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  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
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  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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  • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2330/30—Production chemically synthesised

Definitions

• compositions comprising oligonucleotides, specifically Smad7 antisense oligonucleotides, and methods for preparing the same.
• Oligonucleotides may be used in a variety of biological and biochemical applications.
• oligonucleotides may be used in drug discovery and development, in diagnostic tests, as research agents, such as primers or probes in Polymerase Chain Reaction (PCR), as antisense agents in target validation, as competitive inhibitors of transcription factors, as ribozymes, as aptamers, as stimulators of the immune system, or as therapeutic biological agents to treat disease.
• PCR Polymerase Chain Reaction
• Mongersen (formerly GED-0301) is a 21-base, single-strand phosphorothioate oligonucleotide that hybridizes to the human SMAD7 messenger RNA (mRNA).
• mRNA messenger RNA
• participants with Crohn's disease who received mongersen had significantly higher rates of remission and clinical response than those who received placebo.
• Mongersen is a 21-base oligonucleotide with the sequence 5'-GTC GCC CCT TCT CCC CGC AGC-3' .
• the phosphorothioate chemistry consists of replacement of a nonbonding oxygen with a sulfur atom in each of the internucleotide linkages.
• the cytosine residues at nucleotide positions 3 and 16 are modified by 5-methylation.
• oligonucleotide comprising at least 700 mmol of the oligonucleotide and at most 25 wt.% water; wherein the oligonucleotide has a nucleic acid sequence:
• a single batch composition of an oligonucleotide comprising at least 2 g/mmol of an at least 700 mmol synthesis scale of the oligonucleotide and at most 25 wt.% water; wherein the oligonucleotide has a nucleic acid sequence:
• SEQ ID NO: 3 (5'-GTX GCC CCT TCT CCC XGC AGC-3'), wherein X represents 5-methyl-2'-deoxycytidine.
• SEQ ID NO: 3 (5'-GTX GCC CCT TCT CCC XGC AGC-3'), wherein X represents 5-methyl-2'-deoxycytidine.
• a single batch composition of an oligonucleotide comprising at least 50 mol% of the oligonucleotide output from at least one 700 mmol or greater oligonucleotide synthesis column and at most 25 wt.% water; wherein the oligonucleotide has a nucleic acid sequence:
• SEQ ID NO: 3 (5'-GTX GCC CCT TCT CCC XGC AGC-3'), wherein X represents 5-methyl-2'-deoxycytidine.
• X represents 5-methyl-2'-deoxycytidine.
• SEQ ID NO: 3 (5'-GTX GCC CCT TCT CCC XGC AGC-3'), wherein X represents 5-methyl-2'-deoxycytidine.
• an oligonucleotide composition comprising an oligonucleotide having at most 25 wt.% water and a nucleic acid sequence:
• X represents 5-methyl-2'-deoxycytidine
• the oligonucleotide is prepared according to a method comprising:
• phosphoramidite comprises a protected hydroxyl group and a protected
• the substantially pure oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, disclosed herein at least one of the intemucleotide linkages of the oligonucleotide is an ⁇ , ⁇ -linked phosphorothioate.
• the substantially pure oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, disclosed herein all of the intemucleotide linkages of the oligonucleotide are ⁇ , ⁇ -linked phosphorothioates.
• the substantially pure oligonucleotide composition or of the method of preparing, disclosed herein, the purifying step 1) comprises:
• the substantially pure oligonucleotide composition or of the method of preparing, disclosed herein, the concentrated fully deprotected oligonucleotide resulting from thin film evaporation is further concentrated via a freeze drying process.
• a method of preparing a series of pharmaceutical compositions comprising partitioning a single batch composition or a substantially pure oligonucleotide composition as disclosed herein, or an oligonucleotide composition prepared according to the methods of preparing as disclosed herein, into a series of portions, amounts, or doses, suitable for oral dosage, and combining each portion, amount, or dose, suitable for oral dosage, of the series of portions amounts, or doses, suitable for oral dosage, with a
• a pharmaceutical composition batch comprising at least a portion of a single batch composition or a substantially pure oligonucleotide composition as disclosed herein, or an oligonucleotide composition prepared according to the methods of preparing as disclosed herein, and a pharmaceutically acceptable adjuvant and/or excipient.
• the pharmaceutical composition batch comprises at least one oral dosage form, such as tablet or a coated tablet.
• compositions as disclosed herein, the single batch composition, the
• composition batch the method of preparing the single batch composition, the method of preparing the pharmaceutical composition, or the method of preparing the series of pharmaceutical compositions, complies with the terms batch or lot as defined under 21 CFR 210.3(2) and 21 CFR 210.3(10), respectively.
• the inflammatory bowel disease (IBD) is Crohn's disease (CD).
• the inflammatory bowel disease (IBD) is ulcerative colitis (UC).
• an anti-SMAD7 ODN can reduce expression of an mRNA transcribed from the gene. In certain embodiments, an anti-SMAD7 ODN can reduce expression of a protein encoded by the gene. In certain embodiments, an anti- SMAD7 ODN can reduce secretion of a protein encoded by the gene from the cell into which the anti-SMAD7 ODN was introduced.
• 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleosides of an ODN are linked by phosphorothioate linkages.
• the oligo (or sugar) moiety of the ODN is a naturally occurring sugar, such as furanose ring system (e.g., ribose or 2'-deoxyribose ring systems).
• the ODN is a deoxyribonucleotide (i.e., the oligo (or sugar) moiety of the ODN is a deoxyribose).
• the ODN is a ribonucleotide (i.e., the oligo (or sugar) moiety of the ODN is a ribose).
• the oligo (or sugar) moiety of the nucleoside(s) utilized to prepare the desired ODN is deoxyribose.
• the oligo (or sugar) moiety of the nucleoside(s) utilized to prepare the desired ODN is ribose.
• one or more bases of an ODN are chemically modified, for example, methylated (e.g., 5-methyl-cytosine, 6-O-methyl-guanine, or 7-methyl- guanine). Additional exemplary chemical modifications of an ODN are described, e.g., in Section 5.2.
• composition are understood to refer to an oligonucleotide composition derived from at least one oligonucleotide synthesis column having a solid support (e.g., a solid support with a linker attached thereto) with a loading capacity of a specified quantity, or at least one synthesis run on the oligonucleotide synthesis column having the solid support with a loading capacity of a specified quantity.
• a solid support e.g., a solid support with a linker attached thereto
• the single batch composition refers to an oligonucleotide composition derived from a plurality of oligonucleotide synthesis column(s) having a solid support (e.g., a solid support with a linker attached thereto) with a loading capacity of a specified quantity, or a plurality of synthesis run(s) on the oligonucleotide synthesis column having the solid support with a loading capacity of a specified quantity.
• a solid support e.g., a solid support with a linker attached thereto
• synthesis run(s) on the oligonucleotide synthesis column having the solid support with a loading capacity of a specified quantity e.g., a solid support with a linker attached thereto
• the single batch composition refers to an oligonucleotide composition derived from at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or at most 60 oligonucleotide synthesis columns having a solid support with a loading capacity of a specified quantity or synthesis runs on an oligonucleotide synthesis column(s) (e.g., run on parallel columns or subsequent to each other) with a loading capacity of a specified quantity, for example, no more than between 1-60, 1-55, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 3-10, 3-8, 3-5, 4-6, 5-10, 10-60, 10- 50, 10-40, 10-30, 10-25, 10-20, 10-15, 20-50, 30-60, or 40-55 oligonucleo
• the single batch composition refers to an oligonucleotide composition derived from at least one or a plurality of oligonucleotide synthesis column(s) having a solid support (e.g., a solid support with a linker attached thereto) with a loading capacity of a specified quantity, or at least one or a plurality of synthesis run(s) on the oligonucleotide synthesis column having the solid support with a loading capacity of a specified quantity; wherein the oligonucleotide synthesis output from the at least one, or the plurality of, oligonucleotide synthesis column(s) (or synthesis run(s)) having the solid support with a loading capacity of the specified quantity, may: (1) provide the single batch composition (or single batch oligonucleotide composition) as a liquid oligonucleotide composition or a solid oligonucleotide composition
• an oligonucleotide chemical transformation process e.g., an oligonucleotide deprotection or cleavage from solid support
• an oligonucleotide purification process e.g., ion-exchange chromatography
• an oligonucleotide desalting process e.g., ultrafiltration and/or diafiltration
• a liquid composition concentration process e.g., thin film evaporation
• a drying process e.g., freeze drying
• composition or a solid oligonucleotide composition (b) be pooled or combined (such that the resulting pooled/combined oligonucleotide composition has uniform character and quality, e.g., the physical and chemical properties are distributed throughout said resulting pooled/combined oligonucleotide composition): (i) to provide the single batch composition (or single batch oligonucleotide composition) as a liquid oligonucleotide composition or a solid oligonucleotide composition; (ii) before being processed in a single iteration or in a plurality of parallel iterations (not necessarily conducted simultaneously) by a second, next, or downstream (see, e.g., Figure 1) at least one further process(s) (or combinations thereof); or (iii) followed by partitioning the resulting pooled/combined oligonucleotide composition into smaller portions before being processed in a plurality of parallel iterations (not necessarily conducted simultaneously) by a
• the amount of oligonucleotide synthesized by the at least one, or the plurality of, oligonucleotide synthesis column(s) (or synthesis run(s)) having the solid support with a loading capacity of the specified quantity, and/or contained in the resulting oligonucleotide synthesis output from said at least one, or said plurality of, oligonucleotide synthesis column(s) (or synthesis run(s)), may be a specified quantity of the oligonucleotide.
• one or more of the further processes, or each of the further processes may be performed with an oligonucleotide composition comprising a specified quantity of the oligonucleotide.
• the plurality of parallel iterations may be no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or at most 60 parallel iterations, for example, no more than between 2- 60, 2-55, 2-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3- 10, 3-8, 3-5, 4-6, 5-10, 10-60, 10-50, 10-40, 10-30, 10-25, 10-20, 10-15, 20-50, 30-60, or 40-55 parallel iterations.
• the oligonucleotide synthesis output or the output resulting from the further process may comprise a specified quantity of the oligonucleotide.
• the at least one output, the pooled or combined output, or the partitioned output, that results from the oligonucleotide synthesis process or one or more of the further processes may be (or comprise) a specified quantity of the oligonucleotide.
