Novel method for purifying oligonucleotides

Abstract

Disclosed herein are novel processes for purifying oligonucleotides, such as oligonucleotides for therapeutic use.

Description

Technical Field

[0001] This invention relates to a novel method for purifying oligonucleotides, including single-stranded oligonucleotides, such as therapeutic single-stranded oligonucleotides. Background of the Invention

[0003] Oligonucleotides are typically purified by column chromatography after synthesis. Solid-state supported synthesis is currently the standard method for commercial-scale oligonucleotide synthesis, thus the capacity of the chromatographic system is matched to the synthesis scale. This means that the operating scale of commercial oligonucleotide chromatography ranges from 5 to 10 kg. The chromatographic scale can be scaled up to approximately 20 kg batches. However, the capacity expansion of column chromatography is limited by factors such as column size, flow rate, column packing uniformity, and resin compression.

[0004] As oligonucleotide synthesis technology gradually evolves towards large-scale solid-supported methods, or more likely, liquid-phase synthesis processes, the scale of crude oligonucleotides obtained from single-batch synthesis will exceed the capacity of chromatography-based purification methods. Therefore, the process benefits brought by increased batch size will be lost due to the scale limitations of chromatography. For example, liquid-phase synthesis might enable the synthesis of 100 kg of crude oligonucleotides in a single batch, but if column chromatography's processing scale is limited to 20 kg, it would need to be split into five batches for purification. Thus, the benefits of increased synthesis scale are offset by the batch size limitations of chromatography.

[0005] Column chromatography produces many fractions, each with slightly different product and impurity compositions. To obtain a final product that meets specifications, each fraction needs to be analyzed, and a decision must be made as to whether to add it to the product pool or discard it. This creates a significant analytical burden and is very time-consuming.

[0006] Furthermore, due to the significant waste generated during chromatographic processes, the chromatographic purification of oligonucleotides is considered one of the greatest challenges to sustainable oligonucleotide production. This is particularly pronounced when reversed-phase chromatography is used for purification.

[0007] Therefore, it is necessary to move away from chromatography-based oligonucleotide purification methods and develop new purification methods that are scalable, simpler to operate, less labor-intensive, and more sustainable. Summary of the Invention

[0008] This invention provides a method for purifying a product, wherein the product is a single-stranded oligonucleotide, the method comprising: (a) Obtaining a solution comprising (i) an impurity: template complex and (ii) a product: template complex, wherein the template is an oligonucleotide containing a sequence complementary to the product. (b) The solution is subjected to ultrafiltration / difiltration (UF / DF) to form a first retentate containing the product: template complex and a first permeate containing the impurity, wherein the UF / DF is performed using a first buffer solution with a pH of approximately 9-13. (c) The first retentate from step (b) is subjected to UF / DF to form a second retentate containing the template and a second permeate containing the product, wherein the UF / DF is performed using a second buffer solution with a pH greater than that of the first buffer solution.

[0009] The present invention also provides single-stranded oligonucleotide products obtained by such purification methods.

[0010] Compared with chromatography-based oligonucleotide purification methods, the present invention disclosed herein offers the following advantages: Ultrafiltration can scale up from grams to hundreds of kilograms, or even tons, without any fundamental technological limitations. This method is fundamentally simpler than chromatography, does not involve a solid support, and allows for process control by simply and gradually changing the buffer composition. The number of fractions collected is reduced, or even only a single product fraction needs to be collected, reducing the need for time-consuming analysis of multiple fractions. The desired improvement in the purity of the oligonucleotide product, and Sustainability is improved because simple aqueous buffer solutions are used, and these aqueous buffer solutions are recyclable. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of a tangential flow filtration (TFF) system.

[0012] Figure 2 The HPLC analysis of the retained product during pH screening percolation using K3PO4 buffer is shown.

[0013] Figure 3 The HPLC analysis of the retained product during pH screening percolation using Na3PO4 buffer is shown.

[0014] Figure 4 The HPLC analysis of the retained products during percolation with 200 mM K3PO4 at pH 12.5 is shown.

[0015] Figure 5The HPLC analysis of the permeation of the 13-mer intermediate during percolation with 200 mM or 300 mM K3PO4 at pH 11 is presented.

[0016] Figure 6 The HPLC analysis of the 20-mer product and hub-template during further purification in Experiment 2D is shown (-8 to 0 percolation volume for concentration, 0 to 14 percolation volume for 20-mer recovery).

[0017] Figure 7 This is a schematic diagram illustrating the UF / DF purification process for single-stranded oligonucleotides (20-mer). Detailed Implementation

[0018] definition

[0019] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Unless the context clearly indicates otherwise, the singular forms “an,” “a,” and “the” also include the plural forms. Similarly, unless the context clearly indicates otherwise, the word “or” is intended to include “and.” The term “multiple” means two or more. The term “at least one” means one or more.

[0020] Furthermore, the numerical limits given regarding substance concentrations or levels (such as solution component concentrations or ratios) and reaction conditions (such as temperature, pressure, and cycle time) are intended to be approximate. Unless otherwise stated, when numerical ranges are provided, they are inclusive, meaning they include the endpoints.

[0021] As used herein, the term “about” when referring to measurable values ​​such as quantity means to cover variations of ±20% or ±10% relative to a specified value, including ±5%, ±1% and ±0.1%, such variations apply to the implementation of the disclosed methods.

[0022] As used herein, “purification” refers to the process of removing unwanted components (e.g., impurities) from a solution. In other words, purification refers to increasing the purity of the desired oligonucleotide product. Purification is a relative term and does not require the removal of all trace amounts of unwanted components from the composition. Therefore, the term “purification” does not require absolute purity; rather, it is used as a relative term. A preparation of a purified oligonucleotide product can be purified such that the desired oligonucleotide product constitutes at least 50% of the total oligonucleotide content of the preparation. In some embodiments, the purified oligonucleotide product will constitute at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% or more of the total oligonucleotide content of the preparation.

[0023] "Ultrafiltration" or "UF" refers to a process for separating substances by passing a solution through a semi-permeable membrane or filter having a specific molecular weight cutoff (MWCO) or a specific pore size, wherein larger molecular weight molecules are retained in the retentate, while lower molecular weight molecules pass through the membrane. These lower molecular weight molecules can be products and / or impurities (e.g., shortmers or intermediates). In the purification methods disclosed herein, during ultrafiltration, the template can be present in a form that is substantially retained in the retentate, while the products and / or impurities can be substantially in the permeate. "Permeate" refers to the fraction of solution that passes through the membrane during ultrafiltration. "Retentate" refers to the fraction of solution that does not pass through the membrane during ultrafiltration. Suitable types of UF devices are known to those skilled in the art and can be selected based on various factors, such as the molecular weight of the product to be filtered, the amount and size of the components in the solution to be filtered, and the volume of the solution to be filtered.

[0024] "Ultrafiltration / difiltration" or "UF / DF" refers to an ultrafiltration process in which separation is combined with the addition of a diffusive medium to the retentate. For example, UF / DF allows for the concentration of a solution before its buffer system can be replaced. Additional additives may be introduced, for example, to adjust pH and / or ionic strength. Two basic types of UF / DF can be distinguished: variable-volume UF / DF (sometimes referred to in the art as "discontinuous" UF / DF) and constant-volume UF / DF (sometimes referred to in the art as "continuous" UF / DF).

[0025] As used herein, the term "oligonucleotide" or simply "oligo" refers to a polymer of nucleotide residues that are deoxyribonucleotides (where the resulting oligonucleotide is DNA), ribonucleotides (where the resulting oligonucleotide is RNA), or mixtures thereof. Oligonucleotides may consist entirely of nucleotide residues found in nature, or may contain at least one modified nucleotide or a linking bond between at least one modified nucleotide. Oligonucleotides may be single-stranded or double-stranded. The oligonucleotides of the present invention may be coupled to another chemical moiety, for example, a targeting moiety containing N-acetylgalactosamine (GalNAc) or multiples thereof (GalNAc clusters).

[0026] Oligonucleotides may consist entirely of nucleotide residues found in nature (i.e., "natural nucleotides" or "naturally occurring nucleotides"), or may contain at least one modified nucleotide or a linking bond between at least one modified nucleotide. Examples of naturally occurring nucleotides include deoxyadenosine monophosphate, deoxycytidine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, deoxyuridine monophosphate, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, thymidine monophosphate, and uridine monophosphate. Modified nucleotides are not naturally occurring nucleotides (i.e., they are non-natural nucleotides). Modified nucleotides may contain modified backbones, sugars, and / or nucleobases. It is known that certain modifications occur occasionally in nature, i.e., they exist in naturally occurring nucleotides, such as 2'-OMe or C5 pyrimidine modifications, but in this disclosure, these are considered modified nucleotides.

[0027] As used herein, the term "therapeutic oligonucleotide" refers to an oligonucleotide with therapeutic applications, such as in the prevention or treatment of symptoms or diseases in humans or animals. Such oligonucleotides typically contain one or more modified nucleotide residues or linkages. Therapeutic oligonucleotides function through one of several different mechanisms, including but not limited to antisense, splicing or exon skipping, immune stimulation, and RNA interference (RNAi), for example, via microRNA (miRNA) and small interfering RNA (siRNA).

[0028] As used herein, the term "template" refers to an oligonucleotide containing a sequence complementary to the product described herein. The product may have a desired sequence, and the template contains a sequence complementary to that desired sequence. The template may contain a sequence complementary to the product sequence. The template may consist of sequences complementary to the product sequence. The template may have a longer sequence compared to the product sequence. As used herein, unless otherwise stated, the term "complementary" means 100% complementarity across the entire length of the product.

[0029] As used herein, the term "product" refers to a desired single-stranded oligonucleotide to be purified by the methods disclosed in this disclosure.

[0030] As used herein, the term "complex" in relation to oligonucleotides refers to two or more oligonucleotides bound together in a sequence-specific manner. It is well known that binding can still occur even with some nucleotide mismatches. Complexes can be formed by annealing two or more oligonucleotides. "Annealing" refers to the pairing of an oligonucleotide with another oligonucleotide via hydrogen bonds to form a double-stranded oligonucleotide. Annealing may require specific conditions. Complexes can be formed by hybridization of two or more oligonucleotides. Impurity: Template complex refers to a complex formed by the binding of one or more impurities to a template. Impurity: Template complex may contain one or more short polymers bound to the template. Impurity: Template complex may contain intermediates bound to the template. Impurity: Template complex may contain intermediates partially bound to the template (e.g., where the intermediate contains terminal short polymers or segments that are not bound to the template). Impurity: Template complex may contain one or more intermediates and one or more short polymers bound to the template. Impurity: Template complex may contain an oligonucleotide bound to the template, wherein the oligonucleotide is shorter than the product. Impurities: The template complex may contain oligonucleotides that bind to the template, wherein the oligonucleotides are the same length as the product but contain mismatches. Product: The template complex is the complex formed by the product and the template.

[0031] As used herein, the term “denaturation” in relation to a complex refers to a process that dissociates two or more bound oligonucleotides. Denaturation is initiated by a change in conditions and is sometimes referred to herein as the separation of oligonucleotides. Such separation can be accomplished, for example, by increasing the temperature, changing the pH, or changing the salt concentration of the solution, or a combination of these changes. Denaturing the “complex” produces oligonucleotides that are “released” from the template, which can be either products or impurities.

[0032] As used herein, the term "impurity" refers to an oligonucleotide that does not have the sequence of the desired product. In cases where the product is prepared by the sequential addition of single nucleotides (e.g., solid-phase synthesis), impurities may arise when the chain elongation reaction terminates prematurely. These impurities may include oligonucleotides shorter than the product (e.g., shorter by 1, 2, 3, 4, 5, or more nucleotide residues). In cases where the product preparation process includes a step of forming linking bonds between short polymers or segments, intermediates may be generated if one or more linking bonds are not formed between the short polymers or segments. Intermediates may contain two or more short polymers or segments. Impurities may include short polymers. Impurities may include adenylated short polymers. Impurities may include intermediates. Impurities also include oligonucleotides incorporating incorrect nucleotides, leading to mismatches. Thus, impurities may be the same length as the product but have incorrect sequences compared to the product.

[0033] As used herein, the term "short polymer" or "segment" refers to a smaller portion of a longer oligonucleotide, particularly a smaller portion of a product. For a given product, the product is formed when all its corresponding short polymers are linked together.

[0034] As used herein, the term "enzymatic ligation" refers to the connection between two adjacent nucleotides that is formed enzymatically. This ligation bond can be a naturally occurring phosphodiester bond (PO) or a modified ligation bond, including but not limited to phosphate thioesters (PS) or aminophosphate esters (PA).

[0035] As used herein, the term "ligase" refers to an enzyme that catalyzes the joining (i.e., covalent joining) of two oligonucleotide molecules, for example, by forming a phosphodiester bond between the 3' end of one oligonucleotide (or segment) and the 5' end of another. These enzymes are commonly referred to as DNA ligases or RNA ligases and utilize cofactors: ATP (eukaryotic, viral, and archaea DNA ligases) or NAD (prokaryotic DNA ligases). Although DNA ligases are present in all organisms, they vary significantly in amino acid sequence, molecular size, and properties (Nucleic Acids Research, 2000, Vol. 28, No. 21, 4051-4058). They are typically members of class EC 6.5 of enzymes defined by the International Union of Biochemistry and Molecular Biology, namely ligases for forming phosphodiester bonds. Within the scope of this invention are ligases capable of linking an unmodified oligonucleotide to another unmodified oligonucleotide, ligases capable of linking an unmodified oligonucleotide to a modified oligonucleotide (i.e., linking a modified 5' oligonucleotide to an unmodified 3' oligonucleotide, and linking an unmodified 5' oligonucleotide to a modified 3' oligonucleotide), and ligases capable of linking a modified oligonucleotide to another modified oligonucleotide.

[0036] As used herein, the term "gapmer" refers to an oligonucleotide having an internal "gap segment" flanked by two external "flank segments," wherein the gap segment consists of multiple nucleotides that support RNase H cleavage, and each flanking segment consists of one or more nucleotides that are chemically distinct from the nucleotides within the gap segment.

[0037] As used herein, the term "support material" refers to a chemical compound or material bonded to one or more templates that increases the molecular weight of the resulting template-containing structure, thereby allowing the template to be retained in the retentate during the ultrafiltration process. The resulting template-containing structure may be referred to as a "hub-template".

[0038] Invention Description

[0039] This disclosure provides a method for purifying a product, wherein the product is a single-stranded oligonucleotide, the method comprising: (a) Obtaining a solution comprising (i) an impurity: template complex and (ii) a product: template complex, wherein the template is an oligonucleotide containing a sequence complementary to the product. (b) The solution is subjected to ultrafiltration / difiltration (UF / DF) to form a first retentate containing the product: template complex and a first permeate containing the impurity, wherein the UF / DF is performed using a first buffer solution with a pH of approximately 9-13. (c) The first retentate from step (b) is subjected to UF / DF to form a second retentate containing the template and a second permeate containing the product, wherein the UF / DF is performed using a second buffer solution with a pH greater than that of the first buffer solution.

[0040] Such methods can be used to purify products from impurities, such as single-chain oligonucleotides prepared via phosphoramidite chemical synthesis (e.g., solid-phase chemical synthesis via phosphoramidite chemical synthesis). These methods can also be used to purify products from impurities, such as single-chain oligonucleotides synthesized via enzymatic ligation reactions (e.g., according to the methods described in WO2018 / 011067 and WO 2019 / 121500). Releasing the impurity from the impurity:template complex requires denaturing the impurity:template complex. The impurity can then be separated from the template. Releasing the product from the product:template complex requires denaturing the product:template complex. The product can then be separated from the template. Denaturation of the complex can be achieved by changing the pH of the buffer solution.

