Separation medium and method for purifying nucleotides and nucleotide components using the same

Abstract

The separation medium includes a membrane and a plurality of ligands immobilized on the membrane, the plurality of ligands including anion exchange ligands, cation exchange ligands, thiophilic ligands, hydrophilic ligands, hydrophobic interaction ligands, or combinations thereof. The separation medium may be multimodal. The separation medium may be configured to separate target molecules including nucleic acids, nucleotides, nucleosides, nucleic acid bases, or analogs or derivatives thereof from a reaction mixture. The separation medium may be configured for use with an organic solvent. The separation device includes the separation medium. Materials including nucleic acids, nucleotides, nucleosides, nucleic acid bases, or analogs or derivatives thereof may be purified at high speed using the separation device.

Description

[Technical field]

[0001] The present disclosure relates to separation media useful for separating biomolecules and ions, such as nucleotides, nucleosides, or nucleic acid bases, from a solution, suspension, or dispersion. The separation media of the present disclosure can be used for separations in membrane chromatography. The present disclosure also relates to methods of making and using the separation media.

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

[0003] The use of nucleotides, nucleosides, and their analogs in therapy is a rapidly growing industry sector. As nucleic acid therapeutics are developed and their production scales up, improved separation and purification methods are required. Summary of the Invention [Means for solving the problem]

[0004] Disclosed is a separation medium useful for separating target molecules from a solution, suspension, or dispersion. The target molecules may be biomolecules or ions, including nucleotides or nucleosides. The separation medium of the disclosure can be used for separation in membrane chromatography. The disclosure further relates to methods of making and using the separation medium.

[0005] According to one embodiment, the separation medium includes a membrane and a plurality of ligands immobilized on the membrane. The plurality of ligands may include anion exchange ligands, cation exchange ligands, thiophilic ligands, hydrophobic interaction ligands, hydrophilic ligands, or combinations thereof. The separation medium may be configured to separate target molecules including nucleotides, nucleosides, nucleic acid bases, derivatives and analogs thereof, and combinations thereof from a reaction mixture. The separation medium may be configured for use with an organic solvent.

[0006] The plurality of ligands may include anion exchange ligands including aliphatic diamines or triamines having 1-18 carbons between adjacent amines. The anion exchange ligands may include N,N-dimethylethylenediamine, N,N-dimethylpropylenediamine, N,N-dimethylpropylenediamine, N,N-diethylpropylenediamine, or combinations thereof.

[0007] The plurality of ligands may include cation exchange ligands including amino carboxylic acids, amino sulfonic acids, or combinations thereof. The cation exchange ligands may include amino benzoic acid, amino diacetic acid, amino propanoic acid, 3-amino-1-propanesulfonic acid, 3-amino-1-ethylsulfonic acid, or combinations thereof.

[0008] The plurality of ligands may include two or more of anion exchange ligands, cation exchange ligands, sulfophilic ligands, hydrophilic ligands, and hydrophobic interaction ligands. The plurality of ligands may include ligands having a cation exchange functional group and a sulfophilic functional group. The plurality of ligands may include mercaptobenzoic acid, mercaptosulfonic acid, salts thereof, or combinations thereof. Preferably, the plurality of ligands may include sodium 3-mercapto-1-propanesulfonate.

[0009] The separation 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 immobilized on the membrane, the plurality of ligands may include anion exchange ligands, cation exchange ligands, thiophilic ligands, hydrophobic interaction ligands, hydrophilic ligands, or combinations thereof. The housing may include a cassette or a column. The separation medium may be configured to separate target molecules including nucleotides, nucleosides, nucleic acid bases, derivatives or analogs thereof, or combinations thereof from a reaction mixture. The separation medium may be configured for use with an organic solvent.

[0010] The method of purifying a target molecule may include passing a solution containing the target molecule through a membrane chromatography device. The target molecule may include a nucleotide, a nucleoside, a nucleic acid base, a derivative or analogue 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 immobilized on the membrane, the plurality of ligands may include anion exchange ligands, cation exchange ligands, thiophilic ligands, hydrophobic interaction ligands, hydrophilic ligands, or a combination thereof. The housing may include a cassette or a column. The separation medium may be configured to separate the target molecule, including a nucleotide, a nucleoside, a nucleic acid base, a derivative or analogue thereof, or a combination thereof, from a reaction mixture. The separation medium may be configured for use with an organic solvent.

[0011] The target molecule can be purified from a solution containing a post-synthesis reaction mixture of a nucleotide, a nucleoside, a nucleic acid base, a derivative or analog thereof, or a combination thereof. The solution can contain 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]1 is a schematic diagram of a separation medium according to one embodiment. [Figure 1B] FIG. 1B is a schematic perspective view of a separation device including the separation medium of FIG. 1A. [Diagram 2] 1 is a graphical representation of dynamic binding capacity data from Example 3. [Figure 3A] 1 is a graphical representation of data from Example 4. [Figure 3B] 1 is a graphical representation of data from Example 4. [Figure 3C] 1 is a graphical representation of data from Example 4. [Figure 4] 1 is a graphical representation of dynamic binding capacity and pressure data from Example 4. [Figure 5A] 1 is a graphical representation of the binding-elution data from Example 5. [Figure 5B] 1 is a graphical representation of the binding-elution data from Example 5. [Figure 6A] Chromatogram from Example 6. [Figure 6B] 1 is a graphical representation of DBC10% and recovery data from Example 6. [Figure 7A] 1 is a graphical representation of dynamic binding capacity data from Example 7. [Figure 7B] Chromatogram from Example 7. [Figure 8A] 13 is an overlaid chromatogram from Example 8. [Figure 8B] 1 is a sample TLC plate from Example 8. [Figure 9A] 1 is a graphical representation of the binding-elution data from Example 9. [Figure 9B] 1 is a graphical representation of the binding-elution data from Example 9. [Figure 9C] 1 is a graphical representation of the binding-elution data from Example 9. [Figure 9D] 1 is a comparison of chromatograms from Example 9. [Figure 10] 1 is a graphical representation of the data from Example 10. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] definition All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are intended to facilitate understanding of certain terms used frequently herein and are not intended to limit the scope of the present disclosure.

[0014] Unless otherwise indicated, the terms "polymer" and "polymeric material" include organic homopolymers, copolymers, such as block, graft, random, and alternating copolymers, terpolymers, and blends and modifications thereof. Furthermore, unless otherwise limited, the term "polymer" is intended to include all possible geometric configurations of the material. These configurations include isotactic, syndiotactic, and atactic symmetries.

[0015] 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.

[0016] The term "alkylated" is used in this disclosure to describe a compound that has been reacted to replace a hydrogen atom or negative charge on the compound with an alkyl group, resulting in the alkyl group being covalently bonded to the compound.

[0017] The term "alkyl" is used in this disclosure to represent a monovalent group that is a radical of an alkane, and includes linear, branched, cyclic, and bicyclic alkyl groups, and combinations thereof, including both unsubstituted and substituted alkyl groups. Unless otherwise indicated, alkyl groups typically contain 1 to 30 carbon atoms. In some embodiments, alkyl groups contain 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. Examples of alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, isobutyl, t-butyl, isopropyl, n-octyl, n-heptyl, ethylhexyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.

[0018] The terms "nucleic acid" and / or "oligonucleotide" as used herein refer to a polymer comprising at least two nucleotides (e.g., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form, including DNA and RNA. A "nucleotide" comprises a sugar, a base (sometimes referred to as a nucleobase), and a linking group. In some embodiments, the sugar can be a naturally occurring deoxyribose or naturally occurring ribose (e.g., DNA and RNA, respectively). Nucleotides are linked together through linking groups to form an oligonucleotide. In some embodiments, the linking group can be a phosphate group. A polymer of covalently attached linking groups is sometimes referred to as a backbone. A "nucleoside" is similar to a nucleotide, except that a nucleoside does not include a linking group such as a phosphate group. "Base" or "nucleobase" includes purines and pyrimidines, which further include the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogues, as well as synthetic derivatives of purines and pyrimidines, including modifications that place new reactive groups such as amines, alcohols, thiols, carboxylates, and alkyl halides. Nucleotides include modified or similar nucleobases, modified or similar sugars, and / or modified or similar linking groups. Modified nucleobases, modified sugars, and / or modified linking groups may be non-standard / chemically modified nucleobases, sugars, and / or linking groups that may be synthetic, natural, and / or non-natural, and that have similar binding properties as standard nucleic acids. Examples of such similar and / or modified bases, sugars, and / or linking groups include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, locked nucleic acids (LNA), and peptide nucleic acids (PNA).

[0019] Deoxyribooligonucleotides consist of a five-carbon sugar called deoxyribose covalently linked to phosphates at the 5' and 3' carbons of the sugar to form an alternating, unbranched polymer. The DNA may be in the form of, for example, an antisense molecule, a plasmid DNA, a precondensed DNA, a PCR product, a vector, an expression cassette, a chimeric sequence, a chromosomal DNA, or derivatives and combinations of these groups. Ribooligonucleotides consist of a similar repeating structure in which the five-carbon sugar is ribose. Thus, the terms "polynucleotide" and "oligonucleotide" may refer to polymers or oligomers of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages.

[0020] The terms "polynucleotide" and "oligonucleotide" may also include polymers or oligomers that contain non-natural monomers or portions thereof that function similarly. Such modified or substituted oligonucleotides are often preferred over natural forms due to properties such as, for example, improved 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 and oligomers that contain a combination of both deoxy and ribonucleotides, or variants thereof, in combination with backbone modifications such as those described herein.

[0021] The polynucleotides and oligonucleotides described herein may include one or more nucleotide variants, including non-standard nucleotides, non-natural nucleotides, 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-galactosyl euosine, inosine, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 8-methylguanine, 9-methylguanine, 10-methylguanine, 11-methylguanine, 12-methylguanine, 13-methylguanine, 14-methylguanine, 15-methylguanine, 16-methylguanine, 17-methylguanine, 18-methylguanine, 19-methylguanine, 20-methylguanine, 21-methylguanine, 22-methylguanine, 23-methylguanine, 24-methylguanine, 25-methylguanine, 26-methylguanine, 27-methylguanine, 28-methylguanine, 29-methylguanine, 30-methylguanine, 31-methylguanine, 32-methylguanine, 33-methylguanine, 34-methylguanine, 35-methylguanine, 36-methylguanine, Examples of suitable nucleotides include anine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl eosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine, uracil-5-oxyacetic acid (v), wybutoxocine, pseudouracil, queosin, 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, nucleotides may contain modifications such as modifications to the phosphate moiety at the triphosphate moiety. Non-limiting examples of such modifications include longer phosphate chains (eg, phosphate chains having 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (eg, α-thiotriphosphate and β-thiotriphosphate).