• the further process(s) may be conducted downstream from the synthesis process in the following sequence: an oligonucleotide chemical transformation process (e.g., an oligonucleotide deprotection or cleavage from solid support), an oligonucleotide purification process (e.g., ion-exchange chromatography), and an oligonucleotide desalting process (e.g., ultrafiltration and/or diafiltration); wherein the sequence may be followed by a liquid oligonucleotide chemical transformation process (e.g., an oligonucleotide deprotection or cleavage from solid support), an oligonucleotide purification process (e.g., ion-exchange chromatography), and an oligonucleotide desalting process (e.g., ultrafiltration and/or diafiltration); wherein the sequence may be followed by a liquid oligonucleotide chemical transformation process (e.g., an
• the single batch composition may be a liquid oligonucleotide composition (e.g., a purified, liquid oligonucleotide composition, a desalted and purified, liquid oligonucleotide composition, or a concentrated oligonucleotide liquid composition).
• a liquid oligonucleotide composition e.g., a purified, liquid oligonucleotide composition, a desalted and purified, liquid oligonucleotide composition, or a concentrated oligonucleotide liquid composition.
• the resulting crude oligonucleotide which still has a 5'-hydroxyl protecting group on the terminal oligonucleoside, is loaded onto a preparative anion exchange chromatographic column and the terminal 5'-hydroxyl protecting group is deprotected with a protic acid.
• the deprotected oligonucleotide is purified by anion exchange chromatography, desalted via
• the optional step of capping the unreacted deprotected hydroxyl groups and the optional step of deprotecting the protected hydroxyl group of the nucleoside are performed in each iteration of the reaction cycle, exclusive of the last iteration (i.e., not performed in the last iteration), according to the method of preparing an oligonucleotide described herein.
• the method of preparing an oligonucleotide includes the step of independently capping the unreacted deprotected hydroxyl groups and/or the step of
• step g) optionally, independently deprotecting the protected hydroxyl group of the nucleoside; h) repeating the providing, coupling, thiolating, capping, and deprotecting steps (steps c) through g)) a predetermined number of times to provide a solid support-bound oligonucleotide;
• deprotecting steps (steps c) through g)) a predetermined number of times to provide a solid support-bound oligonucleotide; i) deprotecting the protected phosphorothioate linkages and optional protected phosphate linkages;
• phosphoramidite comprises a protected hydroxyl group and a protected phosphoramidite; d) independently coupling the nucleoside phosphoramidite to the deprotected hydroxyl group of the linker, or to the deprotected hydroxyl group of the nucleoside from the previous iteration of the reaction cycle, thereby creating a phosphite triester linked nucleoside;
• the optional step of capping the unreacted deprotected hydroxyl groups and the optional step of deprotecting the protected hydroxyl group of the nucleoside are performed in each iteration of the reaction cycle, exclusive of the last iteration (i.e., not performed in the last iteration), according to the method of preparing an oligonucleotide described herein.
• the method of preparing an oligonucleotide may comprise or consist of the following steps: a) providing a linker attached to a solid support wherein the linker comprises a protected hydroxyl group; b) deprotecting the protected hydroxyl group of the linker thereby creating a deprotected hydroxyl group; c) independently coupling a nucleoside phosphoramidite to the deprotected hydroxyl group of the linker, or to the deprotected hydroxyl group of the nucleoside from the previous iteration of the reaction cycle, thereby creating a phosphite triester linked nucleoside; d) independently thiolating the protected phosphite triester linkage thereby creating a protected phosphorothioate linkage (or optionally, independently oxidizing the protected phosphite triester linkage thereby creating a protected phosphate linkage); e) optionally, independently capping unreacted deprotecte
• the purifying step k) of the method of preparing an oligonucleotide may comprise 1) loading the oligonucleotide eluate from eluting step j) onto the ion exchange chromatography column; 2) deprotecting the protected hydroxyl group from the terminal nucleoside; 3) eluting the oligonucleotide from the ion exchange chromatography column using a salt gradient; and 4) desalting the oligonucleotide eluate from the ion exchange column via ultrafiltration and/or diafiltration.
• the method of preparing an oligonucleotide includes the step of independently capping the unreacted deprotected hydroxyl groups and/or the step of independently deprotecting the protected hydroxyl group of the nucleoside.
• the optional step of capping the unreacted deprotected hydroxyl groups and the optional step of deprotecting the protected hydroxyl group of the nucleoside are performed in each iteration of the reaction cycle, exclusive of the last iteration (i.e., not performed in the last iteration), according to the method of preparing an oligonucleotide described herein.
• the method of preparing an oligonucleotide may comprise or consist of the following steps: a) deprotecting a protected hydroxyl group of a linker attached to a solid support thereby creating a deprotected hydroxyl group; b) independently coupling a nucleoside phosphoramidite to the deprotected hydroxyl group of the linker, or to the deprotected hydroxyl group of the nucleoside from the previous iteration of the reaction cycle, thereby creating a phosphite triester linked nucleoside; c) independently thiolating the protected phosphite triester linkage thereby creating a protected phosphorothioate linkage (or optionally, independently oxidizing the protected phosphite triester linkage thereby creating a protected phosphate linkage); d) optionally, independently capping unreacted deprotected hydroxyl groups; e) optionally, independently deprotecting the protected hydroxyl groups
• the purifying step j) of the method of preparing an oligonucleotide may comprise 1) loading the oligonucleotide eluate from eluting step i) onto the ion exchange chromatography column; 2) deprotecting the protected hydroxyl group from the terminal nucleoside; 3) eluting the oligonucleotide from the ion exchange chromatography column using a salt gradient; and 4) desalting the oligonucleotide eluate from the ion exchange column via ultrafiltration and/or diafiltration.
• antisense oligonucleotide sequences can hybridize to a complementary region in an mRNA molecule thereby producing a double-strand hybrid that can lead to the activation of ubiquitous catalytic enzymes, such as RNaseH, which degrades DNA/RNA hybrid strands, thus preventing protein translation.
• an antisense oligonucleotide provided herein can hybridize to its target sequence as RNA or DNA. Thus, even if a DNA sequence is provided as target, the corresponding RNA sequence (including uracil instead of thymine) is included.
• the oligonucleotide such as an anti-SMAD7 oligonucleotide or a chemically modified anti-SMAD7 oligonucleotide, may comprise a phosphorothioate backbone, which can be fully or partially protonated to form an acidic form.
• the oligonucleotide such as an anti-SMAD7 oligonucleotide or a chemically modified anti-SMAD7 oligonucleotide, may comprise a non-naturally-occurring sequence tag.
• the oligonucleotide can include nucleobases that can increase the binding affinity of the chemically modified SMAD7 ODN.
• nucleobases can include, e.g., 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosine 5-methylcytosine substitutions.
• the oligonucleotide can include one or more of the above-mentioned modified nucleobases in combination with 2'-0-methoxyethyl sugar modifications.
• the oligonucleotide such as an anti-SMAD7 oligonucleotide or a chemically modified anti-SMAD7 oligonucleotide, that can be synthesized using the methods as described herein, comprises a nucleotide sequence complementary to a region comprising nucleotide 403, 233, 294, 295, 296, 298, 299 or 533 of the human SMAD7 nucleotide sequence of SEQ ID NO: 1, or the corresponding RNA sequence.
• nucleotides X or Y comprises a methylated nitrogenous base; or the complementary sequence thereto.
• X represents 5-methyl-2'-deoxycytidine ("5-Me-dC"); and wherein at least one internucleoside linkage, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of the internucleoside linkage are ⁇ , ⁇ -linked phosphorothioate linkages.
• the oligonucleotide is only partially neutralized, e.g., less than all phosphorothioate linkages are associated with an alkali metal ion or an alkaline earth metal ion (e.g., less than 99%, less than 95%, less than 90%, less than 85%, less than 80%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 3%, or less than 1% are neutralized).
• an alkali metal ion or an alkaline earth metal ion e.g., less than 99%, less than 95%, less than 90%, less than 85%, less than 80%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than
• the loading capacity (or synthesis scale) of a solid support refers to the molar amount of the solid support, such as the solid support having a linker attached thereto, that is available to react to prepare an oligonucleotide, e.g., an equivalent molar amount of the oligonucleotide.
• an oligonucleotide e.g., an equivalent molar amount of the oligonucleotide.
• the amount of the oligonucleotide prepared that is equivalent to the loading capacity of the solid support having a linker attached thereto is considered the theoretical amount.
• the actual amount of the oligonucleotide prepared according to the methods disclosed herein utilizing the solid support having a linker attached thereto may be identical to the theoretical amount, or may be less than the theoretical amount.
• the column housing the solid support having a linker attached thereto that is utilized in the methods disclosed herein has an inner diameter in the range of between 50-100 cm, for example, an inner diameter of 50 cm, 60 cm, 70 cm, 80 cm, 90 cm, or 100 cm, and a column volume in the range of between 20-35 L, for example, a column volume in the range of between 20-25 L, between 23.5-30 L, between 25-30 L, between 30-35 L, or between 20-30 L, such as a column volume of 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 L.
• the support-bound deprotected material is washed with solvent, such as acetonitrile, in preparation for the next step of the method, such as the next reaction on the solid support, for example the support may be washed with between 1-7 column volumes, 1-5 column volumes, 1- 4 column volumes, 1-3 column volumes, or 2-4 column volumes, in preparation for the next step of the method.
• solvent such as acetonitrile
• the coupling comprises providing the nucleoside
• the hydroxyl protecting group can include benzyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, benzoyl, mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl) and 9-(p- methoxyphenyl)xanthine-9-yl (MOX).
• the hydroxyl protecting group can include acetyl, i-butyryl, and dimethoxytrityl (DMT).
• the hydroxyl protecting group is i-butyryl.
• the nucleoside phosphoramidite provided in the coupling step may be selected from the group consisting of:
• the amount of the nucleoside phosphoramidite provided during the coupling step of the method is in the range of 0.1-1 equivalents, relative to the equivalents of activator provided, for example, in the range of 0.1-0.9 equivalents, 0.1-0.8 equivalents, 0.1-0.7 equivalents, 0.1-0.6 equivalents, 0.1-0.5 equivalents, 0.1-0.4 equivalents, 0.1-0.3 equivalents, 0.1-0.2 equivalents, 0.25-0.9 equivalents, 0.25-0.75 equivalents, 0.25-0.5 equivalents, 0.3-0.8 equivalents, 0.3-0.75 equivalents, or 0.5-1 equivalents, relative to the equivalents of activator provided.