[0041] The Watson-Crick base pairing property can be utilized to specifically release impurities from the template before the product is released. The conditions required for denaturation of the impurity:template complex may differ from those required for denaturation of the product:template complex. This difference in denaturation conditions is due to differences in binding strength; for example, the product may bind to the template more strongly than the impurity. Incorrect nucleotides and / or shorter impurities reduce the binding strength to the template. The pH can be adjusted to denature only the impurity:template complex, without denaturing the product:template complex. Impurities can then be separated. Subsequently, the pH can be increased to denature the product:template complex. The product can then be separated.

[0042] In one embodiment, the UF / DF in step (b) is performed using a first buffer solution with a pH greater than or equal to 8. In one embodiment, the UF / DF in step (b) is performed using a first buffer solution with a pH greater than or equal to 9. In one embodiment, the UF / DF in step (b) is performed using a first buffer solution with a pH of about 9-13. In one embodiment, the pH of the first buffer solution is about 9-12.5, about 9-12, about 9-11.5, about 9-11, about 10-12.5, about 10-12, about 10-11.5, about 10-11, or about 10.5-11.5. In one embodiment, the pH of the first buffer solution is 9.5-12.5. In one embodiment, the pH of the first buffer solution is about 9-12. In one embodiment, the pH of the first buffer solution is about 9-11.5. In one embodiment, the pH of the first buffer solution is about 9-11. In one embodiment, the pH of the first buffer solution is 9.5-11.5. In one embodiment, the pH of the first buffer solution is about 10-12.5. In one embodiment, the pH of the first buffer solution is about 10-12. In one embodiment, the pH of the first buffer solution is about 10-11.5. In one embodiment, the pH of the first buffer solution is about 10-11. In one embodiment, the pH of the first buffer solution is about 11.5-12.5. In one embodiment, the pH of the first buffer solution is about 10.5-11.5. In one embodiment, the UF / DF in step (b) is performed using the first buffer solution, wherein the pH of the first buffer solution is selected to specifically denature the impurity:template complex. In one embodiment, the UF / DF is performed using the first buffer solution, wherein the pH of the first buffer solution causes the impurity to be released from the template. In one embodiment, the UF / DF is performed using the first buffer solution, wherein the pH of the first buffer solution does not denature the product:template complex.

[0043] In one embodiment, the UF / DF in step (c) is performed using a second buffer solution with a pH higher than that of the first buffer solution. In one embodiment, the pH of the second buffer solution is about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.0 higher than that of the first buffer solution. In one embodiment, the pH of the second buffer solution is about 0.4 higher than that of the first buffer solution. In one embodiment, the pH of the second buffer solution is about 0.5 higher than that of the first buffer solution. In one embodiment, the pH of the second buffer solution is about 0.6 higher than that of the first buffer solution. In one embodiment, the pH of the second buffer solution is about 0.7 higher than that of the first buffer solution. In one embodiment, the pH of the second buffer solution is about 0.8 higher than that of the first buffer solution. In one embodiment, the pH of the second buffer solution is about 0.9 higher than that of the first buffer solution. In one embodiment, the pH of the second buffer solution is about 1.0 higher than the pH of the first buffer solution. In one embodiment, the pH of the second buffer solution is about 1.1 higher than the pH of the first buffer solution. In one embodiment, the pH of the second buffer solution is about 1.2 higher than the pH of the first buffer solution. In one embodiment, the pH of the second buffer solution is about 1.3 higher than the pH of the first buffer solution. In one embodiment, the pH of the second buffer solution is about 1.4 higher than the pH of the first buffer solution. In one embodiment, the pH of the second buffer solution is about 1.5 higher than the pH of the first buffer solution. In one embodiment, the pH of the second buffer solution is about 1.6 higher than the pH of the first buffer solution. In one embodiment, the pH of the second buffer solution is about 1.7 higher than the pH of the first buffer solution. In one embodiment, the pH of the second buffer solution is about 1.8 higher than the pH of the first buffer solution. In one embodiment, the pH of the second buffer solution is about 1.9 higher than the pH of the first buffer solution. In one embodiment, the pH of the second buffer solution is about 2.0 higher than the pH of the first buffer solution.

[0044] In one embodiment, the pH of the second buffer solution is about 10-14, about 10-13.5, about 10-13, about 11-14, about 11-13.5, about 11-13, about 11-12.5, about 11-12, or about 11.5-12.5. In one embodiment, the pH of the second buffer solution is about 10-14. In one embodiment, the pH of the second buffer solution is about 10-13.5. In one embodiment, the pH of the second buffer solution is about 10-13. In one embodiment, the pH of the second buffer solution is about 10-12.5. In one embodiment, the pH of the second buffer solution is about 11-14. In one embodiment, the pH of the second buffer solution is about 11-13. In one embodiment, the pH of the second buffer solution is about 11-13. In one embodiment, the pH of the second buffer solution is about 11-12.5. In one embodiment, the pH of the second buffer solution is about 11-12. In one embodiment, the pH of the second buffer solution is about 11.5-12.5. In another embodiment, the pH of the second buffer solution is about 12-13.5. In one embodiment, the UF / DF in step (c) is performed using the second buffer solution, wherein the pH of the second buffer solution is selected to specifically denature the product:template complex. In one embodiment, the UF / DF is performed using the second buffer solution, wherein the pH of the second buffer solution causes the product to release from the template. Those skilled in the art will understand that the product:template complex denatures at higher pH levels compared to the impurity:template complex.

[0045] In one embodiment, the first buffer solution and / or the second buffer solution comprises CABS buffer. In one embodiment, the first buffer solution and / or the second buffer solution comprises CAPS buffer. In one embodiment, the first buffer solution and / or the second buffer solution comprises AMP buffer. In one embodiment, the first buffer solution and / or the second buffer solution comprises CAPSO buffer. In one embodiment, the first buffer solution and / or the second buffer solution comprises carbonate buffer. In one embodiment, the first buffer solution and / or the second buffer solution comprises bicarbonate buffer. In one embodiment, the first buffer solution and / or the second buffer solution comprises phosphate buffer. Suitable buffers may be selected respectively based on the pH required to denature the impurity:template complex and / or the product:template complex.

[0046] Those skilled in the art will understand that a buffer solution is a solution whose pH does not change significantly when diluted under constant temperature conditions or when an acid or base is added. Buffer solutions are resistant to pH changes due to the chemical equilibrium between the acid and its conjugate base.

[0047] In the case of phosphate buffer, phosphoric acid (H3PO4) is a triprotic acid that undergoes stepwise dissociation to form H2PO4. - HPO4 2- and PO4 3- .

[0048] Phosphate buffers perform best in the pH range close to their three pK values: pK1 = 2.12, pK2 = 7.21, and pK3 = 12.44. The Henderson-Hasselbalch Equation is a convenient form for buffer calculations, especially when the pH is one unit above or below the pK value. For example, with tribasic phosphate buffers, at pH 12.44 ± 1 (pH 11.44–13.44), the two main components, HPO4, can be calculated. 2- and PO4 3- The proportion.

[0049] Those skilled in the art will understand that buffer solutions with the desired pH can be prepared in a variety of ways.

[0050] For example, a buffer solution can be prepared from 300 mM tripotassium phosphate and titrated with an acid or base to the desired pH. The resulting buffer solution with the desired pH may contain other phosphate substances besides tripotassium phosphate, such as dipotassium phosphate. As used herein, the term "300 mM tripotassium phosphate buffer" refers to a buffer solution prepared by adding an acid (e.g., H3PO4) to 300 mM tripotassium phosphate until the specified pH is reached. Tripotassium phosphate is also known as tribasic potassium phosphate. Trisodium phosphate is also known as tribasic sodium phosphate.

[0051] Alternatively, the same buffer solution can be prepared by mixing two predetermined phosphates (i.e., dipotassium phosphate and tripotassium phosphate) in water in amounts calculated according to the Henderson-Hasselbalch equation to achieve the specified pH.

[0052] In one embodiment, the first buffer solution and / or the second buffer solution comprises phosphate buffer. In one embodiment, the first buffer solution comprises phosphate buffer. In one embodiment, the second buffer solution comprises phosphate buffer. In one embodiment, the first buffer solution and / or the second buffer solution comprises potassium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution comprises sodium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution comprises dipotassium phosphate, tripotassium phosphate, disodium phosphate, and / or trisodium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution comprises dipotassium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution comprises tripotassium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution comprises disodium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution comprises trisodium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution comprises tripotassium phosphate or trisodium phosphate. In one embodiment, the first buffer solution comprises sodium phosphate, and the second buffer solution also comprises sodium phosphate. In one embodiment, the first buffer solution comprises potassium phosphate, and the second buffer solution also comprises potassium phosphate. In one embodiment, the first buffer solution contains trisodium phosphate, and the second buffer solution also contains trisodium phosphate. In one embodiment, the first buffer solution contains tripotassium phosphate, and the second buffer solution also contains tripotassium phosphate. In one embodiment, the first buffer solution contains potassium phosphate, and the second buffer solution contains sodium phosphate. In one embodiment, the first buffer solution contains tripotassium phosphate, and the second buffer solution contains trisodium phosphate.

[0053] In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 100 to about 1000 mM, about 100 to about 800 mM, about 100 to about 600 mM, about 100 to about 500 mM, about 100 to about 450 mM, about 100 to about 400 mM, about 100 to about 350 mM, about 150 to about 300 mM, about 150 to about 350 mM, about 200 to about 500 mM, about 200 to about 400 mM, about 200 to about 350 mM, about 200 to about 300 mM, about 250 to about 500 mM, about 250 to about 400 mM, or about 250 to about 350 mM phosphate solution.

[0054] In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 100 to about 1000 mM, about 100 to about 800 mM, about 100 to about 600 mM, about 100 to about 500 mM, about 100 to about 450 mM, about 100 to 400 mM, about 100 to 350 mM, about 150 to 300 mM, about 150 to about 350 mM, about 200 to about 500 mM, about 200 to about 400 mM, about 200 to about 350 mM, about 200 to about 300 mM, about 250 to about 500 mM, about 250 to about 400 mM, or about 250 to about 350 mM trisodium phosphate.

[0055] In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 100 to about 500 mM trisodium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 100 to about 450 mM trisodium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 100 to about 400 mM trisodium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 150 to about 350 mM trisodium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 200 to about 500 mM trisodium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 200 to about 450 mM trisodium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 200 to about 400 mM trisodium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 200 to about 350 mM trisodium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 200 to about 300 mM trisodium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 200 mM trisodium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 250 mM trisodium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 300 mM trisodium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 350 mM trisodium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 400 mM trisodium phosphate.

[0056] In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 100 to about 1000 mM, about 100 to about 800 mM, about 100 to about 600 mM, about 100 to about 500 mM, about 100 to about 450 mM, about 100 to about 400 mM, about 100 to about 350 mM, about 150 to about 300 mM, about 150 to about 350 mM, about 200 to about 500 mM, about 200 to about 400 mM, about 200 to about 350 mM, about 200 to about 300 mM, about 250 to about 500 mM, about 250 to about 400 mM, or about 250 to about 350 mM tripotassium phosphate.

[0057] In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 100 to about 500 mM tripotassium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 100 to about 450 mM tripotassium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 100 to about 400 mM tripotassium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 150 to about 350 mM tripotassium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 200 to about 500 mM tripotassium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 200 to about 450 mM tripotassium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 200 to about 400 mM tripotassium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 200 to about 350 mM tripotassium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 200 to about 300 mM tripotassium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 200 mM tripotassium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 250 mM tripotassium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 300 mM tripotassium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 350 mM tripotassium phosphate. In one embodiment, the first buffer solution and / or the second buffer solution are derived from about 400 mM tripotassium phosphate.

[0058] In one embodiment, the first buffer solution is derived from about 100 to about 500 mM tripotassium phosphate, and the second buffer solution is derived from about 100 to about 500 mM tripotassium phosphate. In one embodiment, the first buffer solution is derived from about 100 to about 500 mM trisodium phosphate, and the second buffer solution is derived from about 100 to about 500 mM trisodium phosphate. In one embodiment, the first buffer solution is derived from about 100 to about 500 mM tripotassium phosphate, and the second buffer solution is derived from about 100 to about 500 mM trisodium phosphate. In one embodiment, the first buffer solution is derived from about 200 to about 500 mM tripotassium phosphate, and the second buffer solution is derived from about 200 to about 500 mM trisodium phosphate. In one embodiment, the first buffer solution is derived from about 200 to about 500 mM trisodium phosphate, and the second buffer solution is derived from about 200 to about 500 mM trisodium phosphate. In one embodiment, the first buffer solution is derived from about 200 to about 500 mM tripotassium phosphate, and the second buffer solution is derived from about 200 to about 500 mM trisodium phosphate. In one embodiment, the first buffer solution is derived from about 200 to about 400 mM tripotassium phosphate, and the second buffer solution is derived from about 200 to about 400 mM tripotassium phosphate. In one embodiment, the first buffer solution is derived from about 200 to about 400 mM trisodium phosphate, and the second buffer solution contains about 200 to about 400 mM trisodium phosphate. In one embodiment, the first buffer solution is derived from about 200 to about 400 mM tripotassium phosphate, and the second buffer solution is derived from about 200 to about 400 mM trisodium phosphate. In one embodiment, the first buffer solution is derived from about 200 to about 300 mM tripotassium phosphate, and the second buffer solution is derived from about 200 to about 300 mM tripotassium phosphate. In one embodiment, the first buffer solution is derived from about 200 to about 300 mM trisodium phosphate, and the second buffer solution is derived from about 200 to about 300 mM trisodium phosphate. In one embodiment, the first buffer solution is derived from about 200 to about 300 mM tripotassium phosphate, and the second buffer solution is derived from about 200 to about 300 mM trisodium phosphate. In one embodiment, the first buffer solution is derived from about 200 mM tripotassium phosphate, and the second buffer solution is derived from about 200 mM tripotassium phosphate. In one embodiment, the first buffer solution is derived from about 200 mM tripotassium phosphate, and the second buffer solution contains about 200 mM trisodium phosphate. In one embodiment, the first buffer solution is derived from about 300 mM tripotassium phosphate, and the second buffer solution is derived from about 300 mM tripotassium phosphate.In one embodiment, the first buffer solution is derived from about 300 mM trisodium phosphate, and the second buffer solution is derived from about 300 mM trisodium phosphate. In one embodiment, the first buffer solution is derived from about 300 mM tripotassium phosphate, and the second buffer solution is derived from about 300 mM trisodium phosphate. In one embodiment, the first buffer solution is derived from about 400 mM tripotassium phosphate, and the second buffer solution is derived from about 400 mM tripotassium phosphate. In one embodiment, the first buffer solution is derived from about 400 mM trisodium phosphate, and the second buffer solution is derived from about 400 mM trisodium phosphate. In one embodiment, the first buffer solution is derived from about 400 mM tripotassium phosphate, and the second buffer solution is derived from about 400 mM trisodium phosphate.

[0059] In one embodiment, the first buffer solution comprises potassium phosphate with a pH of about 10-12. In one embodiment, the second buffer solution comprises potassium phosphate with a pH of about 11-13. In one embodiment, the second buffer solution comprises sodium phosphate with a pH of about 11-13. In one embodiment, the first buffer solution is derived from about 200 to about 400 mM tripotassium phosphate with a pH of about 10-12. In one embodiment, the first buffer solution is derived from about 200 mM to about 300 mM tripotassium phosphate with a pH of about 10-11.5. In one embodiment, the second buffer solution is derived from about 200 mM to about 400 mM trisodium phosphate with a pH of about 11-13. In one embodiment, the second buffer solution is derived from about 200 mM to about 400 mM tripotassium phosphate with a pH of about 11-13. In one embodiment, the second buffer solution is derived from about 200 to about 300 mM trisodium phosphate with a pH of about 11.5-12.5. In one embodiment, the second buffer solution is derived from about 200 to about 300 mM tripotassium phosphate at a pH of about 11.5-12.5. In one embodiment, the second buffer solution is derived from about 200 to about 300 mM trisodium phosphate at a pH of about 12. In one embodiment, the second buffer solution is derived from about 200 to about 300 mM tripotassium phosphate at a pH of about 12.

