9.

[0025] Fig.9D is a comparison of the chromatograms from Example 9.

[0026] Fig.10 is a graphical representation of the data from Example 10. definition

[0027] Unless otherwise specified, all scientific and technical terms used herein have the meaning commonly used in the art. The definitions provided herein are to aid in the understanding of certain terms frequently used herein and are not meant to limit the scope of the present disclosure.

[0028] Unless otherwise indicated, the terms "polymer" and "polymeric material" include organic homopolymers, copolymers, such as block, graft, random and alternating copolymers, terpolymers, etc., and blends and modifications thereof. Furthermore, unless otherwise specifically limited, the term "polymer" shall include all possible geometric configurations of the material. These configurations include isotactic, syndiotactic and atactic symmetries.

[0029] The term "aromatic ring" is used in this disclosure to refer to a conjugated ring system of an organic compound. The aromatic ring may contain only carbon atoms, or may contain one or more heteroatoms, such as oxygen, nitrogen, or sulfur.

[0030] The term "alkylated" is used in this disclosure to describe a compound that undergoes a reaction to replace a hydrogen atom or a negative charge of the compound with an alkyl group such that the alkyl group is covalently bonded to the compound.

[0031] The term "alkyl" is used to describe the monovalent group as an alkane free radical in this disclosure, which includes straight chain, branched, cyclic and bicyclic alkyl groups and combinations thereof, including both unsubstituted and substituted alkyl groups. Unless otherwise specified, alkyl groups typically contain from 1 to 30 carbon atoms. In certain embodiments, alkyl groups include 1 to 20 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, 1 to 4 carbon atoms or 1 to 3 carbon atoms. The example of alkyl group includes methyl, ethyl, n-propyl, n-butyl, n-pentyl, isobutyl, tert-butyl, isopropyl, n-octyl, n-heptyl, ethylhexyl, cyclopentyl, cyclohexyl, cycloheptyl etc.

[0032] As used herein, the term "nucleic acid" and / or "oligonucleotide" refers to a polymer containing at least two nucleotides (e.g., deoxyribonucleotides or ribonucleotides) in single-stranded or double-stranded form, and includes DNA and RNA." Nucleotide" includes sugar, base (sometimes referred to as a core base) and a linking group. In certain embodiments, sugar can be natural deoxyribose or natural ribose (e.g., DNA and RNA, respectively). Nucleotides are linked together to form oligonucleotides by a linking group. In certain embodiments, the linking group can be a phosphate group. The polymer of the covalently bonded linking group can be referred to as a backbone. "Nucleoside" is similar to nucleotides in other respects, except that nucleoside does not include a linking group, such as a phosphate group. "Base" or "core base" includes purine and pyrimidine, which further includes natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs and synthetic derivatives of purine and pyrimidine, which include the modification of placing new reactive groups such as amines, alcohols, thiols, carboxylates and alkyl halides. Nucleotide comprises modified or similar nucleobase, modified or similar sugar and / or modified or similar linking group.The nucleobase of modification, the sugar of modification and / or the linking group of modification can be non-standard / chemically modified nucleobase, sugar and / or linking group, it can be synthetic, naturally occurring and / or non-naturally occurring, and it has the binding characteristic similar to reference nucleic acid.The example of such analog and / or modified base, sugar and / or linking group includes but is not limited to phosphorothioate, phosphoramidate, methyl phosphonate, chiral-methyl phosphonate, 2'-O-methyl ribonucleotide, locked nucleic acid (LNA) and peptide-nucleic acid (PNA).

[0033] Deoxyribonucleotide is made up of the 5-carbon sugar called deoxyribose, which is covalently attached to phosphate at the 5' and 3' carbon of the sugar to form an alternating non-branched polymer. DNA can be in the form of derivatives and combinations of, for example, antisense molecules, plasmid DNA, pre-condensed DNA, PCR products, vectors, expression cassettes, chimeric sequences, chromosomal DNA or these groups. Ribo-oligonucleotide is made up of similar repeating structures, in which the 5-carbon sugar is ribose. Therefore, the terms "polynucleotide" and "oligonucleotide" can refer to polymers or oligomers of nucleotides or nucleoside monomers composed of (main chain) linkages between naturally occurring bases, sugars and sugars.

[0034] The terms "polynucleotide" and "oligonucleotide" may also include polymers or oligomers comprising non-naturally occurring monomers or portions thereof, which function similarly. Such modified or substituted oligonucleotides are generally preferred over natural forms due to properties such as enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases. It should be understood that the terms "polynucleotide" and "oligonucleotide" may also include polymers or oligomers comprising a combination of deoxy and ribonucleotides or variants thereof in combination with backbone modifications, such as those described herein.

[0035] The polynucleotides and oligonucleotides 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 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-galactosylqueuosine, inosine, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6- Adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl quercetin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine, uracil-5-oxyacetic acid (v), whibutoxin, pseudouracil, quercetin, 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, the nucleotide may include modifications in its phosphate portion, including modifications to the triphosphate portion. 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 with thiol moieties (e.g., α-thiotriphosphates and β-thiotriphosphates).

[0036] Polynucleotides as herein described or oligonucleotides can be modified at base moieties (for example, at one or more atoms that can typically form hydrogen bonds with complementary nucleotides and / or at one or more atoms that typically can not form hydrogen bonds with complementary nucleotides), sugar moieties or linking groups (for example, main chain).Main chain modifications can include phosphorothioate, phosphorodithioate, selenophosphate, diselenose, thioaniline phosphate, aniline phosphate, phosphoramidate and diaminophosphorothioate linking bonds.Phosphorothioate linking bonds replace the non-bridge oxygen in the phosphate backbone with sulfur atoms, and delay the nuclease degradation of oligonucleotides.Diaminophosphorothioate linking bonds (N3'→P5') prevent nuclease recognition and degradation.Replacing the phosphorus in the main chain structure, main chain modifications can also include peptide bonds (for example, N-(2-aminoethyl)-glycine units connected by the 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. [Contemporary Medicinal Chemistry], 8(10): 1157-79, 2001 and Lyer et al., Curr. Opin. Mol. Ther. [Current Opinions in Molecular Therapeutics], 1(3): 344-358, 1999. The nucleic acid molecules described herein may contain sugar moieties including ribose or deoxyribose present in naturally occurring nucleotides, or modified sugar moieties or sugar analogs. Modified sugar moieties include 2'-O-methyl, 2'-O-methoxyethyl, 2'-O-aminoethyl, 2'-fluoro, N3'→P5' phosphoramidate, 2'dimethylaminooxyethoxy, 2'2'dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidiniumethyl, carbamate-modified sugars, and bicyclic-modified sugars. 2'-O-methyl or 2'-O-methoxyethyl modifications promote A-form or RNA-like conformation in oligonucleotides, increase binding affinity to RNA, and have enhanced nuclease resistance. Modified sugar moieties may also include those with additional bridges (e.g., methylene bridges connecting the 2'-O and 4'-C atoms of the ribose in locked nucleic acids) or sugar analogs such as morpholine rings (e.g., in morpholino diaminophosphorothioates).

[0037] 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 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 replaced by mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. [Nucleic Acids Research], 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. [Journal of Biological Chemistry], 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes [Molecular and Cellular Probes], 8: 91-98 (1994)).

[0038] The method of the present disclosure encompasses the separation and / or purification of isolated or substantially purified nucleotides, nucleosides, nucleic acid molecules and compositions containing these molecules. As used herein, "isolated" or "substantially purified" DNA molecules or RNA molecules are DNA molecules or RNA molecules that exist without their natural environment. Isolated DNA molecules or RNA molecules can exist in purified form, or can exist in a non-natural environment, such as a transgenic host cell. For example, "isolated" or "purified" nucleic acid molecules or their biologically active parts are substantially free of other cellular materials or culture media when produced by recombinant technology, or are substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, "isolated" nucleic acids are free of sequences of natural flanking nucleic acids (i.e., sequences at the 5' and 3' ends of nucleic acids) in the genomic DNA of the organism from which the nucleic acid is derived.

[0039] The term "pharmaceutical composition" and its grammatical equivalents as used herein may refer to a mixture or solution containing a therapeutically effective amount of an active pharmaceutical ingredient and one or more pharmaceutically acceptable excipients, carriers and / or therapeutic agents to be administered to a subject (e.g., a human in need thereof).

[0040] The term "kosmotrope" is generally used to refer to a solute that increases the order of water by stabilizing water-water interactions. Kosmotropes can be ionic or non-ionic. In contrast, the term "chaotrope" is generally used to refer to a solute that decreases the order of water by destabilizing water-water interactions. Chaotropes can be ionic or non-ionic.

[0041] The term "substantially" as used herein has the same meaning as "significantly" and may be understood to modify the subsequent term with at least about 90%, at least about 95%, or at least about 98%. The term "substantially free" of a particular compound means that the composition of the present invention contains less than 1,000 parts per million (ppm) of the compound. The term "substantially free" of a particular compound means that the composition of the present invention contains less than 100 parts per million (ppm) of the compound. The term "completely free" of a particular compound means that the composition of the present invention contains less than 20 parts per billion (ppb) of the compound. In the context of the above phrases, the composition of the present invention contains less than the above amount of the compound, whether the compound itself is present in unreacted form or has been reacted with one or more other materials.

[0042] The term "non-substantially" as used herein has the same meaning as "non-significantly" and may be understood to have the opposite meaning of "substantially", i.e., modifying the subsequent term by no more than 25%, no more than 10%, no more than 5%, or no more than 2%.

[0043] The term "about" used herein in conjunction with numerical values ​​is intended to encompass the normal variation in measurements expected by those skilled in the art and should be understood to have the same meaning as "substantially" and encompass a typical error range, such as ±5% of the stated value.

[0044] Terms such as "a," "an," and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used.

[0045] The terms "a", "an" and "the" are used interchangeably with the term "at least one". The phrases "at least one of" and "including at least one of" preceding a list refer to any one item in the list and any combination of two or more items in the list.

[0046] As used herein, the term "or" is generally used in its ordinary sense including "and / or" unless the context clearly dictates otherwise. The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0047] The recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc., or 10 or less includes 10, 9.4, 7.6, 5, 4.3, 2.9, 1.62, 0.3, etc.). Where a range of values ​​is "up to" or "at least" a particular value, that value is included in the range.

[0048] As used herein, "have / having", "include / including", "comprise / comprising", etc. are used in an open-ended sense and generally mean "including but not limited to". It should be understood that the terms "consisting essentially of", "consisting of", etc. are included in "comprising", etc. As used herein, when referring to a composition, product, method, etc., "consisting essentially of" means that the components of the composition, product, method, etc. are limited to the listed components and any other components that do not materially affect the basic characteristic(s) and novel characteristics of the composition, product, method, etc.

[0049] The words "preferred" and "preferably" refer to embodiments that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. In addition, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the present disclosure, including the claims.

[0050] For the sake of clarity, any directions mentioned herein such as "top", "bottom", "left", "right", "upper", "lower" and other directions and orientations are described herein with reference to the accompanying drawings and are not limiting of the actual device or system or use of the device or system. The device or system described herein can be used in multiple directions and orientations. DETAILED DESCRIPTION

[0051] The present disclosure relates to separation media that can be used to separate target molecules from solutions, suspensions or dispersions. The target molecules can be biomolecules or ions, including nucleotides, nucleosides, nucleobases, derivatives or analogs thereof, or combinations thereof. The separation media of the present disclosure can be used for separation in membrane chromatography. The present disclosure further relates to methods for making and using separation media.

[0052] The gene and cell therapy industry is rapidly moving toward commercialization due to its tremendous potential to treat a variety of devastating 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. But pDNA production has become a bottleneck in the industry because it is not simple to scale up pDNA production. Currently, qualified contract manufacturers have long waiting lists and large backlogs to meet the high demand. Like many other biologics manufacturing scenarios, there are multiple steps and unit operations involved in pDNA production.