• the coupling comprises providing the activator and the nucleoside phosphoramidite in a molar ratio of 1 -5 : 1 , for example, in a molar ratio of 2-5 : 1 , such as 3-5: 1, 4: 1, or 3.5: 1.
• the protected phosphite triester linkage such as the protected phosphite triester linkage from the previous iteration of the reaction cycle, is thiolated according to the method of preparing an oligonucleotide disclosed herein, thereby creating a protected phosphorothioate linkage.
• the protected phosphite triester linkage such as the 2-cyanoethoxy-protected phosphite triester linked nucleoside, is thiolated with a thiolating agent, thereby forming a protected phosphorothioate linkage, such as a 2-cyanoethoxy-protected phosphorothioate linkage.
• 2- cyanoethoxy-protected phosphite triester linked nucleoside is oxidized with an oxidizing agent, thereby forming a protected phosphate linkage, such as a 2-cyanoethoxy-protected phosphate linkage.
• the oxidizing agent is provided in an amount in the range of 1-8 equivalents, relative to the equivalents of the solid support, for example, provided in an amount in the range of 2-8 equivalents, such as in the range of 3-8 equivalents, 4-8 equivalents, 5-8 equivalents, 5-7 equivalents, 5-6 equivalents, 1-5 equivalents, or 3-6
• one or more of the cycles of the predetermined number of times performs the step of capping the unreacted deprotected hydroxyl group(s) and deprotecting of the protected 5 '-hydroxyl group of the 5 '-terminal nucleoside of the
• the reaction cycle of performing the steps of providing/coupling of a nucleoside phosphoramidite, thiolating (or oxidizing) of the formed phosphite triester linkage, optionally capping of unreacted deprotected hydroxyl group(s), and optionally deprotecting of the protected 5 '-hydroxyl group of the 5 '-terminal nucleoside of the oligonucleotide are repeated 9-99 times to provide the solid support-bound oligonucleotide, wherein the optional step of capping the unreacted deprotected hydroxyl group(s) and the optional step of deprotecting the protected 5 '-hydroxyl group of the 5 '-terminal nucleoside of the oligonucleotide are excluded during the last reaction cycle iteration, for example, are repeated 9- 89 times, such as 9-79 times, 9-69 times, 9-59 times, 9-49 times, 9-39 times, 9-29 times, 9-24 times,
• the reaction cycle of performing the steps of providing/coupling of a nucleoside phosphoramidite, thiolating (or oxidizing) of the formed phosphite triester linkage, optionally capping of unreacted deprotected hydroxyl group(s), and optionally deprotecting of the protected 5 '-hydroxyl group of the 5'- terminal nucleoside of the oligonucleotide are repeated 14 times to provide the solid support- bound oligonucleotide, for example, are repeated 15 times, such as 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 times, wherein the optional step of capping the unreacted deprotected hydroxyl group(s) and the optional step of deprotecting the protected 5 '-hydroxyl group of the 5 '-terminal nucleo
• the protected phosphorothioate linkages are deprotected with an amine, e.g., triethylamine, for example, 10-50% triethylamine in an aprotic solvent (v/v), for example, with 10-30% triethylamine in an aprotic solvent (v/v), such as with 20% triethylamine in acetonitrile (v/v).
• an amine e.g., triethylamine, for example, 10-50% triethylamine in an aprotic solvent (v/v)
• v/v aprotic solvent
• the step of deprotecting the protected amine e.g., triethylamine, for example, 10-50% triethylamine in an aprotic solvent (v/v), for example, with 10-30% triethylamine in an aprotic solvent (v/v), such as with 20% triethylamine in acetonitrile (v/v).
• the cleaving step of the method disclosed herein comprises providing a 28-30% ammonia aqueous solution (w/w), for example, a heated 28-30% ammonia aqueous solution (w/w), such as a heated solution having a temperature of 40-70°C or 40-60°C, such as a temperature of 40°C, 50°C, 60°C, or 65 °C, for example, providing the heated 28-30%) ammonia aqueous solution (w/w) and recirculating the heated ammonia aqueous solution through the column housing to cleave said deprotected solid support-bound
• the cleaving of the solid support-bound oligonucleotide provides the cleaved oligonucleotide, such as the cleaved oligonucleotide comprising a 5'- hydroxyl protected group on the terminal nucleoside, in an amount (such as a theoretical amount based on the synthesis scale or loading capacity of the solid support having a linker attached thereto) of 300 mmol or greater, for example, in an amount of 600 mmol or greater, 700 mmol or greater, 800 mmol or greater, 900 mmol or greater, 1,000 mmol or greater, 1,100 mmol or greater, 1,200 mmol or greater, 1,300 mmol or greater, 1,400 mmol or greater, 1,500 mmol or greater, 1,600 mmol or greater, 1,700 mmol or greater, 1,800 mmol or greater, 1,900 mmol or greater, 2,000 mmol or greater, 2,100 mmol or greater, 2,200
• the cleaved oligonucleotide is eluted from the solid support according to the method of preparing an oligonucleotide disclosed herein.
• oligonucleotide comprising a 5'-hydroxyl protected group on the terminal nucleoside from the solid support comprises washing the support with water, for example with an aqueous solution or a buffered aqueous solution.
• oligonucleotide such as the cleaved oligonucleotide loaded onto the ion exchange column, can include trityl or dimethoxytrityl (DMT).
• DMT dimethoxytrityl
• the 5'-hydroxyl protecting group of the cleaved oligonucleotide or the loaded, cleaved oligonucleotide is dimethoxytrityl (DMT).
• the amount of the cleaved oligonucleotide contained within the cleaved oligonucleotide eluate that is loaded onto the ion exchange column may be in an amount of 300 mmol (e.g., in terms of purification scale), such as 400 mmol, 500 mmol, 600 mmol, 700 mmol, 800 mmol, 900 mmol, 1,000 mmol, 1, 100 mmol, 1,200 mmol, 1,300 mmol, 1,400 mmol, 1,500 mmol, 1,600 mmol, 1,700 mmol, 1,800 mmol, 1,900 mmol, 2,000 mmol, 2,100 mmol, 2,200 mmol, 2,300 mmol, 2,400 mmol, 2,500 mmol, 2,600 mmol, 2,700 mmol, 2,800 mmol
• the protecting hydroxyl group on the terminal nucleoside of the cleaved oligonucleotide loaded onto the ion exchange column is deprotected, for example, the 5 '-hydroxyl protected group on the terminal nucleoside of the cleaved oligonucleotide loaded onto the anion exchange column is deprotected and then subsequently washed, according to the method of preparing an oligonucleotide disclosed herein.
• the amount of the fully deprotected oligonucleotide eluted from the ion exchange column may be in the range of between 300-5,400 mmol , for example, in the range of between 300-4,500 mmol, 300-4,000 mmol, 300-3,600 mmol, 300-3,000 mmol, 600-3,000 mmol, 700-3,600 mmol, 700-2,700 mmol, 700-2,500 mmol, 700-2,400 mmol, 700- 2,000 mmol, 700-1,900 mmol, 700-1,800 mmol, 700-1,700 mmol, 700-1,600 mmol, 700-1,500 mmol, 700-900 mmol, 800-1,200 mmol, 800-1,000 mmol, 900-1,600 mmol, 900-1,800 mmol, 900-2,400 mmol, 900-2,700 mmol, 900-3,600 mmol, 1,800-2,700 mmol, 1,800-3,
• the process of desalting the eluate comprising the fully deprotected oligonucleotide via an ultrafiltration and/or diafiltration process for example, desalting prior to concentrating (e.g., such as concentrating by thin film evaporation), utilizes a regenerated cellulose membrane to accomplish the desalting process, such as a regenerated cellulose membrane having a 1,000-3,000 Da molecular weight cutoff.
• the amount of the fully deprotected oligonucleotide in the eluate from the ion exchange column that is desalted via an ultrafiltration and/or diafiltration process, for example, that is desalted prior to concentrating may be in an amount of 300 mmol, 400 mmol, 500 mmol, 600 mmol, 700 mmol, 800 mmol, 900 mmol, 1,000 mmol, 1, 100 mmol, 1,200 mmol, 1,300 mmol, 1,400 mmol, 1,500 mmol, 1,600 mmol, 1,700 mmol, 1,800 mmol, 1,900 mmol, 2,000 mmol, 2, 100 mmol, 2,200 mmol, 2,300 mmol, 2,400 mmol, 2,500 mmol, 2,600 mmol, 2,700 mmol, 2,800 mmol, 2,900 mmol, 3,000 mmol, 3,400 mmol, 3,
• the ultrafiltration and/or diafiltration process utilized in the methods disclosed herein has a loading capacity sufficient to provide the desalted fully deprotected oligonucleotide in an amount of 300 mmol, 400 mmol, 500 mmol, 600 mmol, 700 mmol, 800 mmol, 900 mmol, 1,000 mmol, 1,100 mmol, 1,200 mmol, 1,300 mmol, 1,400 mmol, 1,500 mmol, 1,600 mmol, 1,700 mmol, 1,800 mmol, 1,900 mmol, 2,000 mmol, 2,100 mmol, 2,200 mmol, 2,300 mmol, 2,400 mmol, 2,500 mmol, 2,600 mmol, 2,700 mmol, 2,800 mmol, 2,900 mmol, 3,000 mmol, 3, 100 mmol, 3,200 mmol, 3,300 mmol, 3,400 mmol, 3,500 mmol, 3,600 mmol, 4,000 mmol, 4,
• the concentrating of the fully deprotected oligonucleotide eluate, or concentrating the desalted fully deprotected oligonucleotide retentate solution from an ultrafiltration and/or diafiltration process is by thin film evaporation utilizing a thin film evaporation jacket temperature in the range of 60- 90°C and at a pressure in the range of 20-80 Torr.