[0060] In one embodiment, the first buffer solution and / or the second buffer solution comprises disodium phosphate and trisodium phosphate. In another embodiment, the first buffer solution and / or the second buffer solution comprises dipotassium phosphate and tripotassium phosphate. A predetermined ratio of disodium phosphate to trisodium phosphate and dipotassium phosphate to tripotassium phosphate can be used to achieve a desired pH.

[0061] Under alkaline pH conditions, oligonucleotides (e.g., impurities and / or products) can become negatively charged, and membranes used for UF / DF (e.g., membranes containing polyethersulfone) can have a negative surface charge. This negative charge can cause oligonucleotides to repel each other at the membrane surface. This can result in oligonucleotides (e.g., impurities or products) not permeating the membrane or permeating in reduced quantities. Without being bound by any theory, it is believed that the presence of ions at a certain concentration reduces the electrostatic interaction between the oligonucleotides and the membrane surface, thereby enhancing oligonucleotide permeation. Both the ionic strength and conductivity of a solution are related to the concentration of ions present in the solution; therefore, either parameter can be used to determine whether the buffer solution can provide sufficient permeate flux during ultrafiltration.

[0062] In one embodiment, the buffer solution of the first buffer solution and / or the second buffer solution simultaneously provides: (1) a desired pH for denaturing the relevant complex and (2) a desired ionic strength for maintaining sufficient permeation flux during ultrafiltration. In one embodiment, a phosphate buffer provides the desired pH and ionic strength of the first buffer solution and / or the second buffer solution. In one embodiment, dipotassium phosphate and tripotassium phosphate provide the desired pH and ionic strength of the first buffer solution and / or the second buffer solution. In one embodiment, disodium phosphate and trisodium phosphate provide the desired pH and ionic strength of the first and / or second buffer solutions.

[0063] In some embodiments, a single buffer solution may not be sufficient to maintain the desired ionic strength for adequate permeation flux during ultrafiltration. Therefore, in one embodiment, the first buffer solution and / or the second buffer solution also contain an ionic strength modifier. This ionic strength modifier may be a salt. This ionic strength modifier may be a metal salt. This ionic strength modifier may be a sodium salt. This ionic strength modifier may be a potassium salt. This ionic strength modifier may be NaCl. This ionic strength modifier may be KCl.

[0064] In one embodiment, the first buffer solution and / or the second buffer solution comprises about 200 mM to about 1500 mM, about 300 mM to about 1500 mM, about 300 mM to about 1200 mM, or about 600 mM to about 900 mM of a cation, optionally a monovalent cation. In one embodiment, the first buffer solution and / or the second buffer solution comprises about 200 mM to about 1500 mM, about 300 mM to about 1500 mM, about 300 mM to about 1200 mM, or about 600 mM to about 900 mM of K. +Ions. In one embodiment, the first buffer solution and / or the second buffer solution contains 200 mM to about 1500 mM, about 300 mM to about 1500 mM, about 300 mM to about 1200 mM, or about 600 mM to about 900 mM Na. + In one embodiment, the first buffer solution and / or the second buffer solution contain about 300 mM to about 1200 mM K+. + In one embodiment, the first buffer solution and / or the second buffer solution contain about 300 mM to about 1200 mM Na+. + Ions. In one embodiment, the first buffer solution and / or the second buffer solution contain about 300 mM to about 900 mM K+. + In one embodiment, the first buffer solution and / or the second buffer solution contain about 300 mM to about 900 mM Na+. + Ions. The ion concentration described here refers to the total concentration of a specific ion in a buffer solution, for example, derived from both the buffer solution and the ionic strength regulator.

[0065] In one embodiment, the conductivity of the first buffer solution and / or the second buffer solution is greater than or equal to about 20 mS / cm. In one embodiment, the conductivity of the first buffer solution and / or the second buffer solution is greater than or equal to about 25 mS / cm. In one embodiment, the conductivity of the first buffer solution and / or the second buffer solution is greater than or equal to about 30 mS / cm. In one embodiment, the conductivity of the first buffer solution and / or the second buffer solution is greater than or equal to about 40 mS / cm. In one embodiment, the conductivity of the first buffer solution and / or the second buffer solution is approximately 20 mS / cm to approximately 150 mS / cm, approximately 20 mS / cm to approximately 120 mS / cm, approximately 20 mS / cm to approximately 100 mS / cm, approximately 20 mS / cm to approximately 80 mS / cm, approximately 20 mS / cm to approximately 60 mS / cm, approximately 25 mS / cm to approximately 150 mS / cm, approximately 25 mS / cm to approximately 120 mS / cm, approximately 25 mS / cm to approximately 100 mS / cm, approximately 25 mS / cm to approximately 80 mS / cm, approximately 25 mS / cm to approximately 60 mS / cm, approximately 30 mS / cm to approximately 100 mS / cm, approximately 30 mS / cm to approximately 80 mS / cm, approximately 30 mS / cm to approximately 70 mS / cm, approximately 30 mS / cm to approximately 60 mS / cm, approximately 30 mS / cm to approximately 50 mS / cm. The conductivity of the first buffer solution and / or the second buffer solution is approximately 25 mS / cm to 120 mS / cm, approximately 25 mS / cm to 100 mS / cm, approximately 25 mS / cm to 80 mS / cm, approximately 25 mS / cm to 60 mS / cm, approximately 30 mS / cm to 100 mS / cm, approximately 30 mS / cm to 60 mS / cm, approximately 30 mS / cm to 50 mS / cm, or approximately 35 mS / cm to 45 mS / cm. In one embodiment, the conductivity of the first buffer solution and / or the second buffer solution is approximately 25 mS / cm to 120 mS / cm, approximately 25 mS / cm to 100 mS / cm, or approximately 25 mS / cm to 80 mS / cm. In one embodiment, the conductivity of the first buffer solution and / or the second buffer solution is from about 25 mS / cm to about 60 mS / cm, from about 30 mS / cm to about 50 mS / cm, or from about 35 mS / cm to about 45 mS / cm. In another embodiment, the conductivity of the first buffer solution and / or the second buffer solution is from about 25 mS / cm to about 120 mS / cm.In one embodiment, the conductivity of the first buffer solution and / or the second buffer solution is from about 25 mS / cm to about 100 mS / cm. In one embodiment, the conductivity of the first buffer solution and / or the second buffer solution is from about 30 mS / cm to about 120 mS / cm. In one embodiment, the conductivity of the first buffer solution and / or the second buffer solution is from about 30 mS / cm to about 100 mS / cm. In one embodiment, the conductivity of the first buffer solution and / or the second buffer solution is from about 25 mS / cm to about 80 mS / cm. In one embodiment, the conductivity of the first buffer solution and / or the second buffer solution is from about 25 mS / cm to about 60 mS / cm. In one embodiment, the conductivity of the first buffer solution and / or the second buffer solution is from about 30 mS / cm to about 60 mS / cm.

[0066] In one embodiment, the template has additional properties that allow the template to be retained in the retentate. In one embodiment, the template is present in a form that allows the template to be retained in the retentate. In one embodiment, the template is present in a form that allows the template to be retained in the second retentate. In one embodiment, the template is present in a form that allows the template to be retained in the second retentate of step (c) of the disclosed method. It should be understood that if the template is present in a form that allows the template to be retained in the second retentate, then the template in the complex (e.g., impurity:template complex or product:template complex) will also be retained in the retentate. In one embodiment, the product:template complex is retained in the first retentate of step (b) of the disclosed method. In one embodiment, the template is present in a form with a molecular weight greater than the membrane's molecular weight cutoff (MWCO). In one embodiment, the template is present in the form of multiple repeated copies of the template linked together, the copies being separated by a linker sequence or not. In one embodiment, the template is attached to a support material. In one embodiment, the molecular weight of the template and the attached support material is greater than the membrane's molecular weight cutoff (MWCO). In one embodiment, the molecular weight of the template and the attached support material is greater than the molecular weight cutoff (MWCO) of the membrane, so that the template is retained in the retentate.

[0067] The support material can be a soluble support material. Soluble support materials can be selected from the group consisting of: polyethylene glycol, soluble organic polymers, linker sequences, RNA, DNA, proteins, dendritic polymers, polysaccharides, oligosaccharides, and carbohydrates. The support material can be polyethylene glycol (PEG). The support material can also be an insoluble support material. The support material can be a solid support material. Solid support materials can be selected from the group consisting of: glass beads, polymer beads, fibrous supports, membranes, streptavidin-coated beads, and cellulose. Solid support materials can be streptavidin-coated beads. The solid support material can be part of the reaction vessel itself, for example, the reaction wall.

[0068] In one embodiment, one or more copies of the template are attached to a support material. In one embodiment, multiple duplicate copies of the template are attached to the support material in a continuous manner via a single attachment point. The multiple duplicate copies of the template may be spaced apart by joints. The multiple duplicate copies of the template may be direct duplicates, for example, they are not spaced apart by joints. The support material may have multiple attachment points to which the template can be attached. The support material is also referred to as a "hub". Each attachment point may itself have multiple duplicate copies of the template attached in a continuous manner. The multiple duplicate copies of the template may be spaced apart by joints. The multiple duplicate copies of the template may be direct duplicates, for example, they are not spaced apart by joints. In one embodiment, multiple copies of the template are attached to the support material at multiple attachment points. In one embodiment, two or more copies of the template are attached to the support material, for example, attached to multiple attachment points. In one embodiment, two, three, four, five, six, seven, eight, nine, or ten templates are attached to the support material, for example, attached to multiple attachment points. In one embodiment, three copies of the template are attached to the support material, for example, attached to multiple attachment points. In one embodiment, four copies of the template are attached to a support material, such as at multiple attachment points. In one embodiment, five copies of the template are attached to a support material, such as at multiple attachment points. In one embodiment, six copies of the template are attached to a support material, such as at multiple attachment points. In one embodiment, six copies of the template are attached to a support material, wherein the support material is a "hub" containing polyethylene glycol (PEG).

[0069] In one embodiment, the template is recycled for future use. In another embodiment, the template is recycled from the second retentate of step (c) of the method and recycled for future use.

[0070] In one embodiment, UF / DF is performed using a membrane. In one embodiment, the UF / DF in steps (b) and / or (c) is performed using a membrane. The membrane may have a molecular weight cutoff (MWCO) that allows impurities and products to pass through. The membrane may have a molecular weight cutoff (MWCO) that prevents the template from passing through. When the membrane is in a form that retains the template in the retentate, for example, when the template is attached to a support material, the membrane may have a molecular weight cutoff (MWCO) greater than the molecular weight of the impurities and products and less than the molecular weight of the template. The membrane may have a molecular weight cutoff (MWCO) of about 10 to about 100 kDa, about 10 to about 80 kDa, about 10 to about 50 kDa, about 10 to about 40 kDa, about 20 to about 80 kDa, about 20 to about 50 kDa, about 20 to about 40 kDa, about 30 to about 50 kDa, about 25 to about 35 kDa, or about 20 to about 30 kDa. The membrane may have a molecular weight cutoff (MWCO) of about 10 to about 40 kDa, about 20 to about 40 kDa, or about 20 to about 30 kDa. In one embodiment, the membrane has a molecular weight cutoff (MWCO) of about 10 to about 40 kDa. In one embodiment, the membrane has a molecular weight cutoff (MWCO) of about 20 to about 40 kDa. In one embodiment, the membrane has a molecular weight cutoff (MWCO) of about 20 to about 30 kDa. In one embodiment, the membrane has a molecular weight cutoff (MWCO) of about 25 to about 35 kDa. In one embodiment, the membrane has a molecular weight cutoff (MWCO) of about 30 kDa.

[0071] The second membrane may have a molecular weight cutoff (MWCO) that prevents the product from passing through during filtration. Such membranes can be used to replace / remove buffer solutions in the product solution. For example, such membranes can be used when the second permeate from step (c) is subjected to UF / DF to form a third filtrate containing the product and substantially free of the second buffer solution. In one embodiment, the membrane has a molecular weight cutoff (MWCO) of about 1 to about 10 kDa, about 1 to about 7 kDa, about 1 to about 5 kDa, about 2 to about 5 kDa, or about 2 to about 4 kDa. In one embodiment, the membrane has a molecular weight cutoff (MWCO) of about 2 to about 5 kDa. In one embodiment, the membrane has a molecular weight cutoff (MWCO) of about 3 kDa.

[0072] In one embodiment, the membrane comprises polysulfone, polypropylene, cellulose acetate, polylactic acid, cellulose nitrate, mixed cellulose esters, regenerated cellulose, polyethersulfone (PES), polyamide, cellulose derivatives, polyvinylidene fluoride, polytetrafluoroethylene, or polycarbonate track-etched membranes. In one embodiment, the membrane is part of a filtration element, including hollow fiber, tubular, spiral wound, box-type, plate-type, or frame-type membrane types. The membrane may contain polyethersulfone. Commercially available membranes are manufactured by various suppliers, such as Millipore Corporation (Billerica, Mass.), Pall Corporation (East Hills, NY), GE Healthcare Sciences (Piscataway, NJ), and Sartorius Corporation (Goettingen, Germany).

[0073] In one embodiment, the product is synthesized via an enzyme-catalyzed ligation reaction. In another embodiment, the product is synthesized via an enzymatic ligation reaction of short polymers. In yet another embodiment, the enzymatic ligation reaction is carried out using a ligase. The product can be synthesized via a process comprising the following steps: i) Provide a template oligonucleotide (I) that is sequence complementary to the product; ii) Provide an oligonucleotide set containing short-mers (II); iii) Bring (I) and (II) into contact under conditions that allow annealing; and iv) Short polymers are linked by an enzymatic ligation reaction using ligases to form a product.

[0074] In one embodiment, the short polymer is 3 to 20 nucleotides long. In one embodiment, the short polymer is 3 to 15 nucleotides long. In one embodiment, the short polymer is 5 to 10 nucleotides long. In one embodiment, the short polymer is 5 to 8 nucleotides long. In one embodiment, the short polymer is 5, 6, 7, or 8 nucleotides long. In a particular embodiment, there are three short polymers: a 5' short polymer of 7 nucleotides, an intermediate short polymer of 6 nucleotides, and a 3' short polymer of 7 nucleotides, which, when linked together, form a 20-nucleotide-long product (“20-mer”). In a particular embodiment, there are three short polymers: a 5' short polymer of 6 nucleotides, an intermediate short polymer of 8 nucleotides, and a 3' short polymer of 6 nucleotides, which, when linked together, form a 20-nucleotide-long oligonucleotide (“20-mer”). In a particular embodiment, there are three short polymers: a 5' short polymer (5 nucleotides long), an intermediate short polymer (10 nucleotides long), and a 3' short polymer (5 nucleotides long), which, when linked together, form a 20-nucleotide-long product (“20-mer”). In a particular embodiment, there are four short polymers: a 5' short polymer (5 nucleotides long), a 5'-intermediate short polymer (5 nucleotides long), an intermediate-3' short polymer (5 nucleotides long), and a 3'-short polymer (5 nucleotides long), which, when linked together, form a 20-nucleotide-long product (“20-mer”).