[0022] The polynucleotides or oligonucleotides described herein may be modified at the base site (e.g., one or more atoms typically available to form hydrogen bonds with a complementary nucleotide, and / or one or more atoms typically not capable of forming hydrogen bonds with a complementary nucleotide), at the sugar site, or at the linking group (e.g., backbone). Backbone modifications may include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroaniladate, phosphoroamidate, and phosphorodiamidate linkages. Phosphorothioate linkages replace non-bridging oxygens of the phosphate backbone with sulfur atoms, retarding nuclease degradation of the oligonucleotide. Phosphorodiamidate linkages (N3'→P5') prevent nuclease recognition and degradation. Backbone modifications can also include peptide bonds in place of phosphorus in the backbone structure (e.g., N-(2-aminoethyl)-glycine units linked by peptide bonds in peptide nucleic acids), or linking groups including carbamates, amides, and linear and cyclic hydrocarbon groups. Oligonucleotides with modified backbones are reviewed in Micklefield, Curr. Med. Chem., 8(10):1157-79, 2001 and Lyer et al., Curr. Opin. Mol. Ther., 1(3):344-358, 1999. The nucleic acid molecules described herein can include sugar moieties that include 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-guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars. The 2'-O-methyl or 2'-O-methoxyethyl modifications promote an A-form or RNA-like conformation in oligonucleotides, increasing binding affinity to RNA and enhancing nuclease resistance.Modified sugar moieties can also include having an extra bridge bond (e.g., a methylene bridge connecting the 2'-O atom and the 4'-C atom of ribose in locked nucleic acids) or a sugar analog such as a morpholine ring (e.g., as in phosphorodiamidate morpholinos).

[0023] Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the sequence explicitly indicated, but also its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).

[0024] The disclosed methods encompass the separation and / or purification of isolated or substantially purified nucleotides, nucleosides, nucleic acid molecules, and compositions comprising those molecules. As used herein, an "isolated" or "substantially purified" DNA or RNA molecule is a DNA or RNA molecule that exists apart from its native environment. An isolated DNA or RNA molecule may exist in purified form or may exist in a non-native environment, such as, for example, a transgenic host cell. For example, an "isolated" or "purified" nucleic acid molecule or a biologically active portion thereof is substantially free of other cellular materials or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid).

[0025] As used herein, the term "pharmaceutical composition" and its grammatical equivalents may refer to a mixture or solution containing a therapeutically effective amount of an active pharmaceutical ingredient, together with one or more pharma- ceutically acceptable excipients, carriers, and / or therapeutic agents, for administration to a subject, e.g., a human being in need thereof.

[0026] The term "kosmotrope" is generally used to describe a solute that increases the degree of order in water by stabilizing water-water interactions. Kosmotropes may be ionic or non-ionic. In contrast, the term "chaotrope" is generally used to describe a solute that decreases the degree of order in water by destabilizing water-water interactions. Chaotropes may be ionic or non-ionic.

[0027] The term "substantially" as used herein has the same meaning as "significantly" and can be understood to modify the following term by 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 invention contains less than 1,000 parts per million (ppm) of the referenced compound. The term "essentially free" of a particular compound means that the composition of the invention contains less than 100 parts per million (ppm) of the referenced compound. The term "completely free" of a particular compound means that the composition of the invention contains less than 20 parts per billion (ppb) of the referenced compound. In the context of the preceding phrases, the composition of the invention contains less than the amount of the compound as stated above, regardless of whether the compound itself is present in unreacted form or has reacted with one or more other materials.

[0028] The term "not substantially" as used herein has the same meaning as "not significantly" and can be understood to have the opposite meaning of "substantially," i.e., modifying the term that follows it by 25% or less, 10% or less, 5% or less, or 2% or less.

[0029] The term "about" is used herein in conjunction with numerical values ​​to include normal variations in measurement that would be expected by one of ordinary skill in the art and is understood to have the same meaning as "approximately" and encompasses typical error ranges, such as ±5% of the stated value.

[0030] Terms such as "a," "an," and "the" are not intended to refer to only a singular entity, but include general classifications for which specific examples may be used for illustration.

[0031] 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" following a list refer to any one of the listed items, as well as any combination of two or more of the listed items.

[0032] As used herein, the term "or" is generally used in its general sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" refers to one or all of the listed elements or a combination of any two or more of the listed elements.

[0033] 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 up to 10 includes 10, 9.4, 7.6, 5, 4.3, 2.9, 1.62, 0.3, etc.). When a range of values ​​is "up to" or "at least" a particular value, then that value is included within the range.

[0034] As used herein, "have," "having," "include," "including," "comprise," or "comprising" are used in an open-ended sense and generally mean "including, but not limited to." "Consisting essentially of," "consisting of," and the like will be understood to be encompassed by "comprising," and the like. As used herein, "consisting essentially of," with respect to a composition, product, method, or the like, means that the components of the composition, product, method, or the like are limited to the recited components and any other components that do not materially affect the basic and novel characteristics of the composition, product, method, or the like.

[0035] The words "preferred" and "preferably" refer to embodiments that may provide certain benefits, under particular circumstances. However, other embodiments may also be preferred, under the same or different circumstances. Furthermore, 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.

[0036] Any directions referred to herein, such as "up," "down," "left," "right," "upper," "lower," and other directions and orientations, are described herein for clarity with reference to the drawings and are not intended to limit the actual device or system or the use of the device or system. The devices or systems described herein can be used in many directions and orientations.

[0037] Detailed Description The present disclosure relates to a separation medium useful for separating target molecules from a solution, suspension, or dispersion. The target molecules may be biomolecules or ions, including nucleotides, nucleosides, nucleic acid bases, derivatives or analogs thereof, or combinations thereof. The separation medium 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 medium.

[0038] The gene and cell therapy industry is rapidly shifting towards commercial processes due to its promising potential to treat various devastating diseases. Plasmid DNA (pDNA) is a key component in the manufacturing of viral vectors, proteins, and mRNA, which are widely used in gene and cell therapy. There is an urgent need for high-volume, high-quality pDNA manufacturing. However, scaling up pDNA production is not straightforward, making it a bottleneck in the industry. Currently, qualified contract manufacturers have long waiting lists and huge backlogs to meet the high demand. Like many other biologics manufacturing schemes, pDNA manufacturing involves multiple steps and unit operations.

[0039] Nucleotides, nucleosides, nucleic acid bases, their derivatives or analogs, or combinations thereof are increasingly being utilized to prepare pharmaceutical compositions. Derivatization may include, for example, fluorination, sugar substitution, addition of various functional moieties, or many other known or novel modifications. Such derivatization can be designed to convert or modify nucleosides, nucleotides, or nucleic acid bases into key building blocks for nucleic acid therapy or into more tolerable or more effective drugs by improving pharmacokinetics, transport, changing state to a prodrug, or taking advantage of upregulation of enzyme pathways specific to diseased tissues. Such pharmaceutical compositions can be utilized in a variety of contexts, including HIV / AIDS therapy and cancer therapy. Such analogs often include analogs that take advantage of pathways that are upregulated by HIV or cancer-producing cells, or that result in the induction of mismatches or other replication / translation errors, mitotic arrest, and / or induction of cell death.

[0040] Furthermore, with the increase in nucleic acid therapeutics, further substitutions and modifications are being pursued to improve the tolerability of therapeutic agents by improving stability or reducing or upregulating immunogenicity. For example, modifications to cytidine and uridine bases 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-cytidine-5'-triphosphate, 5 ... 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-methylpseudouridine-5'-triphosphate, N 1 -Methylpseudouridine-5'-triphosphate, N 1 -Ethylpseudouridine-5'-triphosphate, N 1 -Methyl-2'-O-methylpseudouridine-5'-triphosphate, N 1 -Methoxymethylpseudouridine-5'-triphosphate, N 1 -propylpseudouridine-5'-triphosphate, or the non-phosphorylated bases thereof. Similar modifications can be made to thymidine and guanidine bases.

[0041] Traditionally, downstream purification has been expensive, time-consuming, and difficult to scale up. A typical purification train for nucleotides, nucleosides, nucleic acid bases, their derivatives or analogs, or combinations thereof includes various steps such as filtration, ultrafiltration, and various chromatographic separations utilizing one or more types of chromatography columns. Typical chromatography columns used in the purification of nucleotides, nucleosides, nucleic acid bases, or nucleic acids can include, for example, packed bed columns with resins configured for size exclusion chromatography or reversed phase chromatography. Resin-based chromatography columns have been the gold standard for the purification of biologics for decades. However, bulk column chromatography can be very time-consuming. It is known that resin columns require long residence times to function adequately.

[0042] According to one embodiment, the separation medium comprises a functionalized substrate. The functionalized substrate may be a functionalized membrane. In contrast to resin columns, membrane sorbers may perform well with short column residence times and rapidly separate biologics. The present disclosure provides membranes suitable for the separation and purification of various nucleotides, nucleosides, nucleic acid bases, derivatives or analogs thereof, or combinations thereof. Target compounds that may be separated using the membranes of the present disclosure, whether in a charged (ionized) or uncharged state, are collectively referred to herein as target molecules. Target molecules may be in a solution, suspension, or dispersion. For brevity, a liquid containing a target molecule is referred to herein as a solution. The liquid may be a reaction mixture. For example, the liquid may be a reaction mixture used to prepare or synthesize a target molecule (e.g., a nucleotide, a nucleoside, a nucleic acid base, a derivative or analog thereof, and combinations thereof). The separation medium may be configured to purify the target molecule from the reaction mixture. The separation medium may be configured to separate the target molecule from starting materials, intermediates, and other reaction products. The reaction mixture may 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 may be configured for use with an organic solvent. The separation medium may be configured to separate or purify a target molecule from a solution that includes an organic solvent.

[0043] The functionalized substrates of the present disclosure include one or more functional groups that interact with target molecules, in some embodiments, the functional groups have an affinity for the target molecule and can bind to or retard migration of the target molecule through or along a membrane.

[0044] According to one embodiment, the target molecule includes a nucleotide, a nucleoside, a nucleobase, a derivative or analog thereof, or a combination thereof. Nucleotides and nucleosides include a nucleobase as their building block. Generally, the present disclosure provides membranes and methods for purifying nucleobases / nucleosides / nucleotides, including natural nucleobases / nucleosides / nucleotides, modified nucleobases / nucleosides / nucleotides, nucleobase / nucleoside / nucleotide analogs, etc.

[0045] 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 trains, including hydrophobic moieties, aromatic moieties, hydrogen bond donors and acceptors, and / or groups that can induce charge. However, modifying the nucleobases / nucleosides / nucleotides can change some of these properties. For example, in isolation, compounds with amine groups may be intentionally or unintentionally protected by agents such as the tert-butyloxycarbonyl protecting group (BOC). Protecting groups are used in synthesis to temporarily mask the characteristic chemical properties of a functional group from preventing another reaction. The protected amine is sterically hindered, resulting in one less hydrogen bond donor site, thus preventing the standard rules of hybridization. However, it is noted that there may be alternative pairing configurations that may or may not be affected by the protected amine group. Often, such bases bearing protecting groups are difficult to separate using traditional chemical separation techniques employed for purification of such nucleobase analogues in pharmaceutical manufacturing settings, such as silica gel chromatography, liquid-liquid extraction, liquid / solid extraction, and distillation (which is often unsafe due to the inhalation hazard of common protecting groups).

[0046] According to one embodiment, the present disclosure provides functionalized substrates (e.g., functionalized membranes) that utilize hybridization and hybridization-based purification methods for purifying nucleosides / nucleotides and their analogs and derivatives. For example, single nucleosides, nucleotides, or oligonucleotides can be effectively purified based on hybridization-based methods such as Watson-Crick base pairing.