• ultrafiltration and/or diafiltration process that is concentrated, such as concentrated by thin film evaporation, may be of 300 mmol or greater, for example, in an amount of 600 mmol or greater, 700 mmol or greater, 800 mmol or greater, 900 mmol or greater, 1,000 mmol or greater, 1, 100 mmol or greater, 1,200 mmol or greater, 1,300 mmol or greater, 1,400 mmol or greater, 1,500 mmol or greater, 1,600 mmol or greater, 1,700 mmol or greater, 1,800 mmol or greater, 1,900 mmol or greater, 2,000 mmol or greater, 2,100 mmol or greater, 2,200 mmol or greater, 2,300 mmol or greater, 2,400 mmol or greater, 2,500 mmol or greater, 2,600 mmol or greater, 2,700 mmol or greater, 2,800 mmol or greater, 2,900 mmol or greater, 3,000 mmol or greater, 3,200 mmol or greater, 3,400 mmol or greater, 3,
• the concentrated oligonucleotide is further subjected to a freeze drying process, wherein the freeze drying process utilizes a vacuum in the range of between 1- 500 millitorr, such as in the range of between 1-450 millitorr, 1-400 millitorr, 1-350 millitorr, 1- 300 millitorr, 1-250 millitorr, 1-200 millitorr, 1-150 millitorr, 1-100 millitorr, 1-50 millitorr, 100- 500 millitorr, 100-400 millitorr, 50-300 millitorr, or 25-250 millitorr, and at a temperature in the range of between -50 to 35°C, such as in the range of -50 to -30°C, -45 to 30°C, -45 to 20°C, -45 to 15°C, -45 to 10°C, -45 to 5°C, -45
• the amount of the concentrated oligonucleotide resulting from the freeze drying process may be 300 mmol, 400 mmol, 500 mmol, 600 mmol, 700 mmol, 800 mmol, 900 mmol, 1,000 mmol, 1, 100 mmol, 1,200 mmol, 1,300 mmol, 1,400 mmol, 1,500 mmol, 1,600 mmol, 1,700 mmol, 1,800 mmol, 1,900 mmol, 2,000 mmol, 2, 100 mmol, 2,200 mmol, 2,300 mmol, 2,400 mmol, 2,500 mmol, 2,600 mmol, 2,700 mmol, 2,800 mmol, 2,900 mmol, 3,000 mmol, 3,400 mmol, 3,600 mmol, 4,000 mmol, 4,200 mmol, 4,500 mmol, 5,000 mmol, or 5,400 mmol.
• the composition may comprise or consist of at least 400 mmol of the oligonucleotide, such as at least 500 mmol, at least 600 mmol, at least 700 mmol, at least 800 at least 900 mmol, at least 1,000 mmol, at least 1, 100 mmol, at least 1,200 mmol, at least 1,300 mmol, at least 1,400 mmol, at least 1,500 mmol, at least 1,600 mmol, at least 1,700 mmol, at least 1,800 mmol, at least 1,900 mmol, at least 2,000 mmol, at least 2, 100 mmol, at least 2,200 mmol, at least 2,300 mmol, at least 2,400 mmol, at least 2,500 mmol, at least 2,600 mmol, at least 2,700 mmol, at least 2,800 mmol, at least 2,900 mmol, at least 3,000 mmol, at least 3,600 mmol, or at least 4,500 mmol, of the oligon
• an oligonucleotide composition such as a single batch composition, a substantially pure oligonucleotide composition, or an oligonucleotide composition prepared according to the method disclosed herein, may comprise or consist of at least 300 mmol, such as at least 700 mmol, of an oligonucleotide (e.g., an anti-SMAD7 oligonucleotide or a chemically modified anti-SMAD7 oligonucleotide, synthesized using the methods as described herein; e.g., an oligonucleotide comprising a portion of at least 10 nucleotides of, consists of, or includes up to 21 nucleotides in length of, having a sequence of any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) and at most 25 wt.% water, wherein the oligonucleotide has a molecular weight of at least 3,000 Da,
• the method disclosed herein provides an oligonucleotide composition, such as a single batch composition, a substantially pure oligonucleotide
• the yield of the composition prepared according to the method disclosed herein may be at least 2.0 g/mmol, such as at least 2.25, 2.5, 2.75, 3.0, 3.25, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or at least 4.0 g/mmol, of an at least 700 mmol synthesis scale, or of two or more pooled at least 700 mmol synthesis scale preparations, such as 3, 4, 5, 6, 7, 8, 9, or 10 or more pooled at least 700 mmol synthesis scale preparations, of the oligonucleotide, wherein the yield may be determined by optical density/mL (OD/mL) at a wavelength at 260 nm or may be determined by dry weight of the oligonucleotide composition corrected for moisture content relative to the synthesis scale to prepare the oligonucleotide, for example, in the range of between 2.0-25 g/mmol synthesis scale of the oligonucleotide, such as in the range of between 2-25
• oligonucleotide synthesis scale preparations of at least 800 mmol, at least 900 mmol, at least 1,000 mmol, at least 1, 100 mmol, at least 1,200 mmol, at least 1,300 mmol, at least 1,400 mmol, at least 1,600 mmol, at least 1,800 mmol, at least 2,400 mmol, at least 2,700 mmol, at least 3,000 mmol, at least 3,600 mmol, or at least 4,500 mmol, or oligonucleotide synthesis scale preparations in the range of between 300-5,400 mmol, such as in the range of between 300-4,500 mmol, 300-4,000 mmol, 300-3,600 mmol, 300-3,000 mmol, 600-3,000 mmol, 700-3,600 mmol, 700-3,000 mmol, 700-2,700 mmol, 700-2,500 mmol, 700- 2,400 mmol, 700-2,000 mmol, 700-1,500
• an oligonucleotide composition such as a single batch composition, a substantially pure oligonucleotide composition, or an oligonucleotide composition prepared according to the method disclosed herein, may comprise or consist of at least 50 mol% of an oligonucleotide output from at least one 300 mmol or greater
• the composition may comprise or consist of at least 50 mol% of the oligonucleotide output (as determined by optical density/mL (OD/mL) or determined by dry weight of the oligonucleotide composition corrected for moisture content relative to the synthesis scale to prepare the oligonucleotide), for example at least 55 mol%, at least 60 mol%, at least 65 mol%, at least 70 mol%, at least 75 mol%, at least 80 mol%, at least 85 mol%, at least 90 mol%, at least 95 mol%, at least 97 mol%, at least 98 mol%, or at least 99 mol%, of the oligonucleotide output, such as in the range of between 50-99 mol%, 60-99 mol%, 70-99 mol%, 80-99 mol%, 90-99 mol%, 95-99 mol%, 75-95 mol%, 80-97 mol%, 85-95 mol%, or
• the composition may comprise or consist of at least 50 mol% of an oligonucleotide output from at least one oligonucleotide synthesis column of 300 mmol or greater (as determined by optical density/mL (OD/mL) or determined by dry weight of the oligonucleotide composition corrected for moisture content relative to the synthesis scale to prepare the oligonucleotide), such as at least one 700 mmol or greater oligonucleotide synthesis column, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in the range of between 1-10, 1-8, 2- 10, 3-9, 4-7, 4-6, 6-10, or 8-10, oligonucleotide synthesis columns of 300 mmol or greater, such as at least one 700 mmol or greater oligonucleotide synthesis column, and at most 25 wt.% water, such as and at most 25 wt.% water, such as at most 24 wt.%, at most 23 w
• the degree of purity of an oligonucleotide composition such as a single batch composition, a substantially pure oligonucleotide composition, or an
• composition prepared according to the method disclosed herein may be derived from the oligonucleotide synthesis output from the at least one, or the plurality of, oligonucleotide synthesis column(s) (or synthesis run(s)) having the solid support with a loading capacity of the specified quantity, wherein said oligonucleotide synthesis output may: (1) provide the single batch composition (or single batch oligonucleotide composition) as a liquid oligonucleotide composition or a solid oligonucleotide composition; or (2) be pooled or combined (such that the resulting pooled/combined oligonucleotide composition has uniform character and quality, e.g., the physical and chemical properties are distributed throughout said resulting pooled/combined oligonucleotide composition) to provide the single batch composition (or single batch oligonucleotide composition) as a liquid oligonucleotide composition or a solid oligonucleotide composition
• composition or a solid oligonucleotide composition (b) be pooled or combined (such that the resulting pooled/combined oligonucleotide composition has uniform character and quality, e.g., the physical and chemical properties are distributed throughout said resulting pooled/combined oligonucleotide composition): (i) to provide the single batch composition (or single batch oligonucleotide composition) as a liquid oligonucleotide composition or a solid oligonucleotide composition; (ii) before being processed in a single iteration or in a plurality of parallel iterations (not necessarily conducted simultaneously) by a second, next, or downstream (see, e.g., Figure 1) at least one further process(s) (or combinations thereof); or (iii) followed by partitioning the resulting pooled/combined oligonucleotide composition into smaller portions before being processed in a plurality of parallel iterations (not necessarily conducted simultaneously) by a
• an oligonucleotide composition such as a single batch composition, a substantially pure oligonucleotide composition, or an oligonucleotide
• composition prepared according to the method disclosed herein may be derived from (or prepared by) subjecting the chemically transformed oligonucleotide output (such as the at least one, the plurality, the partitioned, or the pooled (or combined), chemically transformed oligonucleotide output) to an oligonucleotide purification process (e.g., ion-exchange
• pooled/combined oligonucleotide composition has uniform character and quality, e.g., the physical and chemical properties are distributed throughout said resulting pooled/combined oligonucleotide composition): (a) to provide the single batch composition (or single batch oligonucleotide composition) as a liquid oligonucleotide composition; (b) before being processed in a single iteration or in a plurality of parallel iterations (not necessarily conducted
• the oligonucleotide purification process may be conducted in a single iteration, such as utilizing a single ion-exchange column having an ion exchange capacity sufficient to purify a specified quantity, or may be conducted in a plurality of parallel iterations, such as utilizing a plurality of ion- exchange columns or conducting a plurality of runs on one or more parallel columns or subsequent to each other (not necessarily conducted simultaneously) that have an ion exchange capacity sufficient to purify a specified quantity, for example, may be conducted utilizing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 ion-exchange column(s) having an ion exchange capacity sufficient to purify a specified quantity or purification runs on an ion- exchange column(s) (e.g., run on parallel columns or subsequent to each other) with an ion exchange capacity sufficient to purify a specified quantity, for example,
• oligonucleotide composition (b) before being processed in a single iteration or in a plurality of parallel iterations (not necessarily conducted simultaneously) by at least one further process(s), comprising: a liquid composition concentration process, or a drying process, or combinations thereof; or (c) followed by partitioning the resulting pooled/combined oligonucleotide composition into smaller portions before being processed in a plurality of parallel iterations (not necessarily conducted simultaneously) by the at least one further process(s) (or combinations thereof); (2) further processed in a single iteration or in a plurality of parallel iterations (not necessarily conducted simultaneously) by the at least one further process(s) (or combinations thereof); or (3) partitioned into smaller portions before being processed in a plurality of parallel iterations (not necessarily conducted simultaneously) by the at least one further process(s) (or combinations thereof).