[0053] Nucleotide, nucleoside, core base, its derivative or analog or its combination are increasingly utilized to prepare pharmaceutical composition. Derivatization can include, for example, fluorination, sugar substitution, adding various functional parts or many other known or new modifications. Such derivatization can be designed to be transformed or modified into key structural units for nucleic acid therapy, or more tolerant or more effective medicines by improving pharmacokinetics, transportation, changing prodrug state or utilizing the unique enzymatic pathway of diseased tissue. Such pharmaceutical composition can be used for various situations, including HIV / AIDS treatment and cancer treatment. Such analogs are usually utilized by HIV or the approach of cell upregulation that produces cancer, or include analogs that induce mispairing or other replication / translation errors, prevent division and / or induce cell death.

[0054] In addition, with the rise of nucleic acid therapeutics, additional substitutions and modifications are being sought to improve the tolerability of the therapy by improving stability or reducing or upregulating immunogenic properties. For example, modifications to cytidine and uridine bases may include 5-iodocytidine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 2-thiocytidine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, pseudoisocytidine-5'-triphosphate, N- 4 -methylcytidine-5'-triphosphate, 5-carboxycytidine-5'-triphosphate, 5-formylcytidine-5'-triphosphate, 5-hydroxymethylcytidine-5'-triphosphate, 5-hydroxycytidine-5'-triphosphate, 5-methoxycytidine-5'-triphosphate, thienocytidine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate '-deoxycytidine-5'-triphosphate, 5-methyl-2'-deoxycytidine-5'-triphosphate, 2'-deoxy-P-nucleoside-5'-triphosphate, 5-hydroxy-2'-deoxycytidine-5'-triphosphate, 2-thio-2'-deoxycytidine-5'-triphosphate, 5-aminoallyl-2'-deoxycytidine-5'-triphosphate, pseudouridine-5'-triphosphate, 2'-O-methyl pseudouridine-5'-triphosphate, N 1 -methylpseudouridine-5'-triphosphate, N 1 -ethyl pseudouridine-5'-triphosphate, N 1 -methyl-2'-O-methylpseudouridine-5'-triphosphate, N 1 -methoxymethyl pseudouridine-5'-triphosphate, N 1 -propyl pseudouridine-5'-triphosphate, or its unphosphorylated base. Similar modifications can be made to thymidine and guanidine bases.

[0055] Traditionally, downstream purification is expensive, slow and difficult to scale. Typical purification series of nucleotides, nucleosides, core bases, derivatives or analogs thereof or combinations thereof include various steps, such as filtration, ultrafiltration and various chromatographic separations utilizing one or more types of chromatographic columns. Typical chromatographic columns for nucleotides, nucleosides, core bases or nucleic acid purification can have a packed bed column of resin, which is configured as, for example, size exclusion chromatography or reversed phase chromatography. For decades, chromatographic columns based on resins have been the gold standard for purifying biopharmaceuticals. However, large-volume column chromatography may be very slow. Known resin columns require long residence times to fully function.

[0056] According to an embodiment, the separation medium includes a functionalized substrate. The functionalized substrate can be a functionalized membrane. Compared with a resin column, a membrane adsorber performs well in a shorter column residence time, and it is possible to provide rapid separation for biological agents. The present disclosure provides a membrane suitable for separating and purifying various nucleotides, nucleosides, nucleobases, derivatives or analogs thereof, or combinations thereof. The compounds of interest that can be separated using the membrane of the present disclosure are collectively referred to as target molecules herein, whether they are in a charged (ionized) state or an uncharged state. The target molecule can be present in a solution, a suspension, or a dispersion. For simplicity, the liquid containing the target molecule is referred to as a solution herein. The liquid can be a reaction mixture. For example, the liquid can be a reaction mixture for preparing or synthesizing a target molecule (e.g., a nucleotide, a nucleoside, a nucleobase, a derivative or analog thereof, and a combination thereof). The separation medium can be configured to purify the target molecule from the reaction mixture. The separation medium can be configured to separate the target molecule from starting materials, intermediates, and other reaction products. The reaction mixture can also include a solvent, such as water, an organic solvent, or a combination thereof, and a soluble component dissolved in the solvent. The separation medium can be configured to be used with an organic solvent. The separation medium may be configured to separate or purify a target molecule from a solution comprising an organic solvent.

[0057] The functionalized substrates of the present disclosure comprise one or more functional groups that interact with target molecules. In some embodiments, the functional groups have an affinity for the target molecules and can bind to the target molecules or slow down their transfer across or along the membrane.

[0058] According to an embodiment, the target molecule includes a nucleotide, a nucleoside, a nucleobase, a derivative or an analog thereof, or a combination thereof. Nucleotides and nucleosides include nucleobases as their structural units. Generally, the disclosure provides membranes and methods for purifying nucleobases / nucleosides / nucleotides, which include natural nucleobases / nucleosides / nucleotides, modified nucleobases / nucleosides / nucleotides, nucleobases / nucleosides / nucleotides analogs, etc.

[0059] The four nucleobases in DNA (adenine (A), cytosine (C), guanine (G), thymine (T)) and an additional base (uracil (U)) have unique properties that can be exploited to enhance purification sequences that include hydrophobic components, aromatic components, hydrogen bond donors and acceptors, and / or groups that can induce charges. However, modifications of nucleobases / nucleosides / nucleotides may alter some of these properties. For example, compounds with amine groups may be intentionally or unintentionally protected with reagents such as tert-butyloxycarbonyl protecting groups (BOC) for separation. Protecting groups are used in synthesis to temporarily mask the chemical characteristics of functional groups because they interfere with other reactions. Protected amines are sterically hindered and have one less hydrogen bond donor site, and therefore hinder standard hybridization rules, however it is worth noting that there may be alternative pairing configurations that may or may not be affected by protected amine groups. Typically, such bases with protected groups are difficult to separate using traditional chemical separation techniques used to purify such nucleobase analogs in a pharmaceutical production environment, such as silica gel chromatography, liquid-liquid extraction, liquid / solid extraction, and distillation (which is generally unsafe due to the inhalation hazard of common protecting groups).

[0060] According to embodiments, the present disclosure provides functionalized substrates (e.g., functionalized membranes) for purifying nucleosides / nucleotides and analogs and derivatives thereof using hybridization and purification methods based on hybridization. For example, single nucleosides, nucleotides, or oligonucleotides can be effectively purified based on methods utilizing hybridization such as Watson Crick base pairing.

[0061] In certain embodiments, the use of Watson Crick base pairing provides a high specificity purification of target molecules. In one embodiment, nucleotides, nucleosides or oligonucleotides can be fixed on a substrate to provide a part, and then can be used to capture its complementary base. For example, adenine (A) or its derivatives can be fixed on a substrate to produce a functionalized substrate. Adenine functionalized substrates can be used to capture thymine (T), uracil (U) and derivatives thereof. Thymine (T), uracil (U) or derivatives thereof can be fixed on a support to capture adenine (A) and derivatives thereof. Cytosine (C) can be fixed to capture guanine (G) and derivatives thereof, and vice versa.

[0062] The substrate used as the base material of separation medium can be any suitable material. In certain embodiments, substrate is or includes film, resin, monolith, hydrogel, woven fiber substrate, nonwoven fiber substrate or its combination. In one embodiment, functionalized substrate is or includes film. In one embodiment, functionalized substrate is or includes weaving or nonwoven fiber substrate. Before reacting with the ligand, substrate can be modified to include reactive chemical part. This may be particularly useful in the case of non-reactive substrates such as ePTFE. Modification can include, for example, plasma treatment, dip-coating poly (vinyl alcohol), corona treatment, etc.

[0063] Nonwoven substrates (e.g., webs) are typically produced by meltblowing, wet-laid webs, melt spinning, solution spinning, air-laid webs, or electrostatic spinning. The nonwoven webs can also be processed by post-processing steps such as calendering, embossing, needle punching, or spunlace. The nonwoven substrate can also contain a structural resin with low binding affinity to biomolecules. Such resins are typically used to increase the strength of the nonwoven web. Many nonwoven substrates include a mixture of fiber slurry and fiber material. The fibers used to make nonwoven substrates and woven substrates can include glass, polypropylene, polyamide, polyester, cellulose materials, etc., and combinations thereof. The fiber can have an average fiber size of 0.1 μm or more, 1 μm or more, 2 μm or more, or 3 μm or more. The fiber can 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 can be in the range of 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 can 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 size of a suitable nonwoven substrate can be in the range of 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 a woven substrate can be slightly larger than a nonwoven substrate, and can be in the range of from 1 μm to 100 μm. The basis weight of the fiber substrate can be 1 gsm (grams per square meter) or more, 10 gsm or more, or 20 gsm or more. The basis weight of the fiber substrate can be 200 gsm or less or 80 gsm or less. The basis weight of the fibrous substrate may range from 1 gsm to 200 gsm, or from 20 gsm to 80 gsm.

[0064] According to an embodiment, substrate is or includes film. Film is understood to be a sheet of material having continuous polymer material channels in all dimensions. Examples of membrane materials include polyolefins, polyethersulfones, poly(tetrafluoroethylene), nylon, glass fiber, hydrogels, polyvinyl alcohol, natural polymers such as cellulose, cellulose esters, cellulose acetate, regenerated cellulose, cellulose nanofibers, cellulose derivatives, agarose, chitosan, polyethylene, polyester, polysulfone, expanded polytetrafluoroethylene (ePTFE), polyvinylidene fluoride, polyamide (nylon), polyacrylonitrile, polycarbonate, and combinations thereof.

[0065] According to an embodiment, useful membranes have an average pore size of 10 μm or less, 5 μm or less, 2 μm or less, 1 μm or less, 0.45 μm or less, or 0.2 μm or less as measured by capillary flow porosimetry. The membrane may have an average pore size of 0.1 μm or more, 0.2 μm or more, 0.45 μm or more, 0.7 μm or more, or 1 μm or more. The film can have a mean pore size ranging from about 0.1 μm to 10.0 μm, 0.1 μm to 0.2 μm, 0.1 μm to 0.45 μm, 0.1 μm to 1 μm, 0.1 μm to 2 μm, 0.2 μm to 0.45, 0.2 μm to 1 μm, 0.2 μm to 2 μm, 0.2 μm to 10 μm, 0.45 μm to 1 μm, 0.45 μm to 2 μm, 0.45 μm to 10 μm, 1 μm to 2 μm, or 1 μm to 5 μm. The film can have a thickness of 500 μm or more, 250 μm or more, 100 μm or more, 80 μm or more, 50 μm or more, or 30 μm or more. The film may have a thickness of 2500 μm or less, 1000 μm or less, 500 μm or less, 250 μm or less, or 100 μm or less. The thickness of the film may be in the range of 30 μm to 500 μm, 50 μm to 500 μm, 80 μm to 500 μm, 100 μm to 500 μm, 250 μm to 500 μm, 30 μm to 250 μm, 50 μm to 250 μm, 80 μm to 250 μm, 100 μm to 2500 μm, 30 μm to 100 μm, 50 μm to 100 μm, or 80 μm to 100 μm.

[0066] The membranes can be stacked in a multilayer arrangement to increase the capacity for a given application. In one embodiment, the stacked arrangement of the membranes has a thickness of 70 μm or more, 250 μm or more, or 500 μm or more. The stacked arrangement of the membranes can have a thickness of 10,000 μm or less, 7,500 μm or less, 5,000 μm or less, 4,000 μm or less, 3,000 μm or less, 2,500 μm or less, 2,000 μm or less, 1,000 μm or less, 750 μm or less, 500 μm or less, 400 μm or less, or 300 μm or less. The stacked arrangement of the membranes may have a range from 70 μm to 10,000 μm, 70 μm to 100 μm, 70 μm to 200 μm, 70 μm to 300 μm, 70 μm to 400 μm, 70 μm to 500 μm, 70 μm to 750 μm, 70 μm to 1,000 μm, 70 μm to 2,000 μm, 70 μm to 3,000 μm, 70 μm to 4,000 μm, 70 μm to 5,000 μm, 250 μm to 300 μm, 250 μm to 400 μm, 70 μm to 500 μm, 70 μm to 750 μm, The thickness of the film can be from 250 μm to 400 μm, from 250 μm to 500 μm, from 250 μm to 750 μm, from 250 μm to 1,000 μm, from 250 μm to 2,000 μm, from 250 μm to 3,000 μm, from 250 μm to 4,000 μm, from 250 μm to 5,000 μm, from 500 μm to 1,000 μm, from 500 μm to 2,000 μm, from 500 μm to 3,000 μm, from 500 μm to 4,000 μm, or from 500 μm or 5,000 μm.