[0075] In one embodiment, the disclosed method is used to purify a product, wherein the product is 10 to 200 nucleotides long. In one embodiment, the product is 10 to 150 nucleotides long. In one embodiment, the product is 10 to 100 nucleotides long. In one embodiment, the product is 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, or 10 to 30 nucleotides long. In one embodiment, the product is 10 to 50 nucleotides long. In one embodiment, the product is 10 to 40 nucleotides long. In one embodiment, the product is 10 to 35 nucleotides long. In one embodiment, the product is 10 to 30 nucleotides long. In another embodiment of the invention, the product is 15 to 35 nucleotides long. In another embodiment of the invention, the product is 15 to 30 nucleotides long. In one embodiment, the product is 20 to 30 nucleotides long. In one embodiment, the product is 20 to 35 nucleotides long. In one embodiment, the product is 20 to 32 nucleotides long. In embodiments of the present invention, the product is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In embodiments of the present invention, the product is 20 nucleotides long, "20-mer". In embodiments of the present invention, the product is 21 nucleotides long, "21-mer". In embodiments of the present invention, the product is 22 nucleotides long, "22-mer". In embodiments of the present invention, the product is 23 nucleotides long, "23-mer". In embodiments of the present invention, the product is 24 nucleotides long, "24-mer". In embodiments of the present invention, the product is 25 nucleotides long, "25-mer". In embodiments of the present invention, the product is 26 nucleotides long, "26-mer". In embodiments of the present invention, the product is 27 nucleotides long, "27-mer". In embodiments of the present invention, the product is 28 nucleotides long, "28-mer". In embodiments of the present invention, the product is 29 nucleotides long, "29-mer". In embodiments of the present invention, the product is 30 nucleotides long, "30-mer". In one embodiment of the invention, the product is 31 nucleotides long, "31-mer". In another embodiment of the invention, the product is 32 nucleotides long, "32-mer".

[0076] In an embodiment of the present invention, the method is used to purify a therapeutic product.

[0077] The product according to the invention may have at least one main chain modification and / or at least one sugar modification and / or at least one base modification.

[0078] One embodiment of the present invention provides a method as disclosed herein, wherein the product contains at least one modified nucleotide residue. In one embodiment, the at least one modified nucleotide comprises modifications to the sugar moiety, modifications to the nucleobase, and / or modifications to the backbone. In another embodiment, the modification is located at the 2' position of the sugar moiety.

[0079] The product may contain sugar modifications, i.e., modified forms of the ribosyl moiety, such as 2'-O-modified RNA, such as 2'-O-alkyl or 2'-O-(substituted)alkyl, for example 2'-O-methyl, 2'-O-(2-cyanoethyl), 2'-O-(2-methoxy)ethyl (2'-MOE), 2'-O-(2-thiomethyl)ethyl, 2'-O-butyryl, 2'-O-propynyl, 2'-O-allyl, 2'-O-(3-amino)propyl, 2'-O-(3-(dimethylamino)propyl), 2'-O-(2-amino)ethyl, 2'-O-(2-(dimethylamino)ethyl); 2'-deoxy (DNA); 2'-O-(haloalkoxy)methyl (Arai K). et al. Bioorg. Med. Chem. 2011, 21, 6285), such as 2'-O-(2-chloroethoxy)methyl (MCEM), 2'-O-(2,2-dichloroethoxy)methyl (DCEM); 2'-O-alkoxycarbonyl, such as 2'-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl] (MCE), 2'-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DCME); 2'-halogenated, such as 2'-F, FANA (2'-F arabinosyl nucleic acid); carbasugar and azasugar modifications; 3'-O-alkyl, such as 3'-O-methyl, 3'-O-butyryl, 3'-O-propynyl; and their derivatives.

[0080] In embodiments of the invention, the sugar modification is selected from the group consisting of: 2'-fluoro (2'-F), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), and 2'-amino. In yet another embodiment, the modification is 2'-MOE.

[0081] Other sugar modifications include “bridged” or “bicyclic” nucleic acids (BNAs), such as locked nucleic acids (LNAs), xylo-LNAs, α-L-LNAs, β-D-LNAs, cEt(2'-O, 4'-C-bound ethyl LNAs), cMOEt(2'-O, 4'-C-bound methoxyethyl) LNAs, ethylene-bridged nucleic acids (ENAs), tricyclic DNAs; unlocked nucleic acids (UNAs); cyclohexenyl nucleic acids (CeNAs), atroitol nucleic acids (ANAs), hexitol nucleic acids (HNAs), fluorinated HNAs (F-HNAs), pyranosyl-RNAs (p-RNAs), 3'-deoxypyranosyl-DNAs (p-DNAs); morpholino compounds (e.g., in PMOs, PPMOs, PMOPlus, PMO-Xs); and their derivatives.

[0082] The products of this invention may include other modifications, such as peptide-base nucleic acid (PNA), boron-modified PNA, pyrrolyloxy-peptide nucleic acid (POPNA), ethylene glycol or glycerol-based nucleic acid (GNA), threoyl nucleic acid (TNA), acyclic threonyl nucleic acid (aTNA), oligonucleotides having integrated bases and backbone (ONIB), pyrrolidine-amide oligonucleotides (POM); and derivatives thereof.

[0083] In embodiments of the present invention, the modified product comprises phosphoryldiamine morpholine oligomer (PMO), locked nucleic acid (LNA), peptide nucleic acid (PNA), such as ( S Bridging nucleic acid (BNA) or SPIEGELMER for )-cEt-BNA.

[0084] In a further embodiment, the modification is located in the nucleobase. Base modifications include modified forms of native purine and pyrimidine bases (e.g., adenine, uracil, guanine, cytosine, and thymine), such as inosine, hypoxanthine, orotic acid, agmatidine, lysidine, 2-thiopyrimidines (e.g., 2-thiourea pyrimidine, 2-thiothymine), G-type clips and their derivatives, 5-substituted pyrimidines (e.g., 5-methylcytosine, 5-methyluracil, 5-halouracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, Super... T), 2,6-diaminopurine, 7-deazoguanine, 7-deazoadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazoguanine, 8-aza-7-deazoadenine, 8-aza-7-deazo-2,6-diaminopurine, Super G, Super A and N4-ethylcytosine or derivatives thereof; N 2 -Cyclopentylguanine (cPent-G), N 2-Cyclopentyl-2-aminopurine (cPent-AP) and N 2 -propyl-2-aminopurine (Pr-AP) or its derivatives; and degenerate or universal bases, such as 2,6-difluorotoluene, or absent bases, such as those without a base site (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose; or pyrrolidine derivatives in which the epoxide has been replaced by nitrogen (azaribose)). Examples of derivatives of Super A, Super G, and Super T can be found in US6683173. cPent-G, cPent-AP, and Pr-AP have shown reduced immunostimulatory effects when incorporated into siRNA (Peacock H. et al. J. Am. Chem. Soc. (2011), 133, 9200).

[0085] In embodiments of the invention, the nucleobase modification is selected from the group consisting of 5-methylpyrimidine, 7-dezoguanosine, and abase-free nucleotides. In one embodiment, the modification is 5-methylcytosine.

[0086] The products of this invention may include backbone modifications, for example, modified forms of phosphodiester present in RNA, such as thiophosphate (PS), dithiophosphate (PS2), phosphonoacetate (PACE), phosphonoacetamide (PACA), thiophosphonoacetate, thiophosphonoacetamide, thiophosphate prodrug, H-phosphonate, methylphosphonate, methylthiophosphonate, methylphosphate, methylthiophosphate, ethylphosphate, ethylthiophosphate, boran phosphate, boran thiophosphate, methylborane phosphate, methylborane phosphate, methylborane thiophosphate, methylborane phosphonate, methylborane phosphonate, and derivatives thereof. Further modifications include phosphoramide, aminophosphate, N3'→P5' aminophosphate, phosphoridamide, thiophosphonidamide, aminosulfonate, dimethylene sulfoxide, sulfonate, triazole, oxaloyl, carbamate, methyleneimine (MMI), and thioacetamide nucleic acid (TANA); and derivatives thereof.

[0087] In a further embodiment, the modification is located in the main chain and is selected from the group consisting of phosphate thioesters (PS), aminophosphate esters (PA), and phosphoryldiamine. In an embodiment of the invention, the modified oligonucleotide is a phosphoryldiamine morpholine oligomer (PMO). PMO has a main chain with a methylene morpholine ring and phosphoryldiamine linkages. In an embodiment of the invention, the product has a phosphate thioester (PS) main chain.

[0088] In embodiments of the present invention, the oligonucleotide comprises one or more modifications selected from the group consisting of: 2'-fluoro(2'-F), 2'-O-methyl(2'-OMe), 2'-O-methoxyethyl(2'-MOE), 2'-amino, 5-methylpyrimidine, 7-dezoguanosine, abasic nucleotide, phosphate thioester (PS), aminophosphate (PA), and phosphoric acid diamine. In embodiments of the present invention, the oligonucleotide comprises one or more modifications selected from the group consisting of: 2'-fluoro(2'-F), 2'-O-methyl(2'-OMe), 2'-O-methoxyethyl(2'-MOE), 5-methylpyrimidine, and phosphate thioester (PS). In embodiments of the present invention, the oligonucleotide comprises one or more modifications selected from the group consisting of: 2'-fluoro(2'-F), 2'-O-methyl(2'-OMe), 2'-O-methoxyethyl(2'-MOE), 5-methylcytosine, 5-methyluracil, and phosphate thioester (PS).

[0089] In embodiments of the present invention, the oligonucleotide comprises a combination of two or more modifications as described above. In embodiments of the present invention, the oligonucleotide comprises two or more modifications selected from the group consisting of: 2'-fluoro(2'-F), 2'-O-methyl(2'-OMe), 2'-O-methoxyethyl(2'-MOE), 2'-amino, 5-methylpyrimidine, 7-dezoguanosine, abasic nucleotide, phosphate thioester (PS), aminophosphate (PA), and phosphoric acid diamine. In embodiments of the present invention, the oligonucleotide comprises two or more modifications selected from the group consisting of: 2'-fluoro(2'-F), 2'-O-methyl(2'-OMe), 2'-O-methoxyethyl(2'-MOE), 5-methylpyrimidine, and phosphate thioester (PS). In embodiments of the invention, the oligonucleotide comprises two or more modifications selected from the group consisting of: 2'-fluoro (2'-F), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 5-methylcytosine, 5-methyluracil, and phosphate thioester (PS).

[0090] In embodiments of the invention, the product is a gapmer. In embodiments of the invention, the 5' and 3' flanks of the gapmer comprise, or are composed of, 2'-MOE-modified nucleotides. In embodiments of the invention, the gap segment of the gapmer comprises, or is composed of, a hydrogen-containing nucleotide at the 2' position of the sugar moiety, i.e., DNA-like. In embodiments of the invention, the 5' and 3' flanks of the gapmer are composed of 2'-MOE-modified nucleotides, and the gap segment of the gapmer is composed of a hydrogen-containing nucleotide (i.e., a deoxyribonucleotide) at the 2' position of the sugar moiety. In embodiments of the invention, the 5' and 3' flanks of the gapmer are composed of 2'-MOE-modified nucleotides, and the gap segment of the gapmer is composed of a hydrogen-containing nucleotide (i.e., a deoxyribonucleotide) at the 2' position of the sugar moiety, and all nucleotide linkages are phosphate thioester linkages.

[0091] In one embodiment, the product is the sense strand of a double-stranded oligonucleotide. In another embodiment, the product is the antisense strand of a double-stranded oligonucleotide. The purified sense strand and the purified antisense strand can form a double-stranded oligonucleotide, such as siRNA.

[0092] In one embodiment, the solution of step (a) comprises about 1 to about 100 mg / ml, about 5 to about 100 mg / ml, about 5 to about 80 mg / ml, about 5 to about 60 mg / ml, about 10 to about 80 mg / ml, about 10 to about 60 mg / ml, about 20 to about 60 mg / ml, about 30 to about 60 mg / ml, about 35 to about 55 mg / ml, about 30 to about 50 mg / ml, about 40 to about 50 mg / ml, or about 35 to about 45 mg / ml template. For the avoidance of doubt, the weight of the template does not include the weight of the connector or support material to which the template is attached. In one embodiment, the solution of step (a) comprises about 5 to about 60 mg / ml, about 10 to about 60 mg / ml, about 20 to about 60 mg / ml, about 30 to about 60 mg / ml, about 35 to about 55 mg / ml, or about 35 to about 45 mg / ml template. In one embodiment, the solution of step (a) comprises about 20 to about 60 mg / ml template. In one embodiment, the solution of step (a) contains about 30 to about 60 mg / ml of template. In one embodiment, the solution of step (a) contains about 40 to about 50 mg / ml of template. In one embodiment, the solution of step (a) contains about 40 mg / ml of template. The concentration of template in the solution of step (a) can be based on the initial concentration of template in the enzyme-catalyzed ligation reaction. In one embodiment, the solution of step (a) contains about 4 to about 120 mg / ml, about 5 to about 120 mg / ml, about 10 to about 120 mg / ml, about 20 to about 120 mg / ml, about 40 to about 120 mg / ml, about 60 to about 120 mg / ml, about 70 to about 110 mg / ml, or about 70 to about 90 mg / ml of total oligonucleotides. The total oligonucleotide concentration can be an estimate based on doubling the template concentration.

[0093] In one embodiment, the solution of step (a) comprises about 0.5 to about 10 mM, about 0.5 to about 9 mM, about 0.5 to about 8 mM, about 0.5 to about 7 mM, about 0.5 to about 6 mM, about 0.5 to about 5 mM, about 1 to about 10 mM, about 1 to about 9 mM, about 1 to about 8 mM, about 1 to about 7 mM, about 1 to about 6 mM, about 2 to about 10 mM, about 2 to about 9 mM, about 2 to about 8 mM, about 2 to about 7 mM, about 2 to about 6 mM, about 2 to about 5 mM, about 3 to about 10 mM, about 3 to about 9 mM, about 3 to about 8 mM, about 3 to about 7 mM, about 3 to about 6 mM, or about 3 to about 5 mM template. In one embodiment, the solution of step (a) comprises about 2 to about 8 mM template. In one embodiment, the solution of step (a) comprises about 2 to about 7 mM template. In one embodiment, the solution of step (a) comprises about 3 to about 7 mM template. In one embodiment, the solution of step (a) contains about 2 to about 6 mM of template. In another embodiment, the solution of step (a) contains about 3 to about 6 mM of template.

[0094] In one embodiment, the method is performed at room temperature. In one embodiment, the UF / DF step is performed at room temperature. In one embodiment, the UF / DF of steps (b) and / or (c) is performed at room temperature. In one embodiment, the UF / DF of steps (b) and / or (c) is performed at about 20 to about 30 degrees Celsius, about 22 to about 28 degrees Celsius, about 24 to about 28 degrees Celsius, or about 25 to about 27 degrees Celsius. In one embodiment, the UF / DF of steps (b) and / or (c) is performed at about 25 to about 27 degrees Celsius. One advantage of using such temperatures is that these temperatures have little effect on the stability and / or degradation of the oligonucleotides.

[0095] In one embodiment, the UF / DF in steps (b) and / or (c) is carried out at a temperature below about 70 degrees Celsius, below about 60 degrees Celsius, or below about 50 degrees Celsius. In one embodiment, the UF / DF in steps (b) and / or (c) is carried out at a temperature below about 50 degrees Celsius. In one embodiment, the UF / DF in steps (b) and / or (c) is carried out at a temperature of about 20 to 50 degrees Celsius or about 20 to 49 degrees Celsius. In one embodiment, pH may also be additionally combined with increased temperature to achieve the release of impurities and / or products from the template. For example, the pH of the first and / or second buffer solutions may be 12, and the UF / DF is carried out at 30-40 degrees Celsius.

[0096] In one embodiment, UF / DF is carried out in at least 3, or at least 4, or at least 5 percolation volumes. In another embodiment, UF / DF is carried out in 3 to 30, or 3 to 30, or 3 to 20, or 10 to 20, or 10 to 12, or 3 to 10, or 5 to 10, or 5 to 8 percolation volumes.

[0097] In one embodiment, the maximum transmembrane pressure (TMP) is 5 barg, 4 barg, 3 barg, or 2 barg. In one embodiment, the maximum transmembrane pressure (TMP) is 4 barg.