• an oligonucleotide composition such as a single batch composition, a substantially pure oligonucleotide composition, or an oligonucleotide
• oligonucleotide composition has uniform character and quality, e.g., the physical and chemical properties are distributed throughout said resulting pooled/combined oligonucleotide composition): (a) to provide the single batch composition (or single batch oligonucleotide composition) as a liquid oligonucleotide composition; (b) before being processed in a single iteration or in a plurality of parallel iterations (not necessarily conducted simultaneously) by a drying process; or (c) followed by partitioning the resulting pooled/combined oligonucleotide composition into smaller portions before being processed in a plurality of parallel iterations (not necessarily conducted simultaneously) by the drying process; (2) further processed in a single iteration or in a plurality of parallel iterations (not necessarily conducted simultaneously) by the drying process; or (3) partitioned into smaller portions before being processed in a plurality of parallel iterations (not necessarily conducted simultaneously) by the drying process.
• the amount of oligonucleotide contained within an output oligonucleotide composition may be a specified quantity.
• the plurality of parallel iterations may be no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or at most 60 parallel iterations, for example, no more than between 2-60, 2-55, 2-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4
• the further process(s) may be conducted downstream from the synthesis process in the following sequence: an oligonucleotide chemical transformation process (e.g., an oligonucleotide deprotection or cleavage from solid support), an oligonucleotide purification process (e.g., ion-exchange chromatography), and an oligonucleotide desalting process (e.g., ultrafiltration and/or diafiltration); wherein the sequence may be followed by a liquid composition concentration process (e.g., thin film evaporation) and/or a drying process (e.g., freeze drying); or wherein the sequence may be exclusive of a liquid composition concentration process (e.g., thin film evaporation) and/or a drying process (e.g., freeze drying).
• an oligonucleotide chemical transformation process e.g., an oligonucleotide deprotection or cleavage from solid support
• a single batch composition disclosed herein may involve pooling the output of a plurality of iterations (or runs) of a particular process step before proceeding to the next process step (if any), for example, pooling the output of a plurality of oligonucleotide synthesis column(s) (or synthesis run(s)), of a plurality of ion-exchange purification column(s) (or purification run(s)) of the synthesized oligonucleotide, of a plurality of desalting the purified oligonucleotide via ultrafiltration and/or diafiltration process(s), and/or of a plurality of liquid concentrating the desalted oligonucleotide (such as via a plurality of thin film evaporation process(s)), before proceeding to the next process step.
• a single batch composition disclosed herein may involve directly proceeding to process the output of a particular process step in the next process step (if any), exclusive of a pooling or partitioning of the output from the particular process step, wherein the next process step may involve processing only a single output from the particular process step or may involve processing a plurality of outputs from the particular step in a series of parallel iterations (or runs, not necessarily simultaneously), for example, directly proceeding to process the output of an oligonucleotide synthesis column(s) (or synthesis run(s)), of an ion- exchange purification column(s) (or purification run(s)) of the synthesized oligonucleotide, of a desalting the purified oligonucleotide via ultrafiltration and/or diafiltration process(s), and/or of a liquid concentrating the desalted oligonucleotide (such as via a plurality of thin film evaporation process(s)), in the next process
• a single batch composition disclosed herein may be a solid composition derived from directly proceeding to dry a single output or a plurality of outputs of the liquid concentrated, desalted, oligonucleotide (such as via a single or plurality of freeze drying process(s)).
• synthesis step an oligonucleotide synthesis column(s) is utilized having a solid support with a 900 mmol loading capacity, wherein 3, 4, or 5, oligonucleotide synthesis columns may be run in parallel or as a single column ran in series 3, 4, or 5 times, or combinations thereof, to achieve the 3 x 900 mmol, 4 x 900 mmol, and a 5 x 900 mmol scale synthesis; purification step: an ion-exchange chromatography column(s) is utilized having an ion-exchange capacity to purify an oligonucleotide composition containing 900 mmol of the oligonucleotide, wherein the
• an oligonucleotide composition such as a single batch composition, a substantially pure oligonucleotide composition, or an oligonucleotide
• composition prepared according to the method disclosed herein may be derived from the series of processes that includes a single iteration of an oligonucleotide synthesis process, a single iteration of an oligonucleotide purification process, a single iteration of an oligonucleotide desalting process, and a single iteration of an oligonucleotide liquid composition concentration process, and optionally a single iteration of an oligonucleotide drying process, and thereby providing a purified, desalted, and concentrated (and optionally dried) oligonucleotide composition.
• composition prepared according to the method disclosed herein may be derived from the series of processes that includes a plurality of iterations of a single oligonucleotide synthesis process followed a single oligonucleotide purification process, a single iteration of an oligonucleotide desalting process conducted by desalting the combined or pooled output from the plurality of iterations of the single oligonucleotide synthesis process followed the single oligonucleotide purification process, and a single iteration of an oligonucleotide liquid composition concentration process, and optionally a single iteration of an oligonucleotide drying process thereby, and thereby providing a purified, desalted, and concentrated (and optionally dried) oligonucleotide composition.
• an oligonucleotide composition such as a single batch composition, a substantially pure oligonucleotide composition, or an oligonucleotide
• composition prepared according to the method disclosed herein may comprise or consist of an oligonucleotide at a degree of homogeneity of at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%), at least 96%, at least 97%, at least 98%, or at least 99%, such as a degree of homogeneity in the range of between 65-99%, 70-99%, 75-99%, 80-99%, 85-99%, 90-99%, 95- 99%, 65-95%, 70-95%, 75-95%, 80-97%, or 85-95%, wherein the oligonucleotide may be an anti-SMAD7 oligonucleotide, for example, an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or the complementary sequence thereto, such as Mongersen (formerly
• An oligonucleotide synthesized according to the methods provided herein such as an oligonucleotide obtained from a single batch composition, a substantially pure oligonucleotide composition, or an oligonucleotide composition prepared according to the method disclosed herein, see, e.g., as described in Sections 5-5.16, can be formulated into a pharmaceutical composition, see, e.g., as described in Section 5.18, and published Internation Publication WO 2016/105516, herein incorporated by reference in its entirety.
• the oligonucleotide that can be formulated into a pharmaceutical composition may be a SMAD7 antisense oligonucleotide or a chemically modified SMAD7 antisense oligonucleotide, e.g., an
• Contemplated SMAD7 antisense oligonucleotides comprise oligonucleotides that act against SMAD7 and can be administered orally.
• Disclosed therapies can, when administered orally to a subject suffering from inflammatory bowel disease (IBD), deliver an effective amount of a SMAD7 antisense oligonucleotide to the intestinal system of a patient, e.g. deliver an effective amount of a SMAD7 antisense oligonucleotide to the terminal ileum and/or right colon of a patient.
• IBD inflammatory bowel disease
• a tablet for oral administration can comprise granules (e.g., is at least partially formed from granules) that comprise a disclosed SMAD7 antisense oligonucleotide and pharmaceutically acceptable excipients.
• a tablet can be coated with an enteric coating.
• Contemplated tablets can comprise pharmaceutically acceptable excipients such as fillers, binders, disintegrants, and/or lubricants, as well as coloring agents, release agents, coating agents, sweetening, flavoring such as wintergreen, orange, xylitol, sorbitol, fructose, and maltodextrin, and perfuming agents, preservatives and/or antioxidants.
• the SMAD7 antisense oligonucleotide has a nucleic acid sequence of any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, or 12, preferably having a nucleic acid sequence: SEQ ID NO: 3 : (5'-GTX GCC CCT TCT CCC XGC AGC-3 '), wherein X represents 5-methyl-2'-deoxycytidine.
• contemplated pharmaceutical formulations comprise an intra-granular phase that comprises a contemplated SMAD7 antisense oligonucleotide or a pharmaceutically acceptable salt and a pharmaceutically acceptable filler.
• the oligonucleotide is an oligonucleotide synthesized according to the methods provided herein, such as an oligonucleotide obtained from a single batch composition, a substantially pure
• An anti-SMAD7 therapy formulation can comprise an intragranular phase that comprises a filler.
• exemplary fillers comprise, but are not limited to, cellulose, gelatin, calcium phosphate, lactose, sucrose, glucose, mannitol, sorbitol, microcrystalline cellulose, pectin, polyacrylates, dextrose, cellulose acetate, hydroxypropylmethyl cellulose, partially
• pregelatinized starch calcium carbonate, and others including combinations thereof.
• an anti-SMAD7 therapy formulation can comprise an intragranular phase and/or an extragranular phase that comprises a binder, which can generally function to hold the ingredients of the pharmaceutical formulation together.
• binders comprise, for example, the following: starches, sugars, cellulose or modified cellulose such as hydroxypropyl cellulose, lactose, pregelatinized maize starch, polyvinyl pyrrolidone,
• an intragranular phase and/or an extragranular phase can comprise a disintegrant.
• microcrystalline cellulose sodium starch glycolate, and magnesium stearate, or mixtures thereof.
• a contemplated anti-SMAD7 therapy formulation can comprise a lubricant, e.g., an extra-granular phase can contain a lubricant.
• Lubricants comprise but are not limited to talc, silica, fats, stearin, magnesium stearate, calcium phosphate, silicone dioxide, calcium silicate, calcium phosphate, colloidal silicon dioxide, metallic stearates, hydrogenated vegetable oil, corn starch, sodium benzoate, polyethylene glycols, sodium acetate, calcium stearate, sodium lauryl sulfate, sodium chloride, magnesium lauryl sulfate, talc, and stearic acid.
• the pharmaceutical formulation comprises an enteric coating.
• enteric coatings create a barrier for the oral medication that controls the location at which the drug is absorbed along the digestive track.
• Enteric coatings can comprise a polymer that disintegrates at different rates according to pH.
• Enteric coatings can comprise, for example, cellulose acetate phthalate, methyl aciylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxylpropylmethyl cellulose phthalate, methyl methacrylate-methacrylic acid copolymers, ethylacrylate-methacrylic acid copolymers, methacrylic acid copolymer type C, polyvinyl acetate-phthalate, and cellulose acetate phthalate.
• the enteric coating comprises an anionic, cationic, or neutral copolymer based on methacrylic acid, methaciylic/acrylic esters or their derivatives. In certain embodiments, the enteric coating comprises an ethylacrylate-methacrylic acid copolymer.