[0047] In some embodiments, the use of Watson-Crick base pairing provides highly specific purification of target molecules. In one embodiment, a nucleotide, nucleoside, or oligonucleotide can be immobilized on a substrate to provide a ligand and then utilized to capture its complementary base. For example, adenine (A) or its derivatives can be immobilized on a substrate to produce a functionalized substrate. A substrate functionalized with adenine can be used to capture thymine (T), uracil (U), and their derivatives. Thymine (T), uracil (U), or their derivatives can be immobilized on a support to capture adenine (A) and its derivatives. Cytosine (C) can be immobilized to capture guanine (G) and its derivatives, or vice versa.

[0048] The substrate used as the base material of the separation medium can be any suitable material. In some embodiments, the substrate is or includes a membrane, a resin, a monolith, a hydrogel, a woven fibrous substrate, a nonwoven fibrous substrate, or a combination thereof. In one embodiment, the functionalized substrate is or includes a membrane. In one embodiment, the functionalized substrate is or includes a woven or nonwoven fibrous substrate. The substrate may be modified to include reactive chemical sites prior to reacting with the ligand. This may be particularly useful for non-reactive substrates such as ePTFE. Modification may include, for example, plasma treatment, dip coating with poly(vinyl alcohol), corona treatment, etc.

[0049] Nonwoven substrates (e.g., webs) are typically formed by meltblowing, wetlaying, melt spinning, solution spinning, airlaying, or electrospinning. Nonwoven webs may be further processed through post-treatment steps such as calendaring, embossing, needle punching, or hydroentangling. Nonwoven substrates may also include structural resins that have low binding affinity for biomolecules. Such resins are typically used to increase the strength of nonwoven webs. Many nonwoven substrates include a mix of fiber sizes and fibrous materials. Fibers used to manufacture nonwoven and woven substrates can include glass, polypropylene, polyamide, polyester, cellulosic materials, and the like, and combinations thereof. The fibers may 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 fibers may have an average fiber size of 100 μm or less, 50 μm or less, 25 μm or less, 10 μm or less, or 8 μm or less. The average fiber size may range from 0.1 μm to 50 μm, or from 1 μm to 25 μm. The average pore size measured by a capillary flow porometer may be 1 μm or more, 2 μm or more, or 3 μm or more. The average pore size may be 100 μm or less, 50 μm or less, 25 μm or less, 10 μm or less, or 8 μm or less. The average pore size of a suitable nonwoven substrate may range from 0.1 μm to 50 μm, 1 μm to 10 μm, or 3 μm to 8 μm. The average pore size of the woven substrate may be slightly larger than that of the nonwoven substrate and may range from 1 μm to 100 μm. The basis weight of the fibrous substrate may be 1 gsm (grams per square meter) or more, 10 gsm or more, or 20 gsm or more. The basis weight of the fibrous substrate may be 200 gsm or less, or 80 gsm or less. The basis weight of the fibrous base material may range from 1 gsm to 200 gsm, or from 20 gsm to 80 gsm.

[0050] According to one embodiment, the substrate is or comprises a membrane. A membrane is understood as a sheet of material that has continuous pathways of polymeric material 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.

[0051] According to one embodiment, useful membranes have an average pore size as measured by a capillary flow porometer 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. The membranes 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 membrane may have an average pore size in the range of 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 μm, 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 membrane may 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 membrane 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 membrane thickness 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.

[0052] The membranes can be stacked in multiple layers to increase capacity for a given application. In one embodiment, the membrane stack has a thickness of 70 μm or more, 250 μm or more, or 500 μm or more. The membrane stack 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 film layer structure is as follows: thickness 70μm~10,000μm, 70μm~100μm, 70μm~200μm, 70μm~300μm, 70μm~400μm, 70μm~500μm, 70μm~750μm, 70μm~1,000μm, 70μm~2,000μm, 70μm~3,000μm, 70μm~4,000μm, 70μm~5,000μm, 250μm~300μm, 250μm~ The thickness may be in the range of 400 μm, 250 μm to 500 μm, 250 μm to 750 μm, 250 μm to 1,000 μm, 250 to 2,000 μm, 250 to 3,000 μm, 250 to 4,000 μm, 250 to 5,000 μm, 500 μm to 1,000 μm, 500 μm to 2,000 μm, 500 μm to 3,000 μm, 500 μm to 4000 μm, or 500 μm to 5000 μm.

[0053] In a preferred embodiment, the membrane is a regenerated cellulose membrane having a pore size of 0.2 μm to 5.0 μm, a thickness of 70 μm to 2,000 μm in a laminated configuration with a thickness of about 70 μm to 10,000 μm.

[0054] The substrate may be a microfiltration membrane. Microfiltration membranes are typically formed by a phase inversion process or a swelling process. Typical materials used to make the membrane include PES, nylon, PVDF, cellulose acetate, regenerated cellulose, polypropylene, and expanded PTFE.

[0055] In some cases, the membrane cannot tolerate a wide variety of organic solvents. The membrane and ligands can be selected so that the membrane is insoluble in the solvents used in the separation or purification process.

[0056] In embodiments where the target molecule is a nucleotide or modified nucleotide, a solvent, salt, or other additive can be added to the solution containing the target molecule during the purification process to screen for sufficient repulsion of the associated phosphate group to allow hybridization to occur. Different solvents or low-conductivity buffers can be used to elute the target molecule from the immobilized base by charge repulsion between the target and the ligand. Suitable solvents include, for example, methanol, ethanol, isopropanol, acetonitrile, DMSO, and DMF. In some embodiments, ethanol, isopropanol, or acetonitrile is added to the solution. Suitable solvents can be used during the purification process in an amount of 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more by weight based on the weight of the solution. Suitable solvents can be used in amounts up to 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20% by weight of the solution. Solvents can be used in amounts ranging from 10% to 90%, 20% to 80%, 30% to 70%, or 10% to 50% by weight of the solution.

[0057] Suitable salts that may 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 some embodiments, sodium chloride, potassium chloride, ammonium sulfate, calcium chloride, potassium chloride, or magnesium chloride is added to the solution. The suitable salt may be added in an amount of 2% or more, 5% or more, 10% or more, 15% or more, or 20% or more by weight of the weight of the solution. The suitable salt may be added in an amount of 20% or less, 25% or less, or 30% or less by weight of the weight of the solution. The salt may be added in an amount ranging from 2% to 30%, or 5% to 25%, or 5% to 20% by weight of the weight of the solution.

[0058] 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 smaller factor since nucleosides do not contain phosphate groups, and therefore techniques to mitigate phosphate-phosphate repulsion become less relevant.

[0059] If the target molecule contains modifications at groups involved in hydrogen bonding or at sterically hindered positions, it is possible to separate the unmodified and modified groups using a functionalized substrate (e.g., a functionalized membrane) since the modified groups behave differently in binding in a way that can be used to either capture the modified molecules and allow the unmodified molecules to pass, or capture the unmodified molecules and allow the modified molecules to pass. Additives or solvents that compete with hydrogen bonding during elution may be used. Examples of additives and solvents that compete with hydrogen bonding include acetonitrile, alcohols, water, sugars, and combinations thereof.

[0060] In many embodiments, the target molecule is present in an aqueous solution. However, in some embodiments, the hydrogen bonds between A / T, A / U, and C / G remain effective in non-aqueous environments in various solvents. Examples of such solvents include alcohols, acetonitrile, and combinations thereof. Thus, in some embodiments, the target molecule is present in a solution that includes one or more alcohols or organic solvents, such as acetonitrile. In some such embodiments, the solution includes water and an organic solvent. The majority of the solution may be water. Alternatively, the majority of the solution may be composed of an organic solvent. In some embodiments, the solution is non-aqueous, e.g., composed of an organic solvent.

[0061] In some embodiments, the target molecule contains 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 in the case of hydrophobic modifications) and thus increase the productivity of the process.

[0062] According to one embodiment, the separation medium (functionalized substrate) can be used to purify target molecules at high flow rates. For example, the separation medium can be used to purify target molecules with residence times of 2 minutes or less, 1 minute (60 seconds) or less, 30 seconds or less, 10 seconds or less, or 6 seconds or less. There is no desirable lower limit to the residence time, but in practice residence times are 1 second or more. The separation medium can be arranged as a membrane chromatography column, membrane chromatography cassette, or other membrane chromatography device. A sheet 10 of separation medium is shown diagrammatically in FIG. 1A. The sheet 10 of separation medium may be provided within a separation device 1 (e.g., a chromatography column) as shown in FIG. 1B. The separation device 1 includes a housing 2 with an inlet 4 and an outlet 6 for facilitating flow through the device. The separation device (e.g., a membrane chromatography column, membrane chromatography cassette, or other membrane chromatography device) can provide residence times of 2 minutes or less, 1 minute or less, 30 seconds or less, 10 seconds or less, or 6 seconds or less. According to one embodiment, the productivity can be significantly improved by using a membrane-based purification device.

[0063] The productivity of a process can be defined using the following formula: tot is the total volume of solution passed through the separation medium (e.g., column or cassette) during the entire process, including loading, rinsing, elution, and regeneration steps. BV is the volume of the chromatography medium bed (corresponding to the volume of the separation medium substrate), and τ is the residence time. The loading amount is proportional to the dynamic binding capacity of the chromatography column medium. Therefore, increasing the binding capacity and decreasing the residence time increases the productivity of the process.

number

[0064] According to another embodiment, the separation medium comprises a cation exchange substrate. Such substrates can be used in cation exchange based chromatography to purify target molecules (e.g., nucleosides, nucleotides, nucleic acid bases, or analogs or derivatives thereof). Cation exchange utilizes negatively charged functional groups that target positively charged target molecules.

[0065] The cation exchange ligand can be conjugated to the substrate via a functional group handle. The functional group 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 function as the functional group handle. In general, the ligand including the cation exchange ligand can have the following general formula (I): Fh-Sp-Sg (I) (wherein Fh is a functional handle, Sp is a spacer, and Sg is a functional separation group (e.g., a cation exchange separation group).) The functional handle allows for the conjugation of a ligand (e.g., a cation exchange ligand) to the substrate.

[0066] A cation exchange separation group is a functional group that allows for the separation of nucleic acids, nucleotides, one or more components of nucleotides, analogs thereof, or derivatives thereof. In some embodiments, a cation exchange separation group can include one cation exchange separation site or two cation exchange separation sites.

[0067] The spacer separates the functional handle from the cation exchange separation group. The spacer may be of a length and / or composition that allows the functional handle and / or the cation exchange separation group to function as intended.

[0068] The functional group handle can include any reactive functional group that can react with a chemical site on the substrate to form a covalent bond. The reaction of the functional group handle with the substrate reactive site, which is a reactive site on the substrate, results in a covalent bond of the cation exchange ligand to the substrate, which is also called a conjugated cation exchange ligand. The conjugated cation exchange ligand can be placed on the substrate to allow separation of nucleic acids, nucleotides, one or more components of nucleotides, their analogs, or their derivatives.

[0069] The reactive functional group of the functional handle is understood by the substrate reactive site. That is, the reactive functional handle and the substrate reactive site must be compatible to react and form a covalent bond. Exemplary substrate reactive sites and / or exemplary reactive functional groups include amines; alcohols; activated alcohols, such as tosyl-protected alcohols (e.g., tosyl chloride); epoxides; isocyanates; alkenes; alkynes; cycloalkenes; cyclooctynes; thiols; disulfides; azides; thioisocyanates; N-hydroxysuccinimide; maleimides; and activated esters and / or carboxylic acids, such as 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, metho-p-toluenesulfonate). One of ordinary skill in the art will understand which reactive functional handles are compatible with reactive sites on the substrate.