[0067] In a preferred embodiment, the membrane is a regenerated cellulose membrane having a pore size between 0.2 μm and 5.0 μm, a thickness between 70 μm and 2,000 μm, and a thickness of approximately 70 μm to 10,000 μm in a stacked arrangement.

[0068] The substrate can be a microfiltration membrane. Microfiltration membranes are typically produced by a phase inversion process or an expansion process. Typical materials for preparing membranes include PES, nylon, PVDF, cellulose acetate, regenerated cellulose, polypropylene, and expanded PTFE.

[0069] In some cases, the membrane is not tolerant to a wide range of organic solvents.The membrane and ligand can be selected so that the membrane is insoluble in the solvent used in the separation or purification process.

[0070] In the embodiment that the target molecule is a nucleotide or modified nucleotide, a solvent, a salt or other additives can be added to a solution containing the target molecule during purification to fully screen any repulsion of the relevant phosphate groups, thereby allowing hybridization to occur. Different solvents or low conductivity buffers can be used to elute the target molecule from fixed bases by the charge repulsion between the target and the part. Suitable solvents include, for example, methanol, ethanol, isopropanol and acetonitrile, DMSO and DMF, etc. In certain embodiments, ethanol, isopropanol or acetonitrile are added to the solution. During purification, suitable solvents can be used in an amount of 5wt-% or more, 10wt-% or more, 20wt-% or more, 30wt-% or more, 40wt-% or more, 50wt-% or more, 60wt-% or more, 70wt-% or more, 80wt-% or more or 90wt-% or more by weight of the solution. Suitable solvent can be used in an amount of 90wt-% or less, 80wt-% or less, 70wt-% or less, 60wt-% or less, 50wt-% or less, 40wt-% or less, 30wt-% or less or 20wt-% or less by the weight of the solution. The solvent can be used in an amount ranging from 10wt-% to 90wt-%, 20wt-% to 80wt-%, 30wt-% to 70wt-% or 10wt-% to 50wt-% by the weight of the solution.

[0071] Suitable salts that can be included in the solution include, for example, sodium chloride, potassium chloride, lithium chloride, rubidium chloride, calcium chloride, magnesium chloride, cesium chloride, tris base, sodium phosphate, potassium phosphate, and ammonium sulfate. In certain embodiments, sodium chloride, potassium chloride, ammonium sulfate, calcium chloride, potassium chloride, or magnesium chloride are added to the solution. Suitable salts can be added in an amount of 2wt-% or more, 5wt-% or more, 10wt-% or more, 15wt-% or more, or 20wt-% or more by the weight of the solution. Suitable salts can be added in an amount of 20wt-% or less, 25wt-% or less, or 30wt-% or less by the weight of the solution. Salt can be added in an amount ranging from 2wt-% to 30wt-% or 5wt-% to 25wt-%, or 5wt-% to 20wt-% by the weight of the solution.

[0072] In some embodiments where the target molecule is a nucleoside / nucleobase or modified nucleoside / nucleobase (as opposed to a nucleotide or modified nucleotide), electrostatic repulsion becomes a less influential factor because nucleosides do not contain phosphate groups, and thus techniques to mitigate phosphate-phosphate repulsion become less relevant.

[0073] If the target molecule includes modifications to groups that are involved in hydrogen bonding or sterically hindered positions, a functionalized substrate (e.g., a functionalized membrane) can be used to separate the unmodified and modified groups because the modified groups behave differently when bound in a manner that can be used to capture the modified molecules and allow the unmodified molecules to flow through, or to capture the unmodified molecules and allow the modified molecules to flow through. Hydrogen bonding competitive additives or solvents can be used during the elution process. Examples of hydrogen bonding competitive additives and solvents include acetonitrile, alcohols, water, sugars, and combinations thereof.

[0074] In many embodiments, the target molecule is present in an aqueous solution. However, in some embodiments, even in a non-aqueous environment of a variety of solvents, the hydrogen bonds between A / T, A / U and C / G are still effective. Examples of such solvents include alcohols, acetonitrile, and combinations thereof. Therefore, in some embodiments, the target molecule is present in a solution comprising an organic solvent such as one or more alcohols or acetonitrile. In some such embodiments, the solution comprises water and an organic solvent. Most of the solution can be water. Alternatively, most of the solution can be composed of an organic solvent. In some embodiments, the solution is non-aqueous, for example, composed of an organic solvent.

[0075] In some embodiments, the target molecule includes a modification that reduces the water solubility of the target molecule (e.g., a nucleoside or nucleotide). In such embodiments, an aqueous buffer-organic solvent mixture can be used to help keep the target molecule in solution (especially for hydrophobic modifications) to improve process productivity.

[0076] According to an embodiment, a separation medium (functionalized substrate) can be used to purify a target molecule at a fast flow rate. For example, a separation medium can be used to purify a target molecule at a residence time of 2 minutes or less, 1 minute (60 seconds) or less, 30 seconds or less, 10 seconds or less, or 6 seconds or less. Although there is no desired lower limit for the residence time, in practice the residence time is 1 second or longer. The separation medium can be arranged as a membrane chromatography column, a membrane chromatography box, or other membrane chromatography devices. The sheet 10 of the separation medium is schematically arranged at Figure 1A The sheet 10 of separation medium can be provided in Figure 1B In the separation device 1 (e.g., chromatographic column) shown. The separation device 1 includes a housing 2 with an inlet 4 and an outlet 6, so as to flow through the device. The separation device (e.g., membrane chromatography column, membrane chromatography box or other membrane chromatography device) can provide a residence time of 2 minutes or less, 1 minute or less, 30 seconds or less, 10 seconds or less, or 6 seconds or less. According to an embodiment, the use of a membrane-based purification device can significantly improve productivity.

[0077] The process productivity can be defined using the following equation. In the denominator, V totis the total volume of solution that passes through the separation medium (e.g., column or cartridge) during the entire process including loading, rinsing, elution, and regeneration steps. BV is the chromatographic medium bed volume (corresponding to the volume of the separation medium substrate), and τ is the residence time. The loading volume is proportional to the dynamic binding capacity of the chromatographic column medium. Therefore, process productivity increases with increasing binding capacity and decreasing residence time.

[0078] According to another embodiment, the separation medium includes a cation exchange substrate. Such a substrate can be used for cation exchange-based chromatography to purify a target molecule (e.g., a nucleoside, a nucleotide, a nucleobase, or an analog or derivative thereof). Cation exchange employs negatively charged functional groups that target a positively charged target molecule.

[0079] The cation exchange ligand can be conjugated to the substrate via a functional handle. The functional handle can be covalently bonded to the substrate. In some embodiments, the cation exchange ligand is prepared from a bifunctional molecule. One of the functional groups can serve as the functional handle. Generally, the ligand (including the cation exchange ligand) can have the following general formula (I) Fh-Sp-Sg (I)

[0080] Wherein Fh is a functional handle, Sp is a spacer, and Sg is a functional separation group, such as a cation exchange separation group. The functional handle allows the ligand (eg, a cation exchange ligand) to be conjugated to the substrate.

[0081] A cation exchange separation group is a functional group that allows separation of nucleic acids, nucleotides, one of the various components of nucleotides, analogs thereof, or derivatives thereof. In embodiments, the cation exchange separation group may include one cation exchange separation moiety or two cation exchange separation moieties.

[0082] The spacer separates the functional handle from the cation exchange separation group. The spacer can have a length and / or composition that allows the functional handle and / or the cation exchange separation group to function as intended.

[0083] The functional handle can include any reactive functional group that can react with a chemical moiety on a substrate to form a covalent bond. The reaction of the functional handle with a substrate reactive moiety (a reactive moiety on a substrate) results in the covalent attachment of a cation exchange ligand to the substrate, also referred to as a conjugated cation exchange ligand. The conjugated cation exchange ligand can be displayed on a substrate to allow separation of one of the various components of a nucleic acid, a nucleotide, a nucleotide, an analog thereof, or a derivative thereof.

[0084] The identity of the reactive functional group of the functional handle is informed by the identity of the reactive moiety of the substrate, that is, the reactive functional handle and the reactive moiety of the substrate must be compatible in order to react to form a covalent bond. Exemplary substrate reactive moieties and / or exemplary reactive functional groups include amines; alcohols; activated alcohols, such as tosyl protected alcohols (e.g., tosyl chloride); epoxides; isocyanates; olefins; alkynes; cycloolefins; cyclooctynes; thiols; disulfides; azides; thioisocyanates; N-hydroxysuccinimide; maleimide; and activated esters and / or carboxylic acids, including esters or carboxylic acids activated using carbodiimide compounds (N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-cyclohexyl-(2-morpholinoethyl)carbodiimide, methyl-p-toluenesulfonate. Those skilled in the art will understand which reactive functional handles and substrate reactive moieties are compatible.

[0085] When conjugated to a substrate, the conjugated cation exchange ligand may have the general formula (II):

[0086] Wherein S is substrate, Rp is the reaction product between functional handle and substrate reactive part, Sp is spacer, and Sg is separating group.In certain embodiments, Rp can be the second separating group, which promotes the separation of one of the multiple components of nucleic acid, nucleotide, nucleotide, its analog or its derivative.In some embodiments where Rp is not a separating group and the first separating group (e.g., Sg) includes a single separating part, the part put together is a unimodal part, such as a cation exchange unimodal part. The separating group can be acid, carboxylic acid, sulfonic acid, phosphoric acid, carboxylate, sulfonate or phosphate. In some embodiments where Rp is not a separating group and the first separating group (e.g., Sg) includes two separating parts, the part put together is a bimodal part, such as a cation exchange bimodal part put together. In some embodiments where Rp is the second separating group and the first separating group (e.g., Sg) includes a single separating part, the part put together is a bimodal part, such as a cation exchange bimodal part put together. In some embodiments where Rp is the second separating group and the first separating group (eg, Sg) comprises two separating moieties (eg, a first separating moiety and a second separating moiety), the conjugated ligand is a trimodal ligand, such as a conjugated cation exchange trimodal ligand.

[0087] The properties of the reaction product depend on the properties of the reactive functional groups and the reactive portion of the substrate. Examples of reaction products include, but are not limited to, esters, ethers, thioethers, amides, amines (e.g., primary amines, secondary amines, tertiary amines), olefins, ureas, carbamates, carbonates, thioureas, and triazoles.

[0088] In some embodiments, the separating group (Sg) may include a single separating moiety.

[0089] In some embodiments, the separating group (Sg) may include two separating parts and have the general formula (III) Sm1-Sp2-Sm2 (III)

[0090] Wherein Sm1 is the first separation part, Sp2 is a spacer, and Sm2 is the second separation part.The first separation part and the second separation part can be acid, carboxylic acid, sulfonic acid, phosphoric acid, carboxylate, sulfonic acid ester or phosphoric acid ester.Spacer (Sp / Sp2) can be a carbochain with a length of C1 to C18, C1 to C10, C1 to C6, C1 to C4, C1 to C3 or C2 to C4, which is replaced along carbochain by one or more ethers, ester, benzyl, phenyl or amides alternatively.The example of the separating group comprising two separation parts comprises aminobenzoic acid, aminodiacetic acid, aminopropionic acid, 3-amino-1-propanesulfonic acid and 3-amino-1-ethylsulfonic acid. The cation exchange substrate can be prepared by first subjecting a base substrate (e.g., a membrane or nonwoven substrate) to (e.g., impregnating) a solution of a linker activator and a catalyst, then subjecting the substrate to (e.g., impregnating) a solution containing a ligand and an optional catalyst, and finally immersing the substrate in a quenching buffer solution to passivate unreacted linkers. In an exemplary embodiment, the linker activator is or includes N,N-disuccinimidyl carbonate (DSC), and the catalyst includes triethylamine (TEA).

[0091] In embodiments where the target molecule is a nucleobase / nucleoside or modified nucleobase / nucleoside, suitable solvents, salts or other additives may be added to the solution containing the target molecule to allow the target molecule to dissolve or maintain its stability, or to achieve a desired level of binding and selectivity.