[0098] In one embodiment, UF / DF is performed using a tangential flow filtration system. The method may also include additional steps. In one embodiment, the method further includes a step of replacing the buffer solution with water. This step may be referred to as desalting. In one embodiment, the method further includes step (d): performing UF / DF on the second permeate from step (c) to form a third retentate containing the product and substantially free of the second buffer solution, wherein the UF / DF is performed using water and a membrane having a molecular weight cutoff (MWCO) of about 1 kDa to about 5 kDa. The method further includes lyophilizing the third retentate from step (d) to form a lyophilized composition containing the product.

[0099] In one embodiment, the method further includes a step of recovering the template. The recovered template can be recycled for future use. In one embodiment, the method further includes step (e): subjecting the second retentate from step (c) to UF / DF to form a fourth retentate containing the template, wherein the UF / DF is performed using a third buffer solution with a pH of about 7-8. The third buffer solution may contain tris.HCl. The third buffer solution may contain 100 mM tris.HCl.

[0100] In one embodiment, the method further includes the steps of improving product purity and / or removing residual template. In one embodiment, the method further includes a UF / DF step on the second permeate from step (c), wherein the UF / DF is performed using a second buffer solution.

[0101] In one embodiment, where the product is synthesized via an enzyme-catalyzed ligation reaction, the preparation of the solution in step (a) includes the following steps: (a1) Remove enzymes from the ligation reaction by filtration to provide filtrate; (a2) Treat the filtrate with EDTA to provide a mixture; and (a3) Concentrate the mixture from step (a2) to provide the solution from step (a).

[0102] In one implementation, the steps of the method are performed sequentially. In one implementation, step (a) is performed first, followed by step (b), and then step (c). In another implementation, steps (a1)-(a3) are performed first, followed by step (b), and then step (c).

[0103] In one implementation, steps (d) and (e) are performed sequentially. In another implementation, steps (d) and (e) are performed in parallel. In yet another implementation, step (e) is performed before step (d) but after step (c).

[0104] In one implementation, the method is carried out using a continuous or semi-continuous flow process.

[0105] In embodiments of the invention, this process is used for the large-scale purification of products (particularly therapeutic single-chain oligonucleotides). In the context of the invention, large-scale purification of oligonucleotides refers to purification on a scale of 1 liter or more; for example, the process is carried out with 1 L or more of the solution from step (a). Alternatively or further, in the context of the invention, large-scale purification of oligonucleotides refers to the purification of products in grams, particularly purifying products of 2 grams, 5 grams, or 10 grams or more. In embodiments of the invention, the amount of purified oligonucleotide product is in grams or kilograms or greater. In embodiments of the invention, the amount of purified oligonucleotide product is in grams. In embodiments of the invention, the amount of purified product is greater than or equal to: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 grams. In embodiments of the present invention, the amount of purified oligonucleotide product is greater than or equal to: 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 950 grams. In embodiments of the present invention, the amount of purified oligonucleotide product is 500 grams or more. In embodiments of the present invention, the generated oligonucleotide product is expressed in kilograms. In embodiments of the present invention, the amount of purified oligonucleotide product is 1 kg or more. In embodiments of the present invention, the amount of purified oligonucleotide product is greater than or equal to: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kg. In embodiments of the present invention, the amount of purified oligonucleotide product is greater than or equal to: 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 kg.

[0106] In embodiments of the present invention, the amount of purified product is between 10 grams and 100 kg. In embodiments of the present invention, the amount of purified product is between 10 grams and 50 kg. In embodiments of the present invention, the amount of purified product is between 100 grams and 100 kg. In embodiments of the present invention, the amount of purified product is between 100 grams and 50 kg. In embodiments of the present invention, the amount of purified product is between 500 grams and 100 kg. In embodiments of the present invention, the amount of purified product is between 500 grams and 50 kg. In embodiments of the present invention, the amount of purified product is between 1 kg and 50 kg. In embodiments of the present invention, the amount of purified product is between 10 kg and 50 kg.

[0107] In embodiments of the invention, oligonucleotide purification is carried out on a scale greater than or equal to the following: 2, 3, 4, 5, 6, 7, 8, 9, 10 L, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 L of the solution from step (a). In embodiments of the invention, oligonucleotide purification is carried out on a scale greater than or equal to the following: 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 L, for example, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 L of solution. In embodiments of the present invention, oligonucleotide purification is carried out on a scale greater than or equal to the following: 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 liters, for example, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 L solutions.

[0108] In an embodiment of the invention, for the solution of step (a), the method is carried out in a volume of about 10,000 L, about 5,000 L, about 2,000 L, about 1,000 L, about 500 L, about 125 L, about 50 L, about 20 L, about 10 L, or about 5 L.

[0109] In an embodiment of the invention, for the solution of step (a), the method is carried out in a volume of about 5 to about 10,000 L, about 10 to about 5,000 L, about 20 to about 2,000 L, or 50 to 1,000 L.

[0110] One embodiment of the invention provides a method as described previously herein, wherein the purified product has a purity greater than or equal to about 80%. The purified product refers to the product in the second permeate of step (c) or the product in any subsequent step, and purity is measured by HPLC (% area). Purity can be assessed as a percentage of total oligonucleotides. In a further embodiment, the product purity is greater than or equal to about 90%. In a further embodiment, the product purity is greater than or equal to about 95%. In a further embodiment, the product purity is greater than or equal to about 96%. In a further embodiment, the product purity is greater than or equal to about 97%. In a further embodiment, the product purity is greater than or equal to about 98%. In a further embodiment, the product purity is greater than or equal to about 99%. In a further embodiment, the product purity is greater than or equal to 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. In one embodiment, the purity of the product in the second permeate of step (c) is at least: about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%.

[0111] The present invention disclosed herein provides an improved purification method utilizing the binding properties of oligonucleotides. By providing a template that is 100% complementary to the product and controlling the process conditions, the product can be released and separated under specific conditions, thereby obtaining a product of high purity.

[0112] This invention includes, but is not limited to, the following terms / implementations: 1. A method for purifying a product, wherein the product is a single-stranded oligonucleotide, the method comprising: (a) Obtaining a solution comprising (i) an impurity: template complex and (ii) a product: template complex, wherein the template is an oligonucleotide containing a sequence complementary to the product. (b) The solution is subjected to ultrafiltration / difiltration (UF / DF) to form a first retentate containing the product: template complex and a first permeate containing the impurity, wherein the UF / DF is performed using a first buffer solution with a pH of approximately 9-13. (c) The first retentate from step (b) is subjected to UF / DF to form a second retentate containing the template and a second permeate containing the product, wherein the UF / DF is performed using a second buffer solution with a pH greater than that of the first buffer solution.

[0113] 2. A method for purifying a product, wherein the product is a single-stranded oligonucleotide, the method comprising: (b) subjecting the solution containing (i) the impurity:template complex and (ii) the product:template complex to ultrafiltration / difiltration (UF / DF) to form a first retentate containing the product:template complex and a first permeate containing the impurity, wherein the UF / DF is performed using a first buffer solution with a pH of about 9-13, and wherein the template is an oligonucleotide containing a sequence complementary to the product. (c) The first retentate from step (b) is subjected to UF / DF to form a second retentate containing the template and a second permeate containing the product, wherein the UF / DF is performed using a second buffer solution with a pH greater than that of the first buffer solution.

[0114] 3. The method according to Clause 1 or Clause 2, wherein the pH of the second buffer solution is about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9 or about 2.0 higher than the pH of the first buffer solution.

[0115] 4. The method according to any one of the preceding clauses, wherein the pH of the second buffer solution is about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, or about 1.5 higher than the pH of the first buffer solution.

[0116] 5. The method according to any one of the preceding clauses, wherein the pH of the first buffer solution is about 9-12.5, about 9-12, about 9-11.5, about 9-11, about 10-12.5, about 10-12, about 10-11.5, about 10-11, or about 10.5-11.5.

[0117] 6. The method according to any one of the preceding clauses, wherein the pH of the first buffer solution is about 10-12.

[0118] 7. The method according to any one of the preceding clauses, wherein the pH of the second buffer solution is about 10-14, about 10-13.5, about 10-13, about 11-14, about 11-13.5, about 11-13, about 11-12.5, about 11-12, or about 11.5-12.5.

[0119] 8. The method according to any one of the preceding clauses, wherein the pH of the second buffer solution is about 11-13.

[0120] 9. The method according to any one of the preceding clauses, wherein the conductivity of the first buffer solution and / or the second buffer solution is about 25 mS / cm to about 120 mS / cm, about 25 mS / cm to about 100 mS / cm, about 25 mS / cm to about 80 mS / cm, about 25 mS / cm to about 60 mS / cm, about 30 mS / cm to about 100 mS / cm, or about 30 mS / cm to about 80 mS / cm.

[0121] 10. The method according to any one of the preceding clauses, wherein the conductivity of the first buffer solution and / or the second buffer solution is from about 25 mS / cm to about 100 mS / cm.

[0122] 11. The method according to any one of the preceding clauses, wherein the conductivity of the first buffer solution and / or the second buffer solution is from about 30 mS / cm to about 80 mS / cm.

[0123] 12. The method according to any one of the preceding clauses, wherein the first buffer solution and / or the second buffer solution comprises a phosphate buffer solution.

[0124] 13. The method according to any one of the preceding clauses, wherein the first buffer solution and / or the second buffer solution comprises potassium phosphate, optionally dipotassium phosphate and / or tripotassium phosphate.

[0125] 14. The method according to any one of the preceding clauses, wherein the first buffer solution and / or the second buffer solution comprises tripotassium phosphate.

[0126] 15. The method according to any one of the preceding clauses, wherein the first buffer solution and / or the second buffer solution are derived from about 100 to 500 mM, about 100 to 400 mM, about 150 to 350 mM, 200 mM to about 400 mM, or about 200 to 300 mM tripotassium phosphate.

[0127] 16. The method according to any one of the preceding clauses, wherein the first buffer solution and / or the second buffer solution comprises sodium phosphate, optionally disodium phosphate and / or trisodium phosphate.

[0128] 17. The method according to any one of the preceding clauses, wherein the first buffer solution and / or the second buffer solution comprises trisodium phosphate.

[0129] 18. The method according to any one of the preceding clauses, wherein the first buffer solution and / or the second buffer solution are derived from about 100 to 500 mM, about 100 to 400 mM, about 150 to 350 mM, 200 mM to about 400 mM, or about 200 to 300 mM trisodium phosphate.

[0130] 19. The method according to any one of the preceding clauses, wherein the first buffer solution comprises potassium phosphate and the second buffer solution comprises sodium phosphate.

[0131] 20. The method according to any one of the preceding clauses, wherein the first buffer solution contains potassium phosphate with a pH of about 10-12.

[0132] 21. The method according to any one of the preceding clauses, wherein the second buffer solution comprises sodium phosphate with a pH of about 11-13.

[0133] 22. The method according to any one of the preceding clauses, wherein the first buffer solution comprises tripotassium phosphate and the second buffer solution comprises trisodium phosphate.

[0134] 23. The method according to any one of the preceding clauses, wherein the first buffer solution is derived from about 200 mM to about 400 mM tripotassium phosphate at a pH of about 10-12.

[0135] 24. The method according to any one of the preceding clauses, wherein the second buffer solution is derived from about 200 mM to about 400 mM trisodium phosphate at a pH of about 11-13.

[0136] 25. The method according to any one of the preceding clauses, wherein the first buffer solution and / or the second buffer solution further comprises an ionic strength modifier.

[0137] 26. The method according to Clause 25, wherein the ionic strength modifier comprises a cation, optionally a sodium ion or a potassium ion.

[0138] 27. The method according to any one of the preceding clauses, wherein the first buffer solution and / or the second buffer solution comprises about 200 mM to about 1500 mM, about 200 mM to about 1200 mM, about 300 mM to about 1500 mM, about 300 mM to about 1200 mM, about 400 mM to about 800 mM, or about 600 mM to about 900 mM of cations, optionally sodium or potassium ions.

[0139] 28. The method according to any one of the preceding clauses, wherein the first buffer solution and / or the second buffer solution contains about 300 mM to about 1200 mM sodium ions.

[0140] 29. The method according to any one of the preceding clauses, wherein the first buffer solution and / or the second buffer solution contains about 300 mM to about 1200 mM potassium ions.

[0141] 30. The method according to any one of the preceding clauses, wherein the template exists in a form that allows the template to remain in the second retentate.

[0142] 31. The method according to any one of the preceding clauses, wherein the template is attached to the support material.

[0143] 32. The method according to any one of the preceding clauses, wherein multiple copies of the template are attached to the hub.

[0144] 33. The method according to any one of the preceding clauses, wherein the UF / DF of step (b) and / or step (c) is carried out using a membrane having a molecular weight cutoff (MWCO) of about 5 to about 50 kDa, about 10 to about 40 kDa, about 20 to about 40 kDa, or about 20 to about 30 kDa.

[0145] 34. The method according to any one of the preceding clauses, wherein the UF / DF of step (b) and / or step (c) is carried out using a membrane having a molecular weight cutoff (MWCO) of about 20 to about 40 kDa, optionally about 30 kDa.

[0146] 35. The method according to clause 33 or 34, wherein the membrane comprises polyethersulfone.

[0147] 36. The method according to any one of the preceding clauses, wherein the product comprises about 10-50, about 10-40, about 10-35, about 13-30, about 15-35, about 15-30, about 15-25, about 20-32, or about 20-23 nucleotides.

[0148] 37. The method according to any one of the preceding clauses, wherein the product comprises about 10-35 nucleotides, optionally about 15-35 nucleotides.

[0149] 38. The method according to any one of the preceding clauses, wherein the solution in step (a) comprises about 5 to about 60 mg / ml, about 10 to about 60 mg / ml, about 20 to about 60 mg / ml, about 30 to about 60 mg / ml, about 35 to about 55 mg / ml, or about 35 to about 45 mg / ml template.

[0150] 39. The method according to any one of the preceding clauses, wherein the solution of step (a) contains about 30 to about 60 mg / ml template, optionally about 35 to about 55 mg / ml template.

[0151] 40. The method according to any one of the preceding clauses, wherein the solution in step (a) comprises about 0.5 to about 8 mM, about 0.5 to about 7 mM, about 0.5 to about 6 mM, about 1 to about 8 mM, about 1 to about 7 mM, about 1 to about 6 mM, about 2 to about 8 mM, about 2 to about 7 mM, about 2 to about 6 mM, about 2 to about 5 mM, about 3 to about 7 mM, about 3 to about 6 mM, or about 3 to about 5 mM template.

[0152] 41. The method according to any one of the preceding clauses, wherein the solution of step (a) contains about 2 to about 7 mM template, optionally about 3 to about 6 mM template.

[0153] 42. The method according to any one of the preceding clauses, wherein the impurity is a short polymer or an intermediate.

[0154] 43. The method according to any one of the preceding clauses, wherein the UF / DF of step (b) and / or step (c) is carried out at about 20 to about 30 degrees Celsius, about 22 to about 28 degrees Celsius, about 24 to about 28 degrees Celsius, or about 25 to about 27 degrees Celsius.

[0155] 44. The method according to any one of the preceding clauses, further comprising: (d) The second permeate from step (c) is subjected to UF / DF to form a third retentate containing the product and substantially free of the second buffer solution, wherein the UF / DF is performed using water and a membrane having a molecular weight cutoff (MWCO) of about 1 kDa to about 5 kDa.

[0156] 45. The method according to Clause 44 further comprises lyophilizing the third retentate of step (d) to form a lyophilized composition comprising the product.

[0157] 46. ​​The method according to any one of the preceding clauses, further comprising: (e) The second retentate from step (c) is subjected to UF / DF to form a fourth retentate containing the template, wherein the UF / DF is performed using a third buffer solution with a pH of about 7-8.

[0158] 47. The method according to any one of the preceding clauses, wherein the template is recycled for future use.

[0159] 48. The method according to any one of the preceding clauses, wherein the product is synthesized via an enzyme-catalyzed ligation reaction.