[0070] When conjugated to a substrate, the conjugated cation exchange ligand may have the general formula (II): [ka] (wherein S is a substrate, Rp is a reaction product between the functional group handle and the reactive site of the substrate, Sp is a spacer, and Sg is a separation group). In some embodiments, Rp may be a second separation group that facilitates separation of a nucleic acid, a nucleotide, one or more components of a nucleotide, their analogs, or their derivatives. In some embodiments where Rp is not a separation group and the first separation group (e.g., Sg) comprises a single separation site, the conjugated ligand is a monomodal ligand, such as a cation-exchange monomodal ligand. The separation group may be an acid, a carboxylic acid, a sulfonic acid, a phosphoric acid, a carboxylate, a sulfonate, or a phosphate. In some embodiments where Rp is not a separation group and the first separation group (e.g., Sg) comprises two separation sites, the conjugated ligand is a bimodal ligand, such as a conjugated cation-exchange bimodal ligand. In some embodiments where Rp is a second separation group and the first separation group (e.g., Sg) comprises a single separation site, the conjugated ligand is a bimodal ligand, such as a conjugated cation-exchange bimodal ligand. In some embodiments where Rp is a second separation group and the first separation group (e.g., Sg) comprises two separation sites (e.g., a first separation site and a second separation site), the conjugated ligand is a trimodal ligand, such as a conjugated cation-exchange trimodal ligand.

[0071] The identity of the reaction product depends on the identity of the reactive functional group and reactive sites on the substrate. Exemplary reaction products include, but are not limited to, esters, ethers, thioethers, amides, amines (e.g., primary, secondary, tertiary), alkenes, ureas, carbamates, carbonates, thioureas, and triazoles.

[0072] In some embodiments, the separation group (Sg) may comprise a single separation site.

[0073] In some embodiments, the separation group (Sg) may include two separation sites and may be of the general formula (III): Sm1-Sp2-Sm2 (III) (wherein Sm1 is the first separation moiety, Sp2 is the spacer, and Sm2 is the second separation moiety). The first and second separation moieties may be acid, carboxylic acid, sulfonic acid, phosphoric acid, carboxylate, sulfonate, or phosphate. The spacer (Sp / Sp2) may be a carbon chain of length C1-C18, C1-C10, C1-C6, C1-C4, C1-C3, or C2-C4, optionally substituted with one or more ether, ester, benzyl, phenyl, or amide along the carbon chain. Examples of separation groups containing two separation moieties include aminobenzoic acid, aminodiacetic acid, aminopropanoic acid, 3-amino-1-propanesulfonic acid, and 3-amino-1-ethylsulfonic acid. The cation exchange substrate can be prepared by first exposing (e.g., soaking) a base substrate (e.g., a membrane or nonwoven substrate) to a solution of a linker activator and a catalyst, then exposing (e.g., soaking) the substrate to a solution containing a ligand and an optional catalyst, and finally soaking the substrate in a quenching buffer to inactivate any unreacted linkers. In one exemplary embodiment, the linker activator is or includes N,N-disuccinimidyl carbonate (DSC) and the catalyst includes triethylamine (TEA).

[0074] In embodiments in which the target molecule is a nucleobase / nucleoside or modified nucleobase / nucleoside, appropriate solvents, salts, or other additives may be added to the solution containing the target molecule to dissolve or maintain the stability of the target molecule, or to achieve the desired level of binding and selectivity.

[0075] Suitable salts used during the purification process 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 some 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 amounts of 1 mM or more, 5 mM or more, or 10 mM or more, or 20 mM or more. Suitable salts can be added in amounts of 100 mM or less, 50 mM or less, or 30 mM or less. Salts can be added in amounts ranging from 1 mM to 100 mM, 1 mM to 50 mM, 5 mM to 30 mM, or 5 mM to 20 mM.

[0076] Suitable solvents used during the purification process include, for example, methanol, ethanol, isopropanol, acetonitrile, DMSO, and DMF. In some embodiments, ethanol, isopropanol, or acetonitrile is added to the solution. The suitable solvent can be added in an amount of 5% by weight or more, 10% by weight or more, 20% by weight or more, 30% by weight or more, 40% by weight or more, 50% by weight or more, 60% by weight or more, 70% by weight or more, 80% by weight or more, or 90% by weight or more. The suitable solvent can be added in an amount of 90% by weight or less, 80% by weight or less, 70% by weight or less, 60% by weight or less, 50% by weight or less, 40% by weight or less, 30% by weight or less, or 20% by weight or less. The solvent can be added in an amount ranging from 10% by weight to 90% by weight, 20% by weight to 80% by weight, 30% by weight to 70% by weight, or 10% by weight to 50% by weight.

[0077] A different solvent or a buffer with higher conductivity can be used to elute the target molecule from the immobilized base due to charge repulsion between the target and the ligand.

[0078] Cytosine (C) and guanine (G) bases contain a protonatable primary amine at the 4-position of the pyrimidine ring. The charge state of this amine can be protonated to a positive charge, which can then be used to impart selectivity using a cation exchange chromatography (CEX) substrate (e.g., a membrane). The charge state of other nucleobases can also be manipulated by manipulating 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 pKa of the amine at the 9-position is about 9.2, and the pKa of the amide at the 1-position is about 3.3. The pKa of the amide of thymidine is about 9.96. The pKa of the amine of adenosine is about 3.5. The pH of the solution can be adjusted to be below the pKa of the nucleobase in order to protonate the amine and utilize cation exchange chromatography.

[0079] 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 keep the molecule in a protonated state. The pH can also be maintained above a threshold value to avoid target degradation. The pH threshold can vary from molecule to molecule. For example, BOC is used to protect reactive groups, which can be deprotected with highly acidic conditions such as 4M HCl in dioxane or 1M HCl in acetic acid.

[0080] In some embodiments, alcohol and / or hydroxyl groups on the target molecule can be protected from attack during synthesis to allow amine-targeted conjugation. The protecting groups can typically be removed using an acid solution. During purification, it may be desirable to monitor and control the acidity of the solution to maintain a pH higher than that of deprotection (often performed with trichloroacetic acid or hydrochloric acid). On the other hand, the pH of the solution may be maintained below the pKa of the target molecule to induce or maintain the protonation of the target molecule to utilize cation exchange separation.

[0081] Molecules bound to cation exchange ligands can be eluted, for example, by increasing the conductivity and screening the electrostatic attraction between the CEX chromatography medium and the target molecule. In one embodiment, elution can be performed by changing the pH above the pKa of the amine in the target molecule, creating 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 stepwise isocratic elution.

[0082] The substrate used to make the cation exchange chromatography (CEX) substrate can be any suitable material. In some embodiments, the functionalized substrate is or includes a membrane, a resin, a monolith, a hydrogel, a woven fibrous substrate, a nonwoven fibrous substrate, or a combination thereof. In one embodiment, the functionalized substrate is or includes a membrane. In one embodiment, the functionalized substrate is or includes a woven or nonwoven fibrous substrate. Suitable membranes and nonwoven fibrous substrates are described elsewhere in this disclosure.

[0083] According to one embodiment, the cation exchange substrate can be used to purify target molecules at a high flow rate. For example, the cation exchange substrate can be used to purify target molecules with 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. There is no desirable lower limit to the residence time, but in practice the residence time is 1 second or more. The cation exchange substrate can be arranged as a membrane chromatography column, membrane chromatography cassette, or other membrane chromatography device. The membrane chromatography column, membrane chromatography cassette, 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 one embodiment, the use of a membrane-based purification device can significantly increase productivity.

[0084] In some embodiments, the separation medium comprises an anion exchange substrate. Such substrates can be used in anion exchange-based chromatography to purify target molecules (e.g., nucleosides, nucleotides, nucleic acid bases, or analogs or derivatives thereof). Anion exchange utilizes positively charged functional groups that target negatively charged target molecules.

[0085] The anion-exchange ligand can be conjugated to the substrate via a functional group handle. The functional group 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 function as the functional group handle. The functional group handle can be as described above with respect to formula (I). The conjugated anion-exchange ligand can include a reaction product Rp and a spacer Sp as described above with respect to formula (II). The conjugated anion-exchange ligand further includes a separation group Sg.

[0086] Examples of suitable anion exchange ligands that may be disposed on the separation media substrate include primary, secondary, tertiary, and quaternary amines. Suitable amines may be diamines, triamines, and polyamines. Diamines are generally represented by the formula: [ka]

[0087] 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. In quaternary amines, R 2 , R 3 , R 4 , R 5 , and R 6 are each independently selected from aliphatic straight, branched, or cyclic, substituted or unsubstituted carbon chains having a length of 1 to 10 carbons, 1 to 6 carbons, or 2 to 4 carbons. 5 and R 6does not exist, and R 2 , R 3 , and R 4 is the same as for quaternary amines. For secondary amines, R 5 and R 6 does not exist, and R 2 and R 3 is H and R 4 is the same as for quaternary amines. For primary amines, R 5 and R 6 does not exist, and R 2 , R 3 , and R 4 is H. In some embodiments, the nitrogens of the diamine have different levels of substitution. For example, some amines may be secondary amines and some amines may be primary, tertiary, or quaternary amines. Suitable triamines and polyamines may have similar structures with three or more amine groups (triamines).

[0088] Examples of primary amines include methylenediamine, ethylenediamine, propylenediamine, butylenediamine (putrescine), pentylamine, or any aliphatic diamine having 1-18 carbons between the terminal amines covalently bonded through one of the amines. Such ligands can be made from polyamines such as ethylenediamine, diethylenetriamine, triethylenetetramine, etc., covalently bonded through one of the amines.

[0089] Examples of secondary amines include any of the above-mentioned primary amines immobilized to a substrate, substituted with additional R groups as described above. When a diamine is used, the secondary amine can also be formed by a covalent interaction with the substrate, binding both amines to the substrate. Ligands containing secondary amines with the structure of the ligand, such as linear polyethyleneimine, spermidine, or spermine, can also be immobilized. In addition, groups containing non-terminal primary amines (e.g., 3-aminopentane) can also be conjugated to a substrate to become secondary amines.

[0090] Examples of suitable tertiary amines include N,N-dimethylethylenediamine, N,N-dimethylpropylenediamine, N,N-diethylpropylenediamine, or amines in which one or both amines are substituted with an aliphatic carbon group ranging from 1 to 6 carbons and R 1 The diamine may be any aliphatic diamine having 2 to 18 carbons between the terminal amines.

[0091] Examples of quaternary amines include any of the primary amines previously mentioned that undergo a quaternization reaction to obtain a permanent positive charge. Such reactions can be carried out using alkyl groups, such as methyl iodide, or aryl groups, such as benzyl iodide. Quaternary amines can also include any of the tertiary amines previously mentioned that undergo a quaternization reaction to obtain a permanent positive charge. Such reactions can be illustrated by the Menschutkin reaction, which uses an alkyl halide to form a quaternary ammonium salt from reaction with a tertiary amine. Such reactions can be carried out using alkyl-containing groups of various lengths, such as butyl bromide, or aryl groups, such as benzyl chloride, or combinations thereof. Additionally, compounds containing quaternary amines can also be directly immobilized.

[0092] The ligands may include additional functional groups in addition to the amine group. The ligands may include linkers between the amine and any other functional groups that are 1-18 carbons, 1-10 carbons, 1-6 carbons, or 2-4 carbons in length.