[0092] Suitable salts used in the purification process include, for example, sodium chloride, potassium chloride, lithium chloride, rubidium chloride, calcium chloride, magnesium chloride, cesium chloride, tris, sodium phosphate, potassium phosphate, and ammonium sulfate. In certain embodiments, sodium chloride, potassium chloride, ammonium sulfate, calcium chloride, potassium chloride, or magnesium chloride is added to the solution. Suitable salts can be added in an amount of 1mM or greater, 5mM or greater, or 10mM or greater, or 20mM or greater. Suitable salts can be added in an amount of 100mM or less, 50mM or less, or 30mM or less. Salt can be added in an amount ranging from 1mM to 100mM, 1mM to 50mM, 5mM to 30mM, or 5mM to 20mM.

[0093] The suitable solvent used in the purification process includes, for example, methanol, ethanol, isopropanol and acetonitrile, DMSO and DMF etc. In certain embodiments, ethanol, isopropanol or acetonitrile are added to the solution. Suitable solvent can be added with 5wt-% or more, 10wt-% or more, 20wt-% or more, 30wt-% or more, 40wt-% or more, 50wt-% or more, 60wt-% or more, 70wt-% or more, 80wt-% or more or 90wt-% or more. Suitable solvent can be added with 90wt-% or less, 80wt-% or less, 70wt-% or less, 60wt-% or less, 50wt-% or less, 40wt-% or less, 30wt-% or less or 20wt-% or less. The solvent may be added in an amount ranging from 10 wt-% to 90 wt-%, 20 wt-% to 80 wt-%, 30 wt-% to 70 wt-%, or 10 wt-% to 50 wt-%.

[0094] The target molecule can be eluted from the immobilized base using a different solvent or a higher conductivity buffer via charge repulsion between the target and the ligand.

[0095] Cytosine (C) and guanine (G) bases contain a protonable primary amine at the 4-position of the pyrimidine ring. The charge state of the amine can be protonated to a positive charge, which can then provide selectivity using a cation exchange chromatography (CEX) substrate (e.g., membrane). Other nuclear base charge states can also be controlled by controlling the pH of the solution. For example, the pKa of the amine at the 4-position of cytosine is about 4.45. For guanine, the pKa of the amine at the 2-position is about 12.3, the amine at the 9-position is about 9.2, and the amide at the 1-position is about 3.3. The pKa of the amide in thymidine is about 9.96. The pKa of the amine in adenosine is about 3.5. The pH of the solution can be adjusted to be lower than the pKa of the nuclear base to protonate the amine and utilize cation exchange chromatography.

[0096] When using cation exchange chromatography to separate or purify a target molecule, the pH of the solution can be monitored and controlled to remain below the pKa of the target molecule to maintain the molecule in a protonated state. The pH can also be maintained above a threshold to avoid target decomposition. The pH threshold may vary from molecule to molecule. For example, BOC is used to protect reactive groups that can be deprotected under strongly acidic conditions such as 4 M HCl in dioxane or 1 M HCl in acetic acid.

[0097] In some embodiments, alcohol and / or hydroxyl groups on the target molecule can be protected from synthetic attack to allow targeted conjugation of amines. Typically, acid solutions can be used to remove protecting groups. During purification, it may be desirable to monitor and control the acidity of the solution to maintain the pH above the pH of deprotection (usually performed with trichloroacetic acid or hydrochloric acid). On the other hand, in order to induce or maintain the protonation of the target molecule so as to utilize cation exchange separation, the pH of the solution can be maintained below the pKa of the target molecule.

[0098] Molecules coupled to cation exchange ligands can be eluted, for example, by increasing conductivity, screening electrostatic attraction between CEX chromatography media and target molecules. In one embodiment, elution can be performed by changing the pH to be higher than the pKa of the amine in the target molecule, forming a neutral charge in the target molecule, thereby reducing charge interactions and inducing elution. Different target molecules (or target molecules and other molecules) can be eluted using linear gradient elution or using stepwise isocratic elution.

[0099] The substrate for preparing cation exchange chromatography (CEX) substrate can be any suitable material. In certain embodiments, the functionalized substrate is or includes a film, a resin, a monolith, a hydrogel, a woven fiber substrate, a nonwoven fiber substrate or a combination thereof. In one embodiment, the functionalized substrate is or includes a film. In one embodiment, the functionalized substrate is or includes a woven or nonwoven fiber substrate. Suitable films and nonwoven fiber substrates are discussed elsewhere in this disclosure.

[0100] According to an embodiment, cation exchange substrate can be used for purifying target molecule with fast flow rate.For example, cation exchange substrate can be used for purifying target molecule under 2 minutes or shorter, 1 minute or shorter, 30 seconds or shorter, 10 seconds or shorter or 6 seconds or shorter residence time.Although there is no desired lower limit for residence time, residence time is 1 second or longer in practice.Cation exchange substrate can be arranged as membrane chromatographic column, membrane chromatography box or other membrane chromatography devices.Membrane chromatographic column, membrane chromatography box or other membrane chromatography devices can provide 2 minutes or shorter, 1 minute or shorter, 30 seconds or shorter, 10 seconds or shorter or 6 seconds or shorter residence time.According to an embodiment, use purification device based on membrane can significantly improve productivity.

[0101] In some embodiments, the separation medium comprises an anion exchange substrate. Such a substrate can be used for chromatography based on anion exchange to purify a target molecule (e.g., a nucleoside, a nucleotide, a nucleobase, or an analog or derivative thereof). Anion exchange employs positively charged functional groups that target negatively charged target molecules.

[0102] The anion exchange ligand can be conjugated to the substrate via a functional handle. The functional handle can be covalently bonded to the substrate. In some embodiments, the anion exchange ligand is prepared from a bifunctional, trifunctional or other multifunctional molecule. One of the functional groups can serve as the functional handle. The functional handle can be as described above for formula (I). The conjugated anion exchange ligand can include a reaction product Rp and a spacer Sp as described above for formula (II). The conjugated anion exchange ligand further includes a separation group Sg.

[0103] Examples of suitable anion exchange ligands that can be disposed on the separation medium substrate include primary amines, secondary amines, tertiary amines, and quaternary amines. Suitable amines can be diamines, triamines, and polyamines. Diamines are generally represented by the formula:

[0104] In some embodiments, R 1 is an aliphatic carbon chain of 1 to 18 carbons, 1 to 10 carbons, 1 to 6 carbons, or 2 to 4 carbons. 2 , R 3 , R 4 , R 5 and R 6 Each of is independently selected from aliphatic linear, branched or cyclic substituted or unsubstituted carbon chains of 1 to 10 carbons, 1 to 6 carbons, or 2 to 4 carbons in length. In the tertiary amine, R 5 and R 6 does not exist, and R 2 , R 3 and R 4 Same as in quaternary amines. In secondary amines, R 5 and R 6 Does not exist, R 2 and R 3 is H, and R 4 Same as in quaternary amines. In primary amines, R 5 and R 6 does not exist, and R 2 , R 3 and R 4 is H. In some embodiments, the nitrogen of the diamine has different substitution levels. For example, one amine can be a secondary amine, and one amine can be a primary amine, a tertiary amine, or a quaternary amine. Suitable triamines and polyamines can have similar structures with three (triamine) or more amine groups.

[0105] Examples of primary amines include methylenediamine, ethylenediamine, propylenediamine, butylenediamine (putrescine), pentylamine, or any aliphatic diamine having 1-18 carbon atoms between the terminal amines covalently linked through one of the amines. Such ligands can be prepared from polyamines such as ethylenediamine, diethylenetriamine, triethylenetetramine covalently linked through one of the amines.

[0106] The example of secondary amine can include any above-mentioned primary amine fixed on substrate, and it is replaced by other R groups as mentioned above.In the case of using diamine, two kinds of amine can also be coupled to substrate to form secondary amine by covalent interaction with substrate.The part containing secondary amine with ligand structure can also be fixed, such as linear polyethyleneimine, spermidine or spermine.In addition, the group containing non-terminal primary amine (for example, 3-aminopentane) can also be conjugated to substrate to produce secondary amine.

[0107] Examples of suitable tertiary amines include N,N-dimethylethylenediamine, N,N-dimethylpropylenediamine, N,N-diethylpropylenediamine, or any aliphatic diamine having aliphatic carbon groups ranging from one to six carbons substituted on one or both amines, wherein the R between the terminal amines is 1 Having 2-18 carbons.

[0108] The example of quaternary amine includes any of the above-mentioned primary amines, which have undergone quaternization reaction and have produced a permanent positive charge. Such reactions can be carried out with alkyl groups such as methyl iodide or aryl groups such as benzyl iodide. Quaternary amines can further include any of the above-mentioned primary amines, which have undergone quaternization reaction and have produced a permanent positive charge. Such reactions can be described as Menshutkin reactions, which use alkyl halides to react with tertiary amines to form quaternary ammonium salts. Such reactions can be carried out with alkyl groups such as butyl bromide or aryl groups such as benzyl chloride or combinations thereof of different lengths. In addition, compounds containing quaternary amines can be directly fixed.

[0109] In addition to the amine group, the ligand may also contain additional functional groups. The ligand may contain a linker between the amine and any other functional groups, the linker being 1 to 18 carbons, 1 to 10 carbons, 1 to 6 carbons, or 2 to 4 carbons in length.

[0110] The anion exchange substrate can be prepared by first subjecting a base substrate (e.g., a membrane or nonwoven substrate) to (e.g., impregnating) a solution of a linker activator and a catalyst, then subjecting the substrate to (e.g., impregnating) a solution containing a ligand and an optional catalyst, and finally subjecting the substrate to (e.g., impregnating) a buffer solution. In an exemplary embodiment, the linker activator is or includes N,N-disuccinimidyl carbonate (DSC), and the catalyst includes triethylamine (TEA).

[0111] In an exemplary embodiment, anion exchange substrate is prepared in a three-step method. The first step includes subjecting the base substrate to (e.g., impregnation) a solution of a linker activator and a catalyst. This can include DSC from 0.1 mg / mL to 120 mg / mL and TEA from 5 μL / mL to 100 μL / mL in a solvent. The solvent can include DMSO, acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), hexamethylphosphoramide, sulfolane or any other solvent / solution that makes the substrate (e.g., film) swell. The subjection can be carried out at a temperature between about 10 ° C and 60 ° C for about 1 minute to 1,800 minutes. For example, a film having a diameter of 47 mm and a thickness of 70 μm can be immersed in 300 mg DSC, 139 μL TEA dissolved in 10 mL DMSO for 16 hours at 40 ° C.

[0112] In this exemplary embodiment, the second step includes subjecting the substrate to (e.g., immersing) a solution containing the ligand and optionally the catalyst. This may include from about 1 μL / mL to 100 μL / mL, <100 μL / mL, <75 μL / mL, <50 μL / mL, <20 μL / mL, <10 μL / mL, 1 μL / mL to 10 μL / mL, 1 μL / mL to 20 μL / mL, 1 μL / mL to 50 μL / mL, 1 μL / mL to 75 μL / mL, 1 μL / mL to 100 μL / mL, 10 The DMEDA of μ L / mL to 20 μ L / mL, 10 μ L / mL to 50 μ L / mL, 10 μ L / mL to 75 μ L / mL, 10 μ L / mL to 100 μ L / mL, 20 μ L / mL to 50 μ L / mL, 20 μ L / mL to 75 μ L / mL, 20 μ L / mL to 100 μ L / mL, 50 μ L / mL to 75 μ L / mL or 50 μ L / mL to 100 μ L / mL. The solvent can be DMSO or another organic solvent such as acetonitrile, THF, DMF, hexamethylphosphoramide, sulfolane or any other solvent / solution that makes substrate swell. This withstand can be carried out at a temperature between about 10 ℃ to 60 ℃ for about 1 minute to 24 hours. For example, film can be placed in a solution of 15 μ L / mL of DMEDA in DMSO for 30 minutes at room temperature.

[0113] In this exemplary embodiment, the third step includes subjecting the substrate from step 2 to (e.g., immersing) a buffer solution. This can include 0.05M to 4M Tris at pH 7.0-10.0. The subjection can be performed at a temperature between about 10°C and 60°C for about 1 minute to 24 hours. For example, the membrane is placed in 1M Tris pH 8.0 for 16 hours.

[0114] In some embodiments, the anion exchange substrate is prepared according to Method 1 described in US20200188859A1 (Zhou et al.).