• an anti-SMAD7 therapy in the form of a tablet comprises or consists essentially of about 0.5% to about 70%, e.g., about 0.5% to about 10%, or about 1%) to about 20%, by weight of a SMAD7 antisense oligonucleotide or a pharmaceutically acceptable salt thereof.
• a tablet can comprise for example, about 0.5% to about 60% by weight of mannitol, e.g., about 30% to about 50% by weight mannitol, e.g., about 40% by weight mannitol; and/or about 20% to about 40% by weight of microcrystalline cellulose, or about 10% to about 30%) by weight of microcrystalline cellulose.
• a contemplated tablet can comprise an intragranular phase that comprises about 30% to about 60%, e.g., about 45% to about 65% by weight, or alternatively, about 5 to about 10% by weight of an oligonucleotide that is synthesized according to the methods provided herein, such as an oligonucleotide obtained from a single batch composition, a substantially pure oligonucleotide composition, or an oligonucleotide composition prepared according to the method disclosed herein, wherein the oligonucleotide has a nucleic acid sequence of any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, preferably having a nucleic acid sequence: SEQ ID NO: 3 : (5'-GTX GCC CCT TCT CCC XGC AGC-3'), wherein X represents 5-methyl-2'-deoxycytidine, about 30% to about 50%, or alternatively, about 5% to about 15% by weight mannitol, about 5% to about 15%
• microcrystalline cellulose about 0% to about 4%, or about 1% to about 7%
• hydroxypropylmethyl cellulose and about 0% to about 4%, e.g., about 2% to about 4% sodium starch glycolate by weight.
• Exemplary anti-SMAD7 therapy formulations comprise dosage forms that comprise or consist essentially of about 10 mg to about 500mg of an SMAD7 antisense oligonucleotide that is synthesized according to the methods provided herein, such as obtained from a single batch composition, a substantially pure oligonucleotide composition, or an oligonucleotide composition prepared according to the method disclosed herein, wherein the oligonucleotide has a nucleic acid sequence of SEQ ID NO: 3 : (5'-GTX GCC CCT TCT CCC XGC AGC-3'), wherein X represents 5-methyl-2'-deoxycytidine, for example, tablets that comprise between about 30 mg and about 310 mg, between about 50 mg and about 290 mg, between about 70 mg and about 270 mg, between about 70 mg and about 250 mg, between about 90 mg and about 230 mg, between about 110 mg and about 210 mg, or between 130 mg and about 190 mg, or between 150 mg and about 170
• the tablets comprise between about 5 mg and about 90 mg, between about 10 mg and about 70 mg, or between about 30 mg and about 50 mg of an SMAD7 antisense oligonucleotide that is synthesized according to the methods provided herein, such as obtained from a single batch composition, a substantially pure oligonucleotide composition, or an oligonucleotide composition prepared according to the method disclosed herein, wherein the oligonucleotide has a nucleic acid sequence of SEQ ID NO: 3 : (5'-GTX GCC CCT TCT CCC XGC AGC-3'), wherein X represents 5-methyl-2'-deoxycytidine.
• an SMAD7 antisense oligonucleotide that is synthesized according to the methods provided herein, such as obtained from a single batch composition, a substantially pure oligonucleotide composition, or an oligonucleotide composition prepared according to the method disclosed herein, wherein the oligonucleo
• the tablets comprise about 20 mg, about 40 mg, about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, or about 300 mg of an SMAD7 antisense oligonucleotide that is synthesized according to the methods provided herein, such as obtained from a single batch composition, a substantially pure oligonucleotide composition, or an oligonucleotide
• the anti-SMAD7 therapy can be a tablet for oral use comprising: about 0.5% to about 10% by weight of an SMAD7 antisense oligonucleotide that is synthesized according to the methods provided herein, such as obtained from a single batch composition, a substantially pure oligonucleotide composition, or an oligonucleotide composition prepared according to the method disclosed herein, wherein the oligonucleotide has a nucleic acid sequence of SEQ ID NO: 3 : (5'-GTX GCC CCT TCT CCC XGC AGC-3'), wherein X represents 5-methyl-2'-deoxycytidine, or a pharmaceutically acceptable salt thereof; about 30% to about 50% by weight mannitol; and about 10% to about 30% by weight microcrystalline cellulose.
• an SMAD7 antisense oligonucleotide that is synthesized according to the methods provided herein, such as obtained from a single batch composition, a substantially pure oligonucleotide
• a pharmaceutically acceptable tablet for oral administration comprises an intra-granular phase that can comprise about 50%) by weight of an SMAD7 antisense oligonucleotide that is synthesized according to the methods provided herein, such as obtained from a single batch composition, a substantially pure oligonucleotide composition, or an oligonucleotide composition prepared according to the method disclosed herein, wherein the oligonucleotide has a nucleic acid sequence of SEQ ID NO: 3 : (5'-GTX GCC CCT TCT CCC XGC AGC-3'), wherein X represents 5-methyl-2'- deoxycytidine, (or salt thereof), about 11.5% by weight mannitol, about 10% by weight microcrystalline cellulose, about 3% by weight hydroxypropylmethyl cellulose, and about 2.5% by weight sodium starch glycolate; and an extra-granular phase that can comprise about 20% by weight microcrystalline cellulose, about 2.5% by weight
• a pharmaceutically acceptable tablet for oral administration comprises or consists essentially of: an intra-granular phase that can comprise or consist essentially of about 5% to about 10%, e.g., about 8% by weight of an SMAD7 antisense oligonucleotide that is synthesized according to the methods provided herein, such as obtained from a single batch composition, a substantially pure oligonucleotide composition, or an oligonucleotide composition prepared according to the method disclosed herein, wherein the oligonucleotide has a nucleic acid sequence of SEQ ID NO: 3 : (5'-GTX GCC CCT TCT CCC XGC AGC-3'), wherein X represents 5-methyl-2'-deoxycytidine, about 40% by weight mannitol, about 8% by weight microcrystalline cellulose, about 5% by weight hydroxypropylmethyl cellulose, and about 2% by weight sodium starch glycolate; and an extra- granular phase that can comprise or consist essentially of about 5%
• the anti-SMAD7 therapy can be in the form of a pharmaceutically acceptable tablet for oral use comprising an intra-granular phase and extra-granular phase, wherein for example, the intra-granular phase comprises about 5% to about 10%), by weight (for example about 8% by weight) of an SMAD7 antisense oligonucleotide that is synthesized according to the methods provided herein, such as obtained from a single batch composition, a substantially pure oligonucleotide composition, or an oligonucleotide composition prepared according to the method disclosed herein, wherein the oligonucleotide has a nucleic acid sequence of SEQ ID NO: 3 : (5'-GTX GCC CCT TCT CCC XGC AGC-3'), wherein X represents 5-methyl-2'-deoxycytidine, or a pharmaceutically acceptable salt thereof, about 40% by weight mannitol, about 8% by weight microcrystalline cellulose, about 5% by weight hydroxypropylmethyl cellulose, and about
• the oligonucleotide is an SMAD7 antisense oligonucleotide, for example, wherein the oligonucleotide has a nucleic acid sequence of any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, preferably having a nucleic acid sequence: SEQ ID NO: 3 : (5'-GTX GCC CCT TCT CCC XGC AGC-3'), wherein X represents 5-methyl- 2'-deoxycytidine.
• a method of preparing a series of tablets comprises partitioning a single batch composition of an saccharideoxycytidine.
• oligonucleotide comprising at least 2 kg of the oligonucleotide, e.g., comprising at least 2 kg of the oligonucleotide and at most 25 wt.% water, into one or more portions suitable for oral dosage, and combining at least one, or each, of the one or more portions with a pharmaceutically acceptable adjuvant and/or excipient;
• the oligonucleotide is an SMAD7 antisense oligonucleotide, for example, wherein the oligonucleotide has a nucleic acid sequence of any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, preferably having a nucleic acid sequence: SEQ ID NO: 3 : (5'-GTX GCC CCT TCT CCC XGC AGC-3'), wherein X represents 5-methyl- 2'-deoxycytidine.
• a method of preparing a series of tablets e.g.,
• oligonucleotide comprising at least 50 mol% of the oligonucleotide output from at least one 700 mmol or greater oligonucleotide synthesis column, e.g., comprising at least 50 mol% of the oligonucleotide output from at least one 700 mmol or greater oligonucleotide synthesis column and at most 25 wt.% water, into one or more portions suitable for oral dosage, and combining at least one, or each, of the portions with a pharmaceutically acceptable adjuvant and/or excipient;
• the oligonucleotide is an SMAD7 antisense oligonucleotide, for example, wherein the oligonucleotide has a nucleic acid sequence of any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, preferably having a nucleic acid sequence: SEQ ID NO: 3 : (5'-GTX GCC CCT TCT CCC XGC AGC
• the pharmaceutical composition batch comprises at least 10 wt.%, at least 20 wt.%, at least 30 wt.%, at least 40 wt.%, at least 50 wt.%, at least 60 wt.%, at least 70 wt.%, at least 80 wt.%, at least 90 wt.%, at least 95 wt.%, or 100 wt.%, of the single batch composition, the substantially pure oligonucleotide composition, or the oligonucleotide composition prepared according to the methods of preparing as disclosed herein.
• the pharmaceutical composition batch comprises at least 10 wt.%, at least 20 wt.%, at least 30 wt.%, at least 40 wt.%, at least 50 wt.%, at least 60 wt.%, at least 70 wt.%, at least 80 wt.%, at least 90 wt.%, at least 95 wt.%, or 100 wt.%, of the single
• the pharmaceutical composition batch comprises at least a portion of a single batch composition of an oligonucleotide comprising at least 700 mmol of the oligonucleotide, e.g., at least 700 mmol of the oligonucleotide and at most 25 wt.% water, and a pharmaceutically acceptable adjuvant and/or excipient; wherein the oligonucleotide is an SMAD7 antisense oligonucleotide, for example, wherein the oligonucleotide has a nucleic acid sequence of any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, preferably having a nucleic acid sequence: SEQ ID NO: 3 : (5'-GTX GCC CCT TCT CCC XGC AGC-3'), wherein X represents 5-methyl-2'-deoxycytidine.