[0093] The anion exchange substrate can be prepared by first exposing (e.g., soaking) a base substrate (e.g., a membrane or nonwoven substrate) to a solution of a linker-activating agent and a catalyst, then exposing (e.g., soaking) the substrate to a solution comprising a ligand and an optional catalyst, and finally exposing (e.g., soaking) the substrate to a buffer solution. In one exemplary embodiment, the linker-activating agent is or comprises N,N-disuccinimidyl carbonate (DSC) and the catalyst comprises triethylamine (TEA).

[0094] In one exemplary embodiment, the anion exchange substrate is manufactured in a three-step process. The first step involves exposing (e.g., immersing) the base substrate in a solution of linker activator and catalyst. This can include 0.1 mg / mL to 120 mg / mL DSC and 5 μL / mL to 100 μL / mL TEA in a solvent. The solvent can include DMSO, acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), hexamethylphosphoramide, sulfolane, or any other solvent / solution that swells the substrate (e.g., membrane). The exposure can be performed at a temperature of about 10° C. to 60° C. for about 1 minute to 1,800 minutes. For example, a membrane 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 at 40° C. for 16 hours.

[0095] In this exemplary embodiment, the second step involves exposing (e.g., immersing) the substrate in a solution comprising the ligand and optional catalyst, at a concentration of 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 μL / mL The solvent may be DMSO, or another organic solvent such as acetonitrile, THF, DMF, hexamethylphosphoramide, sulfolane, or any other solvent / solution that swells the substrate. The exposure may be at a temperature of about 10° C. to 60° C. for about 1 minute to 24 hours. For example, the membrane may be placed in a 15 μL / mL solution of DMEDA in DMSO at room temperature for 30 minutes.

[0096] In this exemplary embodiment, the third step involves exposing (e.g., immersing) the substrate from the second step in a buffer, which may include 0.05M to 4M Tris at a pH of 7.0 to 10.0. The exposing may be performed at a temperature of about 10° C. to 60° C. for a period of about 1 minute to 24 hours. For example, the membrane is placed in 1M Tris at pH 8.0 for 16 hours.

[0097] In some embodiments, the anion exchange substrate is prepared according to Method 1 described in US Patent Publication No. 20200188859 A1 (Zhou et al.).

[0098] In embodiments in which 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 dissolve or maintain the stability of the target molecule, or to achieve the desired level of binding and selectivity.

[0099] Suitable salts used during the purification process 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 some 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 amounts of 1 mM or more, 5 mM or more, or 10 mM or more, or 20 mM or more. Suitable salts can be added in amounts of 100 mM or less, 50 mM or less, or 30 mM or less. Salts can be added in amounts ranging from 1 mM to 100 mM, 1 mM to 50 mM, 5 mM to 30 mM, or 5 mM to 20 mM.

[0100] Suitable solvents used during the purification process include, for example, methanol, ethanol, isopropanol, acetonitrile, DMSO, and DMF. In some embodiments, ethanol, isopropanol, or acetonitrile is added to the solution. The suitable solvent can be added in an amount of 5% by weight or more, 10% by weight or more, 20% by weight or more, 30% by weight or more, 40% by weight or more, 50% by weight or more, 60% by weight or more, 70% by weight or more, 80% by weight or more, or 90% by weight or more. The suitable solvent can be added in an amount of 90% by weight or less, 80% by weight or less, 70% by weight or less, 60% by weight or less, 50% by weight or less, 40% by weight or less, 30% by weight or less, or 20% by weight or less. The solvent can be added in an amount ranging from 10% by weight to 90% by weight, 20% by weight to 80% by weight, 30% by weight to 70% by weight, or 10% by weight to 50% by weight.

[0101] A different solvent or a buffer with higher conductivity can be used to elute the target molecule from the immobilized base due to charge repulsion between the target and the ligand.

[0102] According to one embodiment, the pH of the target-containing feed solution is adjusted to maintain the target molecules in a positively charged state, which is usually higher than the pKa of such molecules. On the other hand, the pH of the feed solution is maintained lower than the pKa of the anion exchange membrane ligand to maintain a positive state. For example, when 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.

[0103] In some embodiments, the separation medium comprises a substrate having functional groups that induce hydrophobic interactions with target molecules, impurities, or both. Such substrates can be used in hydrophobic interaction chromatography (HIC) to purify target molecules (e.g., nucleosides, nucleotides, nucleic acid bases, or analogs or derivatives thereof). Hydrophobic interaction chromatography uses hydrophobic functional groups that interact with hydrophobic groups on the target molecule. Hydrophobic interactions exploit the difference in hydrophobicity between the target molecule and possible impurities. Nucleobases contain hydrophobic rings that can be exploited by interacting with HIC ligands on the substrate.

[0104] In one embodiment, such ligands include aliphatic chains having 3 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, sulfur-containing thiophilic ligands such as propanethiol, 2-butanethiol, 3,6-dioxa-1,8-octanedithiol, octanethiol, benzyl mercaptan, 2-mercaptopyridine, thiophenol, 1,2-ethanedithiol, 1,4-benzenedimethanethiol, 2-phenylethanethiol, and the like, and combinations thereof. The hydrophobic interaction ligands can be conjugated to the substrate via functional group handles as described above with respect to formula (I).

[0105] In some embodiments, the target molecule is a nucleic acid base or modified nucleic acid base, a nucleoside or modified nucleoside, or a nucleotide or modified nucleotide. A solvent, salt, or other additive may be added to the solution containing the target molecule to allow binding to occur through the interaction of hydrophobic groups present on both the ligand and the target. In some embodiments, a kosmotropic salt is added to the solution. In some cases, a combination of a kosmotropic salt and a chaotropic salt may be added to the solution. For example, a mixture of a kosmotropic anion and a chaotropic cation may be used. In some embodiments, the proportion of kosmotropic salt is increased and / or the proportion of chaotropic salt is decreased. Cosmotropic salts are known as salts that reduce the solubility of non-polar substances in aqueous solutions, while chaotropic salts increase their solubility. In some embodiments, the proportion of organic solvent in the solution may be increased. In another embodiment, the proportion of organic solvent in the solution may be decreased. In another embodiment, a combination of kosmotropic components, chaotropic components, and / or organic solvent changes may also be used.

[0106] Examples of kosmotropic salts that may be added to the solution containing the target molecule include ammonium sulfate, ammonium phosphate, potassium phosphate, sodium sulfate, sodium chloride, and combinations thereof. Suitable kosmotropic salts may be added in amounts of 0.1M or more, 0.5M or more, or 1.0M or more, or 2.0M or more. Suitable kosmotropic salts may be added in amounts of 6.0M or less, 5.0M or less, or 4.0M or less. Cosmotropic salts may be added in amounts ranging from 0.1M to 6M, 0.5M to 2.5M, or 0.5M to 3.0M.

[0107] 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 chaotropic salt is kept at 1 M or less, 0.5 M or less, or 0.1 M or less. In some embodiments, the solution is free or substantially free of chaotropic salts.

[0108] Suitable solvents used during the purification process include, for example, methanol, ethanol, isopropanol, acetonitrile, DMSO, and DMF. In some embodiments, ethanol, isopropanol, or acetonitrile is added to the solution. The suitable solvent can be added in an amount of 5% by weight or more, 10% by weight or more, 20% by weight or more, 30% by weight or more, 40% by weight or more, 50% by weight or more, 60% by weight or more, 70% by weight or more, 80% by weight or more, or 90% by weight or more. The suitable solvent can be added in an amount of 90% by weight or less, 80% by weight or less, 70% by weight or less, 60% by weight or less, 50% by weight or less, 40% by weight or less, 30% by weight or less, or 20% by weight or less. The solvent can be added in an amount ranging from 10% by weight to 90% by weight, 20% by weight to 80% by weight, 30% by weight to 70% by weight, or 10% by weight to 50% by weight.

[0109] A different solvent or a buffer with low conductivity can be used to elute the target molecule from the immobilized base due to charge repulsion between the target and the ligand.

[0110] The substrate used to prepare the hydrophobic interaction chromatography (HIC) substrate may be any suitable material. In some embodiments, the hydrophobic interaction chromatography (HIC) substrate is or includes a membrane, a resin, a monolith, a hydrogel, a fiber, and the like. 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 fibrous substrate.

[0111] According to one embodiment, the hydrophobic interaction substrate can be used to purify target molecules at a fast flow rate. For example, the hydrophobic interaction substrate can be used to purify target molecules at 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. There is no desirable lower limit to the residence time, but in practice the residence time is 1 second or more. The hydrophobic interaction substrate can be arranged as a membrane chromatography column, membrane chromatography cassette, or other membrane chromatography device. The membrane chromatography column, membrane chromatography cassette, 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 one embodiment, the use of a membrane-based purification device can significantly improve productivity.

[0112] In some embodiments, the separation medium comprises a multimodal medium. A multimodal medium is a medium that comprises two or more types of ligands or functional groups on a substrate. The multimodal medium can enhance the interaction between the ligands and the target molecules. In some embodiments, the multimodal medium comprises an ion exchange ligand or functional group and one other type of ligand or functional group. In some such embodiments, the multimodal medium comprises a cation exchange ligand and at least one other type of functional group. In some embodiments, the 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 may be part of the same ligand as the cation exchange group or anion exchange group, or may be in a separate ligand. For example, the multimodal medium may further comprise a hydrophobic interaction group, a hydrogen bonding group, a sulfophilic group, or a combination thereof, in addition to the cation exchange ligand or anion exchange ligand. In one embodiment, the multimodal medium comprises a combination of a cation exchange ligand and a hydrophobic interaction group. In one embodiment, the multimodal medium comprises a combination of a cation exchange ligand and a hydrogen bonding group. In one embodiment, the multimodal medium comprises a combination of cation exchange ligands and sulfophilic groups. The multimodal medium may also comprise three or more types of functional groups. The multimodal medium can be used to separate or purify nucleotides, nucleosides, nucleobases, and their analogs and derivatives.

[0113] According to an exemplary embodiment, the multimodal medium includes ligands that include cation exchange ligands and hydrophilic ligands.

[0114] According to an exemplary embodiment, the multimodal medium comprises ligands including cation exchange ligands and hydrophobic interaction ligands.

[0115] According to an exemplary embodiment, the multimodal medium includes ligands that include anion exchange ligands and hydrophilic ligands.

[0116] According to an exemplary embodiment, the multimodal medium comprises ligands including anion exchange ligands and hydrophobic interaction ligands.

[0117] In some examples, the multimodal media includes a cation exchange ligand that also includes a sulfophilic functional group. Examples of suitable sulfophilic cation exchange ligands that can be disposed on the separation media substrate include mercaptocarboxylic acids and their salts. The sulfophilic cation exchange ligands can be represented by the formula: [ka] (In the formula, 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 A may be linear, branched, or cyclic. 1 is a carboxylic acid (optionally conjugated to an aromatic group, such as at the benzoic acid or benzylic position) or a sulfonate group.

[0118] Examples of suitable sulfur-philic cation exchange ligands include mercaptobenzoic acids (e.g., 2-mercaptobenzoic acid or 4-mercaptobenzoic acid), and mercaptosulfonic acids and their salts, such as sodium 3-mercapto-1-propanesulfonate. The ligands may contain linkers between the sulfur-containing functional group and the other (e.g., acid) functional group that are 1-18 carbons, 1-10 carbons, 1-6 carbons, or 2-4 carbons in length.