[0160] 49. The method according to Clause 48, wherein the preparation of the solution in step (a) comprises the following steps: (a1) Remove enzymes from the ligation reaction by filtration to provide filtrate; (a2) Treat the filtrate with EDTA to provide a mixture; and (a3) Concentrate the mixture from step (a2) to provide the solution.

[0161] 50. The method according to any one of the preceding clauses, wherein the UF / DF is carried out at a cross-flow rate of about 2 to about 20 L / min / m2, about 2 to about 10 L / min / m2, or about 5 to about 7 L / min / m2.

[0162] 51. The method according to any one of the preceding clauses, wherein the purity of the product in the second permeate of step (c) is at least: about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%.

[0163] 52. The method according to any one of the preceding clauses, wherein the method is carried out on a gram scale or a kilogram scale or larger.

[0164] 53. The method according to any one of the preceding clauses, wherein the product comprises at least one main chain modification and / or at least one sugar modification and / or at least one base modification.

[0165] 54. A single-stranded oligonucleotide product obtained by the method according to any one of the preceding clauses.

[0166] 55. The single-chain oligonucleotide product as described in Clause 54, wherein the single-chain oligonucleotide product is a therapeutic oligonucleotide.

[0167] 56. The single-stranded oligonucleotide product as described in Clause 54 or Clause 55, wherein the single-stranded oligonucleotide product is a gapmer.

[0168] 57. The single-stranded oligonucleotide product as described in Clause 54, wherein the single-stranded oligonucleotide product forms part of a double-stranded oligonucleotide.

[0169] 58. The single-stranded oligonucleotide product as described in Clause 57, wherein the double-stranded oligonucleotide is siRNA.

[0170] Example

[0171] The exemplary methods described below (Examples 1 to 8) use illustrative oligonucleotide products, short polymers, and templates, the sequences of which are shown in Tables 1-3 below.

[0172] Table 1. Single-stranded antisense oligonucleotides, OLIGO-1

[0173] Table 1: Examples of chemical modifications Lowercase letters: nucleotides with ribose substituted with 2'-methoxyethyl (2'-MOE); Italicized capital letters: Nucleotides containing 2'-deoxyribose; -: Phosphodiester linkage; : Thiophosphate ester linkage; All cytidines (c, C All of them are 5-methyl substituted; Ureidine ( u () is 5-methyl substituted.

[0174] Table 2. Double-stranded siRNA - antisense strand, OLIGO-2

[0175] Table 3. Double-stranded siRNA - positive strand, OLIGO-3

[0176] Legends of chemical modifications in Tables 2 and 3: mX: nucleotide modified with 2'-O-methyl; fX: 2'-fluorine-modified nucleotide; invAb: no base in reverse; -: Phosphodiester linkage; : Thiophosphate ester linkage; GalNAc: The targeting portion containing N-acetylgalactosamine.

[0177] Example 1: Determining the optimal pH for separating OLIG-1, short-mers (6-mer / 7-mer), and intermediates (13-mer).

[0178] Using a tangential flow filtration (TFF) system Figure 1 Purifying oligonucleotides. The oligonucleotide solution to be purified is loaded into the feed container (1), and the solution is pumped into the flat membrane cartridge (2) by the feed pump (3), and then returned to the feed container by the pressure control valve (4). The permeate is removed by the permeate pump (5), and liquid is added by a further transfer pump (6) to maintain a constant recirculation volume. A sample of the retentate solution is collected from the feed container.

[0179] Experiment 1A

[0180] Experiments were conducted using the compositions shown in Table 4 below for input reaction mixtures.

[0181] Table 4: Input Compositions

[0182] The TFF system is equipped with a PES membrane cartridge with a MWCO of 30 kDa, prepared by washing with potassium phosphate solution (100 mM, pH 7). Tetrapotassium EDTA solution (approximately 11 g / L total oligosaccharide, approximately 5.5 g / L hub-template retentate) was added to a 100 mM Tris.HCl (pH 7.6) solution containing oligonucleotides, with EDTA at a 1.25 mol equivalent relative to magnesium in the input mixture. The solution was then fed into the system and recirculated through the cartridge at 26°C. Tripotassium phosphate buffer at 200 mM concentrations, with gradually increasing pH (9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, and 13.2), was pumped into the system at a flow rate matched to the permeate flow rate. Retentate samples were analyzed after every two permeate volumes, and the UV absorbance of the permeate was monitored using the TFF system instrument. See [link to TFF system instrumentation]. Figure 2 After every 10 perfiltration volumes, adjust the pH of the buffer solution upwards.

[0183] Experiment 1B

[0184] Experiments were conducted using the compositions shown in Table 5 below for input reaction mixtures.

[0185] Table 5: Input Compositions

[0186] The TFF system is equipped with a PES membrane cartridge with a MWCO of 30 kDa, prepared by washing with potassium phosphate solution (100 mM, pH 7). Tetrapotassium EDTA solution was added to a 100 mM Tris.HCl (pH 7.6) solution containing oligonucleotides (approximately 9.2 g / L total oligos, approximately 4.6 g / L hub-template retentate), with EDTA at a 1.25 mol equivalent relative to magnesium in the input mixture. The solution was then fed into the system and circulated through the cartridge at 26°C. Trisodium phosphate buffer at a concentration of 300 mM, with gradually increasing pH (9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0), was pumped into the system at a flow rate matched to the permeate flow rate. Retentate samples were analyzed after every two permeate volumes, and the UV absorbance of the permeate was monitored using the TFF system instrument. See [link to TFF system instrumentation]. Figure 3 After every 10 perfiltration volumes, adjust the pH of the buffer solution upwards.

[0187] Using tripotassium phosphate and trisodium phosphate, annealed short polymers (6-mer and 7-mer), intermediates (13-mer), and OLIGO-1 (20-mer) can be cleanly separated from the hub-template, which is repelled by the membrane and recovered in the retentate. Furthermore, the annealed short polymers and intermediates are removed from the retentate before the 20-mer product, providing a method for purifying the 20-mer product. Chromatography of the permeate solution shows the presence of the 20-mer product, the absence of the hub-template, and a decrease in the level of 13-mer compared to the input oligonucleotide solution.

[0188] This experiment demonstrates that oligonucleotides of different lengths and molecular weights can be separated using ultrafiltration at low temperatures (approximately 25°C). As shown above, the pH of the buffer used during ultrafiltration affects the level of separation. For example, perfiltration with a pH 11 buffer can separate oligonucleotides of lengths of 6, 7, and 13 nucleotides from the product (OLIGO-1, 20-mer) and the 6-template hub (containing 6 x 26-mers attached to the nucleus), and subsequent perfiltration with a pH 12 buffer can separate the product from the hub-template. Different oligonucleotide sequences may require different buffer strengths and pH conditions, which can be determined using the pH screening experiments described herein.

[0189] Example 2: Determining the optimal ionic strength for OLIG-1, short-mer (6-mer / 7-mer), and intermediate (13-mer) permeation.

[0190] Experiment 2A

[0191] The TFF system is equipped with a PES membrane cartridge with a MWCO of 30 kDa, prepared by washing with 100 mM Tris.HCl (pH 7.6). The input oligo composition is shown in Table 4 above. Tetrapotassium EDTA solution (total oligosaccharide concentration approximately 14 g / L, retentate concentration approximately 7 g / L hub-template) was added to a solution containing oligonucleotides in 100 mM Tris.HCl (pH 7.6), with EDTA at a 1.25 mol equivalent relative to magnesium in the input mixture. The solution was then fed into the system and recirculated through the cartridge at 26°C. Sodium hydroxide solution at gradually increasing concentrations (50 mM, 75 mM, 100 mM) was pumped into the system at a flow rate matched to the permeate flow rate. Once stable retentate pH and conductivity were achieved, retentate samples were analyzed, and the UV absorbance of the permeate was monitored using the TFF system instrument. Comparison of pH and conductivity of sodium hydroxide percolate retentate and HPLC analysis of retentate showed that oligonucleotides did not permeate significantly across the membrane, even though pH was always >12, and conductivity ranged from approximately 18 to 24 mS / cm.

[0192] Experiment 2B

[0193] The TFF system was equipped with a PES membrane cartridge with a MWCO of 30 kDa, prepared by washing with 100 mM Tris.HCl (pH 7.6). The input oligo composition is shown in Table 4 above. Tetrapotassium EDTA solution was added to a solution containing oligonucleotides in 100 mM Tris.HCl (pH 7.6) (total oligosaccharide concentration approximately 12 g / L, retentate concentration approximately 6 g / L hub-template), with EDTA at a 1.25 mol equivalent relative to magnesium in the input mixture. The solution was then fed into the system and recirculated through the cartridge at 26°C. Phosphate buffer (400 mM KH2PO4 / K2HPO4, pH 8) was pumped into the system at a flow rate matched to the permeate flow rate. After 8.5 permeate volumes, the retentate was sampled and analyzed, and the UV absorbance of the permeate was monitored using the TFF system instrument. The percolation buffer was then replaced with potassium tribasic phosphate (200 mM, pH 12.5). After each percolation volume, the retained material was sampled and analyzed, and the UV absorbance of the permeate was monitored using a TFF system. In this experiment, although it had been previously confirmed that high ionic strength (approximately 700–800 mM K+) could permeate unannealed single-stranded oligonucleotides, no oligonucleotide permeation was observed at pH 8. However, when the percolation buffer was replaced with potassium tribasic phosphate (200 mM, pH 12.5), 6-mer, 7-mer, 13-mer, and 20-mer oligonucleotides all permeated the membrane. See [link to relevant documentation] Figure 4 .

[0194] Experiment 2C

[0195] Two large-scale experiments were conducted using the compositions shown in Table 6 for the input reaction mixture.

[0196] Table 6: Input Compositions

[0197] The TFF system was equipped with a PES membrane cartridge (Pall cassette) with a MWCO of 30 kDa, prepared by washing with 100 mM Tris.HCl (pH 7.6). Tetrapotassium EDTA solution was added to 4.5 L of a solution containing oligonucleotides (approximately 80 g) in 100 mM Tris.HCl (pH 7.6), with the EDTA concentration being 1.25 mol equivalents relative to the magnesium content in the input mixture. A portion of this solution (approximately 0.9 L) was then fed into the system and recirculated through the cartridge at 26°C. The remaining oligo solution was pumped into the system at a flow rate matched to the permeate flow rate until all solution was consumed. At this point, the retentate concentration reached approximately 80 g / L total oligos, containing 40 g / L hub-template. The percolation buffer was then replaced with potassium tribasic phosphate (200 mM or 300 mM, pH 11.1). After every two percolation volumes, the retentate was sampled and analyzed, and the UV absorbance of the permeate was monitored using a TFF system. Once analysis showed that the 13-mer intermediate detected by HPLC in the retentate had decreased to <0.2% area (typically after 8-12 percolation volumes), the percolation buffer was replaced with sodium tribasic phosphate (200 mM or 300 mM, pH 12.0). After every two percolation volumes, the retentate was sampled and analyzed, and the UV absorbance of the permeate was monitored using a TFF system. Once analysis shows that the 20-mer product detected by HPLC in the retentate has decreased to <10% area (typically after 4–14 percolation volumes), the percolation buffer is replaced with 100 mM Tris.HCl at pH 7.6, and percolation continues until the pH of the retentate is approximately 7.8. The hub-template can then be recovered for reuse. These two large-scale experiments demonstrate that 6-mer, 7-mer, and 13-mer products can be selectively percolated to achieve separation from the 20-mer product. The yields and purities of these two experiments are shown in Table 7 below.

[0198] Table 7

[0199] Mole % yield based on hub-template charge = (moles of separated API / 6) / moles of HT Furthermore, increasing the concentration while maintaining the same pH to increase the ionic strength of the buffer solution, in turn, increases the permeation rate of the 13-mer intermediate. See also Figure 5 .

[0200] The UF / DF purification process also uses a Merck membrane (Merck Pellicon 3) instead of a Pall membrane cartridge, and the process performance is the same when using a Merck membrane as when using a Pall membrane cartridge.

[0201] Experiment 2D

[0202] The 20-mer product (OLIGO-1) obtained in Experiment 2C-2 was further processed to remove trace amounts of hub-template. The 20-mer product (3 g) was dissolved in trisodium phosphate buffer (2250 mL, 300 mM, pH 12.0) to prepare a solution of approximately 1.1 g / L oligonucleotides. The TFF system was equipped with a PES membrane cartridge with a MWCO of 30 kDa, prepared by washing with 300 mM trisodium phosphate (pH 12). A portion of this solution (approximately 0.25 L) was then fed into the system and recirculated through the cartridge at 26°C. The remaining oligo solution was pumped into the system at a flow rate matched to the permeate flow rate until all solution was consumed. Percolation was then performed, with 300 mM trisodium phosphate (pH 12) pumped into the system at a flow rate matched to the permeate flow rate, and the UV absorbance of the permeate was monitored using the TFF system instrument. Percolation continued until the UV signal of the permeate showed a low level of oligonucleotide percolation. HPLC analysis of the retentate showed retention of the hub-template and permeation of the 20-mer. See also Figure 6 The percolation buffer was replaced with 100 mM Tris.HCl (pH 7.6), and percolation was continued until the pH of the retentate was approximately 7.8, at which point the hub-template could be recovered for reuse. It was observed that as conductivity decreased, the UV signal indicating OLIGO-1 percolation also decreased rapidly.

[0203] The permeates obtained from ultrafiltration and pH 12 percolation were then combined and concentrated using ultrafiltration with a membrane of 3 kDa MWCO, followed by desalination using purified water via percolation. The resulting 20-mer product solution was then lyophilized.

[0204] As shown in Table 7 above, HPLC analysis revealed that the input material contained approximately 0.84% ​​hub-template area. This was completely removed by re-filtration through a 30 kDa membrane, yielding a 20-mer product with HPLC purity of 99.4% area. See Table 8.

[0205] Table 8: Input and output purity of reprocessed OLIGO-1

[0206] Example 3: Purification of crude OLIGO-1 prepared by solid-phase oligonucleotide synthesis

[0207] The TFF system was equipped with a PES membrane cartridge with a MWCO of 30 kDa, prepared by washing with 100 mM Tris.HCl (pH 7.6). A 20-mer (OLIGO-1) solution prepared via solid-phase oligo synthesis (containing 25.9 mg / mL total oligo in 14% wt aqueous ammonium hydroxide) was mixed with a hub-template solution (containing 35 mg / mL total oligo) to achieve a 1:1 ratio of 20-mer product to template. The solution was then fed into the TFF system and recirculated through the cartridge at 26°C. A first percolation was then performed, with 100 mM Tris.HCl (pH 7.6) pumped into the system at a flow rate matched to the permeate until eight percolation volumes were collected. After every two percolation volumes, samples were taken for analysis, and the UV absorbance of the permeate was monitored using the TFF system instruments. A second percolation was then performed, with 300 mM tripotassium phosphate (pH 11.1) pumped into the system at a flow rate matched to the permeate flow rate until 10 percolation volumes were collected. After every two percolation volumes, the retentate was sampled and analyzed, and the UV absorbance of the permeate was monitored using a TFF system. A third percolation was then performed, with 300 mM tripotassium phosphate (pH 12.0) pumped into the system at a flow rate matched to the permeate flow rate until 10 percolation volumes were collected. After every two percolation volumes, the retentate was sampled and analyzed, and the UV absorbance of the permeate was monitored using a TFF system. At the end of the experiment, purified water was used instead of the percolation buffer to desalinate the retentate, yielding an aqueous solution of the hub-template, which was then lyophilized. The permeates obtained from the pH 12 percolation were then combined and concentrated using an ultrafiltration membrane with a MWCO of 3 kDa, followed by desalination using purified water via percolation. The resulting 20-mer product solution was then lyophilized.

[0208] The first percolation with 100 mM Tris.HCl (pH 7.6) did not appear to remove any oligonucleotides from the retentate; the UV signal of the permeate did not increase, and the HPLC analysis showed no change.