[0115] In embodiments where the target molecule is a nucleotide or modified nucleotide, solvents, salts or other additives may be added to the solution containing the target molecule to allow the target molecule to dissolve or maintain its stability, or to achieve a desired level of binding and selectivity.

[0116] Suitable salts used in the purification process include, for example, sodium chloride, potassium chloride, lithium chloride, rubidium chloride, calcium chloride, magnesium chloride, cesium chloride, tris, sodium phosphate, potassium phosphate, and ammonium sulfate. In certain embodiments, sodium chloride, potassium chloride, ammonium sulfate, calcium chloride, potassium chloride, or magnesium chloride is added to the solution. Suitable salts can be added in an amount of 1mM or greater, 5mM or greater, or 10mM or greater, or 20mM or greater. Suitable salts can be added in an amount of 100mM or less, 50mM or less, or 30mM or less. Salt can be added in an amount ranging from 1mM to 100mM, 1mM to 50mM, 5mM to 30mM, or 5mM to 20mM.

[0117] The suitable solvent used in the purification process includes, for example, methanol, ethanol, isopropanol and acetonitrile, DMSO and DMF etc. In certain embodiments, ethanol, isopropanol or acetonitrile are added to the solution. Suitable solvent can be added with 5wt-% or more, 10wt-% or more, 20wt-% or more, 30wt-% or more, 40wt-% or more, 50wt-% or more, 60wt-% or more, 70wt-% or more, 80wt-% or more or 90wt-% or more. Suitable solvent can be added with 90wt-% or less, 80wt-% or less, 70wt-% or less, 60wt-% or less, 50wt-% or less, 40wt-% or less, 30wt-% or less or 20wt-% or less. The solvent may be added in an amount ranging from 10 wt-% to 90 wt-%, 20 wt-% to 80 wt-%, 30 wt-% to 70 wt-%, or 10 wt-% to 50 wt-%.

[0118] The target molecule can be eluted from the immobilized base using a different solvent or a higher conductivity buffer via charge repulsion between the target and the ligand.

[0119] According to an embodiment, the pH of the feed solution containing the target is adjusted to maintain the target molecule positively charged, which is generally higher than the pKa of such a molecule. On the other hand, the pH of the feed solution is maintained below the pKa of the anion exchange membrane ligand, thereby maintaining its positive state. For example, if a weak anion exchange membrane is used to capture adenosine monophosphate from the feed solution, the pH of the feed solution can be adjusted to a range of 3 to 7.

[0120] In some embodiments, the separation medium comprises a substrate having functional groups that induce hydrophobic interactions with a target molecule, an impurity, or both. Such a substrate can be used for hydrophobic interaction chromatography (HIC) to purify a target molecule (e.g., a nucleoside, a nucleotide, a nucleobase, or an analog or derivative thereof). Hydrophobic interaction chromatography employs hydrophobic functional groups that interact with hydrophobic groups on a target molecule. Hydrophobic interactions exploit the difference in hydrophobicity between a target molecule and possible impurities. Nucleobases contain hydrophobic rings that can be utilized by interacting with HIC ligands on a substrate.

[0121] In one embodiment, such ligands include aliphatic chains with three or more carbons (commonly used lengths include butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecyl), benzyl, phenyl, phenol, pyridine, boronic acid groups, branched polymers such as polypropylene glycol, and sulfur-containing thiophilic ligands such as propanethiol, 2-butanethiol, 3,6-dioxa-1,8-octanedithiol, octanethiol, benzylthiol, 2-mercaptopyridine, thiophenol, 1,2-ethanedithiol, 1,4-benzenedimethanethiol, 2-phenylethanethiol, etc., and combinations thereof. The hydrophobic interaction ligand can be conjugated to the substrate via a functional handle as described above for formula (I).

[0122] In some embodiments, the target molecule is a nucleobase or a modified nucleobase, a nucleoside or a modified nucleoside, or a nucleotide or a modified nucleotide. A solvent, a salt or other additive can be added to a solution containing the target molecule to allow binding to occur through the interaction of the hydrophobic groups present on both the ligand and the target. In some embodiments, a lyophilic salt is added to the solution. In some cases, a combination of a lyophilic salt and a chaotropic salt can be added to the solution. For example, a mixture of a lyophilic anion and a chaotropic cation can be used. In some embodiments, the ratio of lyophilic salts is increased and / or the ratio of chaotropic salts is reduced. Lyophilic salts are referred to as salts that reduce the solubility of non-polar substances in aqueous solutions, and chaotropic salts increase their solubility. In some embodiments, the ratio of organic solvents in the solution can be increased. In other embodiments, the ratio of organic solvents in the solution can be reduced. In other embodiments, a combination of changes in lyophilic components, chaotropic components and / or organic solvents can also be used.

[0123] Examples of lyophilic salts that can be added to a solution containing a target molecule include ammonium sulfate, ammonium phosphate, potassium phosphate, sodium sulfate, sodium chloride, and combinations thereof. Suitable lyophilic salts can be added in an amount of 0.1 M or greater, 0.5 M or greater, or 1.0 M or greater, or 2.0 M or greater. Suitable lyophilic salts can be added in an amount of 6.0 M or less, 5.0 M or less, or 4.0 M or less. Lyophilic salts can be added in an amount ranging from 0.1 M to 6 M, 0.5 M to 2.5 M, or 0.5 M to 3.0 M.

[0124] Examples of chaotropic salts that may be present in the solution include sodium chloride, calcium chloride, magnesium chloride, and combinations thereof. In some embodiments, the amount of the chaotropic salt is maintained at 1 M or less, 0.5 M or less, or 0.1 M or less. In some embodiments, the solution is free of or substantially free of chaotropic salts.

[0125] The suitable solvent used in the purification process includes, for example, methanol, ethanol, isopropanol and acetonitrile, DMSO and DMF etc. In certain embodiments, ethanol, isopropanol or acetonitrile are added to the solution. Suitable solvent can be added with 5wt-% or more, 10wt-% or more, 20wt-% or more, 30wt-% or more, 40wt-% or more, 50wt-% or more, 60wt-% or more, 70wt-% or more, 80wt-% or more or 90wt-% or more. Suitable solvent can be added with 90wt-% or less, 80wt-% or less, 70wt-% or less, 60wt-% or less, 50wt-% or less, 40wt-% or less, 30wt-% or less or 20wt-% or less. The solvent may be added in an amount ranging from 10 wt-% to 90 wt-%, 20 wt-% to 80 wt-%, 30 wt-% to 70 wt-%, or 10 wt-% to 50 wt-%.

[0126] The target molecule can be eluted from the immobilized base using different solvents or low conductivity buffers via charge repulsion between the target and the ligand.

[0127] The substrate for preparing the hydrophobic interaction chromatography (HIC) substrate can be any suitable material. In some embodiments, the hydrophobic interaction chromatography (HIC) substrate is or includes a membrane, a resin, a monolith, a hydrogel, and a fiber, etc. In one embodiment, the hydrophobic interaction chromatography (HIC) substrate is or includes a membrane. In one embodiment, the hydrophobic interaction chromatography (HIC) substrate is or includes a nonwoven fiber substrate.

[0128] According to an embodiment, the hydrophobic interaction substrate can be used for purifying target molecules with a fast flow rate. For example, the hydrophobic interaction substrate can be used for purifying target molecules under a residence time of 2 minutes or shorter, 1 minute or shorter, 30 seconds or shorter, 10 seconds or shorter or 6 seconds or shorter. Although there is no desired lower limit for the residence time, the residence time is 1 second or longer in practice. The hydrophobic interaction substrate can be arranged as a membrane chromatographic column, a membrane chromatographic box or other membrane chromatographic devices. Membrane chromatographic column, a membrane chromatographic box or other membrane chromatographic devices can provide a residence time of 2 minutes or shorter, 1 minute or shorter, 30 seconds or shorter, 10 seconds or shorter or 6 seconds or shorter. According to an embodiment, the purification device based on the membrane can significantly increase productivity.

[0129] In some embodiments, the separation medium comprises a multimodal medium. A multimodal medium is a medium comprising two or more types of ligands or functional groups on a substrate. A multimodal medium can enhance the interaction between a ligand and a target molecule. In some embodiments, a multimodal medium comprises an ion exchange ligand or functional group and a ligand or functional group of one other type. In some such embodiments, a multimodal medium comprises a cation exchange ligand and at least one other type of functional group. In some embodiments, a multimodal medium comprises an anion exchange ligand and at least one other type of functional group. The at least one other type of functional group can be part of the same ligand as a cation or anion exchange group, or can be in a separate ligand. For example, in addition to a cation exchange ligand or anion exchange ligand, a multimodal medium can also comprise a hydrophobic interaction group, a hydrogen bonding group, a thiophilic group, or a combination thereof. In one embodiment, a multimodal medium comprises a combination of a cation exchange ligand and a hydrophobic interaction group. In one embodiment, a multimodal medium comprises a combination of a cation exchange ligand and a hydrogen bonding group. In one embodiment, a multimodal medium comprises a combination of a cation exchange ligand and a thiophilic group. A multimodal medium can also comprise three or more types of functional groups. Multimodal media can be used to separate or purify nucleotides, nucleosides, nucleobases, and their analogs and derivatives.

[0130] According to an exemplary embodiment, the multimodal medium comprises ligands comprising cation exchange ligands and hydrophilic ligands.

[0131] According to an exemplary embodiment, the multimodal medium comprises ligands comprising cation exchange ligands and hydrophobic interaction ligands.

[0132] According to an exemplary embodiment, the multimodal medium comprises a ligand comprising an anion exchange ligand and a hydrophilic ligand.

[0133] According to an exemplary embodiment, the multimodal medium includes ligands comprising anion exchange ligands and hydrophobic interaction ligands.

[0134] In some examples, the multimodal medium comprises cation exchange ligands that further comprise sulfophilic functional groups. Examples of suitable sulfophilic cation exchange ligands that can be disposed on the separation medium substrate include mercaptocarboxylic acids and salts thereof. The sulfophilic cation exchange ligands can be represented by the following formula:

[0135] Where R 1 is a spacer group. 1 R may be a substituted or unsubstituted aliphatic or aromatic group containing 1 to 18 carbons, 1 to 10 carbons, 1 to 6 carbons, or 2 to 4 carbons. 1 Can be straight chain, branched chain or cyclic. 1 is a carboxylic acid (optionally conjugated to an aromatic group, for example at the benzoic or benzyl position) or a sulfonate group.

[0136] Examples of suitable sulfur-philic cation exchange ligands include mercaptobenzoic acid (e.g., 2-mercaptobenzoic acid or 4-mercaptobenzoic acid) and mercaptosulfonic acid and salts thereof such as sodium 3-mercapto-1-propanesulfonate. The ligand may include a linker between the sulfur-containing functional group and other (e.g., acid) functional groups having a length of 1 to 18 carbons, 1 to 10 carbons, 1 to 6 carbons, or 2 to 4 carbons.

[0137] The sulfophilic cation exchange substrate can be prepared by first subjecting a base substrate (e.g., a membrane or nonwoven substrate) to (e.g., impregnating) a solution of a linker activator and a catalyst, then subjecting the substrate to (e.g., impregnating) a solution containing a ligand and an optional catalyst, and finally immersing the substrate in a buffer solution. In an exemplary embodiment, the linker activator is or includes N,N-disuccinimidyl carbonate (DSC), and the catalyst includes triethylamine (TEA).

[0138] In an exemplary embodiment, the sulfophilic cation exchange substrate is prepared in a three-step method. The first step includes subjecting the base substrate to (for example, impregnation) a solution of a linker activator and a catalyst. This can include DSC from 0.1mg / mL to 120mg / mL and TEA from 5μL / mL to 100μL / mL in a solvent. The solvent can include DMSO, acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), hexamethylphosphoramide, sulfolane or any other solvent / solution that makes the substrate (for example, film) swell. The subjection can be carried out at a temperature between about 10°C and 60°C for about 1 minute to 1,800 minutes. For example, a film having a diameter of 47mm and a thickness of 70μm can be immersed in 300mg DSC, 139μL TEA dissolved in 10mL DMSO at 40°C for 16 hours.