[0119] The sulfophilic cation exchange substrate can be prepared by first exposing (e.g., immersing) a base substrate (e.g., a membrane or nonwoven substrate) to a solution of a linker activator and a catalyst, then exposing (e.g., immersing) the substrate to a solution containing a ligand and an optional catalyst, and finally immersing the substrate in a buffer solution. In one exemplary embodiment, the linker activator is or includes N,N-disuccinimidyl carbonate (DSC) and the catalyst includes triethylamine (TEA).

[0120] In one exemplary embodiment, the sulfophilic cation exchange substrate is manufactured in a three-step process. The first step involves exposing (e.g., immersing) the base substrate in a solution of linker activator and catalyst. This can include 0.1 mg / mL to 120 mg / mL DSC and 5 μL / mL to 100 μL / mL TEA in a solvent. Solvents include DMSO, acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), hexamethylphosphoramide, sulfolane, or any other solvent / solution that swells the substrate (e.g., membrane). The exposure can be performed at a temperature of about 10° C. to 60° C. for about 1 minute to 1,800 minutes. For example, a membrane 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 at 40° C. for 16 hours.

[0121] In this exemplary embodiment, the second step involves exposing (e.g., immersing) the substrate in a solution containing the ligand and optional catalyst. This may include about 0.1 mg / mL to 150 mg / mL, 1 mg / mL to 100 mg / mL, or 10 mg / mL to 50 mg / mL of sodium 3-mercapto-1-propanesulfonate in a solvent. The solvent may be DMSO, or another organic solvent, such as acetonitrile, THF, DMF, hexamethylphosphoramide, sulfolane, etc., or any other solvent / solution that swells the substrate. The exposing may be performed at a temperature of about 10° C. to 60° C. for about 1 minute to 24 hours. For example, the membrane may be placed in a solution of 300 mg of sodium 3-mercapto-1-propanesulfonate and 1 mL of TEA dissolved in 10 mL of DMSO for 16 hours at 40° C.

[0122] In this exemplary embodiment, the third step involves exposing (e.g., immersing) the substrate from the second step in a buffer, which may include 0.05M to 4M Tris at a pH of 7.0 to 10.0. The exposing may be performed at a temperature of about 10° C. to 60° C. for a period of about 1 minute to 24 hours. For example, the membrane is placed in 1M tris(hydroxymethyl)aminomethane (Tris) at pH 8.0 for 16 hours.

[0123] In some embodiments, the target of interest is a nucleobase or modified nucleobase, a nucleoside or modified nucleoside, or a nucleotide or modified nucleotide. Appropriate solvents, salts, or other additives may be added to the solution to allow the target molecule to dissolve or maintain its stability, or to allow the desired level of binding and selectivity to be achieved.

[0124] Suitable salts used during the purification process 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 some 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 amounts of 1 mM or more, 5 mM or more, or 10 mM or more, or 20 mM or more. Suitable salts can be added in amounts of 100 mM or less, 50 mM or less, or 30 mM or less. Salts can be added in amounts ranging from 1 mM to 100 mM, 1 mM to 50 mM, 5 mM to 30 mM, or 5 mM to 20 mM.

[0125] Suitable solvents used during the purification process include, for example, methanol, ethanol, isopropanol, acetonitrile, DMSO, and DMF. In some embodiments, ethanol, isopropanol, or acetonitrile is added to the solution. The suitable solvent can be added in an amount of 5% by weight or more, 10% by weight or more, 20% by weight or more, 30% by weight or more, 40% by weight or more, 50% by weight or more, 60% by weight or more, 70% by weight or more, 80% by weight or more, or 90% by weight or more. The suitable solvent can be added in an amount of 90% by weight or less, 80% by weight or less, 70% by weight or less, 60% by weight or less, 50% by weight or less, 40% by weight or less, 30% by weight or less, or 20% by weight or less. The solvent can be added in an amount ranging from 10% by weight to 90% by weight, 20% by weight to 80% by weight, 30% by weight to 70% by weight, or 10% by weight to 50% by weight.

[0126] A different solvent or a buffer with higher conductivity can be used to elute the target molecule from the immobilized base due to charge repulsion between the target and the ligand.

[0127] When multimodal media cation exchange chromatography is used 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 value to prevent degradation of the target, which is above the pKa of the multimodal ligand to maintain the ligand in a negatively charged state. Examples of suitable pH ranges include pH 1-3 for cytidine and gemcitabine, with or without a protecting alcohol.

[0128] Molecules bound to the cation exchange ligands of the multimodal medium can be eluted, for example, by increasing the conductivity and screening the electrostatic attraction between the cation exchange ligands and the target molecules. In one embodiment, elution can be performed by changing the pH above the pKa of the amines in the target molecules, creating a neutral charge in the target molecules, thereby reducing charge interactions and inducing elution. Different target molecules (or target molecules and other molecules) can be eluted using linear gradient elution or stepwise isocratic elution.

[0129] When multimodal media anion exchange chromatography is used to separate or purify a target molecule, the pH of the solution can be monitored and controlled to remain above the pKa of the target molecule to maintain the molecule in a deprotonated state. The pH can also be maintained below a threshold value to prevent degradation of the target, which is also below the pKa of the multimodal ligand to maintain the ligand in a positively charged state. Examples of suitable pH ranges include pH 3-10 for adenosine monophosphate purification.

[0130] Molecules bound to the anion exchange ligands of the multimodal medium can be eluted, for example, by increasing the conductivity and screening the electrostatic attraction between the cation exchange ligands and the target molecules. In one embodiment, elution can be performed by changing the pH below 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 stepwise isocratic elution.

[0131] The substrate used to manufacture the multimodal medium may be any suitable material. In some embodiments, the multimodal medium is or includes a membrane, resin, monolith, hydrogel, fiber, and the like. In one embodiment, the multimodal medium is or includes a membrane. In one embodiment, the multimodal medium is or includes a nonwoven fibrous substrate.

[0132] According to one embodiment, the multimodal media can be used to purify target molecules at high flow rates. For example, the multimodal media can be used to purify target molecules with residence times of 2 minutes or less, 1 minute or less, 30 seconds or less, 10 seconds or less, or 6 seconds or less. Residence times depend in part on the size of the separation device, with small devices having residence times as short as 1 second or less. There is no desirable lower limit to residence times, but in practice residence times are 0.1 seconds or more. The multimodal media can be arranged as a membrane chromatography column, membrane chromatography cassette, or other membrane chromatography device. The membrane chromatography column, membrane chromatography cassette, or other membrane chromatography device can provide residence times of 2 minutes or less, 1 minute or less, 30 seconds or less, 10 seconds or less, or 6 seconds or less. According to one embodiment, the use of a membrane-based purification device can significantly increase productivity.

[0133] Exemplary embodiments The following is a non-limiting list of exemplary embodiments according to the present disclosure.

[0134] Embodiment 1 is a separation medium comprising a membrane; and a plurality of ligands immobilized on the membrane, the plurality of ligands comprising anion exchange ligands, cation exchange ligands, thiophilic ligands, hydrophobic interaction ligands, hydrophilic ligands, or combinations thereof.

[0135] Embodiment 2 is the separation medium of embodiment 1, configured to separate target molecules comprising nucleotides, nucleosides, nucleobases, derivatives and analogs thereof, and combinations thereof from a reaction mixture.

[0136] Embodiment 3 is a separation medium of embodiment 1 or 2, configured for use with an organic solvent.

[0137] Embodiment 4 is the separation medium of any one of embodiments 1 to 3, wherein the plurality of ligands comprises anion exchange ligands that include an aliphatic diamine or triamine that includes 1 to 18 carbons between adjacent amines.

[0138] 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.

[0139] Embodiment 6 is the separation medium of any one of embodiments 1 to 5, wherein the plurality of ligands comprises cation exchange ligands comprising amino carboxylic acids, amino sulfonic acids, or combinations thereof.

[0140] Embodiment 7 is the separation medium of embodiment 6, wherein the cation exchange ligand comprises aminobenzoic acid, aminodiacetic acid, aminopropanoic acid, 3-amino-1-propanesulfonic acid, 3-amino-1-ethylsulfonic acid, or combinations thereof, with a spacer between the amino group and the acid or sulfonate group that is 1 to 18 carbons, 1 to 10 carbons, 1 to 6 carbons, or 2 to 4 carbons in length.

[0141] Embodiment 8 is the separation medium of any one of embodiments 1 to 7, wherein the plurality of ligands comprises two or more of anion exchange ligands, cation exchange ligands, sulfophilic ligands, hydrophilic ligands, and hydrophobic interaction ligands.

[0142] Embodiment 9 is the separation medium of any one of Embodiments 1 to 8, wherein the plurality of ligands includes a ligand having a cation exchange functional group and a sulfophilic functional group.

[0143] Embodiment 10 is the separation medium of embodiment 9, wherein the plurality of ligands comprises mercaptobenzoic acid, mercaptosulfonic acid, a salt thereof, or a combination thereof, preferably the plurality of ligands comprises sodium 3-mercapto-1-propanesulfonate.

[0144] Embodiment 11 is a compound according to the present invention, wherein the plurality of ligands is represented by formula (I): Fh-Sp-Sg (I) (wherein Fh is a functional handle, Sp is a spacer, and Sg is a functional separation group). and wherein the ligand has the formula: the functional group handle is selected from amines; alcohols; activated alcohols; epoxides; isocyanates; alkenes; alkynes; cycloalkenes; cyclooctynes; thiols; disulfides; azides; thioisocyanates; N-hydroxysuccinimides; maleimides; activated esters; and activated carboxylic acids; the spacer is a carbon chain of C1-C18, C1-C10, C1-C6, C1-C4, C1-C3, or C2-C4 length, optionally substituted with one or more ether, ester, benzyl, phenyl, or amide along the carbon chain; The functional separation group is a cation exchange separation group, an anion exchange separation group, a hydrophobic separation group, or a sulfophilic separation group; A separation medium according to any one of embodiments 1 to 10.

[0145] 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.

[0146] Embodiment 13 is the separation medium of 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 the functional group handle comprises a secondary amine, a tertiary amine, or a quaternary amine, and 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.

[0147] Embodiment 14 is the separation medium of embodiment 11, wherein the functional separation group comprises an aliphatic chain having a length of 2 or more carbons (optionally butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecyl), benzyl, phenyl, phenol, pyridine, boronic acid, a branched polymer (optionally polypropylene glycol), a sulfur-containing sulfophilic ligand (optionally propanethiol, 2-butanethiol, 3,6-dioxa-1,8-octanedithiol, octanethiol, benzyl mercaptan, 2-mercaptopyridine, thiophenol, 1,2-ethanedithiol, 1,4-benzenedimethanethiol, or 2-phenylethanethiol), or a combination thereof.

[0148] 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.

[0149] Embodiment 16 is a separation device comprising: a housing; and a separation medium disposed within the housing, the separation medium comprising: a membrane; and a plurality of ligands immobilized on the membrane, the plurality of ligands comprising anion exchange ligands, cation exchange ligands, sulfophilic ligands, hydrophobic interaction ligands, hydrophilic ligands, or combinations thereof.

[0150] Embodiment 17 is the separation device of embodiment 16, wherein the housing comprises a cassette or a column.

[0151] Embodiment 18 is the separation device of embodiment 16 or 17, wherein the separation medium is configured to separate a target molecule comprising a nucleobase from the reaction mixture.