• the oligonucleotide is an SMAD7 antisense oligonucleotide, for example
• composition batch comprises at least a portion of a single batch composition of an oligonucleotide comprising at least 50 mol% of the oligonucleotide output from at least one 700 mmol or greater oligonucleotide synthesis column, e.g., comprising at least 50 mol% of the oligonucleotide output from at least one 700 mmol or greater oligonucleotide synthesis column and at most 25 wt.% water, and a pharmaceutically acceptable adjuvant and/or excipient;
• the oligonucleotide is an SMAD7 antisense oligonucleotide, for example, wherein the oligonucleotide has a nucleic acid sequence of any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, preferably having a nucleic acid sequence: SEQ ID NO: 3 : (5'-GTX GCC CCT TCT CCC XGC AGC-3'), wherein X represents 5-methyl-2'-deoxycytidine.
• the pharmaceutical composition batch comprises, or is, a series of oral dosage forms (e.g., tablets or coated tablets).
• the pharmaceutical composition batch comprises, or is, a series of at least 100 tablets (or coated tablets), for example, at least 500, such as at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, at least 100,000, or at least 200,000 tablets (or coated tablets), for example, between 100-1,000,000 tablets (or coated tablets), such as beween 1,000-1,000,000, beween 10,000-1,000,000, beween 50,000-1,000,000, beween 100,000-1,000,000, or beween 500-1,000,000 tablets (or coated tablets).
• a method for treating or managing inflammatory bowel disease (IBD) in a patient having IBD comprises (a) administering to the patient a SMAD7 antisense-oligonucleotide during a first treatment period at a first dose; and (b) administering to the patient the SMAD7 antisense-oligonucleotide during a second treatment period at a second dose.
• IBD inflammatory bowel disease
• the methods provided herein are useful to prevent the reoccurrence of IBD in a patient who has previously received an IBD treatment (e.g., an aminosalicylate treatment or a steroid treatment), which is failing, or in a treatment naive patient who is experiencing a chronic disease with few or no clinical symptoms.
• an IBD treatment e.g., an aminosalicylate treatment or a steroid treatment
• treating of managing IBD comprises improving the quality of life of an IBD patient (e.g., as determined by a patient survey), e.g., by reducing pain in the IBD patient, improving appetite in the IBD patient, or improving the IBD patient's sleep (e.g., length of uninterrupted sleep).
• preventing IBD or preventing the reoccurrence of IBD comprises preventing the occurrence or the reoccurrence of one or more symptoms of UC, such as intestinal swelling, intestinal inflammation, sores in the lining of the large intestine (colon), diarrhea, belly pain, or bleeding from the rectum.
• preventing IBD or preventing the reoccurrence of IBD comprises preventing one or more IBD symptoms during a chronic phase of the disease.
• preventing IBD or preventing the reoccurrence of IBD comprises reducing one or more IBD symptoms during an acute phase of the disease (e.g., during a disease "flare up").
• preventing IBD or preventing the reoccurrence of IBD comprises preventing the occurrence or reoccurrence of a disease flare up or of a disease flare up of a certain intensity. In certain embodiments, preventing IBD or preventing the reoccurrence of IBD comprises preventing the occurrence or reoccurrence of flare ups at a certain frequency (e.g., at a frequency observed in the patient having IBD directly prior to the administration of an IBD treatment regiment or at a (e.g., median, average or mean) frequency observed in a control group of untreated IBD patients).
• a certain frequency e.g., at a frequency observed in the patient having IBD directly prior to the administration of an IBD treatment regiment or at a (e.g., median, average or mean) frequency observed in a control group of untreated IBD patients.
• preventing IBD or preventing the reoccurrence of IBD comprises preventing a (further) deterioration in the quality of life of an IBD patient (e.g., as determined by a patient survey), e.g., by preventing the increase in pain in the IBD patient, preventing (further) loss of appetite in the IBD patient, or preventing a worsening of
• an IBD patient to be treated with a method provided herein is a UC patient or a CD patient.
• the patient having IBD was diagnosed with CD or UC at least 3 months prior to the initial screening period or the first treatment period.
• the patient having IBD was diagnosed with ileitis, or ileocolitis, e.g., as determined by endoscopic, radiographic or another imaging method (e.g., magnetic resonance imaging [MRI], computed tomography [CT] scan), within 2 years prior to the screening period or to the first treatment period.
• the patient has IBD involving the distal to mid transverse colon.
• the patient has extensive colitis.
• Oligonucleotide prepared a deoxyribonucleotide having a nucleic acid sequence:
• oligonucleotide products from the ion-exchange purification processes were then pooled before desalting via the ultrafiltration/diafiltration process, which was conducted at a 2,700-4,500 mmol scale, respectively.
• Synthesizer and Column Synthesis of the oligonucleotide having the nucleic acid sequence of SEQ ID NO: 3 was conducted with a 1454 Synthesizer using a synthesis column having an inner diameter of 50-80 cm that was loaded with 900 mmol of a crosslinked polystyrene solid support (e.g., NITTOPHASE-HL or PRIMER SUPPORT 5G) having attached thereto a linker containing hydroxyl groups protected with 4,4'-dimethoxytrityl group (DMT), that when deprotected were available to initiate assembly of the targeted oligonucleotide (e.g., UNYLINKER).
• a crosslinked polystyrene solid support e.g., NITTOPHASE-HL or PRIMER SUPPORT 5G
• DMT 4,4'-dimethoxytrityl group
• Synthesis Procedure The following synthesis procedure utilized phosphoramidite chemistry for preparing the 21-mer oligonucleotide having the sequence SEQ ID NO: 3 :
• Total Crude OD The amount of crude product cleaved from the synthesis column and collected in the filtrate was measured via optical density (OD) at wavelength 260 nm.
• the Conversion Factor is specific for the target 21-mer oligonucleotide having the sequence SEQ ID NO: 3 (i.e., 21856.3 OD/g). Accordingly, use of this specific coversion factor at the synthesis stage of the process is as a means to estimate or approximate the amount of the DMT-protected target oligonucleotide cleaved and collected from the synthesis column.
• the Conversion Factor was provided as follows: [(extinction coefficient (L*mol “1 *cm “1 ) of target oligonucleotide at wavelength 260 nm) / (molecular weight (g/mol) of sodium salt of the target 21-mer oligonucleotide having the sequence SEQ ID NO: 3)] x 1000; wherein the target 21-mer oligonucleotide having the sequence SEQ ID NO: 3 has a molecular weight of 7044.05 g/mol (as sodium salt) and an extinction coefficient of 153957 L*mol "1 *cm "1 at wavelength 260 nm.
• Step Yield (mol.%) for all recovered oligonucleotide product(s), i.e., the crude yield for the synthesis portion of this process, was determined as follows: [(Total Crude OD) / [(900 mmol scale) x (extinction coefficient (L*mol "1 *cm "1 ) of target oligonucleotide at wavelength 260 nm)]] x 100; wherein the extinction coefficient is 153957 L*mol "1 *cm "1 at wavelength 260 nm.
• FLP Yield (mol. %)
• AktaProcess The crude oligonucleotide, once loaded onto the anion exchange column, was washed with 1-3 column volumes of a mixture of a 25 mM sodium hydroxide solution and a 2 M sodium chloride solution, and then washed with 1-3 column volumes of a 25 mM sodium hydroxide solution.
• the loaded, crude oligonucleotide was detritylated by introducing 80% aqueous acetic acid (about 1 column volume at 1,000-1,200 L/ hr) until the resulting eluent had a pH of about 2 or less, thereby forming a fully deprotected, crude oligonucleotide that was now loaded onto the anion exchange column.
• 80% aqueous acetic acid about 1 column volume at 1,000-1,200 L/ hr
• the pH of the anion exchange column was readjusted to a pH of about 10 or more with a 0.025-0.20 N sodium hydroxide solution (at 1,000-1,200 L/hr) before purifying with a basic salt gradient was initiated (using a gradient of 5%-95% of an aqueous solution containing 20-30 mM sodium hydroxide and 1-3 M sodium chloride; with a gradient slope of about 10-20 column volumes, and using a flow rate of no more than 1,200 L/hr; a salt gradient from 0.2 to 1.8 M sodium chloride in aqueous 25 nM sodium hydroxide solution).
• the final wash to retrieve the purified oligonucleotide product was completed with at least 3 column volumes of an aqueous solution containing 20-50 mM sodium hydroxide and 2-4M sodium chloride.
• % OD Recovery (Step Yield mol.%)
• %FLP mole percent value of the purified target oligonucleotide product recovered from the anion exchange column
• FLP Yield (mol.%)
• the pooled, purified oligonucleotide eluates which were loaded at an initial concentration of about 650-750 OD/mL and had a cross-flow velocity of about 5-6 L/min/m 2 , were desalted by neutralizing to a pH of about 6.5-7.5 (using 0.5-2 M HC1 and 0.05-0.25 M HC1 to achieve pH 6.5-7.5; adjusting with 0.05-0.25 N NaOH solution if necessary), and then diafiltrated with water for at least 7 exchanges until the conductivity of the permeate (diafiltrate) was less than 50 ⁇ 8/ ⁇ , and the resulting concentration of the desalted oligonucleotide in the retentate solution was at least 1,000 OD/mL.
• Total Starting OD optical density
• Total Amount The amount of solid, concentrated target oligonucleotide product recovered from the freeze drying process that is corrected for moisture content, reported in terms of mmol ("Total Amount: mmol"), was determined as follows: [(Total Amount: Harvested Solid Corrected for Moisture (g)) / (molecular weight (g/mol) of sodium salt of the target 21-mer oligonucleotide having the sequence SEQ ID NO: 3)] x 1000; wherein the target 21-mer oligonucleotide having the sequence SEQ ID NO: 3 has a molecular weight of 7044.05 g/mol (as sodium salt).
• Target Oligonucleotide (dry wt.%) 92.4 94.1
• a single batch composition of an oligonucleotide comprises at least 700 mmol of the oligonucleotide and at most 25 wt.% water.
• oligonucleotide synthesis column and at most 25 wt.% water.
• a single batch composition of an oligonucleotide comprises at least 700 mmol of the oligonucleotide, wherein the single batch composition is a liquid composition.
• oligonucleotide synthesis column wherein the single batch composition is a liquid composition.
• an oligonucleotide composition comprises a plurality of at least 700 mmol scale single batch synthetic preparations of an oligonucleotide, wherein the single batch composition is a liquid composition.
• a substantially pure oligonucleotide composition of an oligonucleotide wherein the 5'-hydroxyl group of the 5'-terminal nucleoside is protected is protected.