[0139] In this exemplary embodiment, the second step includes subjecting the substrate to (e.g., impregnation) a solution containing a ligand and an optional catalyst. This can include 3-mercapto-1-sodium propanesulfonate from about 0.1mg / mL to 150mg / mL, from 1mg / mL to 100mg / mL, or from 10mg / mL to 50mg / mL in a solvent. The solvent can be DMSO or another organic solvent such as acetonitrile, THF, DMF, hexamethylphosphoramide, sulfolane, or any other solvent / solution that makes the substrate swell. This subjection can be carried out at a temperature between about 10°C and 60°C for about 1 minute to 24 hours. For example, the film can be placed in a solution of 300mg 3-mercapto-1-sodium propanesulfonate, 1mL TEA dissolved in 10mLDMSO at 40°C for 16 hours.

[0140] In this exemplary embodiment, the third step includes subjecting the substrate from step 2 to (e.g., immersing) a buffer solution. This can include 0.05M to 4M Tris at pH 7.0-10.0. The subjection can be performed at a temperature between about 10°C and 60°C for about 1 minute to 24 hours. For example, the membrane is placed in 1M tris (hydroxymethyl) aminomethane (Tris) pH 8.0 for 16 hours.

[0141] In some embodiments, the desired target is a nucleobase or a modified nucleobase, a nucleoside or a modified nucleoside, or a nucleotide or a modified nucleotide. Suitable solvents, salts or other additives may be added to the solution to allow the target molecule to dissolve or maintain its stability, or to achieve a desired level of binding and selectivity.

[0142] Suitable salts used in the purification process include, for example, sodium chloride, potassium chloride, lithium chloride, rubidium chloride, calcium chloride, magnesium chloride, cesium chloride, tris, sodium phosphate, potassium phosphate, and ammonium sulfate. In certain embodiments, sodium chloride, potassium chloride, ammonium sulfate, calcium chloride, potassium chloride, or magnesium chloride is added to the solution. Suitable salts can be added in an amount of 1mM or greater, 5mM or greater, or 10mM or greater, or 20mM or greater. Suitable salts can be added in an amount of 100mM or less, 50mM or less, or 30mM or less. Salt can be added in an amount ranging from 1mM to 100mM, 1mM to 50mM, 5mM to 30mM, or 5mM to 20mM.

[0143] The suitable solvent used in the purification process includes, for example, methanol, ethanol, isopropanol and acetonitrile, DMSO and DMF etc. In certain embodiments, ethanol, isopropanol or acetonitrile are added to the solution. Suitable solvent can be added with 5wt-% or more, 10wt-% or more, 20wt-% or more, 30wt-% or more, 40wt-% or more, 50wt-% or more, 60wt-% or more, 70wt-% or more, 80wt-% or more or 90wt-% or more. Suitable solvent can be added with 90wt-% or less, 80wt-% or less, 70wt-% or less, 60wt-% or less, 50wt-% or less, 40wt-% or less, 30wt-% or less or 20wt-% or less. The solvent may be added in an amount ranging from 10 wt-% to 90 wt-%, 20 wt-% to 80 wt-%, 30 wt-% to 70 wt-%, or 10 wt-% to 50 wt-%.

[0144] The target molecule can be eluted from the immobilized base using a different solvent or a higher conductivity buffer via charge repulsion between the target and the ligand.

[0145] When using multimodal media cation exchange chromatography to separate or purify a target molecule, the pH of the solution can be monitored and controlled to maintain it below the pKa of the target molecule to maintain the molecule in a protonated state. The pH can also be maintained above a threshold to prevent target decomposition, and above the pKa of the multimodal ligand to maintain the ligand negatively charged. For cytidine and gemcitabine (with or without protected alcohols), examples of suitable pH ranges include pH 1 to 3.

[0146] Molecules coupled to cation exchange ligands of multimodal media can be eluted, for example, by increasing conductivity, screening electrostatic attraction between cation exchange ligands and target molecules. In one embodiment, elution can be performed by changing the pH to be higher than the pKa of the amine in the target molecule, forming a neutral charge in the target molecule, thereby reducing charge interactions and inducing elution. Different target molecules (or target molecules and other molecules) can be eluted using linear gradient elution or using stepwise isocratic elution.

[0147] When using multimodal media anion exchange chromatography to separate or purify a target molecule, the pH of the solution can be monitored and controlled to maintain it above the pKa of the target molecule to maintain the molecule in a deprotonated state. The pH can also be maintained below a threshold to prevent target decomposition and also below the pKa of the multimodal ligand to maintain the ligand positively charged. For adenosine monophosphate purification, examples of suitable pH ranges include pH 3 to 10.

[0148] Molecules coupled to anion exchange ligands of multimodal media can be eluted, for example, by increasing conductivity, screening electrostatic attraction between cation exchange ligands and target molecules. In one embodiment, elution can be performed by changing the pH to be lower than the pKa of the target molecule to reduce charge interactions and induce elution. Different target molecules (or target molecules and other molecules) can be eluted using linear gradient elution or using stepwise isocratic elution.

[0149] The substrate used to prepare the multimodal media can be any suitable material. In some embodiments, the multimodal media is or includes a film, a resin, a monolith, a hydrogel, and a fiber, etc. In one embodiment, the multimodal media is or includes a film. In one embodiment, the multimodal media is or includes a nonwoven fiber substrate.

[0150] According to an embodiment, multimodal medium can be used for purifying target molecules with fast flow rate. For example, multimodal medium can be used for purifying target molecules under 2 minutes or shorter, 1 minute or shorter, 30 seconds or shorter, 10 seconds or shorter or 6 seconds or shorter residence time. The residence time depends on the size of the separation device to some extent, and in small devices, the residence time can be as low as 1 second or shorter. Although there is no desired lower limit for the residence time, the residence time is 0.1 second or longer in practice. Multimodal medium can be arranged as membrane chromatography column, membrane chromatography box or other membrane chromatography devices. Membrane chromatography column, membrane chromatography box or other membrane chromatography devices can provide 2 minutes or shorter, 1 minute or shorter, 30 seconds or shorter, 10 seconds or shorter or 6 seconds or shorter residence time. According to an embodiment, using a purification device based on membrane can significantly improve productivity. Exemplary Embodiments

[0151] The following is a non-limiting list of exemplary embodiments according to the present disclosure.

[0152] Embodiment 1 is a separation medium, which comprises: a membrane; and a plurality of ligands immobilized on the membrane, wherein the plurality of ligands comprises anion exchange ligands, cation exchange ligands, thiophilic ligands, hydrophobic interaction ligands, hydrophilic ligands, or a combination thereof.

[0153] Embodiment 2 is the separation medium of embodiment 1, wherein the separation medium is configured to separate target molecules comprising nucleotides, nucleosides, nucleobases, derivatives and analogs thereof, and combinations thereof from a reaction mixture.

[0154] Embodiment 3 is the separation medium of embodiment 1 or 2, wherein the separation medium is configured for use with an organic solvent.

[0155] Embodiment 4 is a separation medium as described in any one of embodiments 1 to 3, wherein the plurality of ligands comprises anion exchange ligands comprising an aliphatic diamine or triamine comprising 1 to 18 carbons between adjacent amines.

[0156] Embodiment 5 is the separation medium of embodiment 4, wherein the anion exchange ligand comprises N,N-dimethylethylenediamine, N,N-dimethylpropylenediamine, N,N-dimethylpropylenediamine, N,N-diethylpropylenediamine, or a combination thereof.

[0157] Embodiment 6 is the separation medium of any one of embodiments 1 to 5, wherein the plurality of ligands comprises a cation exchange ligand comprising an aminocarboxylic acid, an aminosulfonic acid, or a combination thereof.

[0158] Embodiment 7 is a separation medium as described in embodiment 6, wherein the cation exchange ligand comprises aminobenzoic acid, aminodiacetic acid, aminopropionic acid, 3-amino-1-propanesulfonic acid, 3-amino-1-ethylsulfonic acid, or a combination thereof, and the cation exchange ligand comprises a spacer group with a length of 1 to 18 carbons, 1 to 10 carbons, 1 to 6 carbons, or 2 to 4 carbons between the amino group and the acid or sulfonate group.

[0159] Embodiment 8 is a separation medium as described in any one of embodiments 1 to 7, wherein the plurality of ligands includes two or more of anion exchange ligands, cation exchange ligands, thiophilic ligands, hydrophilic ligands, and hydrophobic interaction ligands.

[0160] Embodiment 9 is the separation medium of any one of embodiments 1 to 8, wherein the plurality of ligands comprises a ligand having a cation exchange functional group and a thiophilic functional group.

[0161] Embodiment 10 is the separation medium of embodiment 9, wherein the plurality of ligands comprises mercaptobenzoic acid, mercaptosulfonic acid, salts thereof, or combinations thereof, preferably wherein the plurality of ligands comprises sodium 3-mercapto-1-propanesulfonate.

[0162] Embodiment 11 is the separation medium of embodiments 1 to 10, wherein the plurality of ligands are formed by ligands having formula (I): Fh-Sp-Sg (I) Wherein Fh is a functional handle, Sp is a spacer, and Sg is a functional separation group, wherein the functional handle is selected from the group consisting of amines; alcohols; activated alcohols; epoxides; isocyanates; olefins; alkynes; cycloolefins; cyclooctynes; thiols; disulfides; azides; thioisocyanates; N-hydroxysuccinimide; maleimide; activated esters; and activated carboxylic acids, wherein the spacer is a carbon chain of length C1 to C18, C1 to C10, C1 to C6, C1 to C4, C1 to C3, or C2 to C4, which is optionally substituted along the carbon chain with one or more ethers, esters, benzyls, phenyls, or amides, and The functional separation group is a cation exchange separation group, an anion exchange separation group, a hydrophobic separation group, or a thiophilic separation group.

[0163] Embodiment 12 is the separation medium of embodiment 11, wherein the functional separation group comprises an acid, a carboxylic acid, a sulfonic acid, a phosphoric acid, a carboxylate, a sulfonate, or a phosphate.

[0164] Embodiment 13 is a separation medium as described in embodiment 11, wherein the functional separation group comprises a primary amine, a secondary amine, a tertiary amine, a quaternary amine, or a combination thereof, and optionally wherein the functional handle comprises a secondary amine, a tertiary amine, or a quaternary amine, and wherein adjacent amines are separated by an aliphatic carbon chain of 1 to 18 carbons, 1 to 10 carbons, 1 to 6 carbons, 1 to 4 carbons, or 2 to 4 carbons.

[0165] Embodiment 14 is a separation medium as described in embodiment 11, wherein the functional separation group comprises an aliphatic chain having two or more carbon atoms (optionally butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecyl), benzyl, phenyl, phenol, pyridine, boric acid, a branched polymer (optionally polypropylene glycol), a sulfur-containing thiophilic ligand (optionally propanethiol, 2-butanethiol, 3,6-dioxa-1,8-octanedithiol, octanethiol, benzyl mercaptan, 2-mercaptopyridine, thiophenol, 1,2-ethanedithiol, 1,4-benzenedithiol, or 2-phenylethanethiol), or a combination thereof.

[0166] Embodiment 15 is the separation medium of embodiment 11, wherein the functional handle comprises a thiol and the functional separation group comprises an acid, a carboxylic acid, a sulfonic acid, a phosphoric acid, a carboxylate, a sulfonate, or a phosphate.

[0167] Example 16 is a separation device, comprising: a shell; and a separation medium disposed in the shell, the separation medium comprising: a membrane; and a plurality of ligands fixed on the membrane, the plurality of ligands comprising anion exchange ligands, cation exchange ligands, thiophilic ligands, hydrophobic interaction ligands, hydrophilic ligands, or a combination thereof.

[0168] Embodiment 17 is the separation device of embodiment 16, wherein the housing comprises a cartridge or a column.

[0169] Embodiment 18 is the separation device of embodiment 16 or 17, wherein the separation medium is configured to separate the target molecule comprising the nucleobase from the reaction mixture.

[0170] Embodiment 19 is the separation device of any one of embodiments 16 to 18, wherein the separation medium is configured for use with an organic solvent.

[0171] Embodiment 20 is a separation device as described in any one of embodiments 16 to 19, wherein the plurality of ligands comprises anion exchange ligands comprising aliphatic diamines or triamines comprising 1 to 18 carbons between adjacent amines.