[0152] Embodiment 19 is the separation device of any one of embodiments 16-18, wherein the separation medium is configured for use with an organic solvent.

[0153] Embodiment 20 is the separation device of any one of embodiments 16-19, wherein the plurality of ligands comprises anion exchange ligands comprising an aliphatic diamine or triamine containing 1 to 18 carbons between adjacent amines.

[0154] Embodiment 21 is the separation device of embodiment 20, comprising N,N-dimethylethylenediamine, N,N-dimethylpropylenediamine, N,N-dimethylpropylenediamine, N,N-diethylpropylenediamine, or a combination thereof.

[0155] Embodiment 22 is the separations device of any one of embodiments 16-21, wherein the plurality of ligands comprises cation exchange ligands comprising amino carboxylic acids, amino sulfonic acids, or combinations thereof.

[0156] Embodiment 23 is the separation device of embodiment 22, wherein the cation exchange ligand comprises aminobenzoic acid, aminodiacetic acid, aminopropanoic acid, 3-amino-1-propanesulfonic acid, 3-amino-1-ethylsulfonic acid, or combinations thereof, comprising a spacer between the amino group and the acid or sulfonate group that is 1 to 18 carbons, 1 to 10 carbons, 1 to 6 carbons, or 2 to 4 carbons in length.

[0157] Embodiment 24 is the separation device of any one of embodiments 16 to 23, wherein the plurality of ligands comprises two or more of anion exchange ligands, cation exchange ligands, sulfophilic ligands, hydrophilic ligands, and hydrophobic interaction ligands.

[0158] Embodiment 25 is the separation device of any one of embodiments 16 to 24, wherein the plurality of ligands includes ligands having a cation exchange functional group and a sulfophilic functional group.

[0159] Embodiment 26 is the separation device of embodiment 25, wherein the plurality of ligands comprises mercaptobenzoic acid, mercaptosulfonic acid, a salt thereof, or a combination thereof, preferably the plurality of ligands comprises sodium 3-mercapto-1-propanesulfonate.

[0160] Embodiment 27 is a compound according to the present invention, wherein the plurality of ligands is represented by formula (I): Fh-Sp-Sg (I) (wherein Fh is a functional handle, Sp is a spacer, and Sg is a functional separation group). and wherein the ligand has the formula: the functional group handle is selected from amines; alcohols; activated alcohols; epoxides; isocyanates; alkenes; alkynes; cycloalkenes; cyclooctynes; thiols; disulfides; azides; thioisocyanates; N-hydroxysuccinimides; maleimides; activated esters; and activated carboxylic acids; the spacer is a carbon chain of C1-C18, C1-C10, C1-C6, C1-C4, C1-C3, or C2-C4 length, optionally substituted with one or more ether, ester, benzyl, phenyl, or amide along the carbon chain; The functional separation group is a cation exchange separation group, an anion exchange separation group, a hydrophobic separation group, or a sulfophilic separation group; 2 is a separation device according to embodiments 16 to 26.

[0161] Embodiment 28 is the separation device of embodiment 27, wherein the functional separating group comprises an acid, a sulfonic acid, or a phosphate.

[0162] Embodiment 29 is the separation device of 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 the functional group handle comprises a secondary amine, a tertiary amine, or a quaternary amine, and 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.

[0163] Embodiment 30 is the separation device of embodiment 27, wherein the functional separation group comprises an aliphatic chain having a length of 2 or more carbons (optionally butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecyl), benzyl, phenyl, phenol, pyridine, boronic acid, a branched polymer (optionally polypropylene glycol), a sulfur-containing sulfophilic ligand (optionally propanethiol, 2-butanethiol, 3,6-dioxa-1,8-octanedithiol, octanethiol, benzyl mercaptan, 2-mercaptopyridine, thiophenol, 1,2-ethanedithiol, 1,4-benzenedimethanethiol, or 2-phenylethanethiol), or a combination thereof.

[0164] Embodiment 31 is the separation device of embodiment 27, wherein the functional handle comprises a thiol and the functional separating group comprises an acid, a sulfonic acid, or a phosphate.

[0165] Embodiment 32 is a method for purifying a target molecule, comprising passing a solution containing the target molecule, the target molecule comprising a nucleic acid, a nucleotide, a nucleoside, a nucleic acid base, or an analog or derivative thereof, through a membrane chromatography device, the membrane chromatography device comprising: a housing; and a separation medium disposed within the housing, the separation medium comprising: a membrane; and a plurality of ligands immobilized on the membrane, the plurality of ligands comprising anion exchange ligands, cation exchange ligands, thiophilic ligands, hydrophobic interaction ligands, hydrophilic ligands, or combinations thereof.

[0166] Embodiment 33 is the method of embodiment 32, wherein the target molecule is purified from a solution comprising the reaction mixture after synthesis of the target molecule.

[0167] Embodiment 34 is the method of embodiment 32 or 33, wherein the separation medium comprises an anion exchange membrane.

[0168] Embodiment 35 is the method of any one of embodiments 32-34, wherein the residence time of the solution in the membrane chromatography device is 60 seconds or less.

[0169] Embodiment 36 is the method of any one of embodiments 32 to 35, wherein the target molecule is a nucleotide or a nucleic acid.

[0170] Embodiment 37 is the method of any one of embodiments 32 to 36, wherein the separation medium comprises a sulfophilic cation exchange membrane.

[0171] Embodiment 38 is the method of any one of embodiments 32-37, wherein the sulfophilic cation exchange membrane comprises cation exchange ligands having sulfophilic functional groups.

[0172] Embodiment 39 is the method of any one of embodiments 32-38, wherein the solution comprises an organic solvent.

[0173] Embodiment 40 is the method of any one of embodiments 32-39, wherein the plurality of ligands comprises a ligand having a cation exchange functional group and a sulfophilic functional group.

[0174] Embodiment 41 is the method of any one of embodiments 32-40, wherein the plurality of ligands comprises mercaptobenzoic acid, mercaptosulfonic acid, a salt thereof, or a combination thereof, preferably the plurality of ligands comprises sodium 3-mercapto-1-propanesulfonate.

[0175] 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

[0176] These examples are for illustrative purposes only and are not intended to limit the scope of the appended claims. All parts, percentages, ratios, etc. in the examples and the remainder of the specification are by weight unless otherwise specified.

[0177] The performance of various types of separation membranes was tested and evaluated against control samples.

[0178] Test Method Dynamic binding capacity at 10% breakthrough (DBC 10% ) can be determined by standard chromatographic methods, such as Cytiva AEKTA Pure Fast Protein Liquid Chromatography (FPLC). First, the separation medium is loaded into a housing unit. The loaded separation medium is then connected to the FPLC. Next, the feed material is passed through the separation medium at a constant column volume per minute flow rate (CV / min) until the target effluent concentration, as determined by a UV signal at an appropriate wavelength, reaches 10% of the feed concentration. Finally, the DBC is calculated based on the retention volume and the volume of the separation medium in the FPLC system. 10% is calculated as follows: ((10% breakthrough - retentate volume) × (feed concentration)) / (separation medium volume) = DBC 10% (This is expressed as mg of target material / mL of chromatography media).

[0179] Sample Test DBC 10% was determined using chromatography as described above. A complete bind-elute chromatograph typically involves the following four steps: Step 1 - Equilibration: The contained separation medium is equilibrated with buffer A. Step 2-Loading: The loading material is injected / pumped through the separation media until 10% breakthrough is achieved. Step 3 - Wash: The medium is washed using a wash buffer. The wash buffer can be a single buffer or multiple buffers to wash away some of the impurities. The wash buffer can include a buffer A or a different buffer B, or a combination of buffers including buffer A and / or B and / or additional buffers (C, D, E, F, etc.), or a gradient transition between buffers A and / or B and / or additional buffers (C, D, E, F, etc.), or a gradient transition between a combination of buffers A and / or B and / or additional buffers (C, D, E, F, etc.). Step 4 - Elution: The loading material (such as the target and a portion of the impurities) is eluted from the separation medium using Buffer C. The elution buffer may include Buffer C or a different Buffer B, or a combination of buffers including Buffer C and / or B and / or additional buffers (A, D, E, F, etc.), or a gradient transition between Buffer C and / or B and / or additional buffers (A, D, E, F, etc.), or a gradient transition between a combination of Buffer C and / or B and / or additional buffers (A, D, E, F, etc.).

[0180] The eluate can be collected for further analysis.

[0181] In addition to the four main steps above, optionally a stripping and / or cleaning in place (CIP) step may be performed before the cycle begins again.

[0182] Example 1 - Preparation of AEX membrane A regenerated cellulose membrane with a diameter of 47 mm and a thickness of 70 μm was immersed in a solution of 300 mg of N,N-disuccinimidyl carbonate (DSC) dissolved in 10 mL of dimethyl sulfoxide (DMSO) and 139 μL of triethylamine (TEA) for 16 hours at 40 °C. The membrane was then placed in a solution of 100 μL of N,N-dimethylethylenediamine (DMEDA) per mL of DMSO for 16 hours at room temperature. Finally, the membrane was placed in 1 M Tris at pH 8.0 for 16 hours.

[0183] Example 2 - Preparation of sulfophilic CEX membrane A regenerated cellulose membrane with a diameter of 47 mm and a thickness of 70 μm was immersed in a solution of 300 mg DSC and 139 μL TEA for 16 h at 40° C. The membrane was then immersed in a solution of 300 mg sodium 3-mercaptopropanesulfonate dissolved in 10 mL DMSO and 0.5 mL TEA for 16 h at 40° C. Finally, the membrane was placed in 200 mM Tris, pH 8.0 for 16 h.

[0184] Example 3 - Separation media DBC The dynamic binding capacity of the thiophilic CEX separation media was tested at various flow rates. The separation membrane was prepared according to Example 2.

[0185] Four layers of 24 mm circular separation membrane were packed into a mini-column (membrane volume = 0.1 mL) and connected to a Cytiva AEKTA Pure to determine the dynamic binding capacity. In this example, the loading material was 0.8 mg / mL cytidine in 10 mM phosphate, pH 2.0. Cytidine solution was applied to the membrane at various flow rates, specified in column volumes / min (CV / min), until 10% breakthrough. DBC 10% The results are shown graphically in Figure 2.

[0186] As can be seen from Figure 2, the DBC of cytidine increases with increasing flow rate. 10% It can be seen that the trans-column pressure (ΔC) is 0.06 MPa at 110 CV / min, which indicates that the flow rate can be further increased.

[0187] Example 4 - Separation of Cytidine Using an Organic Buffer System The ability of the thiophilic CEX separation media to separate cytidine in an organic buffer system was tested. The separation membrane was prepared according to Example 2. Two commercially available products, HITRAP® SP HP cation exchange chromatography column available from Cytiva, Marlborough, MA, and NATRIFLO® HD-Sb column available from Natrix Separations, were used as comparative samples. HITRAP® SP HP is a resin-based strong cation exchange chromatography column product, and NATRIFLO® HD-Sb is a hydrogel-based strong cation exchange column product with enhanced hydrophobic interaction groups.

[0188] For the bind-elute chromatography separation process, eight layers of 24 mm circular separation membrane were packed into a polypropylene column housing (membrane volume = 0.2 mL) and connected to a Cytiva AEKTA Pure. Cytidine was loaded to 10% breakthrough at a determined column volume / min flow rate (CV / min). The list of buffers shown in Table 1 was used in the separation process.