[0209] When the percolation buffer was replaced with 300 mM tripotassium phosphate (pH 11.1), UV signals of the permeate showed some removal of oligos, which was confirmed by the loss of early elution impurities in the retentate.

[0210] Compared to the input oligonucleotide solution, the 20-mer area % in the isolated product increased by approximately 4% (from 87.8% to 91.9%) due to the complete removal of some early elution impurities and the reduction of other impurities.

[0211] Example 4: pH and buffer screening for OLIGO-3 purification

[0212] like Figure 1 The setup shown is a Cytia Akta Crossflow. The feed container contains the oligonucleotide solution to be purified. The solution is pumped into the membrane. Temperature is not controlled throughout the process and is kept at ambient temperature (approximately 25-27°C). Transmembrane pressure, conductivity, and UV absorbance are measured. Refractory and permeate samples are collected at fixed time intervals to track the filtration process. The refractory sample is removed from the refractory container, and the permeate sample is removed from the line flowing to the permeate collection container.

[0213] The reaction mixture containing OLIGO-3 (the product) was treated with 75 mM tetrapotassium EDTA solution (equivalent to 1.25 equivalences of MgCl2 charge in the enzymatic ligation reaction) to chelate magnesium ions from the enzyme. The resulting solution (10⁹ ml = 1 perfiltration volume) was added to the membrane system feed vessel.

[0214] After storing the 30 kDa membrane in 0.1 M NaOH, it was mounted on a membrane rack. The system was first flushed with deionized water until a decrease in conductivity was observed, and then flushed with 200 mM tribasic potassium phosphate at pH 8.5. A solution containing the oligonucleotide product in 100 mMtris (pH 7.6) was fed into the system. The solution was pumped into the system at a feed flow rate of 150 mL / min. The transmembrane pressure was set to 2 barg. Retrieval samples were collected at fixed time intervals for HPLC analysis. Different pH values ​​were screened from 8.5, 9.0, 9.5, 10.0, 10.5, and 11.0. The pH was adjusted based on the HPLC results. It was observed that the intermediate could not permeate the membrane before reaching pH 11.0. When screened again at pH 11, the product was observed to permeate the membrane, so the pH was lowered to 10.7. See the table below.

[0215] Filtration was also performed using 300 mM tribasic potassium phosphate with the pH adjusted to 11.1. Both the product and intermediates permeated through the membrane.

[0216] The product was separated from the hub-template by filtration using 200 mM sodium tribasic phosphate adjusted to pH 12.0. Samples of the retentate and permeate solutions were collected at fixed time intervals for HPLC analysis. The experiment was stopped after 25 permeate volumes. See Table 10.

[0217] Table 9.

[0218] Table 10.

[0219] Table 11.

[0220] In experiments using 200 mM potassium tribasic phosphate (pH 10.7), good separation of short polymers and intermediates was observed, along with good retention of the product and hub-template. Using 200 mM sodium tribasic phosphate at pH 12.0, the product was successfully separated from the template. See Table 10.

[0221] Oligonucleotides of different lengths and molecular weights can be separated using ultrafiltration. As shown above, conditions such as buffer strength and pH affect the level of separation. Experiments showed that segmental oligonucleotides (short polymers of 7 nucleotides in length) and 14-mer intermediates can be separated from the product (OLIGO-3, 21-mer oligonucleotide) and the hub-template (containing 6 x 27-mers attached to a solid support), and subsequently, the product and hub-template separate from each other at appropriate buffer strength and pH.

[0222] Example 5: pH and buffer screening for OLIGO-2 purification

[0223] Input solution reaction mixture

[0224] The oligonucleotide solution reaction mixture (300 mg scale synthesis of OLIGO-2) is a 40 mL reaction solution containing 437 mg hub-template and an estimated 272.70 mg product 21-mer oligonucleotide (OLIGO-2).

[0225] The analysis of the input material is shown in Table 12 below, which shows the area percentage of the identified oligo component peaks. Approximately 4% short polymers and 4% intermediates remain.

[0226] Table 12.

[0227] The reaction mixture was treated with 75 mM tetrapotassium EDTA solution (equivalent to 1.25 equivalences of MgCl2 charge in the enzymatic ligation reaction) to chelate magnesium ions.

[0228] Percolation 1

[0229] The TFF system is equipped with a PES membrane capsule with a MWCO of 30 kDa and is prepared by washing with 250 mM tribasic potassium phosphate (pH 10.5).

[0230] Pour 150 mL of oligonucleotide infusion solution (approximately 6 mg / mL total oligos and approximately 2.9 mg / mL hub-template) into the reservoir. Homogenize the feed solution at a flow rate of 150 mL / min (approximately 8 L / min / m²). Once homogenized, apply a transmembrane pressure of 2 bar.

[0231] Using 250 mM tribasic potassium phosphate percolation buffer, the pH of the buffer was increased from 10.5 to 10.8 in 0.1 increments to determine the pH conditions for percolation of the major impurities, followed by the pH conditions for percolation of the 21-mer product. The retentate volume was kept constant throughout the percolation process.

[0232] The retentate solution was sampled and analyzed at fixed time intervals, with the retentate volume remaining constant throughout the process. The results are shown in Table 1. The percolation volume defined in Table 14 was maintained for each buffer condition. The UV absorbance of the permeate was monitored using a TFF system.

[0233] Percolation 2

[0234] Keep the retentate volume, TMP, flow rate, and membrane constant in percolation 1.

[0235] The buffer solution was switched to 200 mM sodium tribasic phosphate at pH 12.0 and continued for 6 percolation volumes. The sampling intervals and results are shown in Table 13.

[0236] Once approximately 90% of the product (21-mer oligonucleotide) has been observed to have permeated, stop adding sodium phosphate buffer and switch to deionized water to desalt the retentate in order to accurately quantify the remaining oligo species.

[0237] Table 13—HPLC results of the retentates from percolation 1 and percolation 2

[0238] Percolation 3

[0239] (A) Concentration and desalination of 200 mM sodium phosphate permeate (from filtration 2) at pH 12.0

[0240] Equipped with a PES (0.1 m) with a MWCO of 1 kDa. 2 (Surface area) membrane. The system and cartridge were prepared by washing with 200 mM tribasic sodium phosphate buffer at pH 12.0. A flow rate of 450 mL / min and a transmembrane pressure of 2 bar were used. The permeate containing the product (21-mer oligonucleotide) at pH 12.0 was concentrated and then desalted with deionized water for accurate quantification of total oligonucleotides and impurity analysis (see Table 14).

[0241] (B) Concentration and desalination of 250 mM potassium phosphate permeate (from percolation 1) at pH 10.8

[0242] Significant loss of the product (21-mer oligonucleotide) was observed in the permeate at pH 10.8. To adequately quantify this loss, the permeate was concentrated and desalted for accurate quantification of total oligonucleotides and impurity analysis (see Table 14). The same process steps / conditions as in (A) above were followed.

[0243] Table 14—Purity Analysis and Yield

[0244] The data in Table 13 show that, using 250 mM tripotassium phosphate (pH 10.6–10.7), the annealed 7-mer segment and the intermediate 14-mer can be separated from the hub-template. Most of the 21-mer product and the hub-template remain in the annealed state and are repelled by the membrane. At pH 10.8 with 250 mM tripotassium phosphate, the 21-mer product permeates significantly.

[0245] 200 mM sodium tribasic phosphate effectively separated the remaining annealed 21-mer product and facilitated its permeation through the membrane, which was quantified by SoloVPE and purity analysis. Overall, the permeates at pH 10.8 and pH 12.0 contained 68.89% of the 21-mer product with a purity >90%. ~9.4% (remaining peak area) of the 21-mer product was retained in the retentate from percolate 2 with a hub-template.

[0246] Experiments have shown that the segmental oligonucleotides (short polymers of 7 nucleotides in length) and the 14-mer intermediate can be separated from the product oligonucleotides (OLIGO-2, 21-mer oligonucleotides) and the hub-template (containing 6 x 27-mers attached to a solid support), and the product and hub-template can then be separated from each other at appropriate buffer strength and pH.

[0247] Example 6: Purification of single-stranded antisense oligonucleotide (OLIGO-1)

[0248] Ultrafiltration / difiltration (UF / DF) experiments were conducted using an ÄKTA crossflow tangential flow filtration (TFF) system (Cytiva) and a Pall Centramate T-series cartridge with an Omega PES membrane, with the molecular weight cutoff (MWCO) as described below. The TFF system was operated within the manufacturer-specified cartridge range; the crossflow rate was 5–7 L / min / m², and the maximum transmembrane pressure (TMP) was 4 barg. The pH and conductivity of the permeate were measured using instruments integrated into the ÄKTA crossflow system. The pH and conductivity of the retentate were measured using a Mettler-Toledo Seven Compact Duo S213 pH meter and conductivity meter.

[0249] HPLC method: Column: Waters XBridge peptide BEH C18 300A, 3.5 μm, 2.1 mm x 150 mm; Column temperature: 50 °C; Mobile phase A: 5 mM TBuAA and 1 μM EDTA dissolved in 90:10 %v / v water:acetonitrile; Mobile phase B: 5 mM TBuAA and 1 μM EDTA dissolved in 20:80 %v / v water:acetonitrile; Flow rate: 0.5 mL / min; Gradient: time (min) / MPB% , 0 / 35, 18 / 71, 25 / 95, 25.1 / 35, 35 / 35; UV detection wavelength: 260 nm; Injection volume: 7 μL.

[0250] The product (OLIGO-1) solution obtained from the enzymatic ligation reaction (approximately 4.5 L, containing approximately 40 g of 6-mer, 7-mer, 13-mer (intermediate), and 20-mer (product) oligonucleotides annealed in 100 mM tris.HCl (pH 7.6) with approximately 40 g of hub (hub-template) (1 equivalent, 1 wt) containing six 26-mer template oligonucleotides) was filtered to remove the enzyme.

[0251] Step 1: The combined filtrate was mixed with 75 mM tetrapotassium EDTA solution (equivalent to 1.25 equivalences of MgCl2 charge in the enzymatic ligation reaction) to chelate magnesium ions. A portion of the resulting EDTA-treated filtrate (approximately 1 L, this volume = 1 percolation volume, which determines the hub-template concentration in the retentate, with a target of 40 mg / mL) was added to the TFF system, and the remaining EDTA-treated filtrate was then concentrated by ultrafiltration at approximately 25–27 °C using a polyethersulfone (PES) membrane with a MWCO of 30 kDa until the volume was approximately 0.9–1.0 L, thus achieving a total oligo concentration in the retentate of approximately 80 g / L.

[0252] Step 2: Then, perform a first percolation at 25-27°C with 300 mM tripotassium phosphate (pH 11.1). Continue percolating for 10 percolation volumes, and perform HPLC analysis on the retentate at 2-percolation volume intervals to monitor the removal of 6-mer, 7-mer, and 13-mer. Stop the operation when HPLC analysis shows that the level of 13-mer in the retentate has decreased to less than 0.2% area. Approximately 10-20 percolation volumes or 10-20 L of buffer solution are required.

[0253] Step 3: At 25-27°C, perform a second percolation on the permeate from the first percolation using 300 mM sodium tribasic phosphate (pH 12.0). Continue percolating for 4 percolation volumes, and perform HPLC analysis on the retentate at 2-percolation volume intervals to monitor product removal. Stop the operation when HPLC analysis shows that the product level in the retentate has decreased to approximately 8-12% of the area. Approximately 4-14 percolation volumes or 4-14 L of buffer solution are required.

[0254] Step 4: Perfusing the retentate from the second perfusing at 25-27°C with 100 mM tris-HCl buffer (pH 7.6). Continue perfusing for approximately 6 perfusing volumes until the pH of the retentate drops to 7.6-7.8. Concentrate the retentate solution containing the hub-template to approximately 0.5 L.

[0255] Step 5: Optionally, the permeate from the second perfiltration can be further purified to remove residual hub-template. A portion of the permeate from the second perfiltration (0.9–1.0 L, this volume = 1 perfiltration volume) is added to the TFF system, and the remaining permeate is concentrated by ultrafiltration using a polyethersulfone (PES) membrane with a MWCO of 30 kDa at approximately 25–27 °C. During this operation, the product passes through the membrane, and the residual hub-template remains in the retentate. A fourth perfiltration is performed with 300 mM sodium tribasic phosphate (pH 12.0) to recover the product from the retentate. Perfiltration continues for 10–12 perfiltration volumes, and the operation is stopped when the product concentration in the retentate is below 1 mg / mL.

[0256] Step 6: Add a portion of the combined permeate from ultrafiltration and percolation in Step 5 (0.9-1.0 L, this volume = 1 percolation volume, which determines the product concentration in the retentate) to the TFF system. Then, concentrate the remaining combined permeate by ultrafiltration at approximately 25-27°C using a polyethersulfone (PES) membrane with a MWCO of 3 kDa until the product concentration in the retentate reaches 25-50 mg / mL. Then, percolate with purified water to remove trisodium phosphate from the retentate. Continue percolation until the permeate conductivity is below 0.05 mS / cm. Approximately 14-24 percolation volumes are required. Concentrate the product solution until the retentate volume reaches approximately 0.5 L (the product concentration is approximately 40-50 mg / mL based on optical density (OD)).

[0257] A schematic diagram of the purification process for the -20-mer product is shown below. Figure 7 As shown. The resulting product solution was an aqueous solution of the sodium salt (HPLC purity >99%), with a yield of 65-75% based on the input amount of the starting materials (6-mer / 7-mer) for the enzymatic ligation reaction.

[0258] Example 7 Purification of single-stranded positive oligonucleotide (OLIGO-3)

[0259] The product (OLIGO-3) solution obtained from the enzymatic ligation reaction (22 ml reaction volume, diluted to 100 ml with 100 mM tris (pH 7.6), containing approximately 198 mg of 7-mer, 14-mer (intermediate), and 21-mer (product) oligonucleotides annealed in 100 mM tris·HCl (pH 7.6) with approximately 212 mg of hub (hub-template) containing six 27-mer template oligonucleotides) was filtered to remove the enzyme.

[0260] Step 1: Treat the combined filtrate from the enzyme removal step with 75 mM tetrapotassium EDTA solution (equivalent to 1.25 equivalences of MgCl2 charge in the enzymatic ligation reaction) to chelate magnesium ions from the enzyme. Add the resulting solution (109 ml = 1 perfiltration volume) to the membrane system feed vessel.

[0261] Step 2: At approximately 25-27°C, perform the first percolation using a polyethersulfone (PES) membrane with a molecular weight cutoff (MWCO) of 30 kDa and 200 mM tribasic potassium phosphate adjusted to pH 10.7 with phosphoric acid. Continue percolation until HPLC shows that the levels of 7-mer and 14-mer in the filtrate have decreased to <1% area. Perform HPLC analysis at approximately two percolation volumes to monitor impurity removal. Approximately 8-12 percolation volumes are required.

[0262] Step 3: At approximately 25-27°C, perform a second percolation on the permeate obtained from the first percolation using 200 mM sodium tribasic phosphate (pH adjusted to 12.0 with phosphoric acid). Continue percolating for 10-15 percolation volumes, sampling and analyzing the residue at two-percolation-volume intervals to monitor product removal. Stop the operation when HPLC analysis shows that the content of the remaining product has decreased to approximately 2-12% of the area. Approximately 10-15 percolation volumes or 1000-1500 ml of buffer solution is required.

[0263] Step 4: Percolate the retentate obtained from the second percolation with purified water at approximately 25-27°C for a third time. Continue percolation until the conductivity of the retentate decreases to <0.05 mS / cm. Retain 100 ml of the retentate containing the hub-template for reuse in the ligation reaction.