[0172] Embodiment 21 is a separation device as described in embodiment 20, wherein the anion exchange ligand comprises N,N-dimethylethylenediamine, N,N-dimethylpropylenediamine, N,N-dimethylpropylenediamine, N,N-diethylpropylenediamine, or a combination thereof.

[0173] Embodiment 22 is a separation device as described in any of embodiments 16 to 21, wherein the plurality of ligands includes cation exchange ligands, and the cation exchange ligands include aminocarboxylic acids, aminosulfonic acids, or combinations thereof.

[0174] Embodiment 23 is a separation device as described in embodiment 22, wherein the cation exchange ligand comprises aminobenzoic acid, aminodiacetic acid, aminopropionic acid, 3-amino-1-propanesulfonic acid, 3-amino-1-ethylsulfonic acid, or a combination thereof, and the cation exchange ligand comprises a spacer group with a length of 1 to 18 carbons, 1 to 10 carbons, 1 to 6 carbons, or 2 to 4 carbons between the amino group and the acid or sulfonate group.

[0175] Embodiment 24 is a separation device as described in any one of embodiments 16 to 23, wherein the plurality of ligands includes two or more of anion exchange ligands, cation exchange ligands, thiophilic ligands, hydrophilic ligands, and hydrophobic interaction ligands.

[0176] Embodiment 25 is a separation device as described in any one of embodiments 16 to 24, wherein the plurality of ligands includes ligands having a cation exchange functional group and a thiophilic functional group.

[0177] Embodiment 26 is a separation device as described in embodiment 25, wherein the plurality of ligands include mercaptobenzoic acid, mercaptosulfonic acid, salts thereof, or combinations thereof, preferably wherein the plurality of ligands include sodium 3-mercapto-1-propanesulfonate.

[0178] Embodiment 27 is a separation device as described in embodiments 16 to 26, wherein the plurality of ligands are formed by ligands having formula (I): Fh-Sp-Sg (I) Wherein Fh is a functional handle, Sp is a spacer, and Sg is a functional separation group, wherein the functional handle is selected from the group consisting of amines; alcohols; activated alcohols; epoxides; isocyanates; olefins; alkynes; cycloolefins; cyclooctynes; thiols; disulfides; azides; thioisocyanates; N-hydroxysuccinimide; maleimide; activated esters; and activated carboxylic acids, wherein the spacer is a carbon chain of length C1 to C18, C1 to C10, C1 to C6, C1 to C4, C1 to C3, or C2 to C4, which is optionally substituted along the carbon chain with one or more ethers, esters, benzyls, phenyls, or amides, and The functional separation group is a cation exchange separation group, an anion exchange separation group, a hydrophobic separation group, or a thiophilic separation group.

[0179] Embodiment 28 is a separation device as described in embodiment 27, wherein the functional separation group comprises an acid, a sulfonic acid, or a phosphate.

[0180] Embodiment 29 is a separation device as described in Embodiment 27, wherein the functional separation group comprises a primary amine, a secondary amine, a tertiary amine, a quaternary amine, or a combination thereof, and optionally wherein the functional handle comprises a secondary amine, a tertiary amine, or a quaternary amine, and wherein adjacent amines are separated by an aliphatic carbon chain of 1 to 18 carbons, 1 to 10 carbons, 1 to 6 carbons, 1 to 4 carbons, or 2 to 4 carbons.

[0181] Embodiment 30 is a separation device as described in embodiment 27, wherein the functional separation group comprises an aliphatic chain having two or more carbon atoms (optionally butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecyl), benzyl, phenyl, phenol, pyridine, boric acid, a branched polymer (optionally polypropylene glycol), a sulfur-containing thiophilic ligand (optionally propyl mercaptan, 2-butyl mercaptan, 3,6-dioxa-1,8-octanedithiol, octanethiol, benzyl mercaptan, 2-mercaptopyridine, thiophenol, 1,2-ethanedithiol, 1,4-benzenedithiol, or 2-phenylethanethiol), or a combination thereof.

[0182] Embodiment 31 is a separation device as described in embodiment 27, wherein the functional handle comprises a thiol and the functional separation group comprises an acid, a sulfonic acid, or a phosphate.

[0183] Example 32 is a method for purifying a target molecule, the method comprising: passing a solution containing the target molecule through a membrane chromatography device, the target molecule comprising a nucleic acid, a nucleotide, a nucleoside, a nucleobase, or an analog or derivative thereof, and the membrane chromatography device comprising: a shell; and a separation medium disposed in the shell, the separation medium comprising: a membrane; and a plurality of ligands fixed on the membrane, the plurality of ligands comprising anion exchange ligands, cation exchange ligands, thiophilic ligands, hydrophobic interaction ligands, hydrophilic ligands, or a combination thereof.

[0184] Embodiment 33 is a method as described in embodiment 32, wherein after the target molecule is synthesized, the target molecule is purified from the solution containing the reaction mixture.

[0185] Embodiment 34 is a method as described in embodiment 32 or 33, wherein the separation medium comprises an anion exchange membrane.

[0186] Embodiment 35 is the method of any one of embodiments 32 to 34, wherein the residence time of the solution in the membrane chromatography device is 60 seconds or less.

[0187] Embodiment 36 is the method of any one of embodiments 32 to 35, wherein the target molecule is a nucleotide or a nucleic acid.

[0188] Embodiment 37 is the method of any one of embodiments 32 to 36, wherein the separation medium comprises a thiophilic cation exchange membrane.

[0189] Embodiment 38 is the method of any one of embodiments 32 to 37, wherein the sulfophilic cation exchange membrane comprises a cation exchange ligand having a sulfophilic functional group.

[0190] Embodiment 39 is the method of any one of embodiments 32 to 38, wherein the solution comprises an organic solvent.

[0191] Embodiment 40 is the method of any one of embodiments 32 to 39, wherein the plurality of ligands comprises ligands having a cation exchange functional group and a thiophilic functional group.

[0192] Embodiment 41 is the method of any one of embodiments 32 to 40, wherein the plurality of ligands comprises mercaptobenzoic acid, mercaptosulfonic acid, a salt thereof, or a combination thereof, preferably wherein the plurality of ligands comprises sodium 3-mercapto-1-propanesulfonate.

[0193] Embodiment 42 is the method of any one of embodiments 32 to 41, wherein the separation medium is according to any one of embodiments 1 to 15 and / or the separation device is according to any one of embodiments 16 to 31. Examples

[0194] These examples are for illustrative purposes only and are not meant to limit the scope of the appended claims. Unless otherwise indicated, all parts, percentages, ratios, etc. in the examples and the remainder of the specification are by weight.

[0195] The performance of various types of separation membranes was tested and evaluated against control samples. Test Method

[0196] Dynamic binding capacity (DBC) at 10% breakthrough 10％) can be obtained by standard chromatography, for example using Cytiva pure fast protein liquid chromatography (FPLC) assay. First, the separation medium is loaded into a housing unit. Then, the contained separation medium is connected to the FPLC. Next, the feed material is passed through the separation medium at a certain column volume / minute flow rate (CV / min) until the effluent concentration of the target reaches 10% of the feed concentration, as measured by the UV signal at an appropriate wavelength. Finally, based on the hold-up volume in the FPLC system and the separation medium volume, the DBC is calculated as follows 10％ : ((volume to reach 10% breakthrough - hold-up volume) x (feed concentration)) / (volume of separation medium) = DBC 10％ , expressed as mg target material / mL chromatographic medium. Sample testing

[0197] DBC 10％ The chromatographic method described above is used for determination. Complete bind-elute chromatography typically involves four steps:

[0198] Step 1 - Equilibration: Equilibrate the contained separation medium with buffer A.

[0199] Step 2 - Loading: Inject / pump the loading material through the separation medium until a 10% breakthrough is obtained.

[0200] Step 3-Washing: Wash the medium with a wash buffer. The wash buffer can be a single buffer or multiple buffers to wash away some impurities. The wash buffer can include buffer A or different buffers B, or a buffer combination comprising buffer A and / or B and / or another buffer (C, D, E, F, etc.), or a gradient conversion between buffer A and / or B and / or another buffer (C, D, E, F, etc.), or a gradient conversion between a combination of buffer A and / or B and / or another buffer (C, D, E, F, etc.).

[0201] Step 4-Elution: Buffer C is used to elute the loaded material (such as target and some impurities) from the separation medium. The elution buffer may include buffer C or a different buffer B, or a buffer combination comprising buffer C and / or B and / or another buffer (A, D, E, F, etc.), or a gradient switch between buffer C and / or B and / or another buffer (A, D, E, F, etc.), or a gradient switch between a combination of buffer C and / or B and / or another buffer (A, D, E, F, etc.).

[0202] The eluate can be collected for further analysis.

[0203] In addition to the four main steps described above, in some cases, stripping and / or cleaning-in-place (CIP) steps may be performed before restarting the cycle. Example 1 - Preparation of AEX membrane

[0204] At 40°C, a regenerated cellulose membrane with a diameter of 47 mm and a thickness of 70 μm was immersed in a solution of 300 mg N,N-disuccinimidyl carbonate (DSC) and 139 μL triethylamine (TEA) dissolved in 10 mL dimethyl sulfoxide (DMSO) for 16 hours. The membrane was then placed in a solution containing 100 μL N,N-dimethylethylenediamine (DMEDA) per mL DMSO for 16 hours at room temperature. Finally, the membrane was placed in 1 M Tris pH 8.0 for 16 hours. Example 2 - Preparation of thiophilic CEX membrane

[0205] A regenerated cellulose membrane with a diameter of 47 mm and a thickness of 70 μm was soaked in a solution of 300 mg DSC and 139 μL TEA for 16 hours at 40°C. The membrane was soaked in a solution of 300 mg sodium 3-mercaptopropanesulfonate and 0.5 mL TEA dissolved in 10 mL DMSO for 16 hours at 40°C. Finally, the membrane was placed in 200 mM Tris pH 8.0 for 16 hours. Example 3 - DBC of Separation Media

[0206] The dynamic binding capacity of the thiophilic CEX separation medium was tested at different flow rates.

[0207] Four layers of 24 mm circular separation membranes were loaded into a microcolumn (membrane volume = 0.1 mL) and connected to a Cytiva Pure to determine dynamic binding capacity. In this example, the loading material is 0.8 mg / mL cytidine in 10 mM phosphate pH 2.0. The cytidine solution is applied to the membrane at different flow rates as determined by column volume / minute (CV / min) until 10% breakthrough. DBC 10％ The results are presented graphically in Figure 2 middle.

[0208] from Figure 2 As can be seen, increasing the flow rate increases the DBC of cytidine 10％ At 110 CV / min, the cross-column pressure (ΔC) was 0.06 MPa, indicating that further increase in flow rate is possible. Example 4 - Separation of Cytidine Using an Organic Buffer System

[0209] The ability of the thiophilic CEX separation medium to separate cytidine in an organic buffer system was tested. A separation membrane was prepared according to Example 2. The following two commercially available products were used as comparative samples: Cytiva, available from Cytiva, Marlborough, MA; SP HP cation exchange chromatography columns and available from NatrixSeparations HD-Sb column. SP HP is a resin-based strong cation exchange chromatography column product, and HD-Sb is a hydrogel-based strong cation exchange column product enhanced with hydrophobic interaction groups.

[0210] Eight layers of 24 mm circular separation membranes were loaded into a polypropylene column housing (membrane volume = 0.2 mL) and connected to the Cytiva Pure was used for the bind-elute chromatography separation process. Cytidine was loaded at a defined column volume / minute flow rate (CV / min) until 10% breakthrough. The buffer list shown in Table 1 was used during the separation process. Table 1. Buffer list.

[0211] Sample separation membrane, resin ( SP HP) and hydrogel ( The chromatograms of commercial products of HD-Sb are Figure 3A , 3B The operating parameters and outputs are summarized in Table 2, including flow rate, DBC 10％ and the cross-column pressure (ΔC). Table 2. Operating parameters.

[0212] exist Figure 4 The binding capacities of the columns were compared in Table 1. It was observed that the binding capacity of the separation membrane was about 2 times that of the resin and about 18 times that of the hydrogel using the organic buffer system of Table 1. At the same transcolumn pressure, the flow rate of the separation medium was at least 20 times that of the resin. Example 5 - Isolation of Cytidine Using an Aqueous Buffer System

[0213] The ability of CEX separation resin to separate cytidine was tested in an aqueous buffer system. The separation resin was purchased from Stovan Corporation. SP HP resin.