[0189] [Table 1]

[0190] The chromatograms of the sample separation membrane, resin (HITRAP® SP HP), and hydrogel (NATRIX® HD-Sb) commercial products are shown in Figures 3A, 3B, and 3C, respectively. Table 2 shows the flow rate, DBC 10% Operating parameters and outputs including trans-column pressure (ΔC) are summarized.

[0191] [Table 2]

[0192] The binding capacities of the columns are compared in Figure 4. The separation membrane was observed to have a binding capacity approximately twice that of the resin and approximately 18 times that of the hydrogel using the organic buffer system in Table 1. At the same trans-column pressure, the flow rate of the separation medium is at least 20 times faster than that of the resin.

[0193] Example 5 - Separation of Cytidine Using Aqueous Buffer Systems The ability of the CEX separation resin to separate cytidine in an aqueous buffer system was tested. The separation resin was HITRAP® SP HP resin purchased from Cytiva.

[0194] 1 mL of HITRAP® SP HP resin was coupled to a Cytiva AEKTA Pure for a bind-elute chromatographic separation process in a completely aqueous system (20 mM sodium phosphate with various pH values ​​ranging from 2.48 to 1.99).

[0195] The binding-elution profile of cytidine dissolved in 20 mM sodium phosphate in pH 2.48 or pH 1.99 solutions is shown in FIG. 5A.

[0196] The first cycle, loaded at pH 2.48, gave a broadened, bimodal elution peak beginning at the tail end of the wash phase. The bind-elution profile of Figure 5A using 20 mM sodium phosphate at pH 1.99 is shown in Figure 5B.

[0197] It was observed that lowering the loading pH from 2.48 to 1.99 allowed separation of the elution into a single sharp elution peak compared to the broad bimodal peak observed in the bind-elute cycle at pH 2.48. It is hypothesized that the protonated amine on cytidine is more abundant in an acidic environment (low pH) and therefore more strongly adsorbs to the CEX column than the deprotonated cytidine present at a higher pH. Although purification could be performed using the resin, it was performed at significantly slower flow rates, resulting in lower chromatographic productivity for the entire process compared to membranes using the same ligand.

[0198] Example 6 - Recovery of DBC and AEX Anion exchange (AEX) separation media with 1 μm pore size was prepared according to Example 1. Eight layers of 24 mm circular AEX membranes were packed into a housing unit (membrane volume = 0.2 mL) and connected to a Cytiva AEKTA pure to measure the dynamic binding capacity. In this example, the loading material was 0.25 mg / mL adenosine-5'-monophosphate (AMP, 96.98%) dissolved in equilibration buffer. AMP was loaded at different column volumes / min flow rates (CV / min) until 10% breakthrough was reached. A list of buffers used in the separation process is shown in Table 3.

[0199] [Table 3]

[0200] The chromatogram overlay is shown in Figure 6A. 10% and AMP recovery are shown in Figure 6B, which shows that increasing the flow rate does not decrease the binding capacity or AMP recovery of the AEX separation medium.

[0201] Example 7 - DBC of AEX in buffer conditions of different conductivity Eight layers of 24 mm circular AEX membranes were packed in a housing unit (membrane volume = 0.2 mL) and connected to a Cytiva AEKTA pure to determine the dynamic binding capacity. The membranes were prepared according to Example 1. In this example, the feed solution contained 0.25 mg / mL AMP in equilibration buffer supplemented with 0, 100 mM, or 200 mM NaCl. AMP was loaded at different column volumes per minute (CV / min) flow rates until 10% breakthrough. The DBC is shown in Figure 3. A list of buffers used in the separation process is shown in Table 4.

[0202] [Table 4]

[0203] DBC under different flow rates and salt conditions 10% is shown in Figure 7A. An overlay of the chromatograms is shown in Figure 7B. From Figure 3, it can be seen that for each buffer condition, increasing the flow rate does not decrease the binding capacity of the AEX separation medium. However, as shown in Figure 7B, increasing the salt concentration significantly decreases the binding capacity of the AEX separation medium. At 10 CV / mL, the DBC decreases from 38 mg / mL with no added salt to 2 mg / mL with the addition of 200 mM NaCl.

[0204] Example 8 - Isolation of difluoro-substituted nucleoside derivative intermediates with various alcohol and amine protection in organic buffer systems The ability of the thiophilic-CEX separation medium to separate difluoro-substituted nucleoside derivative intermediates having deprotected amines from a mixture in an organic solvent containing a combination of difluoro-substituted nucleoside derivatives having mono- and / or di-protected alcohols and / or protected amines and an excess of a protecting agent (diluted 50-fold with the equilibration buffer described in Table 3) was tested. The separation membrane was prepared according to Example 2.

[0205] Eight layers of 24 mm circular separation membranes were packed into a membrane housing (membrane volume = 0.2 mL) and coupled to a Cytiva AEKTA Pure for bind-elute chromatography separation process. Samples containing a mixture of mono- and / or di-protected alcohols and / or protected amines with excess protecting agent and difluoro-substituted nucleoside derivatives in organic solvent (diluted 50-fold with equilibration buffer listed in Table 3) were loaded at a determined column volume / min flow rate (CV / min) until 10% breakthrough was reached. The buffers listed in Table 5 were used in the separation process.

[0206] [Table 5]

[0207] This process was repeated ten times, and the respective chromatograms are overlaid in Figure 8A.

[0208] The bind-elute purification cycle was found to be consistent between runs. Samples were taken every 5 runs and the concentration was assessed by UV absorbance at 260 nm using a NANODROP™ Lite UV-Vis spectrophotometer (available from Thermo Scientific). The yield was found to be consistent with a coefficient of variation of 5.17%.

[0209] Samples of the feed and eluate were also taken for thin layer chromatography (TLC) analysis. A photograph of the TLC plate is shown in Figure 8B. The left side shows the feed sample, and the right side shows the eluate sample. As shown in Figure 8B, three species in the crude feed (left), representing difluoro-substituted nucleoside derivatives with mono-alcohol protection (bottom), dialcohol protection (middle), and tri- (top) protection, were reduced to a single band of concentrated di-protected species in the eluate (right) at the same position as the band of di-protected species in the feed. This suggests the successful utilization of amine pendant groups on pyrimidines for CEX purification in a partially organic solvent system. The tri-protected species here refers to difluoro-substituted nucleoside derivatives with di-protected alcohol and protected amine. It is hypothesized that the protonated amine is present only in any combination of unprotected alcohol, mono-protected alcohol, and di-protected alcohol where the amine is not protected, and is therefore more strongly attracted to the CEX column than other variants with protected amines. In amine-protected molecules, the pendant amine cannot be protonated, resulting in very poor adsorption to the column via the CEX mechanism.

[0210] Example 9 - Comparative Study Separation of difluoro-substituted nucleoside derivative intermediates having deprotected amines from a mixture in an organic solvent containing difluoro-substituted nucleoside derivatives having a combination of mono- and / or di-protected alcohols and / or protected amines and an excess of a protecting agent (diluted 50-fold with the equilibration buffer described in Table 3) was tested using a cation exchange membrane prepared according to Example 2 and a HITRAP® SP HP cation exchange resin column.

[0211] The test was performed at a flow rate of 1 mL / min with a run time of 12 min. The solution was diluted with a low conductivity ethanol / phosphate mixture (low conductivity) and the column was eluted with 1 M NaCl (high conductivity).

[0212] The resin column had a resin volume of 1 mL with an estimated binding capacity of 8 mg / mL. The expected large-scale residence time is ≥120 min per cycle. The binding-elution data is shown in Figure 9A.

[0213] The media volume of the membrane column was 0.1 mL with an estimated binding capacity of 80 mg / mL. The expected large-scale residence time is approximately 24 minutes per cycle. The binding-elution data is shown in Figure 9B.

[0214] The binding-elution data from both columns are shown overlaid in FIG. 9C.

[0215] Purification could also be achieved using resins, but the flow rates were significantly slower, making the overall process less chromatographically productive compared to separations achieved using membranes.

[0216] A comparison of purity by HPLC showed that the membrane purification gave a slightly purer eluate than the resin purification. Figure 9D shows HPLC chromatograms showing the purity of the eluate pools from the resin and membrane purifications. Furthermore, it was observed that rotary evaporation of the eluate afforded crystals of the target difluoro-substituted nucleoside derivative with a diprotected alcohol and an unprotected amine from the feed solution containing an excess of organic solvent and protecting agent. Rotary evaporation of the unpurified feed containing an excess of organic solvent and protecting agent did not afford a crystalline product, suggesting that the desired target difluoro-substituted nucleoside derivative with an unprotected amine was separated from the other components.

[0217] Elution can be performed by increasing the organic solvent composition in conjunction with increasing salt concentration. This is particularly advantageous for downstream purification before drying, or for subsequent synthesis steps that are performed under low moisture conditions. The use of organic solvents can accelerate the rotary evaporation step, as a larger proportion of solvent is easier to remove and recover. Furthermore, the use of less salt means less material needs to be removed in subsequent purification steps. Elution has been successfully performed with buffers containing as low as 5% 1M NaCl in 95% ethanol (final concentration 50 mM).

[0218] Example 10 - DBC of MCP The ability of resin-based mercaptopyridine (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 and binding was assessed by elution with 20 mM Tris, pH 7.0.

[0219] Binding and elution cycles were successfully performed and the elution peaks were observed as shown in Figure 10. It is expected that increased productivity due to reduced purification time would result from similar purifications using membrane-based MCPs. Furthermore, it is hypothesized that these and similar conditions can be applied to other HIC columns.

[0220] All references and publications cited herein are expressly incorporated by reference in their entirety into this disclosure, except to the extent that they may directly contradict this disclosure. Although specific embodiments have been illustrated and described herein, it will be understood by those skilled in the art that various alternative and / or equivalent implementations may be substituted for the specific embodiments shown and described without departing from the scope of the present disclosure. It should be understood that this disclosure is not intended to be unduly limited by the exemplary embodiments and examples set forth herein, and that such examples and embodiments are presented by way of illustration only, and that the scope of the present disclosure is intended to be limited only by the claims.

Claims

**Claim 1**: A separation medium comprising a membrane and a plurality of ligands immobilized on the membrane, the plurality of ligands including mercaptobenzoic acid, mercaptosulfonic acid, salts thereof, or combinations thereof. **Claim 2**: The separation medium according to claim 1, wherein the plurality of ligands includes sodium 3-mercapto-1-propanesulfonate. **Claim 3**: The separation medium according to claim 1 or 2, wherein the material of the membrane is selected from polyolefin, polyethersulfone, poly(tetrafluoroethylene), nylon, glass fiber, hydrogel, polyvinyl alcohol, natural polymer, cellulose, cellulose ester, cellulose acetate, regenerated cellulose, cellulose nanofiber, cellulose derivative, agarose, chitosan, polyethylene, polyester, polysulfone, expanded polytetrafluoroethylene (ePTFE), polyvinylidene fluoride, polyamide (nylon), polyacrylonitrile, polycarbonate, and combinations thereof. **Claim 4**: A membrane chromatography device comprising a housing and the separation medium according to claim 1 or 2 disposed within the housing. **Claim 5**: A method for purifying a target molecule, comprising passing a solution containing the target molecule through the membrane chromatography device according to claim 4, wherein the target molecule includes nucleic acid, nucleotide, nucleoside, nucleobase, or analogs or derivatives thereof.

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