• phosphoramidite comprises a protected hydroxyl group and a protected phosphoramidite; d) independently coupling a nucleoside phosphoramidite to the deprotected hydroxyl group of the linker, or to the deprotected hydroxyl group of the nucleoside from the previous iteration of the reaction cycle, thereby creating a phosphite triester linked nucleoside; e) independently thiolating the protected phosphite triester linkage thereby creating a
• an oligonucleotide composition comprising at least 700 mmol of an oligonucleotide having at most 25 wt.% water and a nucleic acid sequence:
• SEQ ID NO: 3 (5'-GTX GCC CCT TCT CCC XGC AGC-3'), wherein:
• X represents 5-methyl-2'-deoxycytidine
• the oligonucleotide is prepared according to a method comprising:
• phosphoramidite comprises a protected hydroxyl group and a protected
• step h) repeating the providing, coupling, thiolating, capping, and deprotecting steps (steps c) through g) a predetermined number of times to provide a solid support-bound oligonucleotide;
• one or more than one (including for instance all) of the following further embodiments may comprise each of the other embodiments or parts thereof.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the purifying step 1) comprises: 1) loading the oligonucleotide eluate from eluting step k) onto the ion exchange chromatography column;
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the purifying step 1) comprises:
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the concentrating step m) comprises concentrating the desalted solution of the oligonucleotide compound, such as concentrating with thin film evaporation.
• the concentrating step m) comprises concentrating the desalted solution of the oligonucleotide compound, such as concentrating with thin film evaporation, and further concentrating the solution with a freeze drying process.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the method further comprises independently washing the support with an aprotic solvent, such as acetonitrile, between one or more steps of a reaction cycle iteration, for example washing the support with between 1-10 column volumes, for example, between 1-7 column volumes or between 1-5 column volumes, such as between 2-4 column volumes, between one or more steps of a reaction cycle iteration.
• an aprotic solvent such as acetonitrile
• composition comprises at least 900 mmol of the
• the single batch composition, the substantially pure oligonucleotide composition, or the oligonucleotide composition, of any one of the above embodiments and any one or more of the further embodiments herein is provided by the method disclosed herein at a yield of at least 3.5 g/mmol of an at least 900 mmol synthesis scale of the oligonucleotide, or of a pooled plurality of serial combinations of an oligonucleotide synthesis of an at least 900 mmol synthesis scale via a single oligonucleotide synthesis column (or synthesis run) and a purification of the synthesized oligonucleotide of an at least 900 mmol purification scale via a single ion-exchange chromatography purification column (or purification run) of the oligonucleotide,and at most 25 wt.% water, wherein the yield may be determined by optical density/mL (OD/mL
• the single batch composition, the substantially pure oligonucleotide composition, or the oligonucleotide composition, of any one of the above embodiments and any one or more of the further embodiments herein is provided by the method disclosed herein at a yield of at least 3.5 g/mmol of an at least 900 mmol synthesis scale of the oligonucleotide, or of a pooled plurality of serial combinations of an oligonucleotide synthesis of an at least 900 mmol synthesis scale via a single oligonucleotide synthesis column (or synthesis run) and a purification of the synthesized oligonucleotide of an at least 900 mmol purification scale via a single ion-exchange chromatography purification column (or purification run) of the oligonucleotide, and at most 10 wt.% water, wherein the yield may be determined by optical density/mL (OD/mL
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• composition comprises at least 3 kg of the oligonucleotide and at most 25 wt.% water.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the composition comprises at least 50 mol%, such as between 70- 99 mol%, of the oligonucleotide output from at least one oligonucleotide synthesis column having a capacity of providing at least 300 mmol or greater, such as at least 700 mmol, at least 800 mmol or at least 900 mmol or greater, of the oligonucleotide being synthesized, and at most 25 wt.% water, wherein the output is determined by optical density/mL (OD/mL).
• OD/mL optical density/mL
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the composition comprises at least 50 mol%, such as between 70- 99 mol%, of the oligonucleotide output (as determined by optical density/mL (OD/mL)) from between 4-10 oligonucleotide synthesis columns having a capacity of providing at least 300 mmol or greater, such as at least 700 mmol, at least 800 mmol or at least 900 mmol or greater, of the oligonucleotide being synthesized, and at most 25 wt.% water.
• OD/mL optical density/mL
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the oligonucleotide has 10-100 monomer subunits, for example, 15-25 monomer subunits, such as 20, 21, or 22, monomer subunits.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the oligonucleotide is an anti-SMAD7 oligonucleotide.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the nucleotide sequence has the nucleic acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or the complementary sequence thereto.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the nucleotide sequence has the nucleic acid sequence:
• SEQ ID NO: 3 (5'-GTX GCC CCT TCT CCC XGC AGC-3');
• X represents 5-methyl-2'-deoxycytidine.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further embodiments herein, wherein at least one of the internucleotide linkages of the oligonucleotide is an ⁇ , ⁇ -linked phosphorothioate.
• the single batch composition the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or
• the single batch composition the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or
• oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the molecular weight is a protonated molecular weight.
• the single batch composition the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or
• oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the molecular weight is an alkai metal molecular weight, such as a sodium salt form molecular weight.
• the single batch composition the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or
• the molecular weight is an alkaline metal molecular weight, such as a magnesium salt form molecular weight.
• the single batch composition the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or
• the oligonucleotide composition comprises at most 20 wt.% water, at most 15 wt.% water, or at most 10 wt.% water, such as comprises in the range of between 5-10 wt.%) water, for example, in the range of between 6-8 wt.%> water.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• oligonucleotide is 60%> or greater (as determined, for example, by RP-HPLC).
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the degree of purity of the composition comprising the protected oligonucleotide is 60%> or greater (as determined, for example, by RP-HPLC).
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the protected oligonucleotide has a molecular weight of at least 3,000 Da, such as a molecular weight in the range of between 3,000-20,000 Da.
• the solid support having a linker attached thereto is a controlled pore glass.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the solid support having a linker attached thereto is a crosslinked polystyrene.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• oligonucleotide in an amount of at least 600 mmol, at least 700 mmol, at least 900 mmol, at least 1,600 mmol, or at least 2,400 mmol.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the single batch composition the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or
• the single batch composition the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or
• oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the solid support has a column volume of at least 20 L, such as a column volume in the range of between 20-35 L.
• the single batch composition the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or
• oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the single batch composition the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or
• oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• linker comprises a terminal hydroxyl group.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• linker is a UNYLINKER.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the coupling comprises providing the nucleoside phosphoramidite, for example, providing an excess amount of the nucleoside phosphoramidite, such as 1-8 equivalents of the nucleoside phosphoramidite relative to the equivalents of the solid support.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• nucleoside phosphoramidite is a protected nucleoside phosphoramidite, such as a protected nucleoside phosphoramidite comprising a 5'-hydroxyl protected group and 3'-hydroxyl protected group.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the 5'-hydroxyl protecting group of the protected nucleoside phosphoramidite is a dimethoxytrityl (DMT) group
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the 3'-hydroxyl protecting group of the protected nucleoside phosphoramidite is a phosphoramidite group, such as a (2-cyanoethyl)-N,N-diisopropyl- phosphoramidite group.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the thiolation of the protected phosphite triester linkage with a thiolating agent forms a protected phosphorothioate linkage, such as a 2-cyanoethoxy -protected phosphorothioate linkage.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the thiolating agent is xanthane hydride (XH), such as xanthane hydride (XH) in pyridine.
• XH xanthane hydride
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further embodiments herein, wherein 1-8 equivalents, such as 5-8 equivalents, of the xanthane hydride (XH) is provided, relative to the equivalents of the solid support.
• 1-8 equivalents, such as 5-8 equivalents, of the xanthane hydride (XH) is provided, relative to the equivalents of the solid support.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the single batch composition the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or
• oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• capping of the unreacted deprotected hydroxyl groups comprises adding:
• Cap A a first capping solution comprising N-methylimidazole (NMI), pyridine, and acetonitrile
• Cap B a second capping solution comprising capping agent and acetonitrile.
• the single batch composition the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or
• oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• capping of the unreacted deprotected hydroxyl groups comprises adding a premixed mixture of:
• Cap A a first capping solution comprising N-methylimidazole (NMI), pyridine, and acetonitrile
• Cap B a second capping solution comprising capping agent and acetonitrile.
• the single batch composition the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or
• oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• capping agent is isobutyric anhydride
• the single batch composition the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or
• oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the single batch composition the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or
• oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the steps of providing/coupling of a nucleoside phosphoramidite, thiolating (or oxidizing) the formed phosphite triester linkage, optionally capping the unreacted deprotected hydroxyl group(s), and optionally deprotecting the protected 5 '-hydroxyl group of the 5 '-terminal nucleoside of the oligonucleotide are repeated a predeterminied number of times, for example, repeated 9-99 times, repeated 14-24 times, such as 19, 20, or 21 times, to provide the solid support-bound oligonucleotide in an amount in the range of between 300-3,000 mmol, for example, between 700 -3,000 mmol.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• oligonucleotide comprises providing a solution of ammonium hydroxide, for example, providing a heated solution of ammonium hydroxide, such as providing a heated ammonium hydroxide solution and recirculating the heated ammonium hydroxide solution through the column housing said deprotected solid support-bound oligonucleotide.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• exocyclic amino protecting groups comprises benzoyl and isobutyryl groups.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the provided and/or recirculated heated ammonium hydroxide solution is a 28-30% ammonia aqueous solution (w/w).
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the temperature of the provided and/or recirculated heated ammonium hydroxide solution is 40-70°C or 40-60 °C, such as a temperature of 40°C, 50°C, 60°C, or 65 °C.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further embodiments herein, wherein the heated ammonium hydroxide solution is recirculated through the support for 8-36 hours, for example, 12-36 hours, such as 24 hours.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the cleaved oligonucleotide comprises a 5 '-hydroxyl protected group, such as a dimethoxytrityl (DMT) group
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the eluting of the cleaved and deprotected oligonucleotide from the support comprises washing the support with an aqueous solution.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the amount of the oligonucleotide eluate loaded onto the ion exchange chromatography column is in the range of between 300-3,000 mmol, for example, between 600-3,000 mmol, 700-3,000 mmol, or 1,000-3,000 mmol.
• the single batch composition, the substantially pure oligonucleotide composition, the oligonucleotide composition, the oligonucleotide or oligonucleotide composition prepared according to the methods of preparing, or the method of preparing, of any one of the above embodiments and any one or more of the further
• the oligonucleotide eluate is diluted with an aqueous buffer.

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