[0214] 1mL SP HP resins connected to Cytiva Pure, bind-elute chromatography was performed in an all-water system (20 mM sodium phosphate with different pH values ​​ranging from 2.48 to 1.99).

[0215] The binding-elution curves of cytidine dissolved in 20 mM sodium phosphate solution at pH 2.48 or pH 1.99 are Figure 5A Shown in.

[0216] Initial cycles of loading at pH 2.48 resulted in a broadened bimodal elution peak beginning at the end of the wash phase. Figure 5A The binding-elution curve of Figure 5B Shown in.

[0217] It was observed that lowering the loading pH from 2.48 to 1.99 was able to resolve the elution into a single sharp elution peak, compared to the broad double peak observed in the binding and elution cycles at pH 2.48. It is speculated that the protonated amine on cytidine is more prevalent in an acidic environment (reduced pH), which makes cytidine more strongly adsorbed to the CEX column than deprotonated cytidine at higher pH. Although purification can be performed using the resin, its flow rate is significantly slower than that of a membrane with the same ligand, and therefore results in lower chromatographic productivity for the entire process. Example 6 - DBC and Recovery of AEX

[0218] An anion exchange (AEX) separation medium with a pore size of 1 μm was prepared according to Example 1. Eight layers of 24 mm circular AEX membrane were loaded into a housing unit (membrane volume = 0.2 mL) and connected to a Cytiva pure to determine the dynamic binding capacity. In this example, the loading material is 0.25 mg / mL adenosine-5'-monophosphate (AMP, 96.98%) dissolved in the equilibrium buffer. AMP is loaded at different column volumes / minute flow rates (CV / min) until a breakthrough of 10% is reached. A list of buffers used during the separation process is shown in Table 3. Table 3. Buffer list. step Buffer balance 20mM Tris pH 7 Column washing Deionized water Elution 1M NaCl

[0219] Chromatograms superimposed on Fig. 6A The DBC of AEX separation medium is shown in 10％ and AMP recovery in Figure 6B From Figure 6B As can be seen, increasing the flow rate does not decrease the binding capacity or AMP recovery of the AEX separation medium. Example 7 - DBC of AEX in buffers with different conductivity

[0220] Eight layers of 24 mm circular AEX membrane were loaded into the housing unit (membrane volume = 0.2 mL) and connected to the Cytiva pure to determine the dynamic binding capacity. The membrane was prepared according to Example 1. In this example, the feed was 0.25 mg / mL AMP in an equilibrium buffer supplemented with 0, 100 mM or 200 mM NaCl. AMP was loaded at different column volumes / minute flow rates (CV / min) until 10% breakthrough, and the DBC is shown in Figure 3. A list of buffers used during the separation process is shown in Table 4. Table 4. Buffer list

[0221] DBC under different flow rates and salt conditions 10％ exist Fig. 7A The chromatogram is superimposed on Figure 7B As can be seen in Figure 3, under each buffer condition, increasing the flow rate does not reduce the binding capacity of the AEX separation medium. Figure 7B As shown, increasing salt concentration significantly reduces the binding capacity of the AEX separation medium. At 10 CV / mL, DBC decreases from 38 mg / mL without added salt to 2 mg / mL after addition of 200 mM NaCl. Example 8 - Separation of difluoro-substituted nucleoside derivative intermediates with various alcohol and amine protection in an organic buffer system

[0222] The ability of the thiophilic CEX separation medium to separate difluoro-substituted nucleoside derivative intermediates with deprotected amines from a mixture containing difluoro-substituted nucleoside derivatives with combinations of mono- and / or di-protected alcohols and / or protected amines and excess protecting agent in an organic solvent (diluted 50-fold with the equilibration buffer listed in Table 3) was tested. The separation membrane was prepared according to Example 2.

[0223] Eight layers of 24 mm circular separation membranes were loaded into the membrane housing (membrane volume = 0.2 mL) and connected to the Cytiva Pure was used for the bind-elute chromatography separation process. A sample containing a mixture of difluoro-substituted nucleoside derivatives with a combination of mono- and / or di-protected alcohols and / or protected amines in an organic solvent and an excess of a protective agent (diluted 50 times with the equilibration buffer listed in Table 3) was loaded at a determined column volume / minute flow rate (CV / min) until a 10% permeability was reached. The buffers listed in Table 5 were used during the separation process. Table 5. Buffer list. Buffer 5 mM phosphoric acid pH 2 / 50% EtOH in water 50% EtOH 0.5M NaCl pH 7 / 50% EtOH aqueous solution 30% Isopropyl Alcohol

[0224] This process was repeated 10 times and the corresponding chromatograms were superimposed on Fig. 8A middle.

[0225] The bind-elute purification cycle was found to be consistent from run to run. Samples were taken every 5th run and the NANODROP TM The concentration was assessed by UV absorbance at 260 nm using a Lite UV-Vis spectrophotometer (available from Thermo Scientific).The yield was found to be consistent with a coefficient of variation of 5.17%.

[0226] Feed and eluate samples were also collected for thin layer chromatography (TLC) analysis. Photos of the TLC plates are at Figure 8B , the feed sample is on the left and the eluent sample is on the right. Figure 8B As shown, the three species in the crude feed (left) representing difluoro-substituted nucleoside derivatives with mono-alcohol protection (bottom), di-alcohol protection (middle), and tri-(top) protection are reduced to a single enriched di-protected species band in the eluate (right) at the same position as the di-protected species band in the feed, indicating the successful use of the amine side groups on the pyrimidine for purification by CEX in a partially organic solvent system. The tri-protected species here refers to difluoro-substituted nucleoside derivatives with di-protected alcohols and protected amines. Assuming that the protonated amine is only present in any combination of unprotected, mono-protected, and di-protected alcohols, in the absence of amine protection, the protonated amine has a stronger attraction to the CEX column than other variants with protected amines. For molecules with amine protection, the pendant amine cannot be protonated and is therefore much weaker adsorbed to the column by the CEX mechanism. Example 9 - Comparative Study

[0227] Using the cation exchange membrane prepared according to Example 2 and SP HP cation exchange resin column, tested for separation of difluoro-substituted nucleoside derivative intermediates with deprotected amines from a mixture containing difluoro-substituted nucleoside derivatives with a combination of mono- and / or di-protected alcohols and / or protected amines and excess protecting agent in an organic solvent (diluted 50 times with the equilibration buffer listed in Table 3).

[0228] The test was performed at a flow rate of 1 mL / min and the time for the process was 12 minutes.The solution was diluted in a low conductivity ethanol / phosphate mixture (low conductivity) and the column was eluted with 1 M NaCl (high conductivity).

[0229] The resin column has a 1 mL resin volume and an estimated binding capacity of 8 mg / mL. Large-scale residence times of 120 minutes or more are expected for each cycle. Binding-elution data are available at Fig. 9A Shown in.

[0230] The column had a medium volume of 0.1 mL and an estimated binding capacity of 80 mg / mL. The estimated mass residence time per cycle was approximately 24 minutes. The binding-elution data were Fig. 9B Shown in.

[0231] The binding-elution data from the two columns are superimposed on Fig. 9C middle

[0232] Although purification can be performed using resins, the flow rates are significantly slower than separations using membranes and thus result in lower chromatographic productivity of the overall process.

[0233] The purity of the eluate obtained by membrane purification was slightly higher than that obtained by resin purification. Fig.9D Shown in, it shows the purity of the elution pool of the purification carried out with resin relative to film.In addition, observe that the rotary evaporation of eluent produces the crystal of the nucleoside derivative (with diprotected alcohol and unprotected amine) of targeting difluoro from the charging containing excessive organic solvent and protecting agent.The rotary evaporation of the unpurified charging containing excessive organic solvent and protecting agent can not produce crystalline product, shows that the nucleoside derivative of desired targeting difluoro with unprotected amine is separated with other components.

[0234] Elution can be performed by increasing the organic solvent composition and increasing the salt concentration. This is particularly advantageous for purification prior to downstream drying or for subsequent synthesis steps performed under low water conditions. Using organic solvents makes it easier to remove and recycle a greater proportion of the solvent, thereby potentially accelerating the rotary evaporation step. In addition, using less salt increases the amount of material that needs to be removed in subsequent purification steps. Elution has been successfully performed using as little as 5% 1M NaCl buffer (50mM final concentration) in 95% ethanol. Example 10 - DBC of MCP

[0235] The ability of the resin-based MCP (Cytiva PlasmidSelect) to separate cytidine in an aqueous buffer system was tested. Cytidine at a concentration of 0.3125 mg / mL in 20 mM sodium acetate, 3 M ammonium sulfate pH 4.1 was applied to a resin-based MCP (Cytiva PlasmidSelect) column to assess binding by elution in 20 mM Tris pH 7.0.

[0236] Successful binding and elution cycles were performed and the following observations were made: Fig.10 The elution peaks depicted. It is expected that the increased productivity due to the reduced purification time will be the result of similar purifications using membrane-based MCPs. Furthermore, it is assumed that these and similar conditions will also apply to other HIC columns.

[0237] All references and publications cited herein are expressly incorporated by reference in their entirety into the present disclosure, unless they may directly contradict the present disclosure. Although specific embodiments have been illustrated and described herein, it will be understood by those of ordinary skill in the art that the specific embodiments shown and described may be replaced with a variety of alternative and / or equivalent embodiments without departing from the scope of the present disclosure. It should be understood that the present disclosure is not intended to be overly limited by the illustrative embodiments and examples set forth herein, and such examples and embodiments are presented only by way of example, and the scope of the present disclosure is intended to be limited only by the claims set forth herein below.

Claims

1. A separation medium comprising: membrane; and A plurality of ligands are immobilized on the membrane, wherein the plurality of ligands include anion exchange ligands, cation exchange ligands, thiophilic ligands, hydrophobic interaction ligands, hydrophilic ligands, or a combination thereof.

2. The separation medium according to claim 1, wherein The separation medium is configured to separate target molecules including nucleotides, nucleosides, nucleobases, derivatives and analogs thereof, and combinations thereof from a reaction mixture.

3. The separation medium according to claim 1 or 2, wherein The separation medium is configured for use with an organic solvent.

4. The separation medium according to any one of claims 1 to 3, wherein The plurality of ligands includes anion exchange ligands comprising aliphatic diamines or triamines containing from 1 to 18 carbons between adjacent amines.

5. The separation medium according to any one of claims 1 to 4, wherein The plurality of ligands includes a cation exchange ligand including an aminocarboxylic acid, an aminosulfonic acid, or a combination thereof.

6. The separation medium according to any one of claims 1 to 5, wherein The plurality of ligands include two or more of anion exchange ligands, cation exchange ligands, thiophilic ligands, hydrophilic ligands, and hydrophobic interaction ligands.

7. The separation medium according to any one of claims 1 to 6, wherein The plurality of ligands include ligands having a cation exchange functional group and a thiophilic functional group.

8. The separation medium according to claim 7, wherein The plurality of ligands include mercaptobenzoic acid, mercaptosulfonic acid, salts thereof, or combinations thereof, preferably wherein the plurality of ligands include sodium 3-mercapto-1-propanesulfonate.

9. A separation device comprising: case; and A separation medium is disposed in the housing, the separation medium comprising: membrane; and A plurality of ligands are immobilized on the membrane, wherein the plurality of ligands include anion exchange ligands, cation exchange ligands, thiophilic ligands, hydrophilic ligands, hydrophobic interaction ligands, or a combination thereof.

10. A method for purifying a target molecule, the method comprising: passing a solution containing the target molecule through a membrane chromatography device, The target molecule comprises a nucleic acid, a nucleotide, a nucleoside, a nucleobase, or an analog or derivative thereof, and The membrane chromatography device comprises: a housing; and A separation medium is disposed in the housing, the separation medium comprising: membrane; and A plurality of ligands are immobilized on the membrane, wherein the plurality of ligands include anion exchange ligands, cation exchange ligands, thiophilic ligands, hydrophobic interaction ligands, hydrophilic ligands, or a combination thereof.

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