[0264] Step 5: Optionally, the permeate from the second perfiltration can be further purified to remove residual hub-template. Add a portion of the permeate from the second perfiltration (100 ml, this volume = 1 perfiltration volume) to the TFF system, and then concentrate the remaining permeate by ultrafiltration at approximately 25-27°C using a polyethersulfone (PES) membrane with a MWCO of 30 kDa. During this operation, the product passes through the membrane, and the residual hub-template remains in the retentate. Perform a fourth perfiltration with 200 mM sodium tribasic phosphate (pH 12.0) to recover the product from the retentate. Continue perfiltration for 10-15 perfiltration volumes, stopping the operation when the product concentration in the retentate is below 1 mg / mL.

[0265] Step 6: Add a portion of the combined permeate from ultrafiltration and percolation in Step 5 (100 ml, this volume = 1 percolation volume, which determines the product concentration in the retentate) to the TFF system. Then, concentrate the remaining combined permeate by ultrafiltration at approximately 25-27°C using a polyethersulfone (PES) membrane with a MWCO of 1 kDa until the retentate volume reaches 1-2 mg / ml. Then, percolate with purified water to remove sodium tribasic phosphate from the retentate. Continue percolation until the permeate conductivity is below 0.05 mS / cm. Approximately 29-38 percolation volumes are required. Concentrate the product solution until the retentate volume reaches 1 vol (the product concentration is approximately 1.7 mg / mL based on OD).

[0266] The resulting product solution was an aqueous solution of sodium salt (HPLC purity >84% area), with a yield of 63% based on the input amount of the starting material (7-mer) for the enzymatic ligation reaction.

[0267] Example 8 Purification of single-stranded antisense oligonucleotide (OLIGO-2)

[0268] The product of the enzymatic ligation reaction (OLIGO-2) solution, approximately 40 ml reaction volume, contains approximately 273 mg of 7-mer, 14-mer (intermediate), and 21-mer (product) oligonucleotides annealed in 100 mMtris-HCl buffer at pH 7.6 with approximately 437 mg of hub-template (1 equivalent, 1 wt) containing six 27-mer template oligonucleotides and a hub core. The solution is centrifuged to remove the enzyme.

[0269] Step 1: Treat the filtrate from the enzyme removal step with 75 mM tetrapotassium EDTA solution (equivalent to 1.25 equivalences of MgCl2 charge in the enzymatic ligation reaction) to chelate magnesium ions from the enzyme. Dilute the resulting solution with 80 mL of 100 mM Tris (pH 7.6) and add it to the membrane system feed vessel. After necessary washing, the final volume is (150 mL = 1 perfiltration volume).

[0270] Step 2: At approximately 25-27°C, perform the first percolation using a polyethersulfone (PES) membrane with a molecular weight cutoff (MWCO) of 30 kDa and 250 mM tribasic potassium phosphate adjusted to pH 10.7 with phosphoric acid. Continue percolation until HPLC analysis shows that the levels of 7-mer and 14-mer in the filtrate have decreased to <1% area. Perform HPLC analysis at approximately two percolation volumes to monitor impurity removal. Approximately 8-12 percolation volumes are required.

[0271] Step 3: Perform a second percolation at approximately 25-27°C using 200 mM sodium tribasic phosphate adjusted to pH 12.0 with phosphoric acid. Continue percolating for 10-15 percolation volumes, sampling the residue at two-percolation-volume intervals to monitor product clearance. Stop the process when HPLC analysis shows the retained product level has decreased to approximately 5-15% of the area. Approximately 10-15 percolation volumes or 1500-2250 ml of buffer solution are required.

[0272] Step 4: Perform a third percolation with deionized water at approximately 25-27°C to desalinate the hub-template in the retentate. Continue percolation until the conductivity is <0.05 mS / cm. Retain the retentate containing the hub-template for reuse.

[0273] Step 5: Add a portion of the permeate from Step 3 (100 ml, this volume = 1 percolation volume, which determines the product concentration in the retentate) to the TFF system, and then concentrate the remaining permeate by ultrafiltration at approximately 25-27°C using a polyethersulfone (PES) membrane with a MWCO of 1 kDa until the product concentration in the retentate reaches 1-2 mg / ml. Then percolate with purified water to remove sodium tribasic phosphate from the retentate. Continue percolation until the permeate conductivity is below 0.05 mS / cm. Approximately 29-38 percolation volumes are required. Concentrate the product solution until the retentate volume reaches 1 vol (the product concentration is approximately 1.88 mg / mL based on OD).

[0274] The resulting product solution was an aqueous solution of sodium salt (HPLC purity >90%), with a yield of 68.9% based on the input amount of the starting material (7-mer) for the enzymatic ligation reaction.

[0275] Example 9: Determining the optimal ionic strength for permeation of OLIGO-4 short-term polymers (6-mer / 7-mer) and intermediates (25-mer / 26-mer)

[0276] Example 9 used illustrative oligonucleotide products, short polymers, and templates, the sequences of which are shown in Table 15 below.

[0277] Table 15. Single-stranded antisense oligonucleotides, OLIGO-4

[0278] Table 15: Examples of chemical modifications Lowercase letters: nucleotides with ribose substituted with 2'-methoxyethyl (2'-MOE); Italicized capital letters: Nucleotides containing 2'-deoxyribose; -: Phosphodiester linkage; : Thiophosphate ester linkage; All cytidines (c, C All of them are 5-methyl substituted; Ureidine ( u () is 5-methyl substituted.

[0279] Experiment 9A

[0280] Experiments were conducted using the compositions shown in Table 16 below for input reaction mixtures.

[0281] Table 16: Input Compositions

[0282] The TFF system was equipped with a PES membrane cartridge with a MWCO of 30 kDa, conditioned by washing with 200 mM tribasic potassium phosphate (pH 11.21). The input oligo composition was 4.39 mg / mL total oligos, with a retentate concentration of 1.04 mg / mL hub-template. The oligo solution was fed into the system and recirculated through the cartridge at 25°C. Gradually increasing concentrations (200 mM, 250 mM, 300 mM) of pH 11.21 tribasic potassium phosphate buffer were pumped into the system at a flow rate matched to the permeate flow rate. Retentate samples were analyzed every three percolation volumes, and the UV absorbance of the permeate was monitored using the TFF system instrument. The ionic strength of the buffer was increased every 12 percolation volumes. It was observed that short-mer impurities were completely removed after 6 perfiltration volumes using a lower ionic strength buffer (200 mM tribasic potassium phosphate, pH 11.21), while removal of intermediates required a higher ionic strength buffer (300 mM tribasic potassium phosphate, pH 11.21). Once HPLC analysis showed that the 25-mer intermediate ('5'-short-mer + 'intermediate-short-mer' + 'intermediate-short-mer' + 'intermediate-short-mer') in the retentate decreased to <0.5% area (typically after 12 perfiltration volumes), the perfiltration buffer was switched to a higher pH tribasic potassium phosphate buffer (300 mM, pH 11.88). Retentate samples were analyzed every 2 or 3 perfiltration volumes, with UV absorbance monitored using a TFF system. Once HPLC analysis shows that the 32-mer OLIGO-4 product in the retentate has decreased to <2% area (typically after 12–18 percolation volumes), replace the percolation buffer with purified water to desalinate the hub-template. Pump purified water into the system at a flow rate matched to the permeate flow rate until the retentate conductivity is 43.7 µS / cm. Collect the hub-template from the TFF system and store it at -5°C. Concentrate the permeate containing the 32-mer OLIGO-4 collected from the percolation process to 0.79 mg / mL using an ultrafiltration membrane with a MWCO of 3 kDa, and desalinate it by pumping purified water into the system at a flow rate matched to the permeate flow rate until the retentate conductivity is 41.9 mS / cm. Collect the full-length product from the TFF system and store it at -5°C.

[0283] Experiments show that 6-mer, 7-mer, and 25-mer can selectively permeate to achieve separation from the 32-mer OLIGO-4 product. The purities obtained in this experiment are shown below (Table 17).

[0284] Table 17: Product Compositions

[0285] Experiment 9B

[0286] Experiments were conducted using the compositions shown in Table 18 below for input reaction mixtures.

[0287] Table 18: Input Compositions

[0288] The TFF system was equipped with a PES membrane cartridge with a MWCO of 30 kDa, conditioned by washing with 300 mM tribasic potassium phosphate (pH 11.31). The input oligo composition was maintained at 4.39 mg / mL total oligos and a retentate concentration of 1.42 mg / mL hub-template. The oligo solution was fed into the system and recirculated through the cartridge at room temperature (25°C). Tribasic potassium phosphate buffer (300 mM, pH 11.31) was pumped into the system at a flow rate matched to the permeate flow rate. Retentate samples were analyzed after every three percolation volumes, and the UV absorbance of the permeate was monitored using the TFF system instrument. It was observed that short-polymer impurities were completely removed after three percolation volumes. Once HPLC analysis shows that the 25-mer ('5'-short polymer + 'intermediate polymer' + 'intermediate polymer' + 'intermediate polymer') and 26-mer ('5'-short polymer + 'intermediate polymer' + 'intermediate polymer' + '3'-short polymer) intermediates in the retentate have decreased to <1.0% area (typically after 44 percolation volumes), the percolation buffer is replaced with tribasic potassium phosphate buffer (300 mM, pH 11.95). Retentate samples are analyzed every 3 or 4 percolation volumes, with UV absorbance monitored using a TFF system. Once HPLC analysis shows that the 32-mer OLIGO-4 product in the retentate has decreased to <15% area (typically after 48 percolation volumes), the percolation buffer is replaced with purified water to desalinate the hub-template. Purified water is pumped into the system at a flow rate matched to the permeate flow rate until the retentate conductivity is 31.7 mS / cm. Hub-templates were collected from the TFF system and stored at -5°C. The permeate containing 32-mer OLIGO-4 was concentrated to 0.69 mg / mL via ultrafiltration using a membrane with a MWCO of 3 kDa, and desalinated by pumping purified water into the system at a flow rate matched to the permeate flow rate until the retentate conductivity reached 20.8 mS / cm. 32-mer OLIGO-4 was collected from the TFF system and stored at -5°C.

[0289] Experiments show that 6-mer, 7-mer, 25-mer, and 26-mer can selectively permeate to achieve separation from the 32-mer OLIGO-4 product. The purities obtained in this experiment are shown below (Table 19).

[0290] Table 19: Product Compositions

Claims

1. A method for purifying a product, wherein the product is a single-stranded oligonucleotide, the method comprising: (a) Obtaining a solution comprising (i) an impurity: a template complex and (ii) a product: a template complex, wherein the template is an oligonucleotide comprising a sequence complementary to the product. (b) The solution is subjected to ultrafiltration / difiltration (UF / DF) to form a first retentate comprising the product: template complex and a first permeate comprising the impurities, wherein the UF / DF is performed using a first buffer solution with a pH of about 9-13. (c) Perform UF / DF on the first retentate from step (b) to form a second retentate containing the template and a second permeate containing the product, wherein the UF / DF is performed using a second buffer solution with a pH greater than that of the first buffer solution.

2. The method of claim 1, wherein the pH of the second buffer solution is about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9 or about 2.0 higher than the pH of the first buffer solution.

3. The method according to claim 1 or 2, wherein the pH of the first buffer solution is about 9-12.5, about 9-12, about 9-11.5, about 9-11, about 10-12.5, about 10-12, about 10-11.5, about 10-11, or about 10.5-11.

5.

4. The method according to any one of claims 1 to 3, wherein the pH of the second buffer solution is about 10-14, about 10-13.5, about 10-13, about 11-14, about 11-13.5, about 11-13, about 11-12.5, about 11-12, or about 11.5-12.

5.

5. The method according to any one of the preceding claims, wherein the first buffer solution and / or the second buffer solution has a conductivity of about 25 mS / cm to about 120 mS / cm, about 25 mS / cm to about 100 mS / cm, about 25 mS / cm to about 80 mS / cm, about 25 mS / cm to about 60 mS / cm, about 30 mS / cm to about 100 mS / cm, or about 30 mS / cm to about 80 mS / cm.

6. The method according to any one of the preceding claims, wherein the first buffer solution and / or the second buffer solution comprises a phosphate buffer solution.

7. The method according to any one of the preceding claims, wherein the first buffer solution and / or the second buffer solution comprises potassium phosphate.

8. The method according to any one of the preceding claims, wherein the first buffer solution and / or the second buffer solution comprises sodium phosphate.

9. The method according to any one of the preceding claims, wherein the first buffer solution comprises potassium phosphate and the second buffer solution comprises sodium phosphate.

10. The method according to any one of the preceding claims, wherein the first buffer solution and / or the second buffer solution further comprises an ionic strength modifier.

11. The method of claim 10, wherein the ionic strength modifier is a cation, optionally a sodium ion or a potassium ion.

12. The method according to any one of the preceding claims, wherein the first buffer solution and / or the second buffer solution comprises about 200 mM to about 1500 mM, about 200 mM to about 1200 mM, about 300 mM to about 1500 mM, about 300 mM to about 1200 mM, about 400 mM to about 800 mM, or about 600 mM to about 900 mM of cations, optionally sodium or potassium ions.

13. The method according to any one of the preceding claims, wherein the template exists in a form that allows the template to remain in the second retrieval.

14. The method according to any one of the preceding claims, wherein the template is attached to the support material.

15. The method according to any one of the preceding claims, wherein the UF / DF in step (b) and / or step (c) is performed using a membrane having a molecular weight cutoff (MWCO) of about 5 to about 50 kDa, about 10 to about 40 kDa, about 20 to about 40 kDa, or about 20 to about 30 kDa.

16. The method of claim 15, wherein the membrane comprises polyethersulfone.

17. The method according to any one of the preceding claims, wherein the product comprises about 10-50, about 10-40, about 10-35, about 13-30, about 15-35, about 15-30, about 15-25, about 20-32, or about 20-23 nucleotides.

18. The method according to any one of the preceding claims, wherein the solution in step (a) comprises about 5 to about 60 mg / ml, about 10 to about 60 mg / ml, about 20 to about 60 mg / ml, about 30 to about 60 mg / ml, about 35 to about 55 mg / ml, or about 35 to about 45 mg / ml of template.

19. The method according to any one of the preceding claims, wherein the impurity is a short polymer or an intermediate.

20. The method according to any one of the preceding claims, wherein the UF / DF of step (b) and / or step (c) is performed at about 20 to about 30 degrees Celsius, about 22 to about 28 degrees Celsius, about 24 to about 28 degrees Celsius, or about 25 to about 27 degrees Celsius.

21. The method according to any one of the preceding claims, further comprising: (d) The second permeate from step (c) is subjected to UF / DF to form a third retentate containing the product and substantially free of the second buffer solution, wherein the UF / DF is performed using water and a membrane having a molecular weight cutoff (MWCO) of about 1 kDa to about 5 kDa.

22. The method of claim 21, further comprising lyophilizing the third retentate of step (d) to form a lyophilized composition comprising the product.

23. The method according to any one of the preceding claims, further comprising: (e) The second retentate from step (c) is subjected to UF / DF to form a fourth retentate containing the template, wherein the UF / DF is performed using a third buffer solution with a pH of about 7-8.

24. The method according to any one of the preceding claims, wherein the product is synthesized by an enzyme-catalyzed ligation reaction.

25. The method of claim 24, wherein the preparation of the solution in step (a) comprises the following steps: (a1) The enzyme from the ligation reaction is removed by filtration to provide a filtrate; (a2) Treat the filtrate with EDTA to provide a mixture; and (a3) Concentrate the mixture from step (a2) to provide the solution from step (a).

26. The method according to any one of the preceding claims, wherein the UF / DF is carried out at a cross-flow rate of about 2 to about 20 L / min / m2, about 2 to about 10 L / min / m2, or about 5 to about 7 L / min / m2.

27. The method according to any one of the preceding claims, wherein the purity of the product in the second permeate of step (c) is at least: about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%.

28. A single-stranded oligonucleotide product obtained by the method according to any one of the preceding claims.

Images